stem cells and cancer

 STEM
CELLS
AND
CANCER
7th International
Heinrich F. C. Behr-Symposium
ABSTRACTS
organized by
2012
October 14 –16, 2012
German Cancer Research Center (DKFZ), Heidelberg
STEM
CELLS
AND
CANCER
7th International
Heinrich F. C. Behr-Symposium
October 14 –16, 2012
German Cancer Research Center (DKFZ), Heidelberg
Jointly organized by the German Cancer Research Center (DKFZ), Heidelberg, the Heidelberg Institute for Stem Cell Technology (Hi-STEM), Heidelberg,
the National Cancer Institute (NCI), Bethesda, and the German Academy of
Sciences Leopoldina, Halle/Saale.
2012
ABSTRACT BOOK
1
CONTENTS
ABSTRACTS OF LECTURES (in alphabetical order of speakers)
BARBACID, Mariano............................................................................................................................... 9
BEACHY, Philip...................................................................................................................................... 10
BRABLETZ, Thomas..............................................................................................................................11
CLARKE, Michael F. ..............................................................................................................................12
CLEVERS, Hans C.,................................................................................................................................13
ENVER, Tariq.......................................................................................................................................... 14
FRENETTE, Paul S. ...............................................................................................................................15
FUCHS, Elaine........................................................................................................................................ 16
JAMIESON, Catriona ............................................................................................................................. 17
KNOBLICH, Jürgen A. ...........................................................................................................................18
LIU, Hai-Kun ..........................................................................................................................................19
LYDEN, David C. ...................................................................................................................................20
MARTIN-VILLALBA, Ana........................................................................................................................ 21
MCKAY, Ronald ....................................................................................................................................22
MÜSCHEN, Markus............................................................................................................................... 24
PARADA, Luis F. ...................................................................................................................................25
RAFII, Shahin.........................................................................................................................................26
ROTTER, Varda...................................................................................................................................... 27
SHEN, Michael M. ................................................................................................................................28
TLSTY, Thea...........................................................................................................................................29
TRUMPP, Andreas.................................................................................................................................30
UTIKAL, Jochen..................................................................................................................................... 31
WERNIG, Marius...................................................................................................................................32
WITTBRODT, Joachim...........................................................................................................................33
WITTE, Owen..........................................................................................................................................34
3
ABSTRACTS OF POSTERS (in alphabetical order of authors)
ABERGER, Fritz...................................................................................................................................... 37
AGNUSDEI, Valentina............................................................................................................................38
AHMAD, Aftab.......................................................................................................................................39
ARANDA-ORGILLES, Beatriz.................................................................................................................40
BAGERITZ, Josephine........................................................................................................................... 41
BARTELS, Susanne...............................................................................................................................42
BEYELER, Sarah....................................................................................................................................43
CABEZAS-WALLSCHEID, Nina..............................................................................................................44
CANNISTRACI, Alessio..........................................................................................................................45
CARNEVALLI, Larissa............................................................................................................................46
CHENG, Xinlai........................................................................................................................................ 47
CINGOZ, Ahmet.....................................................................................................................................48
COHEN-KUPIEC, Rachel........................................................................................................................49
COOLEN, Marion...................................................................................................................................50
DE ANGELIS, Maria Laura.................................................................................................................... 51
DRIESSENS, Gregory............................................................................................................................52
EHNINGER, Armin.................................................................................................................................53
4
EIBL, Robert..........................................................................................................................................54
FANG, Liang...........................................................................................................................................55
FASSL, Anne..........................................................................................................................................56
FENG, Weijun......................................................................................................................................... 57
FILATOVA, Alina.....................................................................................................................................58
FORSTER, Tobias...................................................................................................................................59
FRANCES, Daniela................................................................................................................................60
GEISELHART, Anja................................................................................................................................ 61
GOOSSENS, Steven...............................................................................................................................62
HIDE, Takuichiro....................................................................................................................................64
JÄGLE, Sabine.......................................................................................................................................65
KALASAUSKAS, Darius.........................................................................................................................66
KEITEL, Ulrike........................................................................................................................................67
KIM, Ella L..............................................................................................................................................68
KRAFT, Florian.......................................................................................................................................69
LAMERS, Marcelo.................................................................................................................................70
LAUXEN, Isabel...................................................................................................................................... 71
LE MAGNEN, Clémentine......................................................................................................................72
LEE-THEDIECK, Cornelia.......................................................................................................................73
LEVINGER, Irit........................................................................................................................................ 74
LIER, Amelie..........................................................................................................................................75
LIU, Li..................................................................................................................................................... 76
MADDOX, Jessica.................................................................................................................................. 77
MALLM, Jan-Philipp..............................................................................................................................78
MARHOLD, Maximilian.........................................................................................................................79
MATEO, Juan L.......................................................................................................................................80
MCCORKINDALE, Ali............................................................................................................................. 81
MEHBOOB, Riffat..................................................................................................................................82
MEIDHOF, Simone.................................................................................................................................83
MELE, Valentina....................................................................................................................................84
MILANOVIC, Maja..................................................................................................................................85
MOSSNER, Maximilian..........................................................................................................................86
MÜKUSCH, Susanne.............................................................................................................................88
MURARO, Manuele Giuseppe..............................................................................................................89
PASSARO, Diana...................................................................................................................................90
PASTÒ, Anna......................................................................................................................................... 91
PINAZZA, Marica...................................................................................................................................92
PIROZZI, Giuseppe................................................................................................................................93
POPOV, Boris.........................................................................................................................................94
PRECA, Badjak, BOGDAN, Karolina.....................................................................................................95
RIGO-WATERMEIER, Teresa.................................................................................................................96
RÖDLING, Lisa....................................................................................................................................... 97
RÖNSCH, Kerstin...................................................................................................................................98
SALVATI, Valentina................................................................................................................................99
SKODA, Jan..........................................................................................................................................100
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SPECKER, Elisa...................................................................................................................................101
TESIO, Melania....................................................................................................................................102
THAM, Katrin.......................................................................................................................................103
THAMM, Stefanie................................................................................................................................104
UCKELMANN, Hannah........................................................................................................................105
VOLOSHANENKO, Oksana..................................................................................................................106
VONG, Joaquim....................................................................................................................................107
WALTER, Dagmar................................................................................................................................108
WARAKY, Ahmed................................................................................................................................109
WERNER, Fee......................................................................................................................................110
WITTE, Ines..........................................................................................................................................111
WOLF, Jonas........................................................................................................................................112
ZHANG, Ruiyan....................................................................................................................................113
ZHANG, YiYao......................................................................................................................................114
6
ABSTRACTS OF LECTURES
7
Identification of cancer initiating cells in K-Ras oncogene-driven lung adenocarcinoma
Mariano Barbacid, Chiara Ambrogio, Sara Mainardi and David Santamaría
Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
K-RAS oncogenes are involved in about one quarter of lung adenocarcinomas, one of the tumor
types with worst prognosis. We have developed a mouse model that faithfully recapitulates the
natural history of this tumor by inserting an oncogenic G12V mutation in the endogenous K-Ras
locus. Expression of the resulting K-RasG12V oncogene in this model can be temporal and spatially controlled by the presence of a bacterial Cre recombinase (Guerra et al., Cancer Cell, 2003).
Surprisingly, systemic expression of this endogenous oncogene in adult mouse tissues only led to
malignant transformation in lungs. These observations have prompted us to interrogate: (i) the nature of the lung cells susceptible to K-RasG12V transformation and (ii) the molecular bases for their
susceptibility.
First of all, we monitored proliferation of individual lung cells two to four weeks following expression of the resident K-RasG12V oncogene by 4OHT mediated activation of an ubiquitously expressed CreERT2 recombinase. A percentage of K-RasG12V expressing type II alveolar (SPC+) and
Clara (CC10+) cells, as well as an occasional double SPC/CC10+ BASC cell, proliferated for a few
rounds of cell division. However, only the K-RasG12V expressing SPC+ type II alveolar cells displayed
sustained proliferation leading to the generation of hyperplastic areas, followed by the appearance
of adenomas and adenocarcinomas. Interestingly, we also observed occasionally hyperplastic areas of CC10+ cells but only in mice in which expression of the resident K-RasG12V oncogene was mediated by traqueal infection with Cre-expressing adenoviral particles. Thus, suggesting that under
conditions in which the Cre recombinase is preferentially delivered to the CC10+ cells that lined the
bronquiols, they can also be occasionally transformed by K-Ras oncogenes.
Interestingly, the sensitivity of SPC+ type II alveolar cells to K-Ras oncogenes depends on their
developmental stage. Expression of the resident K-RasG12V oncogene during embryonic development in a mouse that expresses a Cre recombinase under a SCA1 promoter leads to the development of CC10+, but not SPC+ hyperplastic areas. The limited survival of these mice (3 months)
has prevented us to determine whether these CC10+ areas will eventually generate more advance
lesions such as adenomas or adenocarcinomas. Again, no lesions were observed in tissues other
than lungs. These observations indicate that lung is the most susceptible tissue to direct transformation by K-Ras oncogenes. Yet, the nature of cell type most susceptible to transformation varies
depending on their developmental stage.
Next, we have isolated early SPC+ hyperplastic lesions (about 500 cells) present in adult mice
and analyze them by gene expression profiling. Surprisingly, these areas, in spite of being morphologically indistinguishable, display two distinct expression profiles designated as H1 and H2 with
similar incidence. Whereas the H1 signature has significant similarities to that of normal areas not
expressing the K-RasG12V oncogene, the H2 signature correlates better with the gene expression
profiling of aggressive human lung adenocarcinomas (Sweet-Cordero et al, Nat. Genetics, 2005).
Detail analysis of these signatures has revealed a subset of genes expressed in H1 lesions that are
silenced in those areas with a H2 signature. We are currently using functional genomics to analyze
the role of representative genes for each signature during tumor development.
9
Hedgehog signaling in regeneration and malignancy
Kunyoo Shin, Agnes Lim, Sally Kawano and Philip A. Beachy
Institute for Stem Cell Biology and Regenerative Medicine, Standford University, Standford, California, USA
The idea that human malignancies may originate from adult tissue stem cells derives from the
intrinsic ability of stem cells to self-renew, from their longevity and consequent ability to accrue
multiple mutations, and from the phenotypic resemblance of tumor-propagating cells to stem cells.
Experimental tests of this hypothesis have relied on cell-specific genetic manipulation to induce
cancer in murine models, and such studies have produced varying conclusions regarding the cancer cell-of-origin.
We have recently identified the urothelial stem cell through lineage tracing and organoid culture,
and have identified some of the signals that control stem cell proliferative activity. Based on these
findings, we examine here the origin of bladder cancer by prospectively marking and tracking these
stem cells in a murine model, selected for its similarity to human carcinogenesis, in which invasive
bladder carcinomas are induced by prolonged exposure to nitrosamine. In this model, we find that
Shh-expressing stem cells in the basal urothelium give rise to the carcinoma, although Shh expression within tumor cells is lost by the time carcinomas develop. This malignant transformation
progresses through epithelial hyperplasia, a precursor lesion consisting of a thickened urothelium
comprising many layers of cytokeratin 5 (CK5)-expressing basal-like cells. Shh expression occurs in
basal but not lumenal layers of this hyperplastic lesion, and these basal cells give rise to the Shhnegative tumor-propagating cells of the full-fledged carcinoma.
10
Confirming that tumor-propagating cells derive from basal stem cells but not more differentiated lumenal cells, we showed that prior ablation of Shh-expressing cells rendered the bladder
resistant to nitrosamine-induced formation of epithelial hyperplasia or carcinoma. Our findings thus
demonstrate that invasive carcinoma is initiated from the basal urothelial stem cell and typically
progresses through a hyperplastic lesion, thus confirming experimentally the view that such lesions
are not only associated with but actually give rise to epithelial cancers. Our findings further suggest
that the phenotypic properties of mature tumor cells, such as absence of Shh expression, may not
provide a sound basis for inferring the identity of the cancer cell-of-origin.
MicroRNAs, EMT and cancer stem cells
Thomas Brabletz
Department of Visceral Surgery and Comprehensive Cancer Center, University of Freiburg, Freiburg, Germany
We have shown, that in particular tumor cells at the invasive front undergo an epithelial-mesenchymal transition (EMT) and aberrantly express EMT-associated transcriptional repressors, such
as ZEB1. The amount of such cells strongly correlates with metastasis formation and poor clinical
outcome. Strikingly, metastases show again a differentiated phenotype, indicating a mesenchymalepithelial re-transition (MET) and support a regulatory role of the tumor environment in metastasis.
We described that the EMT-activator ZEB1 is a crucial promoter of metastasis and demonstrated that ZEB1 inhibits expression of cell polarity factors and the microRNA-200 family, whose
members are strong inducers of epithelial differentiation. These results indicate that ZEB1 triggers
a microRNA-mediated feedback-loop, which stabilizes EMT and promotes dissemination of cancer
cells. Moreover we detected that in addition ZEB1 is necessary for the tumor initiating capacity of
pancreatic and colorectal cancer cells. ZEB1 inhibits expression of miR-200c, miR-203 and miR183, which cooperate to suppress expression of stem cell factors.
We propose that ZEB1 links EMT-activation and stemness-maintenance by suppressing stemness-inhibiting microRNAs and thereby is a promoter of mobile, migrating cancer stem cells with
high phenotypic plasticity. Notably, these cells also acquired a drug-resistance phenotype. Our data
show that breaking the ZEB1 – miR-200 feedback loop is a treatment option for fatal tumors such
as pancreatic cancer, and epigenetic modifications identified at ZEB1 target genes indicate a promising way how to interfere.
11
Deregulation of stem cell self renewal pathways in cancer
Michael F. Clarke
Karel H. and Avice N. Beekhuis Professor in Cancer Biology; Associate Director, Stanford Institute for Stem Cell Biology
and Regenerative Medicine, Stanford, California, USA
Most common cancers, such as cancers of the breast and colon, arise in organs such as the
breast that contain a small population of stem cells that constantly replenish the mature cells of the
tissue. Stem cells are defined by the ability to divide and give rise to a new stem cell (self-renewal),
as well as the ability to give rise to the differentiated cells of an organ, and thus are the only longlived cell population in many tissues. We will present data that the cancer stem cell compartment
of estrogen receptor positive and estrogen receptor negative breast tumors differs.
Identification of stem cell compartments enables the identification of molecular pathways. We
find Bmi-1 is one of the most important players in the maintenance of self-renewal of stem cells
in many adult tissues, including brain, breast and bone marrow. Bmi1 has also been shown to be
important for the maintenance of cancer and cancer stem cells in many tumors. We identified modifier of Bmi1, which is highly expressed in non self renewing progenitor cells, but it is expressed at
low levels in normal stem cells. We found that this modifier can antagonize the function of Bmi-1
in the bone marrow, by reactivating the expression of downstream genes with tumor-suppressor
functions, such as p16INK or p19Arf.
Interestingly, the human Bmi1-modifier is located on chromosome 21, and it is expressed at abnormal high levels in cells from Down Syndrome (DS) patients, which contain three copies (trisomy)
of chromosome 21. In line with our expectations, bone marrow samples from human DS patients
show a reduced number of hematopoietic stem cells. Moreover, DS mice have stem cell defects
in both the bone marrow and the breast. The DS mice have reduced numbers of stem cells with
reduced ability to proliferate in vitro. Since DS patients develop considerably less solid tumors than
expected, we hypothesize that modifiers of Bmi1 could reduce the self-renewal potential not only
of normal stem cells, but also cancer stem cells, providing a protection from tumor development.
Indeed, preliminary data show that this is the case.
12
Lgr5 stem cells in self-renewal and cancer
Hans Clevers
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University Medical Centre Utrecht, Utrecht, The
Netherlands
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using an inducible
Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages
throughout life, implying that it represents the stem cell of the small intestine and colon. Similar
obserations were made in hair follicles and stomach epithelium.
Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids in 3D culture.
Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in these organoids.
We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from
a single stem cell in the absence of a non-epithelial cellular niche. The same technology has now
been developed for the Lgr5+ve stomach stem cells.
Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in
most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other
crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of
adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived
adenomas, lending support to the “cancer stem cell”-concept.
Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed that, as
a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of
1-6 months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a
model in which the resident stem cells double their numbers each day and stochastically adopt
stem or TA fates after cell division. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth cells are
CD24+ and express EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell
maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves
organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt.
Genetic removal of Paneth cells in vivo results in the concomitantloss of Lgr5 stem cells. In colon
crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents.
We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized
daughter cell, the Paneth cell.
13
Genetic and epigenetic sources of heterogeneity in childhood leukaemia
Tariq Enver
Stem Cell Laboratory, UCL Cancer Institute, University College London, London, UK
Relapses after therapy-induced complete clinical remissions remain the most significant challenge in cancer therapy. This suggests that a proportion of cancer cells at presentation, escape
therapy and persist during remission. These cells presumably are the source of relapse. Why are
these cells chemoresistant? We argue that the answer lies in a combination of genetic and epigenetic heterogeneity acting to some degree at the level of ‚cancer stem‘ or ‚tumor propagating‘ cells.
We have obtained evidence in support of this conceptual framework for cancer resistance in the
context or childhood ALL, the commonest cancer of children. Our results encourage a re-positioning
of the cancer stem cell concept as it relates to disease in patients.
14
Neural regulation of stem cell migration in health and cancer
Paul Frenette
Albert Einstein College of Medicine, New York, USA
Hematopoietic stem cell (HSC) migration from and in the bone marrow is regulated by autonomic
nerves from the from the sympathetic nervous system (SNS) that release norepinephrine acting on
beta-adrenoreceptors expressed on the niche cells, thereby controlling the expression retention
factors that regulate stem cell behavior. Further studies have revealed that self-renewing perivascular mesenchymal stem cells (MSC), targeted by the SNS, represent a candidate niche cell that
can be prospectively identified by transgenic expression of GFP under the Nestin promoter. While
SNS signals downregulate the expression of genes that retain HSC in the bone marrow, bone marrow CD169+ macrophages have the opposite effect; they promote the expression of HSC retention
factors by nestin+ niche cells.
Recent data suggest that SNS nerves also regulate expression of adhesion molecules on bone
marrow sinusoids, thereby controlling hematopoietic cell homing. Under steady state, homing and
egress are regulated by circadian oscillations of the sympathetic tone which promote HSC egress
predominantly in the morning in mice, whereas HSC homing is more efficient at night. These observations may have important implications for clinical transplantation of cord blood cells in which the
number of stem cells are limiting. We will also discuss recent results on potential parallels between
the neural influence of HSC migration and cancer cell migration in xenogeneic and transgenic models of prostate cancer. Ultimately, these studies will lead to new insight on the cellular and molecular cues that regulate the tumor microenvironment and allow cancer cells to spread.
15
Stem cells in homeostasis and cancer
Elaine Fuchs
Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065 USA
Stem cells have the ability to self-renew long term and differentiate into one or more tissues.
Typically, stem cells are used sparingly to replenish cells during normal homeostasis. This is particularly true of adult tissues that undergo relatively infrequent or periodic turnover. However, even
stem cells that are quiescent must be able to respond quickly to injury in order to fuel rapid tissue
regeneration. How stem cells balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Moreover, increasing
evidence suggests that the regulatory circuitry governing this balancing act is at the root of some
types of tumors both in mice and in humans.
The skin is an excellent model system to understand how stem cells function in normal tissue
generation and how this process goes awry in cancer. Using skin as our paradigm, we’ve been dissecting how extrinsic signaling to stem sets off a cascade of changes in transcription that governs
the activation of stem cells during tissue development, homeostasis and hair regeneration. Our
findings have provided us with new insights into our understanding of the process of stem cell
activation, and in so doing have revealed mechanisms which are also deregulated in a variety of
different human cancers. As importantly in understanding how stem cells are activated is learning
about the signals that instruct stem cells to stop making tissue.
16
Our recent discoveries on this topic have led us to the realm of identifying cancer stem cells
(tumor-initiating cells) of squamous cell carcinomas of the skin. The second most abundant form of
cancer world-wide, skin squamous cell carcinomas also establish a paradigm for many life-threatening cancers of lung, esophagus, breast, cervix, prostate, throat and oral tissues. We’ve demonstrated that when purified and introduced directly into the skin of a host recipient, a single cancer
stem cell from a skin squamous cell carcinoma can generate a new squamous cell carcinoma that
is similar in properties to the parent tumor. We show that cancer stem cell numbers and aggressiveness are predicated on the tumor microenvironment and on the ability of cancer stem cells to
respond to it. The striking differences between the microenvironments of cancer stem cells and
their normal counterparts seems likely to contribute to why their expression profiles are also markedly distinct. Our findings have major implications for our understanding of stem cells and cancer.
This work was supported by the HHMI and grants from the National Institutes of Health and The Emerald Foundation.
Development of cancer stem cell targeted therapy
Daniel J. Goff, Angela Court Recart, Anil Sadarangani, Alice Y. Shih, Christian L. Barrett, Hye-Jung
Chun, Maryla Krajewska, Wenxue Ma, Heather Leu, Jun Wei, DayongZhai, Ifat Geron, Qingfei
Jiang, Ryan Chuang, Larisa Balaian, Jason Gotlib, Mark Minden6, Giovanni Martinelli, Annelie
E. Schairer, Jessica Rusert, Kim-Hien Dao, Sheldon R.Morris, Thomas J. Hudson, Lawrence
S.B.Goldstein, Kristen M. Smith, Marco Marra, Kelly A. Frazer, Maurizio Pellecchia, John C. Reed,
and Catriona H.M. Jamieson
University of California, Department of Medicine, Division of Hematology-Oncology, San Diego, USA
Human blast crisis leukemia stem cells (BC LSC) evolve from myeloid progenitors that co-opt
stem cell properties such as dormancy, enhanced survival and self-renewal in protective niches.
These BC LSC play a pivotal role in chronic myeloid leukemia (CML) therapeutic resistance and blast
crisis (BC) transformation, in part, through alternative splicing of self-renewal and survival genes. To
elucidate splice isoform regulators of human BC LSC maintenance, we performed whole transcriptome RNA sequencing; splice isoform-specific qRT-PCR, nanoproteomics, stromal co-culture and BC
LSC xenotransplantation analyses.
Previous studies showed that alternative splicing of GSK3b enhances b-catenin and SHH signaling leading to increased self-renewal and survival of myeloid progenitors. Recent RNA Seq analysis demonstrated upregulation of JAK2, isoforms of STAT5A and multiple pro-survival BCL2 family
genes that promote malignant reprogramming of myeloid progenitors into self-renewing LSC that
are quiescent in the marrow niche and contribute to BC transformation. Moreover, enhanced RNA
editing was found to contribute to splice isoform diversity and LSC self-renewal during blastic transformation. Notably, a selective SHH inhibitor reduced BC LSC quiescence in the niche; a JAK2/
FLT3 inhibitor decreased BC LSC self-renewal in RAG2-/-gc-/- mice; and a novel pan-BCL2 inhibitor,
sabutoclax, renders BC LSC sensitive to TKIs atdoses that spare normal progenitors.
These findings underscore the importance of alternative splice isoform expression in the maintenance of BC LSC and suggest that niche targeted inhibition of self-renewal and pro-survival proteins
may represent a key component of therapeutic strategies to eliminate dormant cancer stem cells.
17
Using Drosophila as a model system for tumorigenesis and cancer stem cells
Juergen Knoblich
IMBA – Institute of Molecular Biotechnology; Vienna, Austria
Stem cells are characterized by their ability to generate both self-renewing and differentiating
daughter cells. We use the fruit fly Drosophila as a model system to study how this difference is
established and how disturbing the balance between self-renewal and differentiation can lead to
tumor formation.
In Drosophila neural stem cells, the asymmetric segregation of the fate determinants Numb,
Prospero and Brat restricts self-renewal potential to only one daughter cell and ensures a constant
stem cell pool. In mutants affecting those determinants or their asymmetric segregation, stem
cells overproliferate in an uncontrolled manner, giving rise to gigantic brain tumors. Brain tumors
are lethal and can be transplanted into other animals where they become aneuploid and undergo
metastasis.
The sophisticated genetic tools available in Drosophila allow us to study the process of tumor
formation at a level of detail that is not possible in other organisms. We can generate individual mutant stem cells and then precisely follow the individual steps leading to tumorigenesis. We find that
tumor formation is initiated by a block of differentiation in the transit amplifying compartment of the
mutant stem cell lineage. As a consequence, a differentiation checkpoint initially prevents mitotic
proliferation. Eventually, however, the checkpoint breaks down and the resulting de-differentiated
cells start proliferating in an uncontrolled manner and no longer respond to the signals that control
cell cycle progression in wild type animals. Our analysis indicates that these changes in cellular
behavior are irreversible and are triggered purely by epigenetic mechanisms suggesting a major
change in what is currently believed to promote tumor progression.
18
Genetic dissection of self-renewal and differentiation in normal and malignant brain stem cells
Hai-Kun Liu
Helmholtz Young Investigator Group, Normal and Neoplastic CNS Stem Cells, German Cancer Research Center (DKFZ),
the DKFZ-ZMBH Alliance, Heidelberg, Germany
Self-renewal and differentiation are essential for hemostasis of normal and malignant tissue.
I will discuss how the self-renewal and differentiation processes are regulated in normal and malignant neural stem cells. First I will discuss that the chromatin remodeler CHD7 (chromodomain-helicase–DNA-binding protein 7), which is mutated in human CHARGE syndrome, is a master regulator
of neurogenesis in the mammalian brain. CHD7 is selectively expressed in neurogenic niches in the
adult mouse brain, and inactivation of CHD7 in mouse neural stem cells leads to impairment of neuronal differentiation and maturation. CHD7 expression marks a class of human brain tumors with
a neuronal differentiation molecular signature. We further identified direct target genes of CHD7
which are responsible for the CHD7 mutant phenotype. These results demonstrate the essential
role of CHD7 in neuronal differentiation of NSCs, thus providing strong implications for CHARGE
syndrome and brain tumors.
The second part of my talk will focus on targeting self-renewal in brain tumor stem cells. We
recently established a novel mouse model allows gene targeting in unperturbed high-grade mouse
brain tumors. Using a GFP reporter driven by promoter of the nuclear receptor tailless, which is a
neural stem cell specific transcription factor, we demonstrate that the Tlx positive cells are mostly
quiescent in primary tumors and they are strong candidate for brain tumor stem cells. By introducing a stem cell specific mutation of Tlx gene in the primary mouse tumors, we observe loss of selfrenewal of brain tumor stem cells, which leads to significant prolongation of animal survival. We
further demonstrated that loss of Tlx in brain tumor stem cells leads to induction of essential tumor
suppressor pathways. These results strongly support that there is a cellular hierarchy in primary
brain tumors and the self-renewal pathways are suitable targets for glioma therapy.
19
Tumor-derived exosomes educate bone marrow progenitor cells toward a
pro-metastatic phenotype by upregulation of MET oncoprotein
Héctor Peinado, Masa Aleckovic, Mary S. Brady, Jedd D. Wolchok, Paul B. Chapman, Yibin Kang,
Jacqueline Bromberg, David Lyden
Weill Cornell Medical College, New York, NY, USA
Tumor-derived exosomes are emerging as potential mediators of tumorigenesis. Here we show
that exosomes from highly metastatic melanoma cells increased the metastatic behavior of primary
tumors by educating bone marrow (BM) progenitor cells upregulating MET expression, while exosomes from low metastatic melanoma cells did not alter the incidence of metastases. Melanomaderived exosomes induced vascular leakiness at pre-metastatic sites and promoted BM progenitor
cell education towards a pro-vasculogenic phenotype c-Kit+Tie2+MET+ in the BM. Importantly, we
describe that MET expression is upregulated in circulating BM progenitor cells (CD45-CD117+ and
CD45-CD202B+) isolated from stage III and stage IV melanoma patients. In addition, we found
Rab1a, Rab5b, Rab7, and Rab27a to be highly expressed in melanoma. Rab27a RNA interference
decreased exosome production in melanoma cells, thereby preventing mobilization of BM progenitor cells, tumor growth and metastasis. Finally, we identified a “melanoma signature” in exosomes
isolated from melanoma patients, consisting of TYRP2, VLA-4, Hsp70, an Hsp90 isoform and MET
proto-oncogene, which together with Rab proteins may represent exosome-specific proteins with
prognostic and therapeutic potential.
20
CD95 in tumor initiation and progression
Ana Martin-Villalba
Division of Molecular Neurobiology, German Cancer Research Center (DKFZ), Heidelberg, Germany
Recent studies have provided solid evidence for the presence of a subpopulation of cancer stem
cells (CSCs) in malignant skin, colon and brain tumors. These studies define CSCs as a restricted
population of cells in charge of fueling the tumor that exhibit self-renewal ability and the potential
to differentiate into non-stem cell population of tumor cells. Thus, CSCs and their niche appear as
attractive therapeutic targets to eliminate the repository potential of the tumor. Our lab has identified CD95 as a functional surface marker of adult neural stem cells. This receptor is also found in
a subpopulation of tumor cells within brain tumors and its activation increases their tumorigenic
potential. The talk recapitulates the findings leading to the controversy surrounding manipulation
of the CD95 pathway for cancer therapy and sheds some light on the function of CD95 in normal
and cancer-stem cells.
21
Stem cell technologies & human functional genomics
Ron McKay
Lieber Institute for Brain Development, Baltimore, Maryland, USA
Over the last two decades it has become clear that cells expanded in the laboratory can be used
to generate many, perhaps all, of the cell types in the adult body. Studies on the nervous system
played a prominent role in this epic. The founding observations of the laboratories of Nieuwkoop
and Benzer demonstrated that stem cells generate the nervous system in vivo. Advances in molecular biology allowed the identification of these cells first in vivo and then to the surprising report that
these cells could proliferate and form neurons in the laboratory, in vitro (Frederiksen and McKay,
1988; Lendahl et al., 1990; Cattaneo and McKay, 1990). This was followed by the observation
that neural stem cells and their progeny, neurons and glia, could be efficiently generated from the
most primitive pluripotent embryonic stem cells (Okabe et al., 1996; Brustle et al., 1998; Kim et al.,
2002). Similar approaches generated functional pancreatic and liver cells from both mouse and human pluripotent sources (Lumelsky et al., 2001; Shim et al., 2007; Woo et al., 2012). The high-level
functions of these laboratory generated cells has sparked a growing interest in their use to replace
and restore injured tissues.
There is also great interest in using stem cell based technologies to define disease mechanisms
and identify new pharmaceutical therapies. Our current focus is to develop a deeper understanding
of the mechanisms causing psychiatric disorders using stem cell technologies. The developmental
origin of psychiatric disturbance is established by identification of risk alleles that alter the expression of messenger RNAs expressed in the fetal brain. The concepts and techniques of stem cell biology uniquely give us access to the functional consequences of genetic differences between human
cells during brain development.
22
In principle, pluripotent stem cells provide a powerful platform to link variant genomes and epigenomes to a precise functional effect. However, the extraordinary diversity of the human genome
may make this functional genomic analysis unexpectedly challenging. New insight into how altered
genomes perturb development will only be realized if the genetic and epigenetic status of the cells
is precisely controlled. As a result of our emphasis on quality control, datasets generated by our
group have been used as the gold-standard by multiple groups to qualify new human pluripotent
cells and define their developmental origin. Rigorous quality control can be achieved by applying
the powerful new technologies that to characterize pluripotent cells. We have generated data-sets
on large numbers of human ES and induced pluripotent stem cells. Genetic information has been
obtained using Illumina 1M SNP and DNA methylation 27K beadchips, array comparative genomic
hybridization. DNase hyper-sensitivity assays. Epigenetic and gene expression data is being acquired using sequencing of the DNase hypersensitive sites, ChIP-seq is being used to define the
chromatin state and Illumina paired-end RNA-seq analysis is being used to quantitively define gene
expression levels. These data show that human pluripotent cells can be sustained in a stable state
allowing precise comparison over time.
These high dimensional tools provide extraordinary power and we are using them to focus
on the core developmental mechanisms that establish the human central nervous system: 1. the
transition from the pluripotent state to the somatic neural stem cells that generate the brain, 2.
the lineage processes that generate neurons and glia from neural stem cells and 3. the signaling
mechanisms that link groups of neurons into active circuits.
Our current studies suggest the same molecular device regulates the differentiation of pluripotent stem cells, reprogramming adult cells to the pluripotent state and differentiating neural stem
cells to neurons and glia. By identifying a new pluripotent cell type in the mouse embryo, our labora-
tory showed that two distinct epigenetic states support pluripotency and that one of these, the EpiS
cell, shared defining features with human ES and iPS cells (Tesar et al., 2007; Najm et al., 2011).
RNA-Seq analysis of mouse ES and EpiS cells shows that from this early embryonic stage to the
adult brain, a conserved transcriptional regulatory system controls neural differentiation. We are
developing new imaging tools and cellular assays to determine how differences in human genomes
lead to altered brain circuits.
A deeper understanding of the mechanisms acting from the beginning of embryonic life through
the period when neural circuits are laid down is required if we are to understand how genetic risk
factors lead to altered brain function in later life. These technologies will allow unprecedented
insight into the mechanisms underlying risk of psychiatric dysfunction and suggest new pharmacological therapies to treat brain disorders. An intriguing possibility is that targeting adult neural stem
cells may enhance physiologic function. We have exciting data suggesting that new therapies with
powerful regenerative effects emerge from understanding the mechanisms controlling neural stem
cells (Androutsellis-Theotokis et al., 2009). Even though this prospect may seem ambitious, it is the
natural extension of developing routine technologies to control neural stem cells.
1. Androutsellis-Theotokis et al., Proc. Nat‘l. Acad. Sci. 106:13570 2009
2. Brustle O. et al., Science 285:754 1999
3. Cattaneo E. and McKay R, Nature 347:762 1990
4. Frederiksen K. and McKay R. J. Neuroscience 8:1144 1988
5. Kim et al., Nature 418:50 2002
6. Lendahl. et al., Cell 60:585 1990
7. Lumelsky et al., Science 292:1389 2001
8. Najm et al., Cell Stem Cell 8:318 2011
9. Okabe S. et al., Mechs. Dev. 59: 89 1996
10. Shim et al., Diabetologia 50:1228 2007
11. Tesar et al., Nature 448:196 2007
12. Woo et al., Gastroenterology 142:602 2012
23
Oncogenes and negative feedback in leukemia
Markus Müschen
University of California, San Francisco, USA
Correspondence: E-mail: [email protected], www.lymphoblasts.org
Background: Negative feedback signaling molecules have been shown to limit proliferation. For
instance, DUSP6 and SPRY2 attenuate signaling from surface receptors and thereby function as
tumor suppressors. Here we studied mechanisms of negative feedback signaling in tyrosine kinasedriven (TKD) malignancies.
Results: Studying gene expression changes by microarray analysis, RT-PCR and Western blot,
we found that the DUSP6 and SPRY2 negative feedback regulator molecules are highly expressed
in a wide array of TKD malignancies including solid tumors, myeloid and B cell lineage leukemia. In
contrast to TKD leukemia, Non-TKD tumors lack expression of these genes and DUSP6 and SPRY2
promoters are hypermethylated in Non-TKD tumors. To study the function of DUSP6 and SPRY2 in
a genetic mouse model of TKD-leukemia, we transduced bone marrow pre-B cells from DUSP6-/-,
SPRY1/2-fl/fl mice and respective wildtype controls with retroviral BCR-ABL1. Defects in one of
these negative feedback mediators caused profound signaling imbalances in TKD leukemia cells.
For instance, Dusp6-deficient TKD-leukemia cells rapidly underwent cellular senescence. Lack of
Dusp6 and Spry2 dramatically increased cellular ROS. Owing to excessively high levels of ROS,
TKD-leukemia cells lacking one of these negative feedback mediators failed to initiate colonies in
semisolid agar and failed to initiate leukemia in transplant recipient mice. Inducible activation of
Cre-mediated deletion of Spry1/2 in leukemia cells resulted in rapid cell death.
24
To test whether negative feedback signaling molecules represent a potential target for pharmacological inhibition in the treatment of TKD-leukemia, we tested the Dusp6 small molecule inhibitor
2-benzylidene-3-(cyclohexylamino)-1-Indanone hydrochloride (BCI). At 3μmol/l, BCI induces massive accumulation of ROS and subsequent cell death in 5 patient-derived cases of Ph+ ALL including
two cases with T315I mutation. To test in vivo efficacy of BCI, patient-derived Ph+ ALL cells with
T315I were xenografted into NOD/SCID recipient mice. Mice were treated ten times with either
vehicle or 25 mg/kg BCI (i.p.). In agreement with in vitro results, treatment with BCI resulted in
significant prolongation of overall survival compared to standard TKI-treatment (BCI vs TKI Nilotinib
p=0.01).
Conclusion: Our studies identify negative feedback mediators including DUSP6 and Spry2 as
a novel therapeutic target in TKD-leukemia and TKD solid tumors. Pharmacological inhibition of
negative feedback signaling represents a fundamentally novel and powerful approach to increase
oncogenic signaling beyond a tolerable threshold, thus causing excessive accumulation of ROS and
subsequent cell death. Normal cells lacking oncogenic tyrosine kinase signaling are insensitive to
inhibition of negative feedback signaling.
Genetic mouse models of glioma: Translational tools for therapeutic development
Luis F. Parada
Department of Developmental Biology & Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve
Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Malignant astrocytomas are incurable, locally infiltrative brain tumors for which conventional
anticancer therapy has failed to make significant improvement in prognosis. Although the full spectrum of molecular events that drives tumor initiation and progression has yet to be defined, TCGA
data has revealed frequent mutation in several tumor-relevant genes. We have developed mouse
models of glioma with conditional inactivation of three of the five most frequently mutated genes
in glioma: p53, Nf1, and Pten. These mice develop tumors that histologically and molecularly resemble human astrocytomas with 100% penetrance. These tumors arise from a population of neural stem/progenitor cells that can be propagated in culture with high efficiency, and importantly,
without evidence of undergoing crisis or massive selection. We utilized these tumor-derived cells
for an unbiased, large-scale chemical compound screen to identify compounds that could block
proliferation of these cells. This screen identified a number of interesting hits, many of which have
high activity and specificity for the tumor-derived stem cells. We are currently performing in-depth
characterization of these compound candidates, including target identification, with priority given
to those compounds with the best pharmacokinetic profiles.
25
Angiocrine factors-derived from tumor-specified vascular niche induce and
maintain tumor initiating cells
Shahin Rafii
Weill Cornell Medical College, HHMI, New York, NY, USA
Interaction of tumor cells with their niche cells is essential for initiation and maintenance of
tumor initiating cells. However, the mechanism by which niche cells regulate the progression of
tumor initiating cells remains unknown. We have set forth the concept that endothelial cells (ECs)
within the tumor microenvironment comprise of specialized vascular cells that are not just passive
conduits to deliver oxygen and nutrients. By contrast, tumor ECs establish an instructive vascular
niche, which by elaboration of paracrine factors, known as angiocrine factors, directly promote tumor growth. We have demonstrated that activation of Akt-mTOR pathway in ECs stimulates expression of angiocrine factors that induce expansion of hematopoietic, hepatic, and lung epithelial stem
and progenitor cells. Specifically, angiocrine expression of Jagged-1 and IGFBP2 by marrow ECs
prompted expansion of authentic hematopoietic stem cells (1,2), while angiocrine expression by
liver sinusoids of Wnt2 and hepatocyte growth factor induces hepatic regeneration (3) and MMP14
deployment by lung ECs stimulated the reconstitution of alveolar epithelial cells (4). These data establish the remarkable tissue-specific angiocrine and vascular heterogeneity in orchestrating stem
and progenitor cell expansion that could be applied to tumor initiating cells as well.
26
Therefore, to generate ECs that phenocopy the instructive function of tumor vessels, we have
generated generic naïve ECs by transcriptional reprogramming of amniotic cells into vascular ECs
referred to as rAC-VECs (5). Proper activation of rAC-VECs results in the generation of programmed
ECs that mimic the specialized function of tumor ECs, supporting long-term expansion of hematopoietic and epithelial tumors in xenobiotic-free conditions. To determine the contribution of activated ECs to tumor growth in vivo, we employed a model in which Akt could selectively and conditionally be activated in the ECs. Remarkably, in vivo activation of ECs in the adult mice is sufficient
to initiate and maintain tumor growth through release of angiocrine factors, such as Jagged-1.
Conditional deletion of Jagged-1 selectively in the ECs resulted in a diminished expansion of tumor
initiating cells, such as lymphoma initiating cells. Targeting specific angiocrine factors, (i.e. Jagged-1) in the tumor ECs provides for an effective means to block the propagation and progression
of tumor initiating cells. In addition, co-cultures of tumors initiating cells with rAC-VECs programmed
to phenocopy tumor ECs establish an in vitro model to uncover the angiocrine factors that modulate
tumor progression.
1. Butler JM, … Rafii, S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 3:251-64. 2010.
2. Butler JM, … Rafii S. Development of a vascular niche platform for expansion of repopulating human cord blood stem
and progenitor cells. Blood, In Press 2012.
3. Ding BS,... Rafii, S. Inductive signals from sinusoidal endothelial cells initiate and sustain physiological liver regeneration. Nature. 468(7321):310-5, 2010.
4. Ding BS, … Rafii, S. Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell,
47(3):539-53, 2011.
5. Ginsberg M,… Rafii, S. Efficient direct reprogramming of mature human amniotic cells into endothelial cells by ETS
factors and TGFβ suppression. Cell, In Press, 2012.
The role of p53 in the life of stem cells: p53 counteracts reprogramming by
inhibiting mesenchymal-to-epithelial transition
Varda Rotter
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot. Israel
The process of somatic cell reprogramming is gaining increasing interest as reprogrammed cells
are considered to hold a great therapeutic potential. However, with current technologies this process is extremely inefficient. Recent studies reported that inhibition of the p53 tumor suppressor
gene profoundly facilitates reprogramming and attributed this effect to the ability of p53 to restrict
proliferation, apoptosis, senescence and immortalization. Interestingly, Mesenchymal-to-Epithelial
Transition (MET) was shown to be necessary and a rate-limiting step in the reprogramming of fibroblasts. Since p53 was shown to regulate diverse differentiation pathways, we aimed to investigate
whether p53 counteracts reprogramming by regulating MET. We found that MET is restricted by p53
during the early phases of reprogramming and that this effect is primarily mediated by the ability of
p53 to inhibit Klf4-dependent activation of epithelial genes. We also found that the expression of
E-Cadherin, an epithelial marker, negatively correlates with p53 activity in a variety of mesenchymal
cells. We conclude that loss of p53 in mesenchymal cells predisposes them to the acquisition of
epithelial characteristics and renders them more prone to reprogramming. Our study sheds light on
a novel function by which p53 restrains reprogramming and highlights the role of p53 in the regulation of cell plasticity.
27
Investigating cell types of origin for prostate cancer
Michael M. Shen1,4, Zhu A. Wang1,4, Antonina Mitrofanova2,4, Chee Wai Chua1,4, Ming Lei1,4, Sarah
K. Bergren1,4, Andrea Califano2,4, and Cory Abate-Shen3,4.
1
2
3
4
Departments of Medicine and Genetics & Development
Department of Biomedical Informatics and Center for Computational Biology and Bioinformatics
Departments of Urology and Pathology & Cell Biology
Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, USA.
Understanding the lineage relationship between normal progenitor cells and cell type(s) of origin
for cancer may yield important molecular insights into prostate cancer prognosis and treatment
response. In our previous studies, we have shown that a known regulator of prostate epithelial differentiation, the homeobox gene Nkx3.1, marks a luminal stem cell population that functions during prostate regeneration and is an efficient target for oncogenic transformation in prostate cancer.
Genetic lineage-tracing studies demonstrate that rare cells which express Nkx3.1 in the absence of
testicular androgens (castration-resistant Nkx3.1-expressing cells, CARNs) are bipotential and can
self-renew in vivo, while transplantation assays show that CARNs can reconstitute prostate ducts
in renal grafts. Furthermore, targeted deletion of the Pten tumor suppressor gene in CARNs results
in formation of carcinoma following androgen-mediated regeneration, whereas Pten deletion in
combination with Kras activation results in aggressive cancer in the absence of androgen administration. In ongoing work, we are investigating the functional properties of CARNs during prostate
regeneration and as cells of origin for cancer. In particular, analyses of androgen receptor (AR) in
CARNs suggest that AR is only partially required for the stem cell properties of CARNs.
28
In parallel studies, we have utilized lineage-tracing and transplantation approaches to explore
the potential stem cell properties of prostate basal cells in vivo. Our analyses of untransformed
basal cells demonstrate an unexpected assay-dependence of their stem cell properties in sphere
formation and transplantation assays versus genetic lineage-tracing during prostate regeneration
and adult tissue homeostasis. We also find that both basal and luminal cells can serve as cells
of origin for prostate cancer, giving rise to tumors with similar histological phenotypes. However,
cross-species molecular and bioinformatic analyses show that the luminal origin tumors are more
aggressive than basal origin tumors, and identify a molecular signature that has predictive value
for human patient outcome. These results reveal the inherent plasticity of basal cells, and support
a model in which different cells of origin generate distinct molecular subtypes of prostate cancer.
Generation of multiple cell fates
Thea Tlsty
University of California, San Francisco, USA
Somatic stem cells isolated from adult breast tissues exhibit potency for generating multiple cell
fates. The molecular bases for the control of these processes are fundamental to differentiation
and disease. Several studies have identified specialized populations within human breast tissue
with a reported role in disease progression. We will discuss these cells and their putative roles in
cell-cell interactions.
29
Circulating metastasis-initiating cells in breast cancer
Irène Baccelli1,2, Sabine Riethdorf3, Markus Wallwiener4, Corinna Klein1,2, Klaus Pantel3, Wilko
Weichert5, Andreas Schneeweiss4 and Andreas Trumpp1,2
1
2
Divison of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
HI-STEM – Heidelberg Institute for Stem Cell Technology and Experimental Medicine, Heidelberg, Germany
3
Department of Tumor Biology, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, Hamburg, Germany
4
Nationales Centrum für Tumorerkrankungen (NCT) und Frauenklinik, Universitätsklinikum Heidelberg, Heidelberg, Germany
5
Pathologisches Institut der Universität Heidelberg, Heidelberg, Germany
Metastasis is the foremost cause of cancer-related deaths. Metastatic spread is a complexprocess initiated by the dissemination, seeding and engraftment of malignant cells in sites distant
to the primary tumor. It has been hypothesized that metastasis-initiating cells (MICs) are present
within circulating-tumor-cells (CTCs) in the blood stream of carcinoma patients.
Indeed, the presence of CTCs in metastatic patients correlates with decreased overallsurvival in
several malignancies, including breast cancer. Although these clinical data are consistent with the
hypothesis that CTCs contain MICs, their existence, phenotype and activity has never been demonstrated. We present data showing that as low as 1900 CTCs were able to induce metastases in
mice. Transplantation of primary patient blood derived CTCs induced metastatic growth in bones
and liver, demonstrating the presence of MICs. FACS analysis of primary patient EPCAM+-CTCs revealed heterogeneous inter-patient expression of the metastasis-promoting signaling receptors
CD44, CD47 and MET. While the percentage of EPCAM+CD44+CD47+MET+ CTCs in patient blood
varied between 1.4 and 44%, metastases both from the original patient and those derived experimentally from CTCs showed high levels of all three receptors. The data provide a first demonstration
that EPCAM+-CTCs express CD44, CD47 and MET and contain MICs, providing a molecular basis
for the design of diagnostic tools to detect MICs and for developing rational-based approaches to
target metastasis in breast cancer.
30
Induced pluripotency and cancer cells
Jochen Utikal
Clinical Cooperation Unit Dermato-Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
The ectopic expression of different sets of transcription factors in cells is capable to reprogram
cells into a pluripoptent state- in so called induced pluripotent stem cells (iPSCs). iPSCs have been
reprogrammed from different sources of primary cells. Besides of primary cells also cancer cells
can be converted by the same gene sets into iPS cell-like induced pluripotent cancer cells (iPCCs).
iPCCs and their differentiated progenies provide a valuable experimental platform to model oncogenesis.
31
Direct lineage reprogramming to generate neural cell types
Marius Wernig
Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California, USA
Cellular differentiation and lineage commitment are considered robust and irreversible processes during development. We hypothesized that expression of neural lineage-specific transcription
factors could directly convert fibroblasts into neurons and identified a combination of only three
factors that suffice to rapidly and efficiently convert mouse fibroblasts into functional neurons in
vitro. These induced neuronal (iN) cells expressed multiple neuron-specific proteins, generated action potentials, and formed functional synapses.
One of the outstanding questions in the field was whether a defined non-ectodermal cell can be
converted into iN cells given the heterogeneity of fibroblast cultures. We therefore tested whether
(endoderm-derived) hepatocytes can be reprogrammed to iN cells. Surprisingly, using the same 3
transcription factors primary mouse hepatocytes could be converted very efficiently into iN cells
with the ability to generate action potentials upon depolarization as well as to form synaptic contacts with primary neurons. A genetic lineage-tracing system was employed to prove that the cell of
origin of iN cells were indeed endodermal cells. Moreover, gene expression studies on the global
and single cell level confirmed not only the induction of a neuronal transcriptional program but also
the efficient silencing of the hepatocyte-specific expression pattern. This transcriptional silencing
was extensive but not complete on both population and single cell level. We therefore conclude
that iN cells are not a hybrid cell with equal identities of the starting cell and neurons, but cells with
a predominant neuronal identity with an epigenetic or transcriptional memory of the starting cell
population.
32
Finally, we reasoned it would be desirable to generate a proliferative neural precursor cell population from fibroblasts given the limitations of expandability of iN cells. We followed a similar strategy that was successful to identify the iN cell factors and screened 11 factors to eventually identify
Sox2 and FoxG1 as critical factors that are sufficient to induce neural precursor cells (iNPCs) with
the potential to differntiate into neuronal and astroglial, but not oligodendroglial cell types. The addition of Brn2 confered the ability to differentiate also into oligodendroglial cells. When Sox2 was
omitted from the combination iNPCs were generated with differentiation potential into both glial
cell types but the neuronal differentiation was less robust with the generation of only immature
neuronal cells. However, when transplanted into the myelin-deficient shiverer mouse brain, the cells
incorporated well into the white matter tracts, differentiated into oligodendrocytic cells expressing
an array of lineage specific markers including Olig2, O4, and myelin basic protein and ensheathed
host axons.
Exclusive multipotency in post-embryonic neural stem cells of the fish retina
Jochen Wittbrodt
Center for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany
Adult stem cells self-renew and generate differentiated cells in several tissues and organs.
Stemness is an attribute of an individual cell and can only be addressed in the living, adult organism by following single cells and their descendants. We have recently reported in medaka neural
stem cells (NSCs) of embryonic origin located in the ciliary marginal zone (CMZ) of the retina. Here
we developed inducible clonal labeling followed by non-invasive fate tracking and functionally map
NSCs and transient neural progenitor cells (NPCs) to distinct domains in the CMZ. We identified
retinal homeobox gene-2 (Rx2) as a NSC marker and intriguingly, we found no heterogeneity in the
differentiation potential of individual NSCs. All Rx2+ NSCs generated the complete repertoire of
differentiated cells types in the fish retina. We present a comprehensive analysis of individual postembryonic NSCs in their physiological environment and establish the teleost retina as ideal model
for studying adult stem cell biology at single cell resolution.
33
Stem cells in prostate cancer initiation and progression
Owen N. Witte
Investigator, Howard Hughes Medical Institute; Director, Broad Stem Cell Research Center, Dept. of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA
Major epithelial cancers are often associated with a broad and complex range of genetic and
epigenetic changes that contribute to tumor progression as the disease advances. Our primary goal
has been to understand the relationship of tissue specific stem cells and other cell types in which
these changes occur in the initiation and progression of disease. Using a dissociated cell tissue
regeneration strategy we have defined subpopulations of murine and human prostate stem basal
cells as the primary target for cancer initiation. Cell autonomous changes as well as effects from
the surrounding stroma can play important roles in tumor progression and we observe that generally a combination of two or more signals is required for effective cancer progression. Pathways that
regulate tissue stem cell self renewal continue to play an important role during this process and
may represent useful targets for therapeutic interruption.
34
ABSTRACTS OF POSTERS
35
Hedgehog/GLI and EGFR cooperation response genes in basal cell carcinoma
and tumor-initiating pancreatic cancer cells
Andrea Loipetzberger1, Christina Sternberg1, Markus Eberl1, Doris Mangelberger1, Hendrik
Hache2, Wilfried Nietfeld2, Hans Lehrach2, Christoph Wierling2 and Fritz Aberger1
1
Division of Molecular Tumor Biology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria
2
Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany
Cancer involves the aberrant activation of a limited set of signaling pathways regulating processes such as proliferation, survival, motility and stemness. Cooperative interactions of oncogenic
signals play a critical role in malignant transformation. Our group has previously identified synergistic interactions of two clinically relevant drug targetable pathways, the Hedgehog (HH)/GLI and the
EGFR pathway (Kasper, M., H. Schnidar, et al. (2006). Mol Cell Biol 26(16): 6283-6298.; Schnidar,
H., M. Eberl, et al. (2009). Cancer Res 69(4): 1284-1292.).
Using time resolved transcriptome and proteome analysis, a set of Hedgehog (HH)/GLI-EGFR
signaling “cooperation response genes” (CRG) that regulate the tumor initiating capacity of putative pancreatic cancer stem cells was identified using a systems biology approach. Using pharmacological and RNAi mediated iterative perturbations and systems analysis of the HH/GLI and EGFR
pathway we could confirm the model of biological processes underlying the control of the interacting pathways and could show that the CRGs constitute an interdependent hierarchical regulatory
network (Eberl, M., S. Klingler, et al. (2012). EMBO Mol Med). These novel insights into complex
regulatory interactions in cancer stem cells are important for the development of novel rationale
based combination therapies.
37
Selective Targeting of Notch-1 in T-acute lymphoblastic leukemia
Agnusdei V.1, Minuzzo S.2, Frasson C.3, Axelrod F. 4, Satyal S. 4, Gurney A. 4, Hoey T. 4, Seganfreddo E.3,
Basso G. 3, Moresco R.M. 5, Grassi A.1, Tosello V. 1,6, Ferrando AA. 6, Amadori A.1,2, Indraccolo S. 1
1
Istituto Oncologico Veneto-IRCCS, Padova, Italy
2
Dip. Scienze Chirurgiche, Oncologiche e Gastroenterologiche-Sez. Oncologia e Immunologia- Università di PadovaPadova, Italy
3
Lab. Oncoematologia, Dip. di Salute della Donna e del Bambino, Università di Padova, Padova, Italy
4
OncoMed Pharmaceuticals Inc., Redwood City, USA
5
Nuclear Medicine Department, San Raffaele Scientific Institute, Fondazione Tecnomed, University of Milan Bicocca,
IBFM-CNR, Milan, Italy
6
Dept. of Pediatrics, Columbia University Medical Center, New York, USA
T-acute lymphoblastic leukemia (T-ALL) is an heterogeneous disease, characterized by several
genetic alterations and polymorphic clinical features both in children and adults. The Notch pathway, an evolutionary conserved pathway involved in many biological processes including T cell differentiation and angiogenesis, has been implicated in the pathogenesis of this disease. Notably,
about 60% of T-ALL samples show increased Notch1 activity, due to Notch1/Fbw7 mutations.
Various strategies targeting Notch signaling for therapeutic purposes are currently under development. One approach is to block proteolytic cleavage of Notch receptors by treatment with gamma
secretase inhibitors (GSI) which are, however, poorly selective and rather toxic, due to simultaneous blockade of both Notch1 and Notch2. An alternative route that may overcome this problem is
selective targeting of the Notch pathway by inhibitory antibodies. Here, we investigated the biologic
and therapeutic effects of a human Notch1-specific neutralizing antibody in xenograft models of
pediatric T-ALL.
38
Pediatric patients were classified according to the phenotype, the risk and the response to conventional therapy. T-ALL xenografts were obtained by intravenous injection of primary T-ALL cells in
NOD/SCID mice. T-ALL samples were initially characterized for Notch1 and FBW7 mutations and
expression levels of Notch target genes were determined by quantitative RT- PCR. Anti-Notch1 antibody was weekly administrated and engraftment of T-ALL cells was monitored by serial blood drawings. At sacrifice, infiltration of spleen and bone-marrow by T-ALL cells and levels of apoptosis were
analyzed. Furthermore, we investigated the effects on Notch signaling in human CD5+ cells sorted
from either spleen or BM of anti-Notch1 treated or control mice. To this end, expression levels of 21
Notch-target genes were measured by using TaqMan Low Density Arrays.
A correlation was seen between the Notch/Fbw7 genetic status and expression levels of Notchrelated transcripts in T-ALL xenografts. Anti-Notch1 treatment greatly delayed engraftment of T-ALL
cells bearing an active Notch pathway, including samples derived from poor responders or relapsed
patients. Anti-Notch1-treated mice had a significant reduction in the percentage of blasts both in
the blood, the spleen and the BM. Moreover, we observed an increase in the levels of T-ALL cell
apoptosis and a strong inhibitory effect on Notch transcriptional profile following anti-Notch1 treatment. These results indicate that Notch1/Fbw7 mutated T-ALL samples are suitable candidates
for Notch targeted therapy and highlight the potential of measurements of Notch target genes as
surrogate biomarkers of the therapeutic response.
Anti-proliferative and genotoxic effects of arsenic on primary human breast
cancer cell lines
Aftab Ahmad, A.R. Shakoori
School of Biological Sciences, University of the Punjab, New Campus, Lahore, Pakistan
Breast cancer is one of the most common cancers with high incidence rate among women.
Prevalence of breast cancer in Pakistan is highest in Asia. Arsenic has deleterious effect on human
health as well as arsenic compounds are used for the treatment of different malignancies. Breast
cancer tissues were taken from two hospitals and primary breast cancer cell lines were established
by explants culture method. Neutral red based anti-proliferative assays were done to check the effect of arsenic on Pakistan Breast Cancer INMOL (PBCI) and Pakistan Breast Cancer Jinnah (PBCJ)
cell lines. Comet assay was done to check the genotoxic effect of arsenic. Inhibitory concentration
50 (IC50) for PBCI was 13 µg/ml on 24 h exposure and it shifted to 12 µg/ml when exposed the
cells for 48 h while IC50 for PBCJ was 9 µg/ml. PBCJ cells proved to be more sensitive to arsenic
than PBCI. When 1x104 cells per well in 96 well plate were used IC50 for PBCI was 19 µg/ml. DNA
damage was observed by comet assay in arsenic treated samples as compared to control. Ten different parameters were investigated for arsenic treated and control cells. These results indicate
that arsenic have great cytotoxic and genotoxic effect on breast cancer cells and morphology of
cells was totally changed with higher concentrations (12 µg/ml or higher) of arsenic.
Keywords: breast cancer; primary cell lines; anti-proliferative; cytotoxic; genotoxic.
39
Regulation of stem cell self-renewal, differentiation and transformation by
the ubiquitin-proteasome system
Beatriz Aranda-Orgilles, Shannon Buckley, Alexandros Strikoudis and Iannis Aifantis
Howard Hughes Medical Institute and Department of Pathology, NYU School of Medicine, New York, NY USA
Our lab studies the role of post-translational modifications in the regulation of the intimate balance that stem cells possess and that allows them to be able self-renew and differentiate. In this
study, we focus on ubiquitination and we have used large-scale shotgun proteomics to map for the
first time “ubiquitin signatures” of embryonic stem cells (ESCs) and induced pluripotent stem cells
(iPSCs). These experiments revealed that specific key regulators of pluripotency and reprogramming, such as Oct4, Nanog or c-Myc are ubiquitinated and regulated by this modification. Additionally, we have screened by RNA interference more than 600 members of the ubiquitin proteasome
system (UPS) and have identified several novel candidates that are essential to maintain the pluripotency state of ESCs or to ensure their proper differentiation. We have further focused on two of
two of these candidates, the deubiquitinating enzyme Psmd14 and Fbxw7, an E3 ubiquitin ligase
that our lab recently described as an important tumor suppressor in T-cell acute lymphocytic leukemia. We show that the expression of the E3 ubiquitin ligase Fbxw7 is dynamically regulated during
ESC differentiation, that Fbxw7 silencing favors cellular reprogramming and that it can replace
exogenous expression of its substrate c-myc in the generation of iPSCs.
This is the first global characterization of the UPS as a key regulator of stem cell function, opening the way for future investigations on specific UPS enzymes or ubiquitinated substrates with a role
in stem cell homeostasis, reprogramming or cancer.
40
ENPP1 regulates stem cell gene signatures in glioblastoma stem-like cells
Josephine Bageritz1, Laura Puccio1, Volker Hovestadt1, Teresa Pankert1, Christel Herold-Mende2,
Peter Lichter1 and Violaine Goidts1
1
Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany
2
Division of Neurosurgical Research, University Hospital, Heidelberg, Germany
Glioblastomas are highly aggressive and have the poorest prognosis among brain tumors. A
subpopulation of poorly differentiated glioblastoma stem-like cells (GSCs) has been defined as resistant to therapy and subsequently considered to be responsible for tumor recurrence. We have
used a shRNA mediated loss-of-function screen representing a comprehensive set of phosphatases
to identify genes relevant for the stem cell maintenance. We report of 35 candidate genes, which
upon knockdown induce a significant decrease of the stem cell marker CD133 (p<0.05) suggesting
an impairment of stemness. The shRNA-mediated knockdown of the selected candidate genes was
validated on mRNA level and their impact on CD133 was corroborated in two additional GSCs. We
focused on one of these genes, ENPP1 (ectonucleotide pyrophosphatase/ phosphor-diesterase 1),
as knockdown not only reduced CD133 level, but also further stem cell markers (SSEA-1, A2B5,
MSH1, LHX2). Moreover, gene expression profiling upon ENPP1 knockdown revealed an overall
down regulation of stem-cell associated genes. Further in vitro studies showed a reduced sphere
forming capacity of GSCs in ENPP1 knockdown samples compared to the negative control, which is
partly caused by an increased apoptosis rate. ENPP1 can be considered as a promising therapeutic
target, as a tumor specific expression could be shown when comparing three GSCs with a pool of
normal brain samples. To gain insights into the molecular mechanism leading to a loss of stemness,
detailed pathway analysis is underway.
41
The role of the Angiopoietin/Tie system in regulating hematopoietic stem
cell maintenance and recruitment
Susanne Bartels, Claudia Korn, Carleen Deppermann, and Hellmut Augustin
Department of Vascular Oncology and Metastasis, German Cancer Research Center, Heidelberg, Germany
The Angiopoietin/Tie system controls angiogenesis and vessel maturation. However, signalling
through the receptor tyrosine kinase Tie2 and its ligand Angiopoietin-1 (Ang-1) has recently been
identified to play a crucial role in controlling quiescence and maintenance of hematopoietic stem
cells in the bone marrow niche. Pericytes, smooth muscle cells and fibroblasts express Ang-1 in a
paracrine manner. Recently, bone marrow osteoblasts have also been identified to express Ang-1.
The receptor tyrosine kinases Tie1 and Tie2 are both expressed by hematopoietic stem cells. In contrast, Ang-2 is secreted exclusively by endothelial cells, where it is stored in Weibel-Palade bodies
and released upon endothelial cell activation to act as autocrine antagonist regulator of constitutive
Ang-1/Tie2 signaling. Until now, very little is known about the role of the dynamic modulators Ang-2
and Tie1 within the hematopoietic niche. Preliminary data from our laboratory established a role of
Ang-2 in controlling the hematopoietic stem cell niche. Furthermore, the Tie1 intracellular domain
acts as a Tie2 enhancing co-receptor. Here, we study the expression profile and functional roles of
Ang-2 and Tie1 in controlling the maintenance and egress of hematopoietic stem cells in the bone
marrow niche under steady-state conditions and upon mobilization. Understanding the crosstalk
of the signaling molecules and tightly regulated mechanisms that balance stem cell quiescence
versus proliferation in the hematopoietic niche is of major relevance to improve chemotherapy and
adult stem cell transplantation.
Supported by the DFG (SFB 873).
42
Investigating the role of E proteins as potential therapeutic targets in
experimental gliomas
Beyeler Sarah 1, Joly Sandrine 1, Obermair Franz-Josef 3, Mehmood Rashid 4, Tabatabai Ghazaleh
, Raineteau Olivier 1
2
1
Brain Research Institute, University of Zurich/ Swiss Federal Institute of Technology Zurich, Switzerland
2
Department of Neurology, University Hospital Zurich, Switzerland
3
Institute of Integrative Biology, Swiss Federal Institute of Technology Zurich, Switzerland
4
Children’s Health Research Institute, Department of Paediatrics, University of Western Ontario, London, ON, Canada
Malignant gliomas are aggressive primary brain tumors of glial origin. Helix-loop-helix (HLH, e.g.
Id proteins) and basic HLH (bHLH, e.g. Olig2) transcription factors are expressed in malignant gliomas. High expression of these proteins correlates with unfavorable clinical prognosis. The establishment of novel strategies to interfere with HLH and bHLH transcriptional activity might therefore
be a promising therapeutic approach.
E proteins, such as E47, interact by dimerization with both HLH and bHLH proteins. In this project
we overexpressed wildtype and mutated forms of E47 in order to inhibit HLH and/or bHLH proteins
activity in gliomas and to investigate the anti-glioma effects of these manipulations.
We selected two human glioma cell lines that express Id proteins and low or high levels of the
bHLH protein Olig2, i.e. LN308 and LNT229, respectively. Overexpression of E47 induced pronounced cell death in LN308 but not in LNT229, as quantified by flow cytometry and immunostaining. These results suggest that binding and transactivation of Olig2 bHLH proteins in LNT229 might
counteract the pro-apoptotic effect of E47/Id proteins interactions. Indeed, concomitant overexpression of Olig2 in LN308 cells blocked the pro-apoptotic effect of E47 overexpression. In LNT229
gliomas expressing high level of Olig2, apoptosis could however be induced using a dominant negative form of E47 (dnE47) that lacks the ability to translocate into the nucleus and therefore to induce transcription.
We next assessed the transcriptional consequences of HLH and/or bHLH protein neutralization
in LNT229 glioma cells, by comparing gene-expression by qRT-PCR in cells transfected with E47 or
dnE47. DnE47 overexpression resulted in significant downregulation of the oncogene CDC25a and
the anti-apoptotic gene Bcl-xl.
As brain tumors have been shown to arise from tumor initiating stem cells we tested E47 and
dnE47 overexpression in patient-derived tumor stem cells using lentiviral constructs. Our results
show that these cells highly express both HLH and bHLH proteins and that their sphere formation
capacity is reduced after overexpression of both E47 and dnE47. We are currently investigating
further these findings in vivo.
All together, these original observations suggest that both HLH and bHLH proteins are important
for the viability of glioma cells. Suppressing HLH and bHLH activity by E protein-mediated cytoplasmic sequestration could represent a novel anti-glioma strategy.
43
OMICs of hematopoietic stem cell differentiation: Distinct signatures of
multipotent progenitors and myeloid committed cells
Nina Cabezas-Wallscheid*#, Daniel Klimmeck*+#, Jenny Hansson+#, Simon RaffelY, Lisa Dohrn*,
Sergey Y. Vakhrushev+, Jeroen Krijgsveld+§ and Andreas Trumpp*Y§.
*
44
Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany;
+
Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany;
Y
Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany;
#
These authors contributed equally to this work; § Shared last authorship.
Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent
progenitors that differentiate into lineage-committed progenitors and subsequently mature cells.
In our recently published report(1), we applied an in-depth quantitative proteomic approach to
analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations representing either multipotent hematopoietic stem/progenitor cells (HSPCs, Lin-,Sca-1+,c-Kit+) or myeloid
committed precursors (Lin-,Sca-1-,c-Kit+). By employing stable isotope dimethyl labeling and highresolution tandem mass spectrometry, more than 5,000 proteins were quantified. From biological
triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between HSPCs and myeloid committed cells (1). The differential protein content
in these cell populations points to a distinct structural cytoskeletal organization and remodeling
during differentiation. Strikingly, the data uncover a unique signature related to immune defense
mechanisms, centering around the RIG-I and type-1 interferon response systems, which are employed in HSPCs but not in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature (1). Furthermore, in order to
investigate gene-regulatory mechanisms potentially affecting HSC differentiation on both RNA and
protein level we applied whole transcriptome analysis (RNA-Seq). Strikingly, 6,100 differentially expressed genes were detected. In line with the proteome data, we found that HPSCs were enriched in
processes related to immune response and epithelial-to-mesenchymal transition. In order to better
understand the OMICS landscape of HSPCs at the transcript and protein level, both datasets were
analyzed together. Interestingly, we found diverse cellular processes including iron homeostasis
and response to reactive oxygen species showing anti-correlation between RNA and protein expression indicating post-transcriptional regulation. In conclusion, this study indicates that the transition
of HSPCs towards myeloid commitment is accompanied by a profound change in processing of cellular resources, providing novel insights into the molecular mechanisms at the interface between
multipotency and lineage commitment.
1. Klimmeck D; Hansson J, et al., (2012). Mol Cell Proteomics. Aug;11(8):286-302.
MicroRNAs as predictive and therapeutic tools in prostate cancer
Alessio Cannistraci1, Valeria Coppola1, Michele Patrizii1, Maria Musumeci1, Antonio Addario1,
Matilde Todaro3, Ruggero De Maria2 and Desiree Bonci1
1
Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
2
Regina Elena Cancer Institute, Rome, Italy
3
Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy
Prostate cancer (PCa) is the sixth most common cause of cancer-related deaths in the world.
Curative options for patients with localized disease include radical prostatectomy or radiotherapy,
even though a considerable percentage of these patients experience distant recurrence. Testosterone suppression achieved is the standard initial treatment for men with advanced prostate cancer,
but the majority of tumors evolve, after a median of 1-2 years, towards a castration-resistant state
(CRPC) with consequent development of metastasis, which generally localize in the bone microenvironment. Molecular mechanisms responsible for this incurable form of the disease are still poorly
understood. Emerging evidence demonstrated that Cancer Stem Cells (CSCs) are the critical drivers of tumor progression and metastasis. Deep studies on CSCs intrinsic features, which include
self–renewal and drug resistance abilities, could enhance our knowledge of cancer biology and
consequently improve clinical approaches.
We isolated a putative CD44+/CD24-/CD133-/MET+ stem cell population (PCSC-1) from patientderived tumor. In vitro and in vivo experiments demonstrated that PCSC-1 cells express basal cell
markers, such as p63 and CK5, and they have clonogenic and tumor-forming abilities. Moreover,
they are able to grow within the bone microenvironment and cause osteolytic lesions when injected
intratibially in mice. We also analyzed the PCSC-1 microRNA (miR) expression pattern, comparing
it with microdissected tissue-derived RNA from patients with tumor recurrence or bone metastasis,
trying to find new predictive markers and molecular targets useful for future therapies.
45
Role of Granzyme B in hematopoietic stem cell homeostasis and activation
Larissa S. Carnevalli 1,2, Elisa Laurenti 3, Susann Rahmig 2, Marieke Essers 1,2 Andreas Trumpp 1,2
1
Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany
2
Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
3
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
The serine protease Granzyme B (GzmB) is usually found in the granules of cytotoxic lymphocytes and natural killers (NK) cells. This highly cytotoxic protease has intracellular and extracellular
functions but is mostly known for immune-targeted apoptosis via the granule-exocytosis pathway.
Our group has previously shown that induced loss of the c-Myc and N-Myc oncogenes in the hematopoietic system results in lethal bone marrow cytopenia. This is accompanied by the strong induction of GzmB protein expression in self-renewing hematopoietic stem cells (HSCs) but not other
blood cells (Laurenti et al., Cell Stem Cell 2008), suggesting that GzmB mediates a novel mechanism of stem cell apoptosis upon the simultaneous loss of both Myc genes. In order to genetically
address the potential role of GzmB in HSC cell death we induced the deletion of c-Myc/N-Myc on a
GzmB deficient background by generating a triple knockout mouse line. Deletion of GzmB partially
rescued the apoptotic phenotype in the double KO HSCs, formally demonstrating the crucial role for
GzmB in HSC apoptosis in the context of c-Myc/N-Myc deletion. To address whether physiological
stress signaling pathways also induce GzmB in vivo, we exposed mice to chemotherapeutic agents
and cytokines.
46
Interestingly, in vivo treatment of HSCs with the chemotherapeutic agent 5-FU led to GzmB protein expression in HSCs, which was associated by a moderate increase in cell death. Interestingly,
activation of HSCs by lipopolysaccharide (LPS) also causes a strong upregulation of GzmB protein,
associated with increased HSC cell death and GzmB secretion into the BM environment, indicating that GzmB may perform extracellular roles by modulating the HSC microenvironment or niche.
Finally, GzmB deficient HSCs show increased proliferation and curiously, improved engraftment of
HSC in vivo if in competition with WT cells. Taken together these results demonstrate that GzmB
is part of a stress response cascade in HSCs and suggest that GzmB could be a novel therapeutic
target and could be potentially used to improve the efficiency of blood stem cell transplantation.
Small natural compound derived molecule targets cancer stem cell metabolism
Xinlai Cheng1, Tijen Duvac1, Eleni Dimou1, Pavlo Holenya1, Minh Hoang1, Hamed Alborzinia1, Johannes Fredebohm2, Jörg D. Hoheisel2 and Stefan Wölfl1
1
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
2
Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
Compelling studies evidence the existence of subpopular, self-renewable cells, termed cancer
stem cells (CSCs) within tumors that initiate tumor development, growth and resistant to conventional chemo- and radiation therapy. Therefore, agents aiming at CSCs may show a big advantage
in cancer therapy. Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic
cancer is one of the most uncurable tumors with less than 5% of 5-year survival rate. It has been
reported that some pancreatic cancer cell lines contain high levels of CSC, including Panc1 and
JoPaca-1, which show high expression of CSC markers CD44+/CD24+/ESA+ and expression of
N-Cadherin. E571, an isoindigo derivative, is a small molecule derived from an ingredient of traditional Chinese medicine (TCM) used in the treatment of chronic myeloic leukemia (CML) in China.
We evaluated the anti-proliferative effect of E571 and gemcitabine (Gem) in JoPaca-1, Panc1
and BxPC3 and found that JoPaca-1 and Panc1 were resistant to Gem (JoPaca-1 and Panc1:
IC50>50µM; BxPC3: IC50 1µM), whereas the effect of E571 was two-fold higher in JoPaca-1 (IC50:
5.21µM) and Panc1 (IC50: 4.41µM) than in BxPC3 (IC50: 10µM) suggesting CSC-like cells are more
sensitive to E571. Looking in more detailed we found that E571 induced cell arrest at S and G2/M
phases. E571 also induced PARP cleavage and apoptic cells were detected by annexin V staining.
Both effects could be blocked by caspase inhibition. A concomitant decrease in N-Cadherin levels
indicated a reduction of stem cell behavior upon treatment. Glutathione, a ROS scavenger, protected cells from damage in response to E571. We further analyzed glucose uptake and cellular
respiration. Our results show that the difference between CSCs and somatic cells in metabolism
can be targeted with specific inhibitors, like E571, which alter cell metabolism. This property may
provide new drug candidates for cancer stem cell therapy.
47
Effects of genetically modified human adipose tissue derived mesenchymal
stem cells secreting TRAIL on neuroblastoma cells
Cingoz, A.1,2, Mutlu Altundag, E. 1,2, Cihan, C. 1,2, Corek, C. 1,2, Yaman, K. 1,2, Ucar, K. 1,2, Catalgol, B. 1,2,
Kocturk, S. 1,3, Taga, Y. 1,2
1
Marmara University GEMHAM – Genetic and Metabolic Diseases Research and Investigation Center, Istanbul, Turkey
2
Marmara University F. of Medicine Dep. of Biochemistry, Istanbul, Turkey
3
Dokuz Eylul University F. of Medicine Dep. of Biochemistry, Izmir, Turkey
Neuroblastoma (NB) is the most common solid cancer in childhood and recently the usage of
stem cells brings a new aspect for the effective therapy. The stem cells are present in almost every
tissue and are useful vehicle for cancer therapy since they can be loaded with antitumor agents.
In this direction, TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in
cancer cells which can be inhibited by X-linked inhibitor of apoptosis protein (XIAP).
In this study, human mesenchymal stem cells (MSCs) were isolated from human adipose tissue and characterized. The TRAIL gene vector were transfected to MSCs and shXIAP plasmid were
transfected to SK-N-AS cells (which is an aggressive NB cell line) by Lipofectamine 2000 reagent
and Amaxa Nuclefactor 4D. A GFP transfected group and fibroblast cells were used as control.
These genetically modified MSCs and fibroblasts were co-cultured with SK-N-AS cells for 24 hours.
Culture supernatants were harvested, and secreted TRAIL was measured using the TRAIL ELISA
Kit. Thereafter cell survival of neuroblastoma cells were determined with flow cytometry analysis.
The gene expression levels and western blot analysis of XIAP, TRAIL, caspase 3,-8,-9, Bcl-2 were
assessed. For the evaluation of the NF-κB signal pathways related to SK-N-AS cells by AlphaScreen
Technology (Perkin Elmer).
The results showed that genetically modified MSCs secreting TRAIL reduce the cell survival of
cancer cells by inducing apoptosis and inhibiting the proliferation. By demonstrated that XIAP inhibition sensitizes neuroblastoma cells for TRAIL-induced apoptosis and the NF-κB pathway plays a
critical role in this process.
48
Use of the mesenchymal stem cells as ‘tumor cell killing ligand carrying vehicles like a Trojan
Horse’ is emerging as a new concept in the anti-tumor therapy.
Characterization of DNA methylation patterns in tumor initiating cells
derived from breast tumor subpopulations
Cohen-Kupiec R., Zundelevitch A. and Gal-Yam E.
Cancer Epigenetics Laboratory, Cancer Research Institute, Sheba Medical Center, Tel-Hashomer, Israel
Background: Individual breast-cancer tumors are classified into subtypes according to their proliferation rate, metastatic potential and relative sensitivity to therapy. This inter-tumor variation is
further complicated by intra-tumor variability between individual cancer cells, leading to profound
implications on tumor development and therapeutic outcomes. One common characteristic however between tumors is the existence of tumor initiating cells (TICs) that help propagate cancer and
survive chemotherapy. Efforts are made in recent years to characterize TICs that share molecular
traits with stem-cells, such as the expression of specific cell surface markers and the ability to self
renew.
Objectives: We hypothesize that the epigenetic makeup (specifically DNA methylation patterns)
of tumor initiating cells from different breast cancer subtypes dictates the phenotypically diverse
metastatic progeny that will eventually develop from these TICs. We therefore aim to develop a
methodology that will enable the isolation of breast TICs from different tumor subtypes and the
characterization of DNA methylation heterogeneity in these metastatic TICs. We will use these
methods to examine how particular epigenetic subpopulations correlate with tumor subtype.
Methods: We FACS-sort breast cancer cells using CD24 and CD44 surface markers and functionally select cells with self-renewal potential via mammospheres growth. These cells are examined
for their malignancy potential. We study the distribution and heterogeneity of DNA methylation in
CpG islands of selected amplicons in the resulting TICs subpopulations, searching for correlations
between breast tumor subpopulations; functional behavior (sphere growth and differentiation potential) and DNA methylation profiles.
Preliminary results: To develop our proposed methodology we use breast cancer cell lines from
3 different subtypes: ER/PR+, Her2+ and TN (triple negative) as well as normal breast mammary cell
lines. Cells were FACS-analyzed and grown as mammospheres to enrich for TICs. The characterization for expressing TICs markers and in parallel, DNA methylation experiments of selected amplicons are in progress. The methods we are developing will serve to analyze both metastatic cells and
primary tumors collected from breast cancer patients.
49
miR-9 balances proliferation, differentiation and quiescence in neural
progenitors
Marion Coolen1, Shauna Katz1, Denis Thieffry2, Øyvind Drivenes3, Thomas Becker3,4, Laure Bally-Cuif1
1
Zebrafish Neurogenetics Group, Laboratory of Neurobiology and Development (N&D), Institute of Neurobiology Alfred
Fessard, Gif-sur-Yvette, France
2
Institute of Biology of the Ecole Normale Supérieure (IBENS) Paris, France
3
Sars Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway
4
Brain and Mind Research Institute, Sydney Medical School, University of Sydney, Camperdown, NSW, Australia
The maintenance of neural progenitors throughout embryogenesis and adult life requires the
balanced regulation of their proliferation, differentiation and quiescence. Our current results in the
zebrafish embryonic neural tube identify a microRNA, miR-9, as a key factor in this process. Across
the ventricular zone of the late embryonic hindbrain, miR-9 expression occurs across a range of
progenitor commitment stages. Target protection analyses in vivo identify the progenitor-promoting
genes her6 and zic5 and the cell cycle exit-promoting gene elavl3/HuC as sequential targets of
miR-9 as neurogenesis proceeds. Through deregulation of these targets, abrogating miR-9 function
transiently delays cell cycle exit, leading to the increased generation of late-born neuronal populations. Our results, corroborated by logical modeling, suggest that, through this direct inhibition of
targets with antagonistic functions, miR-9 activity generates an ambivalent progenitor state at the
transition between proliferation and differentiation and poised to respond to both progenitor maintenance and commitment cues. We are currently investigating whether this function of miR-9 can
be extended to other neural progenitor contexts, such as adult neural stem cells and tumor stem
cells. In particular, we observed that miR-9 is expressed in a subset of radial glial progenitors in the
zebrafish adult telencephalon, and is required to maintain these cells in a quiescent state. We also
wish to analyze the implication of miR-9 in regulating glioblastoma stem cells in vitro and in vivo in
the zebrafish model.
50
De Angelis ML*, **, De Luca G*, Simeone P*, Stassi G*, Ricci-­‐Vitiani L*, Baiocchi M*, De Maria R*,*** * Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy. **
Scuola Superiore di Catania, Piazza dell’Università 2, Catania 95124, Italy. *** Istituto Regina Elena Nationl Cancer Institute, Via Elio Chianesi 53, Rome 00144, Italy.
In vitro and in vivo antitumor activity of cetuximab in human colon cancer
Background: stem cell lines (cCSCs)
in correlation to mutational state
Targeting the epidermal growth factor receptor (EGFR) pathway is an important therapeutic approach in
*,***
variety f tumors. IPn * ,cStassi
olon cancer, responsiveness cetuximab been put R
into relationship to De Angelis ML*, **, De Luca
G*, oSimeone
G*, Ricci-Vitiani
L*,to Baiocchi
M*h,as De
Maria
*
f EGFR pathway olecules. In particular, patients carrying mutated forms of K-­‐RAS ar
Istituto Superiore di Sanità,mutational Viale Reginastatus Elena o299,
Rome
00161,mItaly
**
currently excluded from etuximab therapy Scuola Superiore di Catania,
Piazza dell’Università
2, cCatania
95124,
Italy (1). Colon CSCs may represent predictive tools for patient’s ***
therapeutic response (2). Chianesi
In this study we analyze of a panel of patient-­‐derived cCSC to inhibiti
Istituto Regina Elena Nationl
Cancer Institute,
Via Elio
53, Rome
00144,sensitivity Italy
by cetuximab, both in vitro and in mouse xenografts, versus their K-­‐RAS and BRAF mutational status. Background: Targeting
the
epidermal
growth
factor
receptor (EGFR) pathway is an important
Methods and Results: therapeutic approach in aFive variety
tumors.
In colon cancer,
responsiveness
to acetuximab
hasstatus beenof K-­‐RAS and BRAF cCSC lof
ines were independently generated from patients, nd mutational put into relationship to mutational
status
of EGFR
molecules.
In particular,
patients
carmutations was assessed by PCR pathway
and sequencing. Cell proliferation in the presence or the absence of rying mutated forms of K-RAS
are (currently
fromin cetuximab
therapy
(1).assay. Colon
CSCs may tumors derived from
cetuximab 100 µg/ml) excluded
was evaluated vitro by Cell-­‐Titer Glow Subcutaneous represent predictive toolsCSCs for patient’s
therapeutic
(2).
Intumor this study
we
analyze
sensitivity 100 mm3 , the anim
were established in NSG response
mice. When the volume reached approximately of a panel of patient-derived
cCSC
to inhibition
cetuximab,
in vitro
and Tinumor mouse
xenografts,
received cetuximab (10 mby
g/kg, i.v., twice aboth
week, 4 weeks). size w
as measured every 2-­‐3 days by versus their K-RAS and BRAF
mutational
status.
caliper. results summarized in the table. The growth of Cfrom
TSC1, patients,
wild type fand
or both K-­‐RAS and BRAF, is fully
Methods and results:The Five
cCSCare lines
were independently
generated
mutainhibited b
y c
etuximab b
oth i
n v
itro a
nd i
n v
ivo. C
onversely, t
he G
12V-­‐ K
-­‐RAS m
utated tional status of K-RAS and BRAF mutations was assessed by PCR and sequencing. Cell proliferation CTSC2 shows very
little sensitivity to the drug in bµg/ml)
oth assays. mutated (Glow
CTSC3 and CTSC4) or V60
in the presence or the absence
of cetuximab
(100
wasInterestingly, evaluated G
in13D-­‐ vitroK-­‐RAS by Cell-Titer
BRAF m
utated (
CTSC 5
) l
ines s
howed i
ntermediate, d
ifferent d
egrees o
f r
esponse to cetuximab. assay. Subcutaneous tumors derived from CSCs were established in NSG mice. When the tumor
3
volume reached approximately
100 mm ,
the animals received cetuximab (10 mg/kg,
i.v., twice a week, 4 weeks). Tumor size was
measured every 2-3 days by caliper.
The results are summarized in the table.
The growth of CTSC1, wild type for both KRAS and BRAF, is fully inhibited by cetuximab both in vitro and in vivo. Conversely,
the G12V- K-RAS mutated CTSC2 shows
very little sensitivity to the drug in both assays. Interestingly, G13D- K-RAS mutated
(CTSC3 and CTSC4) or V600E BRAF mutated (CTSC 5) lines showed intermediate,
different degrees of response
to cetuximab.
% inhibition (cetuximab-­‐treated over controls) in vitro Xenograft proliferation volume KRAS BRAF CTSC1 WT WT 100% Full regression CTSC2 G12V WT 7±7 28±11 CTSC3 G13D WT 2±2 55±4 CTSC4 G13D WT 36±7 59±6 CTSC5 WT V600E 15±3 58±4 Conclusions: K-RAS and BRAF mutational status correlates with sensitivity to cetuximab in cCSC.
In fact, full proliferation inhibition and xenograft remission was achieved in a KRAS WT/BRAF WT
line, while little if any effect was observed in a line carrying the G12V K-RAS mutation. Lines carrying either a G13D mutated K-RAS, or a V600E mutated BRAF showed different degrees of response
to cetuximab in vivo, even though lower or no inhibition was observed in vitro. Recent data suggest
that patients carrying these mutations may not be actually quite unresponsive to cetuximab, as
previously believed (2;3). Our data would indeed support this hypotheses. The fact that a higher
sensitivity is observed for individual lines in xenografts as compared to their responsiveness in vitro
may possibly be explained by host-mediated effects. This point is currently under investigation.
1. Murphy JE et al. Expert Rev Anticancer Ther. 2010 Sep;10(9):1371-3.
2. Baiocchi et al. Curr Opin Pharmacol. 2010 Aug;10(4):380-4.
3. Bokemeyer C et al. Eur J Cancer. 2012 Jul;48(10):1466-75.
4. De Roock W et al. JAMA. 2010 Oct 27;304(16):1812-20.
51
Defining the mode of tumor growth by clonal analysis
Gregory Driessens1, Benjamin Beck1, Amélie Caauwe1, Benjamin D. Simons2,3 and Cédric Blanpain1,4
1
Université Libre de Bruxelles, IRIBHM, Brussels Belgium
2
Cavendish Laboratory, Department of Physics, Cambridge, UK
3
The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK
4
WELBIO, Brussels, Belgium
One of the key questions in cancer is to understand how tumors grow. Recent studies suggest
that cancer can be hierarchically organized as normal tissues containing cancer stem cells at the
top of the cellular hierarchy. Cancer stem cells have been described in different human cancers
including skin cancers. Cancer stem cells have been hypothesized to sustain tumor growth, to resist
to chemo and radiotherapy and to be responsible for tumor relapse. Until now, cancer stem cells
have been demonstrated by their ability to reform tumor upon transplantation into severely immunodeficient mice. These studies clearly show the potential of cancer cells in these experimental
conditions but not necessarily reflect the actual fate of tumor cells in their native environment, and
the existence of cancer stem cells during unperturbed tumor growth remained unproven.
52
In this study, we made use of clonal analysis to unravel the mode of tumor growth in vivo in its
natural environment1. To this end, we used a genetic labelling strategy that allows individual tumor cells to express the YFP reporter gene and follow their fate as well as their progeny over time
at different stages of tumor progression. Interestingly, we found that in benign skin tumors, the
majority of tumor cells have limited proliferative potential, while only a minority have the capacity
to persist long term and divide rapidly, giving rise to progeny that occupy a big part of the tumor,
consistent with the marking of long lived cancer stem cells. Quantitative analysis of clonal fate data
supports the existence of rapidly cycling cancer stem-like cells and a second population of more
slowly cycling committed progenitors, mirroring the composition, hierarchy, and fate behaviour of
the normal epidermis2. Such behaviour is shown to be consistent with double-labelling experiments
and detailed clonal fate characteristics. By contrast, clonal dynamic in invasive squamous cell carcinoma is consistent with emergence of a single CSC population with limited potential for terminal
differentiation. This study presents the first experimental evidence for the existence of CSC during
unperturbed solid tumor growth.
1. Driessens G, Beck B, Caauwe A, Simons BD, Blanpain C. Defining the mode of tumor growth by clonal analysis.
Nature, 2012; 488 : 527-30.
2. Mascre G, Dekoninck S, Drogat B, Youssef KK, Brohe S, Sotiropoulou PA, Simons BD, Blanpain C. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature, 2012; in press.
The matricellular protein SPARC inhibits the induction of HSC quiescence
after stress-induced myelosuppresion
Armin Ehninger1,2, Tobias Boch1,2, Gertraud Orend3, Andreas Trumpp1,2
1
Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
2
HI-STEM, Heidelberg Institute for Stem Cell Technology and Experimental Medicine, Heidelberg, Germany
3
INSERM Unit 682, Strasbourg, France
To better understand the process of homing and engraftment of HSCs to/in the bone marrow
that occurs around birth, we performed a comparative microarray analysis of HSCs from the liver
and the bone marrow of newborn mice. Interestingly, gene ontology analysis revealed an enrichment of genes involved in extracellular matrix biology and bone development. Among these genes,
the matricellular protein SPARC was highly enriched in HSCs that just colonized the bone marrow,
implying a role for SPARC in homing and engraftment of HSCs to this site. However, HSC numbers
in newborn and adult SPARC-deficient mice are unchanged and SPARC-deficient HSCs perform as
well as their wildtype counterparts in competitive transplantation assays. These results suggest
that cell-autonomous SPARC is dispensable for HSC function or that its lack is substituted by other
factors.
Intriguingly, SPARC-deficient mice have a reduced sensitivity to serial treatment with the chemotherapeutic agent 5-Fluorouracil (5-FU) in 10-day intervals. While the speed of recovery of the
blood system is normal, HSCs in a SPARC-deficient niche show an accelerated return to quiescence,
which seems to protect them from the lethal effects of serial 5-FU treatment. We are currently investigating the mechanism underlying the observed phenotype.
53
Rapid adhesion modulation of metastatic stem cells at the single-molecule
level
Robert Eibl
Stanford University (I.L. Weissman lab; and E.C. Butcher lab), California, USA; Technical University of Munich, Pathology;
University of Munich, Biophysics; University of Miami, Biophysics
Adhesion phenomena at the cellular level, such as rolling, chemokine-mediated integrin activation with rapid arrest under shear, and sticking on endothelium, can easily be observed on lymphocytes, lymphoma cells, and, to a lesser extend on metastasizing tumor cells using a flow chamber
assay. It appears to be more challenging to investigate such phenomena at the single molecule
level between two living cells. To the best of his knowledge to date, the author became the first
to 1) transfer his observed metastasis model of rapidly arresting B16 melanoma cells to level of
single-molecule adhesion, i.e. measuring the molecular forces between individual VLA-4/VCAM-1
adhesion receptors between two living cells; 2) to detect similar adhesion events at the single-molecule level between living lymphoma and either endothelial cells or immobilized VCAM-1 counterreceptors, respectively; and 3) to detect with his system at the single-molecule level the immediate
activation of VLA-4 integrin receptors by a chemokine (SDF-1) at the single-molecule level and at
the same time, on a living cell. This very unique tool of measuring the activation state of metastasissupporting cell adhesion receptors, as well as metastasis inhibiting repellent molecules) will be
evaluated for its use in the clinical management of metastatic solid tumors and leukemias.
1. Eibl RH, Single-Molecule Studies of Integrins by AFM-Based Force Spectroscopy on Living Cells. In: Scanning Probe
Microscopy in Nanoscience and Nanotechnology 3, Bhushan B. (Editor), Springer (2012)
2. Eibl RH, Cell adhesion receptors studied by AFM-based single-molecule force spectroscopy. In: Scanning Probe
Microscopy in Nanoscience and Nanotechnology 2, Bhushan B. (Editor), Springer, (2011)
3. Eibl RH, Direct Force Measurements of Receptor-Ligand Interactions on living cells. In: Applied Scanning Probe
Methods XII - Characterization. Bhushan B, Fuchs H (Editors), Springer, pp. 1-31, (2009)
54
4. Eibl RH and Moy VT, Atomic force microscopy measurements of protein-ligand interactions on living cells. In:
Protein-Ligand Interactions, (Editor: G.Ulrich Nienhaus), Humana Press, Totowa, NJ, U.S.A., pp. 437-448, ISBN
1588293726 (2005)
5. Eibl RH and Benoit M, Molecular resolution of cell adhesion forces. IEE - Nanobiotechnology 151(3):128-132
(2004)
Novel inhibitor of Wnt/β-catenin signaling targeting cancer stem cells
Liang Fang1, Edgar Specker2, Jens von Kries2 and Walter Birchmeier1
1
Max Delbrück Center for Molecular Medicine, Berlin, Germany
2
Leibniz Institute for Molecular Pharmacology, Berlin, Germany
Aberrant activation of the Wnt/β-catenin pathway has long been associated with various cancers, especially colon cancer. Inhibition of Wnt signaling has shown promising results on Wnt addicted cancers both in the clinic and laboratory. However, the limited inhibitory effect from nonspecific therapy (e.g., NSAIDs and vitamins) doesn’t improve the outcome for most of the patients in
the clinic, and genetic methods that attenuate Wnt signaling efficiently in the lab are not feasible in
the clinic. All these aspects encourage basic scientists and pharmaceutical companies to develop
specific small-molecule inhibitors of the Wnt pathway. In this study, using AlphaScreen and ELISA,
we identified a small-molecule compound that inhibits the interaction between TCF4 and β-catenin.
This compound, LF3, interferes only with the interaction between TCF4 and β-catenin, not between
E-cadherin and β-catenin, and specifically inhibits Wnt signaling in reporter assays and in various
colon cancer cell lines. It reduces proliferation and migration of colon cancer cells and arrests the
cell cycle of colon cancer cells in G1 phase. Remarkably, LF3 also inhibits sphere formation of colon cancer stem cells and induces differentiation of mouse salivary gland cancer stem cells. These
data demonstrate that LF3 is a specific Wnt/β-catenin inhibitor, affects various aspects of cancer
cells and cancer stem cells and has the potential to be developed further for clinical therapy.
55
Notch1 inhibition and the death ligand TRAIL – together against glioblastoma
Anne Fassl1, Katrin E. Tagscherer1, Jutta Richter1, Christel Herold-Mende2 and Wilfried Roth1,3
1
Molecular Tumor-Pathology, German Cancer Research Center, Heidelberg, Germany
2
Division of Neurosurgical Research, Department of Neurosurgery
3
Institute of Pathology, University of Heidelberg, Germany
Glioblastoma is the most frequent and malignant human primary brain tumor. Despite multimodal therapy, tumor occurrence is almost universally fatal with a median patient survival of 12
to 15 months after diagnosis. Effective anti-glioblastoma therapies are therefore urgently needed.
Several studies in recent years suggested the death ligand TRAIL to be effective in killing glioblastoma cells. However, glioblastomas exhibit a strong internal resistance to TRAIL treatment which
has to be overcome for a successful TRAIL application. We found that the inhibition of the Notch1
pathway can abrogate TRAIL resistance in glioblastoma cells as Notch1 knockdown sensitizes them
for TRAIL-induced apoptosis. Our data demonstrate that the anti-apoptotic effect of Notch1 signaling is hereby based on the Notch1-mediated upregulation of the anti-apoptotic Mcl-1 protein and
downregulation of the death receptor TRAIL-R2, the main TRAIL receptor in glioblastomas. Notch1dependent induction of Mcl-1 results from an increase in EGFR pathway activity. The regulation of
TRAIL-R2 occurs via Notch1-mediated inhibition of the transcription factor Sp1 causing a decreased
transcription from the TRAIL-R2 promoter. The effects of Notch1 signaling on Mcl-1 and TRAIL-R2
expression are present in glioblastoma long-term cell lines, primary cultures and glioblastoma stem
cell-like cells. Furthermore, a Notch1-knockdown accompanied increase in apoptosis following
TRAIL treatment was also observed in tissue slices of glioblastoma xenografts. The therapeutic
targeting of Notch1 in combination with TRAIL might therefore represent a promising novel strategy
in the treatment of glioblastomas.
56
CHD7 is essential for neurogenesis
Weijun Feng 1, Zhe Zhu 1, Amir Muhammad Khan 1, Olga Bernhardt 1, Christel Herold-Mende 2,
Hai-Kun Liu 1
1
Helmholtz Young Investigator Group-Normal and Neoplastic CNS Stem Cells, German Cancer Research Center (DKFZ),
DKFZ-ZMBH Alliance, Heidelberg, Germany
2
Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany
Chromatin factors regulating neurogenesis in the brain remain largely unexplored. Here we demonstrate that the chromatin remodeler CHD7 (chromodomain-helicase–DNA-binding protein 7),
which is mutated in human CHARGE syndrome, is a master regulator of neurogenesis in the mammalian brain. CHD7 is selectively expressed in neurogenic niches in the adult mouse brain, and
inactivation of CHD7 in mouse neural stem cells (NSCs) leads to impairment of neuronal differentiation and maturation. Strikingly, neurogenesis defect in the hippocampus of CHD7 mutants can be
rescued by physical exercise. CHD7 expression marks a class of human brain tumors with a neuronal differentiation molecular signature. We further identified that Sox4 and Sox11 are direct target
genes of CHD7 that are responsible for the CHD7 mutant phenotype. Our study demonstrates the
essential role of CHD7 in controlling neuronal differentiation, thus providing strong implications for
CHARGE syndrome and brain tumors.
57
Cancer stem cell maintenance is synergistically controlled by hypoxia and
acidosis through Hsp90
Alina Filatova*, Boyan K. Garvalov*, Sascha Seidel, Till Acker
Institute of Neuropathology, Justus Liebig University, Giessen/Germany
* These authors contributed equally to the work.
The growth of gliomas and other tumor types is thought to be driven by a population of cells with
stem cell character, termed cancer stem cells (CSCs). However, the mechanisms that regulate CSC
maintenance within tumors are insufficiently understood. One prominent characteristic of solid tumors is the prevalence of regions with low oxygen tension (hypoxia). Hypoxia induces a set of adaptive responses that ultimately promote a more aggressive tumor phenotype. Among the changes induced by hypoxia is a shift towards a glycolytic metabolism, coupled to synthesis of acidic products
and alterations in regulators of cellular pH, which result in acidification of the tumor environment.
The aim of our study was to characterize the interplay between tumor hypoxia and acidosis in regulating CSC maintenance and tumor progression. We have demonstrated that the CSCs phenotype
is maintained in a hypoxic niche in gliomas through hypoxia-inducible factor 2a (HIF-2a) function
(Seidel et al, Brain 2010, 133:983-95). We now identify acidosis as a further crucial component of
the hypoxic niche to control CSCs. Thus, hypoxia and acidosis synergized in increasing HIF levels
and the expression of HIF target genes, including genes involved in CSC maintenance. Importantly,
decreased pH promoted the CSC phenotype under hypoxia and lead to enhanced tumorigenicity.
Interestingly, the effects of acidosis on HIF and CSC function were not dependent on the classical
PHD/VHL pathway, which controls HIF stability, but were instead mediated by Hsp90. Hsp90 inhibition abolished the enhanced self-renewal of CSCs in vitro and tumorigenicity in vivo induced by low
pH and hypoxia. Our findings highlight the role of tumor acidification within the hypoxic niche in the
regulation of CSC function and point to potential therapeutic strategies for targeting this critical
tumor cell population.
58
Impaired gap junctional intercellular communication by lack of connexin 43
enhances aggressivity of pancreatic cancer
T. Forster1,2, V. Rausch1,2, L. Lui1,2, S. Labsch1,2, J. Mattern1,2, L. Disch1,2, J. Werner2, M.M. Gebhard 2,3,
M.W. Büchler2, M. Schäfer2,3 and I. Herr1,2
1
Molecular OncoSurgery,
2
General Surgery,
3
Experimental Surgery, University of Heidelberg and German Cancer Research Center, Heidelberg, Germany
Background: The aggressiveness of pancreatic cancer may be due to cancer stem cells (CSCs)
conferring therapy resistance and metastasis. In solid tumors, it is believed that gap junctional
intercellular communication (GJIC) is responsible for growth control, differentiation and apoptosis.
Major characteristics of cancer cells are the lack of growth control, normal differentiation, immortality and the absence of functional GJIC. Furthermore, recent studies suggest that stem cells do
not express Connexins, the protein family involved in gap junction formation and intercellular communication. Thus, CSCs seem to be responsible for pronounced therapy resistance due to impaired
GJIC.
Hypothesis: GJIC may influence CSC characteristics of highly aggressive pancreatic cancer cells.
Methods: We examined the expression of Connexins by western blot analysis and immunofluorescence stainings. Functional GJIC in the pancreatic cancer cell lines BxPc-3, BxPc-3 Gemcitabine
resistant and Capan-1 cells was investigated by Lucifer Yellow microinjection and double-dye flow
cytometry experiments. The correlation of impaired GJIC, Gemcitabine bystander effect and therapy
resistance was evaluated by viability assay and FACS measurements.
Results: Strong GJIC and Gemcitabine bystander effects were observed in BxPc-3, which were
diminished in BxPc-3 Gemcitabine resistant and completely missing in Capan-1 cells. These results
correlated with the pronounced reduction of viability and induction of apoptosis in BxPc-3, which
was weaker in BxPc-3 Gemcitabine resistant and nearly absent in Capan-1 cells. Analysis of cell
lines individual drug sensitivity revealed that gemcitabine resistance was associated with dysfunctional GJIC and the presence of pancreatic cancer cells with stem cell characteristics. Connexin
43 was identified as a key candidate mediating GJIC and bystander effects. Its high expression
was observed in normal pancreatic epithelial and BxPc-3 cells, while expression was diminished in
BxPc-3 Gemcitabine resistant and totally downregulated in Capan-1 cells. Correspondingly, siRNA
mediated knock down of Cx43 in BxPc-3 cells prevented GJIC and Gemcitabine bystander effect.
Conclusions: Our data suggest that targeted induction of Connexin 43 may be a promising approach for restoring GJIC and enhancing treatment effect of pancreatic cancer.
59
Progenitor cell dynamics of sebaceous gland development in mammalian skin
Daniela Frances and Catherin Niemann
Center for Molecular Medicine Cologne (CMMC), University of Cologne, Germany
Mammalian epidermis protects the body against environmental assaults and consists of the
interfollicular epidermis (IFE) with associated hair follicles (HF) and sebaceous glands (SG). HFs,
IFE and SGs undergo constant cellular renewal. This implies stem cells (SC) to be involved in the
process of epidermal regeneration. Indeed, various stem and progenitor cell compartments have
been described in epidermal subcomponents during the past years. In addition to the essential role
of HFSC for tissue regeneration it has recently been recognised that they also have an important
role in the process of tumor initiation.
However, the specific role of individual stem cell pools is not well understood. Therefore, it is crucial to investigate the intrinsic properties, biological potential and behaviour of the individual stem
cells during development, homeostasis and disease.
Important questions in the field are: when are diverse epidermal stem cell compartments are
established during skin morphogenesis and when is their function initiated?
To study the formation of epidermal stem cell compartments, we have investigated the development of HF and SG in great detail. Therefore, analysis of the spatio-temporal organisation of
stem and progenitor compartments was performed during skin morphogenesis. Interestingly, SC
markers Sox9 and Lrig1 are co expressed by HF cells at early stages of development before SG
development is initiated. Later, each marker is confined to a particular zone of the HF suggesting a
common precursor population of different HFSC compartments. Our data show that Lrig1 positive
SCs proliferate giving rise to sebocytes of the future SG whereas Sox9 positive cells localise to the
future HFSC region.
60
Our results could be of major importance for understanding initial steps of skin tumor development as it has been proposed that during tumor formation, basics steps of morphogenesis are
recapitulated.
Insertional mutagenesis to screen for factors which impact upon hematopoietic stem cell self-renewal
Anja Geiselhart1, Sina Huntscha2, Martijn Brugman3, Dagmar Walter2, Amelie Lier2, Christopher
Baum4, David A. Williams5,6, Andreas Trumpp1,2 and Michael D. Milsom1,2
1
German Cancer Research Center (DKFZ), Division of Stem Cells and Cancer, Experimental Hematology (A012), Heidelberg, Germany
2
Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Experimental Hematology Group,
Heidelberg, Germany
3
Leiden University Medical Center, Department of Immunohematology and Blood Transfusion, ZA Leiden, The Netherlands
4
Hannover Medical School, Institute of Experimental Hematology, Hannover, Germany
5
Children’s Hospital Boston and Dana-Farber Cancer Institute, Harvard Medical School, Division of Hematology/Oncology, Boston, Massachusetts, USA
6
Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
Correspondence: E-mail: [email protected]
Transplantation of hematopoietic stem cells (HSCs) is a potential curative therapy for many inherited blood disorders and, in concert with high dose chemo- or radiotherapy, can be effective in
the treatment of a range of cancers such as leukaemia. However, this approach is mainly restricted
to the availability of an immunologically matched HSC donor. Whilst HSCs are able to differentiate
into all mature hematopoietic lineages they also retain self-renewing potential, which is essential
for the maintenance and expansion of HSC numbers and is also deregulated in leukemic stem cells.
Therefore, dissecting the biology of HSCs might reveal novel therapeutic modalities that facilitate
the production of transplantable HSCs from novel sources such as umbilical cord blood.
To interrogate HSC self-renewal, we have designed a forward genetic screen for cellular factors
that have a major impact on the HSC engraftment potential using the Fanconi Anemia (FA) HSC
disease model. FA patients display an early onset of leukaemia and are predisposed towards myeloid dysplastic syndrome and some solid tumors. Mouse models deleted for components in the FA
signalling pathway demonstrate compromised HSC self-renewal as seen by a pronounced depletion
of HSCs during ex vivo expansion and a severe engraftment defect in vivo.
To screen for factors that are involved in the process of HSC self-renewal, we have transplanted
retroviral marked FA HSCs, which are deficient in the Fanca, Fancc and Fancg genes, into lethally
irradiated WT mice in order to analyse their ability to serially repopulate the recipients. In the absence of a functional FA pathway, HSCs have a severe engraftment defect. Therefore we would predict that if the semi-random mutagenesis induced by the retroviral vector results in a gain of function mutation that rescues this phenotype, the progeny of this clone will become over-represented
in the transplanted recipient mouse. In line with this hypothesis, we are able to demonstrate the
emergence of these so-called dominant clones in recipient mice that have been transplanted with
retroviral transduced FA HSCs. We are now in the process of determining which cellular genes have
been mutated in these dominant clones and validating whether they are able to impact upon HSC
self-renewal.
61
Overexpression of the EMT regulator Zeb2/Sip1 results in a block in T cell
development and identifies this gene as a new driver for T cell lymphoblastic
leukemia
Steven Goossens1, Tom Taghon2, Odile Blanchet3, Joni Van der Meulen4, Morvarid Farhang
Ghahremani1, Sonia Bartunkova1, Katharina Haigh1, Lieven Haenebalcke1, Pieter Rondou4, Geert
Berx1, Viktor Janzen5, Jules Meijerink6, Danny Huylebroeck7, Jean Soulier3, Frank Speleman4 and
Jody J. Haigh 1
1
Dept. for Molecular Biomedical Research, VIB-UGhent, Ghent, Belgium
2
Dept. of Clinical Chemistry, Microbiology and Immunology, UGhent, Ghent, Belgium
3
Institut Universitaire d’Hématologie and U944 INSERM, Hopital Saint-Louis, Paris, France
4
Center for Medical Genetics, UZ Ghent, Belgium
5
Dept. of Internal Medicine III, Hematology and Oncology, University of Bonn, Germany
6
Dept. of Pediatric Oncology/Haematology, Erasmus MC Rotterdam - Sophia Children’s Hospital, Rotterdam, The Netherlands
7
Dept. of Development and Regeneration, KULeuven, Leuven, Belgium
Zeb2 (Sip1/Zfhx1b) is a member of the ZEB family of transcriptional repressors demonstrated
to regulate epithelial to mesenchymal transition (EMT) processes during embryogenesis and tumor
progression. The expression of some EMT regulators is correlated with the formation and/or function of tumor-initiating cells or cancer stem cells in solid epithelial tumors. Using a loss-of-function
approach, we have previously demonstrated that Zeb2 is highly expressed in the hematopoietic system (Goossens et al., Blood 2010) and evidence from mouse retroviral mutagenesis studies points
to a putative role of Zeb2 in initiation and/or progression of leukemia/lymphoma.
62
In this study we examined the roles of Zeb2 in the stem cells of the hematopoietic system and
in leukemia formation through a conditional gain-of-function approach in the mouse. For this, we inserted a Zeb2 encoding cDNA preceded by a floxed transcriptional stop cassette within the Rosa26
locus (See Nyabi et al., NAR 2009). Using the Tie2-cre line (that is active in the endothelium and
entire hematopoietic system), we were able investigate the effects of Rosa26 locus based overexpression of Zeb2 in HSC biology and during leukemic transformation. Bi-allelic overexpression of
Zeb2 from the Rosa26 locus resulted in altered T cell development. A partial block in differentiation was observed both in vivo as in vitro, using an OP9-DL1 coculture system, at the DN3 (CD4/8-,
CD44-,CD25+) pre-T cell stage, at the beta-selection checkpoint. In addition, Zeb2 overexpressing
mice spontaneously develop thymic lymphomas starting at 6 months of age, indicating that Zeb2
acts as a driver in T cell malignancies, most likely due to the disruption of normal thymocyte differentiation. To further investigate the role of Zeb2 in T-ALL progression, we have bred these mice
onto a tumor-prone background (p53flox/flox) and observed a significant decrease in T-cell lymphoma/
leukemia latency and an increase of the stem/progenitor markers c-Kit and CD44, suggesting an
increase in leukemic stem cells. Indeed, using a minimal dilution series of tumor cells into NOD/
SCID mice we could demonstrate a 10-100 fold increase in leukemia initiating cells in the Zeb2
overexpressing tumors.
To assess the relevance of these findings with human disease, we screened a cohort of T-ALL
patients and found increased expression of ZEB2 predominantly associated within immature/ETPALL patients. Compared to other T-ALL subgroups (cortical or mature), ETP-ALLs are defined by a
gene-expression profile similar to Early Thymic Precursor (ETP) cells, a subset of thymocytes with
stem cell-like features, and a striking feature is that these patients have poor response to conventional therapies with a high risk of remission failure or hematological relapse. The basis of the poor
prognosis in ETP-ALL patients has yet to be identified, although one potential contributing factor
is the stem cell properties of this aggressive leukemia. Importantly, we could identify two ETP-ALL
patients who presented a unique chromosomal translocation t(2;14)(q22;q32) on karyotype. This
new translocation involves the ZEB2 locus and the BCL11B locus as confirmed by FISH analysis.
Such BCL11B-associated translocations lead to the overexpression of the partner gene, thereby
identifying ZEB2 deregulation as a molecular driving force for the development of ETP-ALL in these
two patients.
In conclusion, we have shown that Zeb2 overexpression affects early T cell development and
predisposes mice to develop an aggressive form of T-ALL with increased stem cell properties. This
mimics patients with aggressive ETP-ALL driven by ZEB2 translocations, demonstrating that the
EMT regulator ZEB2 is an oncogene for T-ALL.
63
Identification of new treatment targets of GBM from researching on glioma
initiating cells
Takuichiro Hide1,2, Tatsuya Takezaki1,2, Hideo Nakamura2, Keishi Makino, Jun-ichi Kuratsu2 and
Toru Kondo1
1
Laboratory for Cell Lineage Modulation, Center for Developmental Biology, RIKEN, Kobe, Japan
2
Department of Neurosurgery, Kumamoto University Graduate School of Medical Science, Kumamoto, Japan
Purpose: Malignant tumors, including glioblastoma multiforme (GBM), contain cancer stem cells
(CSCs), which self-renew indefinitely and are tumorigenic. The mean survival time of GBM patients
is still about one year. To improve survival time of GBM patients, we are searching new therapeutic
targets of GBM using GICs.
Methods: Neurosphere cells (NSC), oligoderndrocyte precursor cells (OPC) and astrocytes (AST)
derived from p53-/- embryonic mouse brain at 13.5 dpc. were prepared and established artificial
GICs (NSCL61, OPCL61 and ASTL61) by overexpression of active form of Hras (HrasL61) with GFP.
To identify candidate targets, microarray analysis were carried out.
Results: NSCL61 and OPCL61 formed GBM even by ten cell injection but ASTL61 form anaplastic astrocytoma by 10,000 cell injection. To characterize the gene expression changes that
occurred during the transformation of NSCs and OPCs, We used DNA microarray technique and
found some candidate genes. The cyclooxygenese (Cox)-2 and epidermal growth factor (EGF) ligands are expressed in the transformed cells and human GICs. The combination of a Cox2 inhibitor and an EGFR inhibitor significantly inhibits tumorigenicity in artificial GIC transformed OPCs
and one of human GIC.
Next we established subline of NSCL61 by single cell culture and pick Sox11 up from microarray
data. Sox11is highly expressed in non-tumorigenic sublines, but not in tumorigenic sublines. Overexpression of Sox11 prevented tumorigenesity of NSCL61and tumorigenesity in non-tumorigenic
subclones were induced by knockdown of Sox11 by shRNA.
64
Conclusion: From GIC research, we identify that the combination of Cox2 inhibitor and EGFR
inhibitor is a new therapeutic strategy against malignant gliomas and that Sox11 is the potential
target of treatment focusing on GICs.
Regulation of the tumor suppressor EPHB3 reveals anti-tumorigenic
functions of Notch signalling in colorectal cancer cells
Sabine Jägle1, Kerstin Rönsch1, Marcel Jäger1, Amelie Proske1, Sarah Ortolf1, Afsheen Yousaf2,
Sylvia Timme3, Tom Michoel2, Silke Laßmann3 and Andreas Hecht1,4
1
Institute of Molecular Medicine and Cell Research, Faculty of Biology, Albert-Ludwigs-University Freiburg, Germany
2
FRIAS - Freiburg Institute for Advanced Studies, Albert-Ludwigs-University Freiburg, Germany
3
Institute of Pathology, Freiburg University Medical Center, Albert-Ludwigs-University Freiburg, Germany
BIOSS - Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Germany
4
The EPHB3 receptor tyrosine kinase acts as tumor suppressor by locally restricting tumor cell
spreading through repulsive cell-cell interactions. In colorectal tumorigenesis, mutations leading to
constitutive activation of Wnt/β-catenin signalling initially result in upregulation of the EPHB3 gene.
However, EPHB3 expression is frequently lost in colorectal carcinomas. Molecular mechanisms
underlying this secondary silencing are largely unknown. Here, we identify a transcriptional enhancer critical for EPHB3 expression. Activity of the EPHB3 enhancer varies in different colorectal
cancer (CRC) cell lines and loss of EPHB3 enhancer function appears to result from reduced Notch
pathway activity. Accordingly, reconstitution of Notch pathway activity in CRC cells restores EPHB3
expression. Microarray expression data from CRC patients and immunohistochemical analyses of
human tissue specimens further support the importance of Notch pathway activity for EPHB3 expression. Moreover, reconstituted Notch activity induces cell cycle arrest and apoptosis. Overall
our results demonstrate a simultaneous requirement of the Wnt/β-catenin and Notch pathways
for EPHB3 expression and argue for a tumor suppressive function of Notch signalling in CRC cells.
65
Stem-like glioma cells comprise a non-homogeneous population of cells
with varying degrees of stemness and tumorigenicity
Darius Kalasauskas1, Anne Rupp1, Mirjam Renovanz1, Bettina Sprang1, Marina Janocha1, Walter
Schulz-Schaeffer2, Alf Giese1, Ella L. Kim1
1
The Translational Neurooncology Research Group, Department of Neurosurgery, University Medical Centre, Johannes
Gutenberg University, Mainz, Germany
2
Department of Neuropathology, University Medical Centre Göttingen, Göttingen, Germany
Glioblastoma multiforme (GBM) is one of the most challenging human cancers. The slow progress in developing effective therapies for GBM has been explained by the lack of appropriate paradigm for the pathogenesis and progression of this devastating disease. Recently, a new conception
of GBM has emerged based on the identification of a distinct population of glioma cells (generally
called Brain Tumor Initiating Cells, BTICs) that possess properties of stem cells. According to the
BTIC concept, stem-like glioma cells have the highest tumorigenic potential among all cell types
comprising the tumor. While the BTIC paradigm is rapidly gaining wide-spread acceptance, there is
still considerable uncertainty as to the identity of stem-like glioma cells and their precise roles in
the initiation, maintenance and progression of GBM.
Fundamental remaining questions include: Is there a universal type of stem-like glioma cells?
What criteria define glioma cell stemness? How does the degree of glioma cell stemness relate to
the clinicopathological criteria of glioma aggressiveness? To address these questions, the relationship between tumorigenicity, proliferative potential and differentiation was examined through a
combinatorial approach based on in vitro and in vivo analyses of different types of stem-like glioma
cells. Our study provides evidence that, in GBM, the type of cells collectively called BTICs comprises
heterogeneous subtypes of phenotypically and functionally distinct cells with varying tumorigenic
potential. Our findings urge a reconsideration of some of the key assumptions of the BTIC paradigm
and provide important insights into the roles of different types of stem-like cells in GBM.
66
Chemoresistance as a consequence of epithelial-mesenchymal transition
Ulrike Keitel1, Christina Scheel2, Cathrin Hippel1, Antje Dickmanns1, Jürgen Thomale3, Robert A.
Weinberg4, Matthias Dobbelstein1
1
Department for Molecular Oncology, Göttingen Center of Molecular Biosciences, Germany
2
Department for Normal and Malignant Breast Stem Cells, Helmholtz Center Munich, Germany
3
Department for DNA Repair, University of Essen, Germany
4
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, USA
During tumor progression, carcinoma-derived cells frequently adopt the properties of mesenchymal cells, including increased motility and invasion. This Epithelial-Mesenchymal Transition (EMT)
is initiated and executed by different mechanisms such as the Wnt- and TGFβ-signaling pathways
or via the activation of multiple tyrosine kinase receptors. More recently, a system of human mammary epithelial cells was established by Weinberg and colleagues, allowing the isolation of a mesenchymal subset from a generally epithelial population of cells.
We found that EMT renders the cells resistant towards Cisplatin treatment, whereas the sensitivities towards other chemotherapeutics remained similar. The difference in sensitivity towards
cisplatin was not only observed with regard to apoptosis, but also through differential accumulation
of phosphorylated histone 2AX (gammaH2AX), a hallmark of the early DNA damage response. Strikingly, even the adducts formed by Cisplatin and DNA, were not found to accumulate differentially
before and after EMT. We propose that different chromatin structures before and after EMT lead
to differential DNA damage response. Treatment of the mesenchymal subpopulation with histone
deacetylases inhibitors partially overcame the resistant phenotype towards Cisplatin.
67
Chloroquine affects chromatin structure and sensitizes stem-like glioma
cells to ionizing radiation
Ella L. Kim 1, Anne Rupp 1, Gabriela Salinas-Riester 2, Darius Kalasauskas 1, Mirjam Renovanz 1,
Bettina Sprang 1, Marina Janocha 1, Walter Schulz-Schaeffer 3, Alf Giese 1
1
The Translational Neurooncology Research Group, Department of Neurosurgery, University Medical Centre, Johannes
Gutenberg University, Mainz, Germany
2
The Transcriptome Analysis Laboratory, University Medical Centre Göttingen, Göttingen, Germany
3
Department of Neuropathology, University Medical Centre Göttingen, Göttingen, Germany
Glioblastoma multiforme (GBM) is notoriously resistant to conventional treatments comprised by
surgery and combined radio- and chemotherapy. The underlying reason for is an impaired function
of multiple signalling pathways involved in the regulation of cell survival. A further level of complexity is added by the existence of a distinct type of glioma cells possessing properties of cancer stem
cells. A growing body of evidence indicates that such cells collectively called Brain Tumor Initiating
Cells (BTICs) comprise the most aggressive type of cells in GBM. Thus, the development of novel
therapies that could effectively target the BTIC compartment in GBM is the matter of high priority.
There is a growing evidence that chroloquine (ClQ), a derivative of 4-aminoquinoline has potent
antineoplastic effects. We have previously shown that ClQ exerts growth suppressive effects and
activates apoptotic response through the p53 pathway in conventional glioma cell lines lacking
stemness properties. This study was carried out to explore the effects of ClQ on glioma stem-like
cells (GSCs) with mutant p53.
68
The most significant findings is that ClQ sensitizes GSCs to ionizing radiation in vitro and in
vivo. Genome-wide gene expression analyses revealed that ClQ induces dramatic changes in global
gene expression patterns in GSCs. Among the genes whose expression is affected by ClQ, prime
therapeutic targets Aurora A/B and TGF-beta2 have been identified in silico and validated by qRTPCR and western blot. Chromatin immunoprecipitation (ChIP) analyses revealed that ClQ induces
structural changes in the chromatin and affects the binding of several DNA binding proteins including mutp53 and ets-1. Our results indicate that modulation of chromatin structure is an important
mechanism underlying antineoplastic effects of ClQ and provide the first evidence for the therapeutic potential of ClQ in overcoming radioresistance mediated by GSCs.
Thyroid hormone induces cellular senesce
F. Kraft*, W. Hessenkemper*, W. Hong+, V. Lorenz* and A. Baniahmad*
* Institute of Human Genetics, Jena University Hospital, Jena, Germany
+
Department of Immunology, Tianjin Medical University, Tianjin, China
Aims: To analyze the signaling pathway of thyroid hormone-induced growth inhibition in neuroblastoma cells, to investigate the expression of potential mediators of growth inhibition on both
mRNA and protein level, to identify key proteins in the signaling pathway
Methods: The expression and function of proteins which could be involved in the induction of
growth inhibition were analyzed by qRT-PCR, Western blotting, reporter gene assays and chromatin
immunoprecipitation (ChIP). The induction of cellular senescence was visualized by SA-β-gal and
DAPI staining. Cell cycle arrest was measured with flow cytometry and growth curves.
Results: Thyroid hormone (T3) induces cellular senescence, which is a major contributor of cell
growth inhibition and tumor suppression. The thyroid hormone receptor β (TRβ) is known as a potent tumor suppressor and leads in neuroblastoma N2a cells expressing TRβ to a growth arrest mediated by its ligand T3. We show that T3-treatment of N2a β cells results in TRβ-depended cellular
senescence. The expression of different specific cellular senescence markers such as senescenceassociated β galactosidase (SA-β-Gal) and senescence-associated heterochromatic foci (SAHF)
could be detected. On molecular level, treatment with T3 leads to repression of the cell cycle regulating factor cyclin D1 on both mRNA and protein level. In addition, a decrease of E2f1 and c-myc
and an increase of p16 and p19 mRNA after treatment with T3 could be detected. We could also
observe an induction of Dec1 on mRNA and protein level, which could be a mediator of senescence.
Moreover, we show the binding of the TRβ to the Dec1 promotor by ChIP and functionally by reporter
gene assays. Furthermore, our results demonstrate a repression of FoxO-mediated transactivation
and therefore another possible mechanism of senescence induction via the TRβ.
Conclusions: T3 treatment of N2a β cells leads to the induction of cellular senescence and thus
to the inhibition of growth in these tumor cells. This process seems to be mediated by the TRβ dependent regulation of selected cell cycle regulators.
69
Myosin II expression and regulation on oral squamous cell carcinoma
Otavio Fancisco Gomes Dias, Bernardo Salim Silveira, Grasieli Ramos, Isabel da Silva Lauxen,
Manoel Sant’Ana Filho, Pantelis Varvaki Rados, Marcelo Lazzaron Lamers
Federal University of Rio Grande do Sul, Porto Alegre, Brazil
Cell migration is a prominent feature in cancer metastasis and the characterization of migration-related proteins might be considered as potential targets for therapeutics and for diagnostic
intervention. Among the proteins involved on cell migration, the non-muscle myosin type II isoforms
play a pivotal role in several events related to cell migration, as cell adhesion, cell signaling and
cell contractility. There are three isoforms of myosin type II which can be expressed in mammalian
cells: myosin IIA (MIIA), myosin IIB (MIIB) and myosin IIC (MIIC). However, few studies have been conducted to characterize their expression and distribution patterns in cells from different tumor types,
including oral squamous cell carcinoma. The aim of the study was to analyze the expression and
distribution of myosin II isoforms (A, B and C) in biopsies samples of oral squamous cell carcinoma
with a high level of aggressiveness and differentiation. Tongue and lip biopsies were collected from
distinct regions of the tumor: the free zone, the centre zone and the invasion zone. These samples
(n=4) were fixed, crioprotected, cut on criostat, submitted to immunolocalization of MIIA, MIIB and
MIIC and analyzed on confocal microscopy. The three non-muscle myosin II isoforms were differentially expressed and distributed according to the region of the tumor sample. MIIA was distributes
mainly at the epithelium basal layer at the free zone of the tumor. The MIIB and MIIC were overexpressed at the center zone and both isoforms presented a perinuclear distribution as well a strong
colocalization with the actin cytoskeleton at the cell cortex. These results suggest that MIIA and
MIIC are related to tumor proliferation while MIIB is involved on tumor invasiveness.
Keywords: cell migration, cytoskeleton, metastasis.
Funding Support: UFRGS (Propesq and Relinter), CAPES, CNPq
70
ALDH1 in head and neck cancer and the immunohistochemical profile of a
collective cell migration event
Isabel da Silva Lauxen 1, Ana Luísa Homem de Carvalho 1, Laura de Campos Hildebrand 1, Marcelo Lazzaron Lamers 1, Jacques Eduardo Nör 2, Manoel Sant’Ana Filho 1
1
Oral Pathology, School of Dentistry, UFRGS Patologia – Faculdade de Odontologia da UFRGS, Porto Alegre, Brasil
2
Angiogenesis Research Laboratory, School of Dentistry, UMICH, Ann Arbor, MI, USA
Background: Epithelial cells of carcinomas can invade and metastasize as multicellular aggregates in a process known as collective cell migration and evidence has shown that only a minority of
these cancer cells have stem properties. Cancer high tumorigenic cells (CHTC) are responsible for
tumor maintenance, growth and metastases and the presence of CHTC antigens, as ALDH1, can be
evidenced in some human tumors by phenotypic analysis through immunostaining.
Aims: To identify CHTC through ALDH1 immunostaining in three different areas of head and neck
primary squamous cell carcinomas - tumor center, invasive tumor front and non-tumor epithelium and analyze the immunoprofile of a group of collective migratory cells invading a blood vessel.
Methods: Non-tumor epithelium was classified in dysplastic and non-dysplastic. Tumor histological grading was categorized in: low risk, moderate risk and high risk. The ALDH 1 immunolabeled
cells were grading negative (<5%) or positive (>5%). Collective migratory cells phenotype analysis
was assessed by E-Cadherin, N-Cadherin and vimentin immunostaining.
Results: No statistical relationship between ALDH1 immunostaining and tumor morphological
grading was found. There was a positive correlation between ALDH1+, tumor size and lymph node
compromise at non-tumor epithelium and invasive front. The 2 years of follow up showed high level
of metastasis on patients with ALDH+ at non-tumor epithelium. The collective migratory cells immunostaining showed a decrease on E-Cadherin expression associated to a randomic vimentin
labeling and a strong N-Cadherin staining.
Conclusion: Head and neck derived squamous cell carcinoma biopsies presented ALDH1 staining in both tumor and non-tumor epithelia which correlated with clinical parameters. The collective
migratory cells associated to this tumor type showed simultaneously epithelial-mesenchyme transition biomarkers. These results suggest that positive immunostaining for ALDH1 can identify more
aggressive tumors, as well as it may be a predictive method of disturbance in non-tumor epithelium.
Ethical approval by Comitê de Ética em Pesquisa do HCPA (GPPG n°09-315).
Conflict of interest statement: None declared.
71
Characterization and clinical relevance of ALDHbright populations in
prostate cancer
Clémentine Le Magnen 1, Lukas Bubendorf 4, Cyrill A. Rentsch 2, Chantal Mengus 1, Joel Gsponer 4,
Tobias Zellweger 3, Malte Rieken 1,2, Alexander Bachmann 2, Stephen Wyler 2, Michael Heberer 1,
and Giulio Spagnoli 1
1
ICFS, Departments of Surgery and Biomedicine, Basel University Hospital, Basel, Switzerland
2
Department of Urology, Basel University Hospital, Basel, Switzerland
3
Division of Urology, St Claraspital, Basel, Switzerland
4
Institute for Pathology, Basel University Hospital, Switzerland
High aldehyde dehydrogenase (ALDH) activity has been suggested to select for cells endowed
with tumor-initiating capacity in various tumors including prostate cancer (PCa). Yet existence of
cells with high ALDH activity (ALDHbright) in fresh PCa primary specimens and their potential clinical
significance have not been demonstrated so far. In this study, we investigated presence, phenotype,
and clinical significance of ALDHbright populations in clinical PCa specimens.
72
Taking profit of the Aldefluor™ technology, we assessed ALDH activity in cells derived from freshly excised PCa specimens (n= 39). Remarkably, ALDHbright populations were present and heterogeneously distributed across PCa specimens, and were mainly included within the EpCAM(+) and
Trop2(+) cell populations. Additionally, genes encoding several ALDH specific isoforms, previously
shown to be involved in tumor initiation/progression, were highly expressed in PCa tissues (n>50).
Among these isoforms, only ALDH1A1 gene expression significantly correlated with ALDH activity
(p<0.01), and was increased in cancers with high Gleason scores (p=0.03). ALDH1A1 protein expression, location, and prognostic significance were then assessed by staining two tissue microarrays (TMA) including over 500 samples of benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and multi-stage PCa. Strikingly, ALDH1A1 protein was expressed significantly
more frequently and to higher extents in advanced as compared to low stage PCa and BPH. Convincingly, ALDH1A1 positivity was also associated with poor survival (p=0.02) in “hormone therapy
naïve” patients.
Taken together, our data indicate that ALDH contributes to the identification of subsets of PCa
cells of potentially high clinical relevance.
Sensitivity of hematopoietic stem and progenitor cells to biofunctionalized
nanopatterns and substrate elasticity
Cornelia Lee-Thedieck 1,2, Christine A. Muth 2, Eva Altrock 2, Carolin Steinl 3, Gerd Klein 3, Joachim P.
Spatz 2
1
Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, Eggenstein-Leopoldshafen, Germany
2
Max Planck Institute for Intelligent Systems, Department of New Materials and Biosystems, Stuttgart, Germany, and
University of Heidelberg, Department of Biophysical Chemistry, Heidelberg, Germany
3
University of Tübingen, Center for Medical Research, Section for Transplantation Immunology and Immunohematology,
Tübingen, Germany
Human hematopoietic stem cells (HSCs) are indispensible for regenerative therapies of hematological disorders such as leukemia. However, the clinical application of HSCs is limited by the availability, because there are fewer donors than patients in need. Thus, a targeted in vitro proliferation
of HSCs would be a great achievement for patients suffering from hematological disorders. The
fundamental problem in this purpose is the fast differentiation of HSCs in culture. In the body HSCs
reside in the bone marrow in specific stem cell niches. These niches possess unique microenvironments that allow the maintenance and self-renewal of HSCs. They have a characteristic biochemical composition and defined physical properties such as substrate nanostructure and elasticity,
which are both known to influence cell behaviour. The aim of our study was to study the influence
of ligand type, substrate nanostructure and elasticity on HSCs.
Human hematopoietic stem and progenitor cells (HSPCs) were isolated from umbilical cord
blood and applied to nanopatterned, biofunctionalized gold nanoparticle arrays. With this approach
we could show that HSPC adhesion, signal transduction and gene expression depend on the lateral
nanometer-scale distance between integrin ligands. Furthermore, we studied the role of substrate
stiffness in the HSC niche. Based on our data, we developed a new model that describes (i) how
substrate stiffness is modulated in the context of a HSC niche during physiological processes and
(ii) how the stiffness, in concert with other factors, influences HSC adhesion and migration in the
niche. In conclusion, our work reveals that beside the biochemical composition also the physical
properties of the microenvironment influence HSC behavior.
73
Endothelial differentiated MSCs alter tumorgenic potential of fibrosarcoma
in co-culture
Irit Levinger, Razi Vago
Avram and Stella Goldstein-Goren, Department of Biotechnology Engineering, Ben-Gurion University of The Negev, Beer
Sheva, Israel
In order to develop and metastasize malignant tumors relays on array of signals and interactions. Angiogenesis and vasculogenesis are pivotal among these processes. Mesenchymal stem
cells (MSCs) are triggered by cancer cells to participate mainly in the tumor’s vasculogenesis. In
recent years it had became apparent that MSCs are intimately involved with tumor progression. However, their role remains controversial, with the majority of the studies claims that MSCs are recruited
by tumor cells to somehow maintain tumor growth and seed new tissue for further metastases and
development. Here we applied a three-dimensional in vitro model to study the interactions between
endothelial differentiated MSCs (EDMSCs) on the nature and tumorgenicity of the culture.
We showed that the presence of MSCs within the fibrosarcoma elevated the tumorgenic potential of the culture, having high expression of tumor-related markers such as: CCL5, CCL2, c-Myc;
increased proliferation rate; and stronger migration abilities. However, when EDMSCs were introduced to the microenvironment, the tumorgenic potential of the culture was decreased in comparison with co-culture of fibrosarcoma and undifferentiated MSCs. We also found that expression of
VEGF was decreased in co-culture with MSCs and further decreased in co-culture with EDMSCs. Our
3D in vitro study supports the previous findings regarding the role of MSCs in tumor development.
We suggest that along with MSCs contribution to tumor vasculogenesis, via paracrine signaling and
direct cell-cell interactions, the presence of EDMSCs restrain tumorgenic capacity of the co-culture.
74
The role of pro-inflammatory cytokines in governing hematopoietic stem
cell self-renewal
Amelie Lier 1,4, Dagmar Walter 1,4, Anja Geiselhart 1, Sina Huntscha 1,4, Irem Bayindir 1, David Brocks 1,
Marieke Essers 2,4, Andreas Trumpp 3,4 and Michael D. Milsom 1,4
1
Experimental Hematology Group (A012), German Cancer Research Center (DKFZ), Heidelberg, Germany
2
Hematopoietic Stem Cells and Stress Group (A011), German Cancer Research Center (DKFZ), Heidelberg, Germany
3
Division of Stem Cells and Cancer (A010); German Cancer Research Center (DKFZ), Heidelberg, Germany
4
Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany
Correspondence: E-mail: [email protected]
Hematopoietic stem cells (HSCs) are responsible for the life-long production of all blood cell
lineages. To maintain HSC numbers, a tightly controlled balance must exist between the differentiation of HSCs into lineage-committed progenitor cells and the production of daughter cells that retain
stem cell properties (self-renewal). HSC fate decisions are governed by the complex interaction of
numerous intrinsic and extrinsic signaling factors. Recently, pro-inflammatory cytokines such as
interferons and tumor necrosis factor-alpha have been proposed to act as physiologic mediators of
a range of biological responses in HSCs, although their mechanism of action is still poorly defined.
The goal of this project is to dissect the role of pro-inflammatory cytokines in governing HSC selfrenewal, using a murine model of the progressive bone marrow failure syndrome, Fanconi anemia
(FA). FA HSCs are severely compromised in their ability to self-renew and are predisposed towards
leukemic transformation. FA HSCs are also hypersensitive to the inhibitory action of a number of
pro-inflammatory cytokines.
Previous studies have shown that exposure to polyinosinic-polycytidylic acid (pI:pC), which mimics a RNA virus and thereby induces release of pro-inflammatory cytokines, pushes HSCs into proliferation. Our results demonstrate that the exit of HSCs from a dormant state coincides with the
induction of a DNA damage response as shown by examining DNA double strand breaks in highly
purified HSCs. In addition, we are able to detect increased expression levels of mRNAs encoding FA
DNA repair genes as well as elevated activation of the FA signaling pathway at the post-translational
level. Notably, the phenotype of FA HSCs is more severe since FA long-term HSCs show more DNA
damage than their normal counterparts at homeostasis and this differential is greatly increased
following HSC activation.
Our data provide a potential mechanism linking DNA damage following HSC activation with the
exposure of HSCs to pro-inflammatory cytokines. Thus, pro-inflammatory cytokines may comprise a
physiologic mediator of bone marrow failure in FA. Complementing the knowledge about the effect
of pro-inflammatory cytokines in regulating normal and diseased HSC biology may lead to targets
for therapeutic intervention.
75
Triptolide from TCM abolishes NF-κB-signaling, EMT and stem-like
features in a hypoxic microenvironment of pancreatic cancer
Li Liu 1,2, Alexei V. Salnikov 1,3, Markus W. Büchler 2, Ingrid Herr 1,2
76
1
Molecular OncoSurgery Group, Department of General Surgery, University of Heidelberg and German Cancer Research
Center (DKFZ), Heidelberg, Germany,
2
Department of General Surgery, University of Heidelberg, Heidelberg, Germany,
3
Department of Translational Immunology, German Cancer Research Center (DKFZ) and National Center for Tumor
Diseases, Heidelberg, Germany
Pancreatic cancer is characterized by a pronounced hypoxic microenvironment, which may contribute to metastasis via the induction of NF-κB signaling and epithelial-to-mesenchymal transition (EMT). Since only cancer stem cells (CSC) are described to have the ability to metastasize we
investigated the influence of hypoxia to CSC-like features and EMT before and after inhibition of
NF-κB activity. We show that hypoxia induces colony formation, sphere, ALDH1 activity and expression of CSC-related proteins (Notch1, Jagged1, Nanog and Sox2). These changes were associated
with a switch in morphology from an epithelial to a more fibroblastoid/mesenchymal phenotype
and protein expression typical for EMT, namely down-regulation of E-cadherin and up-regulation
of Vimentin, Slug, Snail and Twist2. The migratory potential was also increased upon hypoxia as
measured by a wounding assay. Inhibition of NF-κB signaling by siRNA against c-Rel or the antiinflammatory and immunosuppressive compound Triptolide from chinese herbal medicine reduced
Twist2 expression, prevented morphological changes, decreased migration and down-regulated
NF-κB binding activity and expression of the NF-κB subunits c-Rel and Rel-A. Triptolide inhibited
CSC-like features including ALDH1 activity, CSC marker expression and colony formation, sphere
despite hypoxia. To involve the pronounced tumor stroma of pancreatic cancer in our analysis we
are now about to perform in vivo xenotransplantation experiments using primary CSC isolated from
patient tumors. Our preliminary data suggest that hypoxia-induced NF-κB signaling is an important
mediator of CSC-like cells by induction of EMT signaling. Targeting of NF-κB by specific inhibition of
c-Rel or Triptolide could be beneficial for strategies aiming to eliminate CSC-like cells in the hypoxic
microenvironment of pancreatic cancer.
Transcription factor Oct1 as a somatic and cancer stem cell determinant
Jessica Maddox, Arvind Shakya, Samuel South, Jared Andersen, Dawne Shelton, Stephanie
Chidester, David A. Jones, Jinsuk Kang, Gerald Spangrude, Bryan E. Welm, and Dean Tantin
Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA
Correspondence: E-mail: [email protected]
Oct1 is a member of the POU transcription factor family, a large family of octamer sequence
binding transcription factors. Oct1 is widely expressed in adult tissue and when knocked out in mice
results in late gestational embryonic lethality (Wang et al, 2004). Oct1 is a stress sensing transcription factor functioning by directly recognizing genotoxic and oxidative stress and altering expression
of multiple targets (Kang et al, 2009) required for a normal stress response (Tantin et al, 2005).
Oct1 responds to genotoxic and oxidative stress by associating with alternative regulatory targets
(Kang, 2009), and also responds to cyclic AMP (cAMP) levels by translocating between the cytoplasm and nucleus (Wang, 2010). Studies on Oct1 deficient MEFs have shown that Oct1 normally
maintains high levels of glycolysis and dampens mitochondrial function (Shayka et al, 2009). Oct1
also enforces poised transcriptional states (Shakya, 2011) and promotes a glycolytic metabolic
profile associated with dampened mitochondrial function and reactive oxygen species (ROS) levels
(Shakya, 2009). Loss of Oct1 has little impact on cell growth and viability in culture, or on immortalization by serial passage, but antagonizes oncogenic transformation in vitro and tumorigenicity in
vivo (Shakya, 2009). These studies contribute to the mounting evidence supporting a role for Oct1
in controlling stem cell phenotypes.
Defining master transcription factors governing somatic and cancer stem cell identity is essential to targeted therapeutic development, not only for cancer, but also aging related diseases.
Here we show that Oct1 regulates normal and pathologic stem cell function. Oct1HI cells in the colon
and small intestine co-express stem cell markers. In primary malignant tissue, high Oct1 protein but
not mRNA levels strongly correlate with the frequency of CD24LOCD44HI cancer initiating cells. Modulating Oct1 alters the proportion of ALDHHI and dye effluxHI cells, and ALDHHI cells harbor elevated
Oct1 protein but not mRNA levels. We show that Oct1 promotes tumor engraftment frequency and
haematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to
previously described targets, we identify four Oct1 targets associated with the stem cell phenotype.
The data indicate that Oct1 regulates normal and cancer stem cell function.
77
Glioblastoma initiating cells can be selectively driven into apoptosis by
treatment with a histone demethylase inhibitor
Jan-Philipp Mallm 1, Benito Campos 2, Christel Herold-Mende 2 and Karsten Rippe 1
1
Deutsches Krebsforschungszentrum & Bioquant, Heidelberg, Germany
2
Sektion Neurochirurgische Forschung, Neurochirurgische Universitätsklinik, Heidelberg, Germany
We have conducted a comparative study of mouse embryonic stem cells (mESCs) and human
glioblastoma initiating cells (hGICs) to identify chromatin features that are associated with pluripotency and inhibit the transformation into a differentiated cell state. In agreement with previous
studies, chromatin in mouse embryonic stem cells was found to be in an open and transcriptionally
permissive conformation. Upon differentiation, dense pericentric heterochromatin foci formed that
were enriched with Heterochromatin Protein 1 (HP1), the histone methyltransferase Suv39h1 and
the trimethylation of lysine 9 in histone H3 (H3K9me3) that is set by this enzyme. These changes
were accompanied by an increased chromatin binding affinity of both proteins as judged by fluorescence recovery after photobleaching experiments.
We report here that similar functionally relevant rearrangements of the chromatin state occur
also in hGICs. Upon differentiation of these cells, HP1 and Suv39h1 form dense chromatin clusters. We find that chromatin binding of HP1 and Suv39h1 is weak in hGICs and that the H3K9me3
level is reduced as compared to differentiated cells. Since knockdown of the histone demethylases
Jmjd1a and Jmj2dc in mESCs has been reported to induce differentiation, we induced an increase
of histone H3K9me3 levels with a histone demethylase inhibitor. This resulted in cell death of only
hGICs but not of their differentiated counterparts. By ChIP-seq and RNA-seq we find that the underlying molecular mechanism involves downregulation of hGIC promoters that were previously active
and were carrying the H3K9 acetylation (H3K9ac) mark. The corresponding genes are involved in
cell proliferation, transcription and DNA repair. Thus, the H3K9me3-H3K9ac equilibrium at these
promoters is crucial to maintain hGICs, and we identify an enrichment of the H3K9me3 state as a
novel approach to specifically target hGICs.
78
Hypoxic signaling leads to deregulation of the AKT/mTOR pathway in prostate
cancer stem cells
Maximilian Marhold, Erwin Tomasich, Michael Krainer, Peter Horak
Department for Internal Medicine I, Medical University of Vienna, Vienna, Austria
Prostate cancer (PCa) is the most widespread malignant disease amongst men in developed
countries. Tumor-initiating subpopulations of carcinoma cells, also known as cancer stem cells
(CSCs), were identified and characterized in PCa. We hypothesized that the PI3K/AKT/mTOR pathway, which is known to be frequently altered within PCa cells, and one of its main components, the
mammalian target of rapamycin (mTOR), play a role in PCa stem cell maintenance.
We isolated CSC like subpopulations from the androgen independent human prostate cancer
cell line DU145 using fluorescence activated cell sorting (FACS) according to their expression of the
stem cell markers CD44 and CD49f. Further, we sorted the murine prostate cancer cell line TRAMPC1 based on the expression of the murine stem cell markers Sca-1 and CD49f. We used sphere
formation assays to confirm the stem and progenitor cell properties of the sorted subpopulations.
We observe lower mTOR and higher AKT activity in prostate cancer stem cells isolated from
DU145 and TRAMP-C1 cell lines compared to non-CSCs in vitro. Hypoxia is known to regulate CSC
maintenance, thus we evaluated the hypoxic signaling in prostate CSCs. Hypoxic treatment leads to
a more pronounced decrease in mTOR activity and an increase in AKT activity in CSC like subpopulations. Normoxic and hypoxic CSCs display higher levels of HIF1α in comparison to the non-CSC
subpopulation. HIF-mediated negative regulation of mTOR activity and consequent deregulation
of the S6K/IRS/PI3K negative feedback loop might offer an explanation for these effects. Indeed,
HIF targets involved in negative regulation of mTOR, such as REDD1 and REDD2 are upregulated
in prostate CSCs. Further, IRS phosphorylation is lost in CSCs, leading to activation of the PI3K/
AKT pathway. Flow cytometric measurements of the AKT and S6 phosphorylation status in prostate
tumors isolated from TRAMP mice confirm our in vitro findings, hence showing elevated AKT and
decreased S6 phosphorylation within the CSC subpopulations.
In addition, we evaluated the effects of mTOR inhibitors rapamycin and everolimus as well as
of the PI3K/mTOR inhibitor NVP-BEZ235 under normoxic (20% O2) and hypoxic (3% O2) conditions
in the CSC and non-CSC cell populations. Our results suggest that CSC like prostate cancer cells
are more resistant to mTOR inhibitors when compared to their non-CSC counterparts. Most interestingly, while the non-CSC population has a higher sensitivity to mTOR inhibitors under hypoxic
conditions, the viability of CSCs remains unaffected. Small molecule inhibitors of multiple kinases
along the PI3K/AKT pathway, such as NVP-BEZ235, are successful in eradicating the CSC-like cell
populations in hypoxia as well as normoxia.
Conclusively, we define a deregulation of mTOR signaling in prostate cancer stem cells, leading
to increased AKT activity and survival. In view of our data, we suggest that prostate cancer stem
cells might be resistant to mTOR inhibitors and that targeting multiple kinases along the PI3K/
AKT/mTOR axis would be more effective in this setting. Our findings could be helpful to assess the
impact of mTOR targeted therapy in prostate cancer, especially in light of ongoing clinical trials of
mTOR inhibitors such as everolimus and temsirolimus in castration resistant prostate cancer.
79
Elucidating the gene regulatory network governing the eye development in
fish
Juan L. Mateo 1, Ana Fernández-Miñán 2, Ina Weisswange 1, Juan Ramón Martinez-Morales 2, Beate
Wittbrodt 1, José Luis Gómez-Skarmeta 2, Joachim Wittbrodt 1
1
Center for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany
2
Centro Andaluz de Biología del Desarrollo (CABD), Consejo Superior de Investigaciones Científicas/Universidad Pablo
de Olavide, Sevilla, Spain
The eye, as part of the central nervous system, represents an invaluable system to study neurogenesis. In addition, the eye of some animals, like fish for instance, presents the amazing ability of
regeneration and continuous growth even during adulthood. The mechanisms of how the delicate
balance between proliferation and differentiation is achieved, both during development and in the
adult organism, are currently not completely understood.
We aim to unravel the principles of such control at transcriptional level using medaka, a Japanese killifish, as model system.
We have developed a bioinformatic pipeline using Hight Throughput Sequencing data, RNA-seq
and ChIP-seq, in order to identify the key genes involved in the eye development, in one side, and
putative CREs (Cis-Regulatory Elements), in the other. The integration of these data in the next level
allows us to determine a network of interactions of transcription factors and the association of distal regulatory elements, or enhancers, with proximal ones, or promoters. In this way we will be able
to elucidate the transcriptional regulatory network that governs the trade-off between proliferation
and differentiation.
We validate these predictions in vivo using medaka embryos taking benefit of the enhancer assay developed previously in the lab.
80
Piwi: Could the “guardian of the genome” act as the villain in cancer?
Ali McCorkindale 1,2, Mehnaaz Lomas 1,2, Jennifer Cropley 1,2, Romaric Bouveret 1,2, Catherine Suter 1,2
1
Molecular Genetics Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia
2
Faculty of Medicine, University of New South Wales, NSW, Australia
In eukaryotic cells, small (~24-30nt) RNAs associate with and act as guides for the Piwi proteins,
a clade of the Argonaute family specific to animals with three known functional members in mice.
The Piwi-associated (pi)RNAs appear to “direct” transcriptional silencing via DNA methylation of homologous transposon DNA sequence in germ cells. The function of the Piwi/piRNA pathway has not
been thoroughly explored in somatic cells as it has generally been assumed to be germline-specific.
However, there is ancient evidence for a secondary function of Piwi in the maintenance of germline
and somatic stem cell self-renewal. We have demonstrated that the murine Piwi, Miwi2, is highly
expressed and functional in somatic stem cells of the mouse. It appears that expression of Miwi2
correlates with potency, with peak expression observed in pluripotent embryonic stem (ES) cells
and enriched expression in stem-like progenitors present in whole tissue. The rapid downregulation
of miwi2 in differentiated cells and observed growth arrest following gene knockdown in pluripotent cells suggests that this pathway serves a role in the normal maintenance of potency and selfrenewal of stem cells. This “stemness” function could also be relevant in the context of disease. We
have shown that a number of murine cancer cell lines derived from a single parent mammary tumor
show variable expression of miwi2, with peak expression in 4T1 cells, with the greatest propensity
for metastasis. Knockdown of miwi2 in vitro induces an immediate and dramatic phenotype and
a reduced growth rate of 4T1 cells. In vivo experiments are in progress to investigate the effect of
miwi2 knockdown on 4T1 tumor growth and metastasis. We postulate that the Piwi/piRNA pathway
is aberrantly activated in cancer to promote the self-renewal of a highly aggressive cancer stem cell
population with a propensity for metastasis. Our group is working towards identifying putative targets of Piwi and its associated piRNA in cancer, and this will give great insight into the precise role
of this ancient pathway in normal homeostasis and disease in higher organisms.
The authors would like to acknowledge funding from the National Health and Medical Research Council of Australia
(NHMRC #1025210).
81
p53 tumor suppressor gene protein expression in normal, cirrhotic and
carcinomatous liver – a comparative cross-sectional study
Shahida Parveen 1, Shahzad Ahmad, Riffat Mehboob 1*
Department of Pathology, King Edward Medical University, Lahore, Pakistan
Introduction: Hepatocellular carcinoma is among the most common malignancies worldwide,
particularly in oriental region but there is insufficient information available regarding its diagnosis
due to lack of facilities and awareness, causative agents and no radical treatment available other
than resection. Aim of the present study was to determine immunohistochemically the expression
of p53 in hepatocellular carcinoma and cirrhosis of liver to provide an effective diagnostic tool.
Material and Methods: A total of one hundred liver biopsies were studied, it included twenty
cases of hepatocellular carcinoma, sixty cases of cirrhosis of the liver and twenty cases of normal
liver from autopsy specimen as a control. H&E staining and immunohistochemistry for p53 was
done for all the biopsy specimens.
Results: Out of 20 cases of hepatocellular carcinoma, 13 were positive for p53 stain and three
were negative. In rest of the 4 cases tissue was washed during processing. None of the sixty cases
of cirrhosis or 20 with normal histology reveals p53 expression. Immunohistochemistry also helps
in differentiation from well differentiated hepatocellular carcinoma to moderately differentiated hepatocellular carcinoma and to poorly differentiated hepatocellular carcinoma.
Conclusion: In the present study p53 expression was positively related with different grades of
hepatocellular carcinoma as all the poorly differentiated hepatocellular carcinoma cases were positive for p53 immunohistochemistry.
82
Epigenetics and therapeutic options of EMT- associated drug resistance in
pancreatic cancer
Simone Meidhof 1,2,3, Maria Berthold 1, Waltraut Lehmann 1, Tiberius Preca 1, Simone Brabletz 1 and
Thomas Brabletz 1,4
1
Department of Visceral Surgery, Freiburg, Germany
2
Faculty of Biology, University of Freiburg, Germany
3
Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany
4
Comprehensive Cancer Center Freiburg, University of Freiburg, Freiburg, Germany
Two major clinical problems impede a cure or a long time survival of patients with solid tumors:
Metastasis and resistance to radio-/chemotherapy. Of all human cancers, pancreatic cancer has
the worst prognosis with an average 5 year survival rate below 2%. The only option to improve the
patients’ situation is the utilization of novel specific drugs fighting these two fatal processes. Recent
breakthroughs in understanding mechanisms and functions of epigenetic modifications indicate
the avenue of new therapeutic strategies. We have shown that drug resistance in cancer cells is
linked to the activation of an epithelial-mesenchymal transition (EMT) and is conferred by the EMT
activator and transcriptional repressor ZEB1. We analyzed epigenetic modifications associated with
ZEB1 expression or drug resistance. Knockdown of ZEB1 in cancer cells results in a re-expression
of its target genes accompanied by increased levels of the permissive histone marks trimethylated
lysine 4 of histone 3 (H3K4me3) and acetylated histone 3 (H3ac). This was paralleled by a removal
of repressing DNA methylation on their promoter CpG islands. Therefore, epigenetic chemotherapeutics were tested for their potential to re-activate ZEB1 target genes and thus overcome the drug
resistance. Preliminary results with certain HDAC inhibitors show promising results.
83
Mesenchymal stromal cells increase the aggressiveness of human colorectal
cancers by inducing epithelial-to-mesenchymal transition
Valentina Mele 1,2, Manuele G. Muraro 1, Diego Calabrese 2, Nunzia Amatruda 1,3, Francesca Amicarella 1,
Dennis Pfaff 4, Brynn Kvinlaug 1, Chiara Bocelli-Tyndall 5, Ivan Martin 1, Therese J. Resink 4,
Luigi Terracciano 2, Giulio C. Spagnoli 1 and Giandomenica Iezzi 1.
1
Institute of Surgical Research and Hospital Management (ICFS) and Department of Biomedicine, University of Basel,
Basel, Switzerland;
2
Institute of Pathology, University of Basel, Basel, Switzerland;
3
Department of Anatomy, University of Naples “Federico II”, Naples, Italy;
4
Department of Biomedicine, University of Basel,
5
Department of Rheumatology, University of Basel, Basel, Switzerland.
Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to
home to inflammatory areas and tumors. Because of these features, they are currently being used
in preclinical models and in clinical trials as potential vehicles for delivery of targeted therapies.
Clinical applications of MSC however, require a deeper understanding of their biology and, in particular, of their interaction with cancer cells. In this study we have investigated the effects mediated
by human bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo.
We found that MSC promoted CRC cell proliferation in vitro through the release of soluble factors, in
particular of IL-6. Most importantly, MSC induced upregulation of epithelial-to-mesenchymal (EMT)related genes and downregulation of E-cadherin in tumor cells. These effects required cell-to-cell
contact and were mediated by expression of surface-bound TGF-β, induced in MSC upon coculture
with tumor cells. CRC cells exposed to MSC formed larger tumor masses in vivo, characterized by
higher vessel density and increased mesenchymal marker expression, and displayed enhanced
invasive capacity. Thus, by promoting cell expansion, EMT-related phenomena and tumor angiogenesis, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells. These findings
raise concerns regarding the safety of MSC administration for clinical purposes in patients with
cancer.
84
Senescence-related reprogramming promotes cancer stemness
Maja Milanovic 1, Jan R. Dörr1, Yong Yu 2, Dido Lenze 3, Michael Hummel 3, Zhen Zhao 4, Scott
Lowe 4 and Clemens A. Schmitt 1,2
1
Charité - Universitätsmedizin Berlin, Hematology, Oncology and Tumor Immunology, and Molekulares Krebsforschungszentrum, Berlin, Germany
2
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
3
Charité - Universitätsmedizin Berlin, Pathology, Berlin, Germany
4
Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, USA
Premature senescence is a stress-inducible terminal cell-cycle arrest, which, in addition to apoptosis, prevents propagation of aberrantly proliferating cells. The connection between senescence
and stem cell-related features (“stemness”) was recently suggested, where several key senescence
factors like p53, Ink4aARF or Suv39h1 limit reprogramming of somatic cells to pluripotency. In turn,
senescence mediators such as p16INK4a, p21CIP1 or FoxO transcription factors protect the stem cell
compartment from premature exhaustion. Hence, the molecular machinery executing cellular senescence stands in close relation to stem cell properties of both normal and cancer cells but, it
remains unclear whether it interferes or synergises with their stemness capacity. We investigated
here the possibility that tumor cells with inherent self-renewing potential may latently augment their
stemness through therapy induced senescence (TIS), which could become visible upon senescence
reversal.
To this end, we analyzed stem cell-related features in Eµ-myc driven B-cell lymphomas (apoptosis-protected by Bcl2 overexpression), which massively undergo TIS under chemotherapy. Proliferation-dependant stem cell capacities were tested in lymphomas genetically designed to provide
mandatory senescence-inducing factors (p53, Suv39h1 or Klf4) in strictly conditional fashion, allowing conditional TIS induction and reversal of the cell cycle block upon achieving full senescence
phenotype. The clonogenicity, proliferative and repopulating assays were performed in vitro and in
vivo, comparing the cells grown out of senescence (Previously Senescent, PS) with the counterparts
receiving chemotherapy, but never entering TIS (Never Senescent, NS).
Results show that TIS in Eµ-Myc lymphomas strongly increases expression of adult tissue stem
cell signature, surface stem-cell markers and enhances ALDH and ABC-transporter activity. Unrelated lineage signatures are established in TIS, suggesting genomic plasticity and transdifferentiation
potential. Upon releasing from TIS by acute deactivation of Suv39h1, p53 or Klf4, PS cells resumed
proliferating and eventually exceeded proliferative, clonogenic and tumor initiating capacity of NS
cells. Interrogation of self-renewal-relevant cascades revealed activation of and dependence on
canonical Wnt-signalling in TIS, as blocking this pathway led to apoptosis and reduced growth of
PS, but not NS cells. Temporary TIS enforcement also reprogrammed the bulk T-ALL cells to cancer
stem cells, suggesting that TIS execution not only augments the inherent stem cell features, but
also endows de novo tumor-initiating capacity in bulk cancer cells devoid of this potential. Quantitative and qualitative changes of stem cell-related features were furthermore confirmed in human
B-cell lymphoma patient samples and non-hematological cancer cell lines.
Our results indicate that TIS-activated senescence-like machinery associated with the cell cycle
arrest on one side, but also with latent induction of the functional stem cell properties, which became evident upon reversal of the senescence cell cycle arrest and resulted in very aggressive cell
behaviour. These findings raise a fundamental question about the plasticity of (cancer) stem cells
and bear a critical concern for senescence-inducing cancer therapies.
85
Skewed X-inactivation patterns in aging healthy and myelodysplastic
hematopoiesis determined by a novel transcriptional clonality assay
Maximilian Mossner 1,4, Florian Nolte 1, Gero Hütter 2, Jana Reins 3, Marion Klaumünzer 1, Verena
Nowak 1, Julia Obländer 1, Katrin Ackermann 1, Silke Will 1, Susanne Brendel 1, Uwe Neumann 5,
Martin Neumann 3, Olaf Hopfer 3, Claudia D. Baldus 3, Eckhard Thiel 3, Wolf-Karsten Hofmann 1, and
Daniel Nowak 1
1
Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany
2
Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, German Red
Cross Blood Service of Baden-Württemberg – Hessen, Mannheim, Germany
3
Department of Hematology, Oncology and Transfusion Medicine, Charité - University Hospital Benjamin Franklin, Berlin,
Germany
4
Institute of Chemistry and Biochemistry, Free University Berlin, Berlin, Germany
5
Department of Traumatology, Community Hospital, Reichenbach, Germany
Correspondence: E-mail: [email protected]
Objectives and background: X-chromosome inactivation is a well described process that occurs
randomly during human female embryogenesis in order to maintain gene dosage equivalence between male (XY) and female (XX) cells. Investigation of X-chromosome inactivation patterns (XCIP)
by determination of differential CpG-methylation has been widely applied for investigation of female
cell clonality. Using this approach the clonal origin of various tumors has been corroborated via
detection of skewed XCIPs. Controversially, strong age-related increase of peripheral blood (PB) cell
clonality in hematologically healthy females was reported. Recently, transcriptional XCIP ratio analysis challenged these results and questioned the suitability of methylation-based clonality assays. To
reinvestigate XCIP-skewing in hematopoiesis of healthy females and patients with myelodysplastic
syndrome (MDS), we established a novel transcriptional assay utilizing pyrosequencing technique
for quantification of SNP allele frequencies, representative for XCIP ratios.
86
Patients and methods: BM, CD34+, PB cells and granulocytes were obtained from patients
with MDS (IPSS-low/int-1-risk BM: n=25, CD34+: n=13, PB: n=21, IPSS-int-2/high-risk BM: n=16,
CD34+: n=9, PB: n=12) and age related healthy donors (BM: n=19, CD34+: n=15, PB: n=154,
granulocytes: n=34; age range: 0-97) after informed consent. Genomic DNA SNP genotyping was
carried out in order to screen for heterozygous XCIP marker genes located on the X-chromosome,
namely BTK, FHL1, IDS, MPP1 and G6PD. For assessment of XCIP skewing we developed pyrosequencing assays for each marker gene. After PCR-amplification of informative marker loci from
cDNA transcripts, SNP allele ratios, representative for XCIP, were quantified using the PyroMark
ID system (Qiagen, Hilden, Germany). Skewing was assumed for samples exhibiting skewed XCIP
ratios >80% (allelic ratio of >4:1).
Results: Standard curves from pyrosequencing reactions with predefined allelic ratios revealed
strong correlations for assessment of XCIP ratios in all markers (R²>0.99). Furthermore, high correlations have been detected for inter-marker XCIP ratios from individuals with multiple informative
markers ranging from R²>0.83 to R²<0.98 as well as for marker results between matched PB and
granulocytes (R2 = 0.95–0.99) emphasizing the suitability of the method.
XCIP skewing incidences were strongly elevated in PB cells of hematologically healthy old (mean:
51%, age >65 years) as compared to young females (mean: 14%, age <40 years, p=0.0002). MDS
patients exhibited strongly increased skewing incidences of 90-100% in CD34+, BM and PB cell
fractions as compared to healthy old and young females with 43-44% and 20-25% in the BM and
CD34+ compartment. Excessive skewing (XCIP ratios >95%) was frequently detected in 50-67% of
all MDS cell types and almost absent in healthy specimen except for BM and CD34+ cells of healthy
old females (22-29%).
Summary: Significant age-related increase of XCIP skewing in PB cells from healthy elderly females was confirmed. Moreover, XCIP ratio analysis suggests even stronger clonal manifestation in
aged BM and CD34+ cells. In MDS, XCIP skewing levels were distinctively elevated as compared to
age-matched controls and higher degrees were associated with poor clinical outcome. In conclusion, our pyrosequencing based XCIP analysis approach allows accurate assessment of XCIP ratios,
reveals novel insights into aging healthy and myelodysplastic hematopoiesis and should be easily
applicable to other fields of clonality research.
87
ZEB1 in glioma and neural stem cells
Mükusch, Susanne 1, Castro, Diogo 2, Calogero, Raffaele 3, Harter, Patrick 1, Plate, Karl-Heinz 1,
Momma, Stefan 1
1
Neurological Institute, Frankfurt University Hospital, Germany
2
IGC, Oeiras, Portugal
3
Deptartment of Clinical and Biological Sciences, Torino University, Italy
Correspondence: E-mail: [email protected]
Glioblastoma is the most frequent and most aggressive type of brain tumor in adults. Its infiltrative nature prevents effective therapy and cure. We identified the transcription factor ZEB1 as an
important promoter of glioblastoma invasiveness.
Whereas ZEB1 as an inducer of epithelial mesenchymal transition in carcinomas is well described, nothing is known about ZEB1 in the human neural system. We find that ZEB1 is highly
expressed both in glioma and in the neural stem cell niche in human fetal brains. To investigate
ZEB1 function, we performed a ZEB1 knockdown in a glioblastoma stem like cell line and a fetal
neural stem cell line. Microarray expression analysis showed that regulated genes in the two cell
lines overlap significantly but differ greatly from the ZEB1 regulated genes in carcinoma.
Xenotransplantation of glioblastoma stem like cells revealed that ZEB1 knockdown cells were
less tumorigenic than control cells and tumors were significantly smaller and less invasive than
control tumors.
We conclude that ZEB1 plays an important role both in human neural stem cells during development and in glioma.
88
Differential gene expression patterns in 3D cultures of human colon cancer
cells in the presence of hypoxic and/or necrotic cores
Silvio Däster 1,#, Nunzia Amatruda 1,#, Diego Calabrese 2, Paul Zajac 1, Giulio C. Spagnoli 1, Giandomenica Iezzi 1, Valentina Mele 1,*, Manuele G. Muraro 1,*
1
2
#
*
Institute of Surgical Research and Hospital Management ICFS, University Hospital Basel, Basel, Switzerland
Institute of Pathology, University of Basel, Basel, Switzerland
These authors contributed equally to this work.
Shared last authors
Background: The maintenance of three-dimensionality is fundamental for understanding and
replicating in vitro the behavior of cells within tissue. Anti-tumor drug development and testing are
conventionally conducted on established human cell lines cultured in two-dimensional (2D) systems and it has been extensively demonstrated a pre-screening high attrition rates. The multicellular tumor spheroid (MCTS) model is one of the most simple and easily reproducible tool widely used
for studying tumor biology in 3D. Similar to primary tumors, in MCTS, depending on the spheroid
size, hypoxia and necrosis occur due to oxygen and nutrient gradients. However, the impact that
these phenomena may have on gene expression profile of tumor cells has not yet been elucidated.
Methods: We have established and standardized an evolution model, based on the hanging-drop
system, to study different stages of growth of MCTS of colorectal cancer (CRC) cell lines HT29 and
HCT116, depending on the presence or absence of hypoxic and/or necrotic cores. The expression
of a panel of 94 genes related to tumor progression, cancer stem cells, metastatic behavior, and
drug resistance, growth factors, and chemokines was assessed on MCTS at different growth stages
in comparison to conventional 2D cultures and xenograft.
Results: Our results showed that CRC cells growing in 3D acquire the ability to generate acinuslike structures similar to primary tissue. More importantly, the gene expression profile of MCTS
containing hypoxic and necrotic areas appear to be the most closely related to in vivo xenograft
cultures.
Conclusions: We highlighted for the first time the need to include the hypoxia and necrosis in a
3D system to better mimic the in vivo condition of CRC.
89
Calcineurin is required for leukemia-initiating cell activity in a mouse model
of T-cell lymphoblastic leukemia
S Gachet 1-3,*, E Genesca 1-3,*, D Passaro 1-3, M Irigoyen 1-3, H Alcalde 1-3, C Clémenson 1-3, A Janin 4-6,
C Lasgi 1-3, S Dodier 1-3, M Soyer 7,8, G Dumenil 7,8 and J. Ghysdael 1-3
Institut Curie
CNRS UMR3306
3
INSERM U1005, Centre Universitaire, Bat 110 Orsay, France
4
Hôpital Saint-Louis AP-HP
5
INSERM U728
6 Université Paris Diderot, Paris, France
7
Paris Cardiovascular Research Center INSERM U970
8
Université Paris Descartes, Paris, France
1
2
* SG and EG contributed equally to this work.
Background: Engagement of cell surface receptors coupled to the activation of the phospholipase C leads to an increase in intracellular calcium that results in the activation of calcineurin (Cn),
a serine/threonine phosphatase composed of a catalytic (CnA) and a regulatory (CnB) subunit. Calcium-dependent activation of Cn leads to dephosphorylation of its substrates, including the NFAT
family of transcription factors (NFATc1-c4). Cn/NFAT signaling plays critical role in a number of developmental processes, but is best characterized for its implication in controlling many aspects of T
cell biology. We have previously reported the sustained activation of Cn in human lymphoma, acute
lymphoblastic leukemia (ALL) and mouse models of lymphoid leukemias/lymphomas. Pre-clinical
studies showed that pharmacological targeting of Cn with cyclosporine A (CsA) and FK506 has clear
anti-leukemic effects in T-ALL mouse models. Besides its inhibitory activity towards leukemic cells,
systemic delivery of these compounds is likely to inhibit Cn in cells of the tumor microenvironment,
possibly contributing to their anti-leukemic activity.
90
Aims: To investigate the intrinsic function of calcineurin in leukemic cells, we generated mouse
T-ALL in which Cn can be specifically inactivated in tumor cells without affecting the tumor microenvironment.
Methods: We generated mouse models of human T-ALL induced by either an activated Notch1
allele (ICN1) or the TEL-JAK2 fusion oncoprotein in which Cn inactivation is specifically obtained in
leukemic cells by Cre-mediated deletion of a conditional, floxed CnB1 allele. We examined consequences of Cn inactivation on leukemia dissemination and reinitiation in these models.
Results: We found that Cn activation in leukemic cells is under stromal control and that Cn inactivation alters physical and functional interactions that leukemic cells establish with their microenvironment, including their abnormal adhesion, migration and defective leukemic cells survival and
proliferation. We show that Cn favors but is not required for in vivo expansion of leukemic blasts.
In contrast, Cn is critical for leukemia-initiating cell activity as analyzed in transplantation studies.
Importantly, Cn ablation in de novo homed leukemic cells also abolished disease re-initiation.
Summary and conclusions: These results demonstrate that calcineurin has an intrinsic prooncogenic function in leukemic cells that affects several phenotypic traits of tumor cells that ultimately control leukemia dissemination and expansion in vivo. Besides, these findings indicate
that calcineurin is a promising target to prevent T-ALL relapse and call for clinical trials combining
conventional debulking therapies with- or followed by Cn inhibitors during consolidation therapy.
PKH26 staining defines distinct subsets of normal human colon epithelial
cells at different maturation stages
Anna Pastò 1, Maddalena Marchesi 1, Adamo Diamantini 1, Chiara Frasson 2, Matteo Curtarello 3,
Claudia Lago 1, Giorgia Pilotto 1, Anna Rosita Parenti 4, Giovanni Esposito 3, Marco Agostini 1,
Donato Nitti 1 and Alberto Amadori 1,3
1
Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy
2
Hemato-Oncology Laboratory, Department of Pediatrics, University of Padova, Padova, Italy
3
IRCCS Istituto Oncologico Veneto, Padova, Italy
4
Department of Diagnostic Sciences and Special Therapies, University of Padova, Padova, Italy
Background and aim: Colon crypts are characterized by a hierarchy of cells distributed along
the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell
subsets in different maturation stages from normal human colon.
Methodology and major findings: Dissociated colonic epithelial cells were stained with PKH26,
which allows identification of distinct populations based on their proliferation rate, and cultured in
vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell
populations and in three colon crypt populations isolated by microdissection. A PKHpos population
survived in culture and formed spheroids; this population included subsets with slow (PKHhigh) and
rapid (PKHlow) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and
Lgr-5 in PKHhigh cells; by cytofluorimetric analysis, Msi-1+/Lgr5+ cells were only found within PKHhigh
cells, whereas Msi-1+/Lgr5- cells were also observed in the PKHlow population. As judged by qRT-PCR
analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1)
was highly enriched in Msi-1+/Lgr5+ cells. While CK20 expression was mainly found in PKHlow and
PKHneg cells, a small PKHhigh subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with
these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results
mirrored those found in cells isolated from different crypt portions by microdissection, and based
on proliferation rates and marker expression they allowed to define several subsets at different
maturation stages: PKHhigh/Lgr5+/Msi-1+/CK20-, PKHhigh/Lgr5-/Msi-1+/CK20+, PKHlow/Lgr5-/Msi-1+/
Ck20-, and PKHlow/ Lgr5-/Msi-1-/ CK20+ cells.
Conclusions: Our data show the possibility of deriving in vitro, without any selection strategy,
several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and
molecular profile of cells in a discrete crypt location.
91
Epigenetic regulation of Notch and c-Myb in T-acute lymphoblastic leukemia
Pinazza M. 1, Ghisi,M. 2, Minuzzo S. 1, Agnusdei V. 3 and Indraccolo S 3
1
Department of Surgery, Oncology and Gastroenterological Sciences, University of Padova, Padova, Italy
2
Peter MacCallum Cancer Center, Melbourne, Australia
3
Istituto Oncologico Veneto-IRCCS, Padova, Italy
Histone deacetylases (HDACs) are enzymes involved in the remodeling of chromatin. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer. In fact, HDAC inhibitors promote apoptosis, induce cell cycle
arrest and differentiation of tumor cells, by mechanisms which remain in part unknown. In our
studies we observed that treatment of T-Acute Lymphoblastic Leukemia (T-ALL) cell lines with the
pan-HDAC inhibitor Trichostatin A (TSA), caused marked reduction of Notch1, Notch3 and c-Myb
protein levels. The mRNA expression levels of the two Notch receptors did not change, on the contrary c-Myb transcript decreased. This result suggests that Notch1 and Notch3 could be regulated
post-transcriptionally and/or post-translationally following TSA treatment. Blockage of HDAC activity
also decreased the expression of Notch target transcripts such as pTα, CR2 and DTX-1, indicating a general down-regulation of Notch signaling. Moreover, inhibition of HDACs exerts strong proapoptotic effects in all cell lines tested (n=3). These findings are confirmed in a panel of primary
T-ALL cells from xenografts (n=7) and treated in vitro with TSA, albeit heterogeneous responses
were observed. To identify HDAC family member(s) responsible for these effects, we are currently
exploiting class specific-HDACi as well as shRNA approaches. At the same time, we are investigating
whether increased protein degradation may account for Notch reduction. following TSA treatment.
To this end, we treated cells with proteasome and lysosome inhibitors, after TSA treatment. Protein
levels of the two Notch receptors were rescued using the lysosome inhibitor chloroquine, suggesting involvement of the endocytic pathway, whereas proteasome inhibitors had minimal effects.
Prospectively, HDAC inhibitors could represent a novel therapeutic approach for poor prognosis
T-ALL patients, alone or in combination with conventional chemotherapy.
92
TGFβ1 increased migration characteristics and induced different stemness
“degrees” in A549 cells
Virginia Tirino 1, Rosa Camerlingo 2, Elena Irollo 2, Giuseppe Sessa 2, Francesca Paino 1, Maria
Vincenza Carriero 2, Nicola Normanno 2, Gaetano Rocco 3 and Giuseppe Pirozzi 2
1
Department of Experimental Medicine, Second University of Naples, Naples, Italy
2
Department of Experimental Oncology, National Cancer Institute, Naples, Italy
3
Department of Thoracic Surgery and Oncology, National Cancer Institute, Naples, Italy
Objectives: Metastasis is the leading cause of death by cancer. It involves many steps: local
invasion of cancer cells into the surrounding tissue, transport through the microvasculature of the
lymph and blood systems, translocation, mainly through the bloodstream, to microvessels of distant tissues, exit from the bloodstream, survival and adaptation in the distant microenvironment,
and formation of a secondary tumor. Epithelial-to-mesenchymal transition (EMT) is a process that
plays essential roles in cancer invasion and migration characterized by loss of epithelial properties
and acquisition of mesenchymal traits. EMT is associated with a poor clinical outcome and cancer
progression and metastasis. TGFβ1 is the major inductor of EMT. Aim of this study was to investigate the role of TGFβ1 on cancer stem cells (CSCs) and non-CSCs starting from A549 Non Small
cell Lung Cancer (NSCLC) cell line.
Methods: A549 cell line was sorted for CD133 surface expression and Side Population (SP)
profile by flow cytometry. A549 cells and A549 CD133+ (CSCs), A549 CD133- (not-CSCs), A549
SP+ (CSCs) and A549 SP- (not-CSCs) cells were treated with 2ng/ml of TGFβ1 for 48 hours. After
incubation, all cell fractions were analyzed for epithelial markers expression such as e-cadherin
and cytokeratins and mesenchymal markers expression such as vimentin by immunofluorescence
and RT-PCR. RT-PCR for Snail, main EMT marker, and MMP9 were performed. In addition, stemness
transcriptional factor such as OCT4, and epigenetic modifications of H3K4me3 and H3K27me3
were analyzed. Migration was performed.
Results: TGFβ1 induced EMT in all fractions of A549 tested both CSCs and not-CSCs by overexpression of Snail, and vimentin and down-regulation of e-cadherin and cytokeratins. Morphological changes by epithelial to fibroblast like shape were also observed.
In addition, TGFβ1 increased the percentage of cells migrating respect to those untreated in all
cell fractions except for CD133- fraction. Interestingly, the migration of SP- cell fraction is stronger
than of its untreated corresponding cells. Although this, CD133+ cell fraction responded more effectively than other fractions to the TGFβ1 treatment. Regarding to stemness, TGFβ1 induced an
increase of OCT4 m-RNA levels in all fractions except for that of CD133-, an increase of H3K4me3
and down-regulation of H3K27me3 only in SP+ cell fraction.
Conclusion: TGFβ1 induced EMT both in CSCs and not-CSCs in A549 cell line. In particular,
TGFβ1 highlights different stemness subpopulations with different characteristics: i) a stationary
CSCs subpopulation identified as SP+ cell subset and ii) a migratory CSCs subpopulation identified
as CD133+ cell subset. Noteworthy and interestingly, also SP- showed strong migratory capacity
indicating TGFβ1 could induce cell reprogram on non-CSCs by EMT leading to coexistion by switching between the two phenotypic states. This study could highlight the possibility to address a novel
pharmacological approach versus TGFβ1 pathway and/or/in combination with those of SP+/- and
CD133+.
93
Study the functional role of the p130/Gsk3β/β-catenin complex in long
term culture of mesenchymal stem cells
Boris Popov, Nikolay Petrov, Valery Zenin
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
94
Here we showed that in mesenchymal stem cells (MSC) the pRb and Wnt/β-catenin signal
pathways interact each other by formation of the complex included the p130 and β-catenin. Both
these proteins under normal conditions are phosphorylated at G0/G1 by Gsk3β, which may serve
as a matrix for their physical interaction. In the co-culture with the А-549 cells of epithelial origin
or treatment with lithium 2+ MSC activate Wnt/β-catenin signal pathway and express some epithelial markers that is combined with formation of the p130/E2f4 complex. This complex inhibits
exit from quiescence in differentiated somatic T98G cells, but not in MSC. In MSC the p130/E2f4
includes Gsk3β and β-catenin and is detected in asynchronously growing or synchronized cells.
The levels and phosphorylation patterns of p130, E2f4 and β-catenin in MSC are not changed
during cell cycle progression. In contrast, control T98G cells reveal hyperphosphorylation of p130
at G1/S transition followed by sharp decrease in the protein levels at S and G2/M phases. Activation of the Wnt/β-catenin pathway in MSC is associated with increase in the p3 hyper- and p1
hypophosphorylated forms of p130 included in the complex. The same p130 molecular forms are
co-precipitated with Gsk3β from nuclear extracts of MSC. In contrast, the p2 form producing due to
the p130 phosphorylation by Gsk3β is preferentially coupled to β-catenin in cytosol. This suggests
that Gsk3β mediated phosphorylation of p130 does not change its functional status. The р130/
Gsk3β/β-complex in nucleus includes the Tcf3,4 proteins and reveals transcriptional activity in the
cells expressed stabilized exogenous β-catenin. This transactivation is partly inhibited after expression of exogenous р130 when detected by reporter assay with the constructs encompassing a
luciferase reporter driven by artificial promoter with several LEF/TCF binding sites. During long term
passaging the MSC show activation of proliferation, inhibition of differentiation and adhesion. MSC
of 43-47 passages injected subcutaneously or intramuscular to syngeneic recipients induce tumor
formation. Tumorigenic ability in MSC is detected in parallel with an increase in the nuclear levels of
both total β-catenin and Tcf3,4. The results obtained suggest that Wnt/β-catenin and pRb pathways
interact in coarse of the cell cycle and transformation of MSC by formation of the р130/Gsk3β/βcomplex which may play mechanistical role in regulation of their stem and tumorigenic abilities.
This work was supported by the RFBR grant # 12-04-00252
CD44s is required for EMT and stemness properties via ZEB1 activation
Karolina Bajdak 1, Bogdan-Tiberius Preca 1, Jessica Pfannstiel 1 and Thomas Brabletz 1,2
1
Department of Visceral Surgery, Albert-Ludwigs-University Freiburg, Germany
2
Comprehensive Cancer Center Freiburg, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Correspondence: E-mail: [email protected]
Invasion and metastasis of carcinomas are often promoted by the aberrant epithelial to mesenchymal transition (EMT) program at the invasive front of the tumor. The transcription factor ZEB1 is
a crucial promotor of metastasis formation by inducing EMT and tumor initiating capacity. CD44, a
cell surface protein, is used as marker to identify tumor initiating cells in many cancer types. It is involved in cell-cell interactions, cell adhesion and migration. Through alternative splicing, controlled
by the epithelial splicing factor ESRP1, CD44 is differentially regulated during EMT, resulting in a
switch from the variable exon-containing CD44v isoforms to the standard isoform CD44s, which is
devoid of all CD44 variable exons.
We examine the reciprocal interaction of CD44s and ZEB1 in their function as tumor initiation
markers. CD44s and ZEB1 are co-expressed in poorly differentiated human cancer cells.Transient
down regulation of CD44s results in decrease of ZEB1, which in turn leads to reduced sphere formation ability. Likewise, inhibition of CD44s increases ESRP1. Knockdown of ESRP1 decreases
expression of CD44v isoforms, increases CD44s and ZEB1 and the sphere forming capacity. Additional knockdown of CD44s reduces the sphere formation. Furthermore, knockdown of ZEB1
inhibits ESRP1 expression, which leads to upregulation of CD44s.
In summary we identified a positive feedback loop maintaining stemness and mesenchymal
features in cancer cells: CD44s activates expression of ZEB1, which suppresses transcription of the
splicing factor ESRP1, resulting in further increase in CD44s expression.
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Identification of novel therapeutic targets in advanced patient-matched
renal cell carcinoma models
Teresa Rigo-Watermeier 1, Corinna Klein 2, Vanessa Vogel 2,3, Christian Eisen 1,2, Thomas Höfner 2,4,
Wilko Weichert 3, Sascha Pahernik 4, Markus Hohenfellner 4, Martin R. Sprick 1,2 and Andreas Trumpp 1,2
1
Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
2
Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany
3
Department of Pathology, University Heidelberg, Heidelberg, Germany
4
Department of Urology, University Heidelberg, Heidelberg, Germany
Clear-cell renal cell carcinoma (ccRCC) is highly resistant to conventional treatments. Pre-clinical
in vitro and in vivo models accurately mimicking this disease are critical for developing more effective therapies. Since previous attempts to culture these cells were mostly unsuccessful, only few
RCC cell lines are available. In addition most of them have changed during long-term FCS-culturing
and do not resemble the original tumor anymore. This might explain why drugs identified by screening these cell lines cannot be successfully transferred into the clinic and reveals the necessity for
the development and characterization of clinically relevant ccRCC-models.
Therefore we established a ‘patient-matched’ serum-free spheroid culture system that can be
used to propagate ccRCC cells in vitro. The success of the culture establishment was highly dependant on the tumor grade of the corresponding patient. The ccRCC cells retained their tumor-initiating potential and mimicked the human malignancy upon orthotopic injection into immunodeficient
mice. Further these cells showed de novo metastasis to the lungs, the most common metastatic
site for ccRCC. The established ccRCC cultures can be used as a screening platform for novel
subpopulation-specific surface proteins by FACS analysis and mass spectrometry. Our analyses
revealed marker heterogeneity determining different subpopulations. Further in vitro and in vivo
assays showed, that the identified subpopulations also displayed distinct functional characteristics.
Currently these markers are validated for their suitability as diagnostic and therapeutic targets and
the subpopulations are analyzed for their role in tumor-initiation, metastasis and drug-resistance.
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Engineering of a three-dimensional biomimetic niche for the in vitro expansion
of hematopoietic stem cells
Annamarija Raic 1, Lisa Rödling 2 & Cornelia Lee-Thedieck 1,2
1
Max Planck Institute for Intelligent Systems, Department of New Materials and Biosystems, Stuttgart, Germany, &
Department of Biophysical Chemistry, University of Heidelberg, Heidelberg, Germany
2
Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany
Hematopoietic stem cells (HSC) hold great promise for the treatment of diseases of the hematopoietic system. The application of HSCs, however, is restricted due to their limited availability and
in vitro expansion of HSCs has not yet been achieved to the necessary extend. In vivo HSCs persist
in the bone marrow in their niche and renew themselves for an individual’s life-time. In order to
overcome the restriction of HSC accessibility, our goal is to construct an artificial stem cell niche
in which HSCs can be expanded while preserving their stem cell character. The natural HSC niche
is a complex, three-dimensional (3D) environment that cannot be imitated by standard cell culture
in two-dimensional (2D) tissue culture plates. Therefore, 3D culture systems are needed for the
expansion of HSCs by biomimetic approaches.
For this purpose polyethylene glycol (PEG) based 3D-hydrogels with interconnecting pores, which
mimic the architecture of the HSC niche, were developed and produced via a salt-leaching-technique. Functionalization of the hydrogel with RGD-peptides equipped the hydrogels with an adhesive, biologically active surface. Hematopoietic stem and progenitor cells (HSPCs) were isolated
from human umbilical cord blood and mesenchymal stem cells were used as supportive cells in
order to mimic the cellular components of the niche. The positive effect of mesenchymal stem
cells on the expansion and preservation of CD34+ cells was confirmed in a standard 2D co-culture
setup. The co-culture conditions were optimized in 2D and then transferred to the engineered 3D
hydrogel system. The efficient seeding of the 3D hydrogels with both cell types was confirmed by
electron and confocal microscopy. CD34-expression, proliferation and differentiation of HSPCs were
monitored over a period of 4 days. CD34+ HSPCs survived and proliferated in the biomimetic, porous hydrogels. The supportive effect of mesenchymal stem cells on HSCPs appeared even more
pronounced in 3D than in 2D.
These results are the promising basis for the reconstruction of the HSC niche and contribute to
bridging the gap between laboratory studies and clinical application. A scale-up of such 3D-systems
could compensate the limited number of donor HSCs by an in vitro expansion in a high throughput
manner.
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The EMT inducer SNAIL1 is a repressor of the EPHB3 tumor suppressor
gene in colorectal cancer cell lines
Kerstin Rönsch 1, Sabine Jägle 1, Katja Bruser 1, Vivien Freihen 1 and Andreas Hecht 1
1
Institute of Molecular Medicine and Cell Research, and Faculty of Biology, Albert-Ludwigs-University Freiburg, Germany
Aberrant Wnt/β-catenin signaling is a driving force during initiation and progression of colorectal
cancer. Initially, mutations in Wnt/β-catenin pathway components lead to cellular hyperproliferation
and the formation of benign polyps which exhibit spatially restricted growth. One major effector of
cell positioning within the intestinal crypts is the Wnt/β-catenin target gene EPHB3. However, at
the adenoma-carcinoma transition, when dysplastic cells gain features of epithelial-mesenchymal
transition (EMT) and become invasive, EPHB3 is frequently transcriptionally silenced. The molecular mechanisms underlying the apparent loss of expression despite persistent pathway activity are
largely unknown. To gain insight into this, we characterized a panel of colorectal carcinoma (CRC)
cell lines with divergent expression of EPHB3. Interestingly, we found that the expression of EPHB3
is inversely correlated with the expression of ZEB1 and SNAIL1 in our panel of CRC cell lines.
ZEB1 and SNAIL1 are inducers of EMT – a process which is implicated in tumor progression and
enhanced invasiveness. In vitro, SNAIL1, but not ZEB1, mediates repression of an EPHB3 luciferase reporter construct and binds to an E-box in a distal regulatory region of EPHB3. This binding is
abolished by a mutation in this E-box. Induction of SNAIL1 in living cells leads to its binding to the
endogenous EPHB3 gene and to downregulation of EPHB3. Simultaneously, induction of SNAIL1
displaces activators like p300 and TCF4 from the EPHB3 regulatory region and leads to loss of the
active histone marks H3K4 trimethylation and H3K9/K14 acetylation. Taken together, our results
show that SNAIL1 is a transcriptional repressor of the tumor suppressor EPHB3. Further analyses
have to reveal how downregulation of EPHB3 is linked to EMT processes which potentially have
implications for progression of colorectal tumorigenesis.
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Phosphorylation of EGFR predicts EGFR-TKIs sensitivity in EGFR mutationnegative NSCLC stem cells
Valentina Salvati*, Giovanni Sette* Fiorenza Lotti*, Paola Di Matteo*, Emanuela Pilozzi**, Enrico
Duranti**, Paolo Visca***, Ruggero De Maria*** and Adriana Eramo*
*
Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
** Department of Experimental Medicine, Sant’ Andrea Hospital, University ‘La Sapienza’, Rome, Italy
*** Regina Elena National Cancer Institute, Rome, Italy
Increasing evidence indicates that cancer can arise from a cancer stem cell (CSC), a tumorinitiating cell that has properties similar to those of stem cells. Although conventional anti-cancer
treatments might eradicate most malignant cells in a tumor, they may be ineffective against chemoresistant CSCs, thus this cellular subpopulation should represent the primary target for innovative and most effective experimental therapeutic strategies. Recent perspectives in cancer therapy
consider altered protein signaling networks as the main signature underlying neoplastic transformation. In addition, they emphasize the importance of studying signaling pathways active in tumor
cells to develop effective antitumor drugs.
The epidermal growth factor receptor is frequently over-expressed in non-small cell lung cancer
(NSCLC) and is taken as a promising target for NSCLC treatment.
Here, we examined the most frequent EGFR mutations in lung-CSC that we previously isolated
from lung cancer patients and extensively characterized, and we found that all lines were wild type
for the region encompassing the most common and clinically relevant mutations (exons 18, 19
and 21), but they showed phosphorylation of EGFR. NSCLC patients bearing activating mutations
of the EGFR are generally the best responders to EGFR inhibition, thus the EGFR inhibitor Erlotinib
(Tarceva®) has become the treatment of choice in mutated EGFR patients. EGFR mutation negative
patients are currently treated with chemotherapy with scarce efficacy, however it has been shown
that about 10% of them may respond to Erlotinib even in the absence of mutation.
We found that EGFR mutation-negative lung-CSCs with activated receptor, were efficiently killed
by Erlotinib in vitro, differently from the Erlotinib resistant lung-CSC where phosporylated EGFR was
not detected. Erlotinib citotoxicity occurred through decrease of phosphorylated STAT3 and BCL-XL
down-modulation in lung CSCs. In vivo, EGFR phosphorylation in lung-CSC derived xenografts paralleled that of the lung-CSC.
In vivo treatment of mice bearing lung CSC-derived tumor xenografts determined a marked inhibition of tumor growth, EGFR and STAT3 dephosphorylation and decrease of BCL-XL.
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Expression of CD133 and identification of CD133/nestin positive cells in
rhabdomyosarcomas and rhabdomyosarcoma cell lines
J. Skoda 1,2,3, J. Neradil 1,6, J. Sana 1, I. Zambo2, P. Chlapek 1,2, M. Hermanova 2, P. Mudry 3, A. Vasikova 4,
K. Zitterbart 3, A. Hampl 5, J. Sterba 3,6, R. Veselska 1,3
1
Laboratory of Tumor Biology, Department of Experimental Biology, School of Science, Masaryk University, Brno, Czech
Republic
2
1st Institute of Pathologic Anatomy, St. Anne’s University Hospital and School of Medicine, Masaryk University, Brno,
Czech Republic
3
Department of Pediatric Oncology, University Hospital Brno and School of Medicine, Masaryk University, Brno, Czech
Republic
4
Centre of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital
Brno and School of Medicine, Masaryk University, Brno, Czech Republic
5
Department of Histology and Embryology, School of Medicine, Masaryk University, Brno, Czech Republic
6
Masaryk Memorial Cancer Institute, Brno, Czech Republic
Co-expression of CD133, a cell surface glycoprotein, and nestin, an intermediate filament protein, is considered as a marker of neural stem cells and a marker of cancer stem cells (CSCs) in
neurogenic tumors. The aim of this study was to determine whether CD133 and nestin could also
serve as the markers of CSCs in rhabdomyosarcomas.
For evaluation of CD133 and nestin expression we used 10 tumor tissue samples taken from patients with rhabdomyosarcoma and 5 rhabdomyosarcoma-derived cell lines. Formalin-fixed paraffinembedded tissue samples were analyzed using immunohistochemistry and immunofluorescence.
The cell lines were examined with use of immunofluorescence, immunoblotting, flow cytometry and
RT-PCR. The cell lines were also used in the functional analyses – in vitro clonogenic assay and in
vivo tumorigenic assay.
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CD133 and nestin were detected in all the examined tumor tissue samples and in all the cell
lines. However, the incidence of CD133+, Nes+ and CD133/Nes+ cells as well as the intensity of
fluorescence signal varied among individual samples and the cell lines. The expression of CD133
and nestin was subsequently verified by immunoblotting in all of the cell lines. Furthermore, RT-PCR
revealed expression of stem cells specific markers: Oct3/4 and nucleostemin. Functional analysis
confirmed clonogenic potential of NSTS-11 cell line in vitro as well as its ability to form tumors in
vivo after injection into NOD/SCID mice. Imunohistochemistry of these tumors revealed presence
of myogenic markers (myoglobin, desmin, muscle actin) and again confirmed the expression of
CD133 and nestin.
Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and
rhabdomyosarcoma cell lines. Co-expression of CD133 and nestin together with the results of the
functional analysis suggest, that cell with CSCs phenotype are present in rhabdomyosarcomas.
Financial support: IGA MZCR NT13443-4 and RECAMO CZ 1.05/2.1.00/03.0101
The epidermal stem cell – A new approach to identify stem cells as viable
label-retaining cells
Elisa Specker, Hans-Jürgen Stark and Petra Boukamp
Division of Genetics of Skin Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
Stem cells in the human interfollicular epidermis are still difficult to identify, mainly due to a lack
of definitive surface markers. Consequently, it is a major challenge to understand how epidermal
stem cells are regulated to generate a healthy and constantly regenerating epidermis. Since stem
cells rarely divide, they can be functionally identified as label retaining cells (LRCs). In particular,
it is possible to label the cells with nucleotide analogues like IdU (Iododeoxyuridine) and to detect
infrequently dividing cells with pulse-chase experiments. Until now, the isolation of DNA labelled
vital LRCs is restricted due to the need of fixation to visualize the DNA label. To bypass this, cellintrinsic fluorescent dyes are required. With the generation of a lentiviral based vector expressing a
Tet-Off controlled histone 2B-GFP (Tet-Off-H2B-GFP) reporter gene, a new tool for the detection and
isolation of viable LRCs was established. In initial experiments, the vector was successfully used to
infect human malignant keratinocytes (HaCaT ras II-4).
The aim is now to analyse the regulation of human epidermal stem cells and to identify more reliable surface markers for their unbiased identification. For this latter, normal human keratinocytes
are infected, cultivated in OTCs and are allowed to establish LRCs during long-term regeneration.
These LRCs will be isolated and purified via FACS sorting and used for the analysis of their expression profile and functional competence.
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The tumor suppressor CYLD regulates hematopoietic stem cell dormancy
Melania Tesio 1, Hind Medyouf 1, Lisa Dohrn 1, Anne Wilson 1, Ari Waisman 3, Mannolis Pasparakis 4,
and Andreas Trumpp 1,2
1
Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany
2
Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany
3
Medical Department, Johannes Gutenberg-University Mainz, Mainz, Germany
4
Institute of Genetics, Centre for Molecular Medicine (CMMC), and Cologne Excellence Cluster on Cellular Stress
Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
The most potent hematopoietic cells (HSCs) in the healthy adult mouse are located as single
cells in niches of the bone marrow and are in a status of deep long-term quiescence or dormancy
(1). Dormant HSCs (dHSCs) have a lin−Sca1+cKit+CD150+CD48−CD34− phenotype and can be identified by long-term label retention. Mathematical modeling combined with reconstitution assays
have revealed a proliferation frequency during homeostasis of about 5 divisions per lifetime. While
dHSCs form a silent reservoir of stem cells during homeostasis, they are efficiently activated to
proliferate and self-renew in response to bone marrow injury (1-3). cDNA and real time PCR analysis showed that CYLD (cylindromatosis), a tumor suppressor gene with deubiquitinase activity was
strongly up-regulated in dHSCs. CYLD mutant mice showed a 2-fold decrease in dormant label
retaining HSCs, and a concomitant increase in the numbers of activated non label retaining HSCs.
Furthermore, in competitive transplantation experiments mutant cells were not able to efficiently
reconstitute hematopoiesis. Cell cycle analysis indicated that CYLD DUB deletion severely impairs
quiescence of l lin−Sca1+cKit+CD150+CD48−CD34− by regulating the CDK inhibitor p57 and by preventing deubiquitination of NF-KB signaling components. In summary, these data indicate that
CYLD enzymatic activity is an important regulator of HSC dormancy during homeostasis.
1. Wilson et al., (2008). Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis
and repair. Cell 135, 1118-1129.
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2. Essers M. et al., (2009). IFNalpha activates dormant haematopoietic stem cells in vivo. Nature 458, 904-908.
3. Trumpp et al., (2010). Awakening dormant haematopoietic stem cells. Nat Rev Immunol. 2010 Mar;10(3):201-9.
Wnt/β-catenin-activated stromal fibroblasts: A decisive role for tumor-stroma
cross-talk in human skin carcinogenesis
Marius Tham, Katrin Sobel, Hermann Stammer, Petra Boukamp
Genetics of Skin Carcinogenesis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany,
Correspondence: E-Mail: [email protected], phone: +49-6221-424516
Human skin squamous cell carcinomas (SCCs) are the second most frequent skin cancer type
and numbers are even increasing due to immunosuppressed transplant recipients, who contract
SCCs. Underlying mechanisms of SCC development are not fully understood, but involve the misregulation of signaling pathways including aberrant Wnt/β-catenin activation, which is a hallmark of
many cancers. In particular, previous studies have demonstrated the translocation of β-catenin to
nuclei of SCC tumor cells. By analyzing primary SCCs, we show in addition to Wnt-deregulation in tumor cells also Wnt-activation in the tumor-underlying stroma and particularly in fibroblasts. In vitro,
HaCaT keratinocytes, a model for pre-malignant SCCs, respond only weakly to direct Wnt pathway
activation and show no functional consequences. However, in tissue-like organotypic co-cultures
(OTCs) with human dermal fibroblasts, which respond strongly to Wnt 3a, HaCaT keratinocytes
show a significant increase in proliferation. To determine the critical mediators in this fibroblastepithelial cross-talk, we performed a global gene expression analysis of Wnt 3a-stimulated fibroblasts, identifying that fibroblasts secrete high amounts of the cytokines interleukin-8 (IL-8) and
chemokine (C-C motif) ligand 2 (CCL2). Blocking IL-8 and CCL2 in OTCs prevents the Wnt-induced
increase in HaCaT cell proliferation, showing that both chemokines are indeed the responsible factors. Additionally, fibroblasts express matrix metalloprotease 1 (MMP1) upon Wnt 3a stimulation,
which assists keratinocytes in modulating the extracellular matrix. Thus, these findings suggest a
Wnt/β-catenin pathway-dependent fibroblast-epithelial cross-talk and demonstrate a novel role of
Wnt signaling in the microenvironment of developing SCCs, underlining how misregulation influences tumor progression.
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LPS induced activation of haematopoietic stem cells
Stefanie Thamm 1, Raphael Lutz 2, Andrea Kuck 1, Stephan Wurzer 1, Andreas Trumpp 1,2, Marieke
Essers 1,2
1
Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
2
HI-STEM – Heidelberg Institute for Stem Cell Technologies and Experimental Medicine gGmbH, Heidelberg, Germany
Correspondence: E-mail: [email protected]
Mouse haematopoietic stem cells (HSCs) are one of the best-characterized stem cells in the
adult body. Most of the time self-renewing HSCs are in a dormant state, but can be activated in
response to bone marrow injury. Recently, we showed that Interferon-α (IFNα), a cytokine produced
by the immune system in response to infections, can efficiently induce activation of HSCs (Essers
et al., 2009). Activated HSCs start to proliferate in vivo and up-regulate stem cell antigen 1 (Sca-1).
These results were surprising, as it has been shown that upon virus infections IFNs have an antiproliferative effect on most cells of the blood system. In order to explain this opposite effect of IFNα
on HSCs we wondered whether during infection IFNα might be part of a feedback loop leading to
the activation of HSCs. Using a reporter mouse to monitor IFNα production in the bone marrow,
several forms of bone marrow stress were tested. Interestingly, injection of mice with lipopolysaccharide (LPS) leads to increased IFNα production. Furthermore, LPS treatment also induced proliferation of quiescent HSCs in a TLR4-dependent manner. Similar to IFNα, LPS induced activation
was accompanied by, and dependent on, up-regulation of Sca-1 on the surface of HSCs. However,
though IFNα has a direct effect on HSCs, both in vivo and in vitro experiments showed that LPS has
an indirect effect on HSCs. Using co-culture experiments of sorted HSCs and other bone marrow
cells we could show that only CD11b+CD115+Ly-6C+ cells of the bone marrow are able to initiate an
up-regulation of Sca-1 on HSCs in response to LPS. We are currently unravelling the mechanism
underlying the indirect activation of HSCs in response to LPS. These data will further increase our
knowledge on the mechanism of activation of HSCs under stress conditions.
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Role of Sca-1 in IFNα induced activation of hematopoietic stem cells
Hannah Uckelmann 1, Stephan Wurzer 1,2, and Marieke Essers 1,2
1
German Cancer Research Center (DKFZ), Division of Stem Cells and Cancer, Heidelberg, Germany
2
HI-STEM - Heidelberg Institute for Stem Cell Technologies and Experimental Medicine gGmbH, Heidelberg, Germany
Correspondence: E-mail: [email protected]
The life-long maintenance of the blood system is accomplished by a pool of self-renewing and
pluripotent hematopoietic stem cells (HSCs). Adult HSCs are found in a dormant state for most of
their lifetime, entering cell cycle only to maintain homeostatic blood supply. This balance between
dormancy and activation is controlled by external factors such as chemokines and cytokines as well
as the interaction of HCSs with cells of the stem cell niche. Under stress conditions such as chemotherapy or bleeding, HSCs are stimulated to proliferate and give rise to various specialized blood
and immune cells. The molecular mechanisms underlying the processes of activation of HSCs are
still largely unknown.
Our group has previously shown that HSCs proliferation can be induced by in vivo IFNα treatment of mice. This response is dependent on signaling via the IFNα receptor (IFNAR) and STAT1
leading to downstream induction of IFNα target gene expression (Essers et al., 2009). Furthermore,
expression of the cell surface protein Stem cell antigen-1 (Sca-1) is highly induced upon IFNα treatment. Interestingly, treatment with IFNα of mice lacking Sca-1 showed no increased proliferation
of HSCs, demonstrating the requirement of Sca-1 for the IFNα induced activation of HSCs (Essers
et al., 2009). To gain insight into the mechanism of activation and into the role of Sca-1, we now
compared the transcriptional response of HSCs of C57Bl/6 wildtype and Sca-1 KO mice treated
with PBS or IFNα by microarray expression analysis. Interestingly, we discovered several cell cycle
inhibitors, such as p57 to be down regulated upon IFNα treatment, which might enable the cells to
exit G0 and start cycling. Furthermore, several components of the extracellular matrix and the stem
cell niche were differentially regulated upon IFNα treatment, pointing towards a remodeling of the
HSC environment. Surprisingly only few differences were detected between wildtype and Sca-1 KO
HSCs upon IFNα treatment. We are currently validating the regulation of these targets on the protein level and their functional role in HSC activation. This will allow us to unravel the mechanism of
IFNα induced activation of HSCs and will tell us more about the role of Sca-1 in this process.
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Wnt secretion is required for colon cancer cell survival
Oksana Voloshanenko 1*, Gerrit Erdmann 1*, Iris Augustin 1, and Michael Boutros 1
German Cancer Research Center (DKFZ), Div. Signalling and Functional Genomics and University of Heidelberg, Heidelberg, Germany
1
* These authors contributed equally to this work
Sporadic colorectal carcinomas are associated with mutations in APC or β-catenin, leading to a
constitutive activation by preventing the degradation of β-catenin, allowing its accumulation in the
nucleus and drive the expression of transcriptional target for growth and proliferation. Recently, it
was shown that independent of APC/β-catenin mutations colon cancer stem cells are characterized
by high canonical Wnt pathway activity, when the mechanism of this action is still not clear.
Here we show that colon cancer cells require Wnt secretion for survival and can be regulated
Wnts independent of mutations status. We demonstrate that Evi/Wls, a protein required for the
secretion of Wnt proteins, impairs the expression of β-catenin dependent transcriptional targets.
Blocking Wnt secretion is sufficient to inhibit Wnt signalling and cell survival of colon cancer cell
lines ex vivo and in vivo, independent of mutations in APC and β-catenin.
We conclude that mutations in APC and β-catenin do not render colon cancers independent of
autocrine activation through Wnt ligands, they just sensitize cells to Wnts. These results may open
new avenues for therapeutic interventions in colon cancer by antagonists of Wnt signalling that act
at or upstream of the receptor level and explain previous observations that in colon cancers nuclear
localization of β-catenin is still heterogeneous.
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The role of lamins in lung cancer development
Joaquim Vong, Gergana Dobreva
Emmy-Noether Junior Research Group, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany
Lung cancer is the leading cause of cancer-related death. Therefore, it is important to find new
molecular markers that will facilitate the early detection of the tumor and to understand the molecular mechanisms responsible for lung cancer initiation, progression and metastasis. In lung cancer
cells there is dramatic alteration of the nuclear structure – e.g. the nuclear and nucleolar size and
shape and the chromatin appearance. These alterations are characteristic for a certain tumor type
and can be used in cancer diagnostics. Lamins maintain nuclear structure and directly associate
with chromatin, suggesting that they may play a role in chromatin organization and positioning of
genes in the nucleus, thus setting up a particular gene expression pattern during differentiation
and malignancy. The nuclear lamina is comprised from Lamin A/C and Lamin B proteins. Interestingly, ablation of Lmnb1 gene in mice leads to perinatal lethality, caused by respiratory failure. Here
we show that Lamin B1 is highly expressed in bronchioalveolar (BASC) cells, which are capable to
self-renewal after injury and are stem cells at the distal terminal of the lung. Furthermore, silencing
of Lamin B1 results in defects in nuclear integrity and dramatic changes of chromatin structure of
mouse lung epithelial cells. Microarray, followed by quantitative PCR analysis revealed a significant
amount of genes related to tumor formation, proliferation and metastasis to be deregulated upon
loss of Lamin B1. Moreover, we found that Lamin B1 expression is highly upregulated and aberrantly localized in different types of lung cancer, however, its expression is reverse proportional to
tumor malignancy. Consistent with this, loss of Lamin B1 results in increased migration ability and
metastasis formation.
Key words: lung carcinoma, nuclear matrix, lamins, SUMO
107
Activation of Fanconi anemia hematopoietic stem cells induces DNA damage
Dagmar Walter 1,4, Amelie Lier 1,4, David Brocks 1, Anja Geiselhart 1, Sina Huntscha 1,4, Irem Bayindir 1,
Marieke A Essers 2,4, Andreas Trumpp 3,4, Michael D Milsom 1,4
1
Experimental Hematology Group (A012),German Cancer Research Center (DKFZ), Heidelberg, Germany
2
Hematopoietic Stem Cells and Stress Group (A011), German Cancer Research Center (DKFZ), Heidelberg, Germany
3
Division of Stem Cells and Cancer (A010); German Cancer Research Center (DKFZ), Heidelberg, Germany
4
Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany
Correspondence: E-mail: [email protected]
Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome, resulting
from mutations in one of 15 known genes that cooperate in a DNA repair pathway. FA patients suffer to varying degrees from a heterogeneous range of developmental defects and, in addition, have
an increased likelihood of developing cancer. Almost all FA patients develop severe, progressive
bone marrow failure, which impacts upon production of all hematopoietic lineages and hence is
thought to be driven by a defect at the level of hematopoietic stem cell (HSC). In addition, FA patients and cells are hypersensitive to interstrand crosslinking agents; pro-inflammatory cytokines;
and oxidative stress, leading to chromosomal instability.
Our primary objective was to address whether cell cycle activation of FA HSCs induces DNA damage resulting in bone marrow failure and/or cellular transformation.
So far, we have observed that activation of FA HSCs via induction of an interferon-α-mediated
stress response or an interferon-α independent one lead to an increased DNA damage response
that we hypothesize is dependent on HSC proliferation. Furthermore, we could show that upon serial activation, HSCs are depleted in FA mice compared to the wild type setting.
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In summary, our data are consistent with the hypothesis that when dormant HSCs are induced
into cell cycle via exposure to pro-inflammatory cytokines, a DNA damage response is provoked
which can lead to HSC depletion in the absence of a functional FA DNA repair pathway.
The role of insulin-like growth factor-I receptor in hepatocyte differentiation
A. Waraky 1, O. Larsson 1, E. Aleem 1,2
1
Cancer Center Karolinska, Karolinska Institutet, Department of Oncology Pathology, Stockholm, Sweden
2
Alexandria University, Faculty of Science, Division of Molecular Biology, Alexandria, Egypt
The type-I insulin-like growth factor receptor (IGF-IR) is a receptor tyrosine kinase (RTK), when activated by ligand binding it controls the proliferation of cells in a variety of ways, namely: (1) It sends
a mitogenic signal, (2) it promotes growth in cell size, (3) it protects cells from a variety of apoptotic
injuries, and (4) it regulates cell adhesion and cell motility. Furthermore, IGF-IR has been shown to
be sumoylated and translocated into the nucleus, where it may affect transcription. Although, the
mitogenicity of the IGF-IR has been known for a long time, there are recent indications on its ability
to send differentiation signals. In addition, the IGF-IR has been shown to be expressed at significant
levels in the undifferentiated human embryonic stem cells (hESc), and inhibition of the IGF-IR reduced hESc self renewal. Furthermore, other studies have shown that IGF-IR expression is elevated
with Nanog (key regulator for stem self-renewal) expression in hepatocellular carcinoma (HCC).
Taken together, the goal of the present study is to investigate the role of IGF-IR in stem cell differentiation and the significance of IGF-IR overexpression in HCC. The role of the IGF-IR in differentiation will be studied in hESc and normal hepatocytes (nHeps). nHeps do not express any detectable
levels of IGF-IR mRNA or protein, while hESc have been shown to express high levels of IGF-IR.
To achieve our goal, IGF-IR expression will be investigated in different stages of hepatocyte differentiation along with stem cell markers. In order to investigate genes regulated by the IGF-IR, we
will follow two approaches; 1) Knockdown of IGF-IR in hESc using shRNA, and 2) overexpression
of IGF-IR in nHeps, followed by gene expression profiling using microarrays. In order to investigate
potential transcriptional role of IGF-IR, the following approaches will be used: 1) Identification of
IGF-IR-DNA interactions using electrophoretic mobility shift (EMSA) and Chromatin immuneprecipitation (ChIP) assays. 2) Identification of proteins binding in complex with IGF-IR using Co-Immunoprecipitation (Co-IP) and proximity ligation assay (PLA). Currently we have detected the expression
of IGF-IR, Nanog, Sox17and FoxA2 in the early stages of hESc differentiation.
The outcome of the present study may provide more insight into the clinical importance of IGF-IR
overexpression in cancer.
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Lysosomal proteases and cancer stem cells in the PyMT mouse model of
metastatic breast cancer
Fee Werner 1,2, Jan Hülsdünker 1, Julia Bender 1, Marlene Tacke 1, Marie Follo 3, Thomas Reinheckel 1
1
Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
2
Speman graduate School of Biology and Medicine, Freiburg, Germany
3
Department of Hematology and Oncology, Freiburg University Medical Center, Freiburg, Germany
Cathepsins are cysteine proteases of the lysosome that promote tumor metastasis. In the Polyoma Middle T (PyMT) mouse model of invasive breast cancer, an overexpression of human cathepsin B (TghCTSB) resulted in higher metastatic burden in the lungs. A combined knockout of the
cathepsins B and Z (CBCZko) reduced lung metastasis. Therefore we asked the question whether
the stem cell pool differs in size or functionality between the different cathepsin genotypes. Furthermore, we analyzed the function of lysosomal proteases in tumor stem cells.
Cancer stem cells were defined as CD90+CD24+CD45- tumor cells. The stem cell population
size was assessed by analytic FACS. For functional analysis sorted cancer stem cells were employed
in colony formation assay and liquid culture. Expression of proteases was analyzed by RT-PCR and
proteolytic activity was shown in enzyme activity assay with cell lysates and by microscopic imaging
of matrix proteolysis by living cells.
The proportion of CD90+CD24+CD45- in primary tumors of 12 week old female PyMT mice did
not significantly differ between the genotypes WT, TghCTSB, CBCZko. Colony formation capacity of
sorted cells did not show a difference either. Expression of the cathepsins B, Z, L could be shown
in cancer stem cells. CD90+CD24+CD45- cells exhibited enzymatic activity of cysteine cathepsins.
We conclude that in the PyMT mouse model the CD90+CD24+CD45- stem cell population size
and colony formation capacity does not account for the phenotypic differences between cathepsin
genotypes. However, cathepsins were shown to be present and active in cancer stem cells and their
proteolytic function could be promoting lung metastasis.
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The anti-apoptotic enzyme paraoxonase-2 modulates hematopoetic stem
cell development
Ines Witte 1, Andrea Schüler 2, Thomas Kindler 2, Ulrich Förstermann 1 and Sven Horke 1
1
2
Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
Medical Department III, Oncology and Hematology, University Medical Center of the Johannes Gutenberg University
Mainz, Mainz, Germany
Apoptosis resistance is a serious complication of cancer therapies. As many malignant cells
are under oxidative stress, interference of redox-balance in cancer and stem cells gained increasing attention. Here we addressed the weakly characterized anti-oxidative enzyme, paraoxonase-2
(PON2). Recently we found that PON2 grants robust anti-oxidative and anti-apoptotic benefit to
tumor cells due to enhanced evasion from cell death and resistance to chemotherapeutics. The
enzyme locates to the endoplasmic reticulum and mitochondria where it fulfills vital functions in
the control of ROS generation. In particular, PON2 interacts with coenzyme Q10 causing a diminishment of mitochondrial ROS formation. This anti-oxidative effect resulted in lowered mitochondrial
cytochrome C release, caspase activation and cell death. Consistently, we could demonstrate that
PON2 was upregulated in many malignancies, including leukemias. Intriguingly, PON2 knock-down
increased cell death, particularly spontaneous apoptosis of K562 CML cells as well as their sensitivity to imatinib (Gleevec®). This is in line with the association of PON2 levels with response to
front-line therapies in pediatric ALL and imatinib resistance in CML patients. These findings and the
known impact of redox signaling on quiescence, apoptosis, differentiation and self-renewal of hematopoetic stem cells (HSCs) prompted us to analyze hematopoiesis in PON2-/- mice. These mice
showed severe alterations of the hematopoetic stem cell compartment, i.e. an increased number
of long-term HSCs, an imbalance in granulocyte/monocyte progenitors and structural aspects of
erythrocytes and thrombocytes. Additionally, these knock-out animals had significant changes in
blood cell counts and older female PON2-/- mice developed a pronounced splenomegaly. Our studies provide evidence that the anti-oxidative enzyme PON2 plays a yet undiscovered role in HSCs,
which underlines the importance of redox signaling and proposes PON2 as new potential target in
cancer therapies.
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In vivo RNAi screening identifies tumor suppressors in breast cancer
Jonas Wolf 1, Fan Zhang 4, Maria Goli Shahmorad 4, Christa Flechtenmacher 3, Gordon Mills 4, Karin
Müller-Decker 2, Jörg Hoheisel 1, Michael Böttcher 1
1
Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
2
Core Facility Tumor Models, German Cancer Research Center (DKFZ), Heidelberg, Germany
3
Institute of Pathology, University Hospital, Heidelberg, Germany
4
Department of Systems Biology, M.D. Anderson Cancer Center, Houston, Texas, USA
Given the limitations of cell culture based assays to recapitulate an appropriate microenvironment, we adapted RNAi technology to screen for genes involved in the development of breast cancer in mice. Therefore we use short hairpin RNAs (shRNAs) to suppress gene expression in order to
analyze the resulting loss-of-function phenotypes. The advent of pooled barcoded shRNA libraries
enables us to perform highly parallel genome-wide screens in vivo.
We discovered 16 unknown tumor suppressor genes that inhibit the tumor growth in SCID mice
and we functional validated 3 candidate genes in xenograft experiments. Further, breast cancer
database analysis indicates their clinical relevance for the overall survival of patients. Next, we
performed a gene expression profile of one of our candidates, a transcription factor, to indentify
regulated pathways. This helps us to find the underlying molecular mechanisms how the validated
candidates act as a tumor suppressor. This approach yields important information about new unknown functions of genes involved in cancerogenesis at the site of tumor development.
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How molecular modelling tools support stem cell research in neurooncology
Ruiyan Zhang 1, Melitta Schachner 2, Oksana Raabe 3, Stefan Arnhold 3, Athanasios K. Petridis 4,
Hubertus Maximilian Mehdorn 4, Roland Schauer 5, Hans-Christian Siebert 1
1
RI-B-NT - Research Institute of Bioinformatics and Nanotechnology, Schauenburgerstrasse 116, 24118 Kiel, Germany
2
Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany
3
Institute of Veterinary Anatomy, Histology, and Embryology, Justus-Liebig University of Giessen, Frankfurterstrasse 98,
35392 Giessen, Germany
4
Department of Neurosurgery, University Hospital of Schleswig-Holstein, Campus Kiel, Schittenhelmstrasse 10, 24105
Kiel
5
Biochemisches Institut, Universität Kiel, Olshausenstrasse 40, 24098 Kiel, Germany
The discovery of an overexpression concerning polysialic acids (PolySia) in malignant astrocytomas provides new therapeutic strategies in the field of neurooncology (1). Sialic acid receptors
from various origin have structural similarities in the architectures of their carbohydrate recognition
domains (CRDs) (2). Of high interest are PolySia receptors interacting with NCAMs (Neural Cell Adhesion Molecules) which contain alpha2,8-linked sialic acids.
Structural similarities between the CRDs of sialic acid receptors and the CRDs harboring other
negatively charged carbohydrates are found. These receptor structures are relevant for specific interactions with negatively charged sulfated saccharides e. g. the HNK-1 (Human Natural Killercell-1)
epitope. This epitope which is responsible for neuronal differentiation processes interacts with certain amino acid residues (3). The three-dimensional patterns of binding-relevant amino acids especially their functional groups in the CRDs are characteristic for NCAM- and HNK-1-receptors. Since
we have described that small peptide structures (collagen-fragments) can trigger the differentiation of stem cells (4) we are now testing the potential of diverse carbohydrate epitopes in a similar
way. It has been described that molecular modelling tools (in silico techniques such as molecular
dynamics simulations and molecular docking calculations) can provide important contributions to
understand the biological function of carbohydrate epitopes (5). Using suited biophysical methods
such as NMR and SPR (3, 6) we are now able to answer with suited molecular modelling techniques
crucial questions of clinical relevance concerning the design of artificial PolySia receptors.
1. A. K. Petridis, H. Wedderkopp, H. H. Hugo, H. M. Mehdorn (2009). Polysialic acid overexpression in malignant astrocytomas. Acta Neurochir. 151, 601-604.
2. H.-C. Siebert, S.-Y. Lu, R. Wechselberger, K. Born, T. Eckert, S. Liang, C.-W. von der Lieth, J. J. Barbero, R. Schauer, J. F.
G. Vliegenthart, T. Lütteke, S. André, H. Kaltner, H.-J. Gabius, T. Kožár (2009). A lectin from the Chinese bird-hunting
spider binds sialic acids. Carbohydr. Res. 344, 1515-1525.
3. A. Bhunia, S. Vivekanandan, T. Eckert, M. Burg-Roderfeld, R. Wechselberger, J. Romanuka, D. Bächle, A. V. Kornilov,
C.-W. von der Lieth, J. J. Barbero, N. E. Nifantiev, M. Schachner, N. Sewald, T. Lütteke, H.-J. Gabius, H.-C. Siebert
(2010). Why structurally different cyclic peptides can be glycomimetics of HNK-1. J. Am. Chem. Soc. 132, 96-105.
4. O. Raabe, C. Reich, S. Wenisch, A. Hild, M. Burg-Roderfeld, H.-C. Siebert, S. Arnhold (2010). Hydrolyzed fish collagen
induced chondrogenic differentiation of equine adipose tissue-derived stromal cells. Histochem. & Cell Biol. 134,
545-554.
5. A. M. Wu, T. Singh, J.-H. Liu, S. André, M. Lensch, H.-C. Siebert, M. Krzeminski, A. M. J. J. Bonvin, H. Kaltner, J. H. Wu,
H.-J. Gabius (2011). Adhesion/growth-regulatory galectins: Insights into their ligand selectivity using natural glycoproteins and glycotopes. Adv. Exp. Me5d. Biol. 705, 117-141.
6. Y. E. Tsvetkov, M. Burg-Roderfeld, G. Loers, A. Ardá, E. V. Sukhova, E. A. Khatuntseva, A. A. Grachev, A. O. Chizhov,
H.-C. Siebert, M. Schachner, J. Jiménez-Barbero, N. E. Nifantiev (2012). Synthesis and molecular recognition studies
of the HNK-1 trisaccharide and related oligo-saccharides. The specificity of monoclonal anti-HNK-1 antibodies as
assessed by surface plasmon resonance and STD NMR. J. Am. Chem. Soc. 134, 426-435.
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Aspirin sensitizes pancreatic cancer to chemotherapeutic drugs by inhibition of cancer stem cell features
Yiyao Zhang 1,2, Li Liu 1,2, Ewa Aleksandrowicz 1,2, Sabrina Labsch 1,2, Frank Schönsiegel 1,2, Vanessa
Rausch 1,2, Adam Kaczorowski 1,2, Alexei V. Salnikov 1,3, Eduard Ryschich 2, Markus W. Büchler 2,
Ingrid Herr 1,2
1
Molecular OncoSurgery Group, Department of General Surgery, University of Heidelberg and German Cancer Research
Center (DKFZ), Heidelberg, Germany
2
Department of General Surgery, University of Heidelberg, Heidelberg, Germany
3
Department of Translational Immunology, German Cancer Research Center (DKFZ) and National Center for Tumor
Diseases, Heidelberg, Germany
According to the cancer stem cell (CSC) theory it is believed that solely the small subset of CSC
is responsible for self-renewal, invasive potential and therapy resistance of pancreatic cancer and
other tumor entities. Recent clinical and epidemiological investigation demonstrate that daily intake
of Aspirin significantly prevents the risk for colorectal and breast cancer, inhibits metastasis, and
prolongs life of patients. Since metastasis is associated with CSC these recent data suggest that
Aspirin may target CSC although this issue has not been examined in pancreatic cancer so far.
By the use of primary and established models of pancreatic CSC we demonstrate that Aspirin
inhibits viability, induces apoptosis, even in gemcitabine-resistant pancreatic cancer cells, and enhances efficacy of gemcitabine as measured by MTT assay as well as by annexin and PI staining
followed by FACS-analysis. Aspirin inhibited self-renewal potential as shown by blocking colony and
sphere formation along with activity of ALDH1. Also, the migratory potential was decreased at early
time points after treatment in living cells. Most importantly Aspirin affected solely the highly malignant cells and had no toxic effects to non-malignant primary pancreatic ductal cells or mesenchymal stem cells.
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In ongoing studies we analyze involvement of downregulation of typical stem cell-associated
factors by Western blot analysis, protein arrays and electrophoretic mobility shift assay. To confirm
these in vitro data and to involve the microenvironment xenotransplantation studies with primary
pancreatic CSC isolated from patient tumors are in preparation. Finally, we will correlate identified
key factors targeted by Aspirin to stainings of patient tissue and treatment schedules of patients.
These studies may provide the experimental basis for a new strategy for prevention and co-treatment of pancreatic cancer.