PROGRAMM 5. Symposium Urologische Forschung

Transcription

5. Symposium
Urologische Forschung
der Deutschen Gesellschaft für Urologie
CME
Zellbiologie des Urogenitalsystems
Entwicklung, Homöostase, Pathogenese
PROGRAMM
In Kooperation mit
der Arbeitsgemeinschaft Uropathologie
der Deutschen Gesellschaft für Pathologie
14. bis 16. November 2013
Aula im Hauptgebäude
der JLU-Gießen
Inhalt
GRUßWORT .............................................................................................................................................3
HINWEISE ................................................................................................................................................5
ALLGEMEINE HINWEISE ................................................................................................................................................................. 5
LAGEPLAN ...................................................................................................................................................................................... 6
AUF-FORSCHUNGSPREISE ............................................................................................................................................................. 7
PUBLIKATION DER ABSTRACTS ....................................................................................................................................................... 7
CME-ZERTIFIZIERUNG ................................................................................................................................................................... 7
HINWEISE FÜR REFERENTEN & MODERATOREN ............................................................................................................................ 8
Vorträge .........................................................................................................................................................................................8
Poster..............................................................................................................................................................................................8
SCIENTIFIC PROGRAM.............................................................................................................................9
Thursday, 14.11.2013 .............................................................................................................................................................. 11
Friday, 15.11.2013 ................................................................................................................................................................... 13
Saturday, 16.11.2013............................................................................................................................................................... 20
SOCIAL PROGRAM ................................................................................................................................25
Begrüßungsabend...................................................................................................................................................................... 27
Experimenteller Abend ............................................................................................................................................................. 27
ABSTRACTS............................................................................................................................................29
ANHANG ...............................................................................................................................................87
AUF-PREISTRÄGER .......................................................................................................................................................................89
SPONSOREN .................................................................................................................................................................................91
AUSBLICK: 6. AUF-SYMPOSIUM 2014 IN HOMBURG/SAAR ......................................................................................................93
2
Grußwort
Liebe Kolleginnen und Kollegen,
wir freuen uns, Ihnen den Tagungsband für das nunmehr 5. Symposium der
Arbeitsgruppe urologische Forschung (AuF) der Deutschen Gesellschaft für Urologie
überreichen zu können. Diese Veranstaltung steht unter dem Leitthema „Zellbiologie
des Urogenitalsystems - Entwicklung, Homöostase und Pathogenese“ und wird in
guter Tradition gemeinsam mit der Arbeitsgemeinschaft Uropathologie durchgeführt.
Die Funktion des Urogenitalsystems wird durch seine vielfältigen, oft
hochspezialisierten und im Körper einzigartigen Zellen sichergestellt. Störungen in
der Funktion, Interaktion und Homöstase dieser Zellen sind die Ursache vieler
Erkrankungen, mit denen die Urologie befasst ist. Das Symposium soll daher die
normale Funktion des Urogenitalsystems und seine Erkrankungen aus dem
Blickwinkel der Zellbiologie beleuchten. Ziel ist es, die Fortschritte auf diesem Gebiet
zu präsentieren und zu diskutieren, wie sie in der urologischen Forschung
weitergeführt und in der klinischen Praxis aufgenommen werden können. Auch
methodische Entwicklungen sollen nicht zu kurz kommen.
Für das 5. AuF-Symposium konnten wir namhafte Wissenschaftler als Referenten
gewinnen. Neben der Möglichkeit mit diesen ausgewiesenen Experten ins direkte
Gespräch zu kommen, bieten wir insbesondere den urologischen
Nachwuchswissenschaftlerinnen und Nachwuchswissenschaftlern mit moderierten
Vortrags- und Postersitzungen geeignete Foren zur Präsentation ihrer eigenen
Arbeiten. Wir freuen uns, auch diesmal wieder ehemalige Eisenberger-Stipendiaten
begrüßen zu können, die über ihre Projekte sowie gemeinsam mit ihren Betreuern
über Ablauf und Bedeutung ihrer Stipendien sprechen werden.
Wir heißen Sie herzlich willkommen in Gießen und wünschen Ihnen einen
angenehmen Aufenthalt!
3
4
Allgemeine Hinweise
5
Lageplan
3
5
1
4
2
11
6
AuF-Forschungspreise
Die Arbeitsgruppe Urologische Forschung der DGU prämiert auf seinem Symposium 2013
wieder vier herausragende Vortrags- und Posterpräsentationen mit je 500 €. Die von der
Deutschen Gesellschaft für Urologie e.V. gestifteten Forschungspreise werden in den beiden
Kategorien jeweils an einen Mediziner und einen Naturwissenschaftler vergeben.
Über die Preisvergabe entscheidet eine Jury:




Prof. Dr. rer. nat. Gerhard Unteregger
PD Dr. med. Frederik Roos
Prof. Dr. med. Ruth Knüchel-Clarke
Prof. Dr. med. Sven Perner
Die Preisverleihung findet am Samstag, den 16.11.2013 im Rahmen der Abschlusssitzung um
12:45 Uhr statt.
Publikation der Abstracts
Alle Abstracts der präsentierten Beiträge werden in der Januar-Ausgabe 2013 der Zeitschrift
„Der Urologe“ zitierfähig veröffentlicht.
Die Abstacts der vergangenen drei AuF-Symposien können unter den folgenden InternetAdressen abgerufen werden:
1. Symposium: http://link.springer.com/article/10.1007/s00120-009-2165-3
2. Sympoisum: http://link.springer.com/article/10.1007/s00120-010-2485-3
CME-Zertifizierung
Das 5. Symposium Urologische Forschung der DGU ist eine von der Akademie der Deutschen
Urologen in Zusammenarbeit mit der Landesärztekammer Hessen mit 16 CME-Punkten
zertifizierte und evaluierte Veranstaltung. Die gemäß den Fortbildungskriterien vergebenen
CME-Punkte werden bundesweit von allen Landesärztekammern anerkannt.
Die CME-Registrierung der Teilnehmer erfolgt täglich an den Empfangstischen des
Symposiums. Bitte halten Sie dazu Ihre EFN-Nummer (Barcode-Aufkleber) bereit. Die
Akademie übernimmt die Meldung der über EFN registrierten Teilnehmer an die einzelnen
Landesärztekammern.
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Hinweise für Referenten & Moderatoren
VORTRÄGE
Vorträge sind auf 7 Min. plus 3 Min. Diskussion begrenzt. Wir möchten Sie freundlich bitten,
Ihre Redezeit strikt einzuhalten. Tagungssprache ist grundsätzlich Deutsch. Es kann aber
wegen der internationalen Beteiligung auch gerne auf Englisch vorgetragen werden. Wir
möchten Sie in jedem Fall darum bitten, Ihre Vortragsfolien in englischer Sprache zu
erstellen.
Ihnen steht im Hörsaal ein Windows-Laptop mit einer aktuellen Microsoft PowerPointVersion zur Verfügung. Bitte achten Sie auf Windows-Kompatibilität Ihrer ppt-Dateien, da
wir kein individuelles Anschliessen von Apple-Laptops ermöglichen können.
Die Medieannahme befindet sich im Tagungshörsaal. Reichen Sie bitte Ihre
Präsentationsdatei möglichst eine halbe Stunde vor Beginn Ihrer Session dort ein. Sollte Ihr
Vortrag Videosequenzen enthalten, achten Sie auch darauf, die entsprechenden Dateien
zusätzlich zu Ihrer ppt-Datei abzugeben.
POSTER
Poster sind hochformatig bis DIN A0 zu erstellen. Die Präsentation der Poster erfolgt auf
Deutsch oder wahlweise auf Englisch. Auf jeden Fall sollen die gedruckten Poster wegen der
internationalen Beteiligung in englischer Sprache erstellt sein. Bitte hängen Sie Ihr Poster
rechtzeitig in der Pause vor Ihrer Session auf und anschließend wieder ab.
In den moderierten Posterpräsentationen erklären die Autoren - am Poster stehend - kurz
und knapp ihre Arbeiten. Die Redezeit ist dabei auf 3 Min. plus 2 Min. Diskussion begrenzt.
Unsere Moderatoren sind angewiesen, streng auf die Einhaltung der Redezeit zu achten und
die Vortragenden zugunsten eines harmonischen Programmablaufs nötigenfalls zu
unterbrechen.
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SCIENTIFIC
PROGRAM
9
10
THURSDAY, 14.11.2013
17:0017:20
Welcome
Florian Wagenlehner & Wolfgang Schulz, Task Force Urological Research (AuF)
Joybrato Mukherjee, President of the Justus-Liebig-University Giessen
Wolfgang Weidner, Director of the Clinic of Urology, University of Giessen
Jan Fichtner, President of the German Society of Urology
Ruth Knüchel-Clarke, Chairwoman of the Working Group Uropathology
1st
Symposium
Focus: Tumour stem cells and circulating tumour cells
Moderation: Bernd Wullich & Wolfgang Schulz
17:2017:50
V1.1
The molecular characterisation of disseminated cancer cells - Is it clinically
relevant?
Nikolas Stöcklein
Dept. of Experimental Surgical Oncology, University of Duesseldorf
V1.2
Connecting the machineries of asymmetric division and tumor suppression in
stem cells
Salvatore Pece
Institute FIRC of Molecular Oncology, Milan, I
Short lectures
17:5018:20
18:2019:30
V1.3
Targeting a cancer stem cell population in renal cell cancer using small
molecule inhibitors
Annika Fendler
Clinic of Urology, Charité Berlin
V1.4
Epigenetic silencing of ITIH5 is associated with bladder cancer progression
and early relapse of pT1 high grade urothelial tumors
Michael Rose
Institute of Pathology, RWTH Aachen
V1.5
Expression changes of long noncoding RNA HOTAIR have tissue specific
effects on HOX gene expression and phenotype in urothelial carcinomas
Michèle Hoffmann
Clinic of Urology, University of Duesseldorf
11
V1.6
Prevalence and clinical features of HOXB13 mutation carriers in German
prostate cancer patients
Manuel Lüdeke
Clinic of Urology, University of Ulm
V1.7
Immunohistochemical study on invasive tumor cells of clear cell renal cell
carcinoma
Anja Rabien
from 19:45
12
Get Together at Dach Café
Ludwigsplatz 11, Giessen; http://dachcafe.com
FRIDAY, 15.11.2013
Keynote
Lecture
Jenny Southgate
Moderation: Wolfgang Weidner & Klaus Steger
08:3009:00
K1
Developmental aspects of the urinary tract
Jenny Southgate
Jack Birch Unit, Dept. of Biology, University of York, UK
2nd
Symposium
Focus: Andrology
Moderation: Wolfgang Weidner & Klaus Steger
09:0009:30
V2.1
Role of epigenetic in reproduction
Klaus Steger
Clinic of Urology, University of Giessen
V2.2
Testicular stem cells
Stefan Schlatt
Center of Reproduction Medicine und Andrology, University of Muenster
Short lectures
09:3010:00
10:0010:30
V2.3
Transcriptional control of tubular lumen size control in normal and diseased
kidneys
Zeliha Yesmin Yurtdas
Max Delbrueck Center for Molecular Medicine, Berlin
V2.4
Influence of testosterone on the regulation of inflammatory responses in
testicular and immune cells
Monika Fijak
Institute for Anatomy and Cell Biology, University of Giessen
V2.5
New biomarkers to differentiate malignant germ cell tumours of the testis
established by the SILAC-method in combination with high-resolution mass
spectrometry
Felix Bremmer
Institute of Pathology, University of Goettingen
10:3011:00
Coffe Break / Poster Exhibition
13
Poster I
1st Poster Presentation
Moderation: Walburgis Brenner, Jörg Ellinger & Helge Taubert
11:0012:15
Brenner
15 Posters
Ellinger
14
P1.1
Transcript levels of Piwi-like 1-4 genes are associated with clinicopathological
parameters in renal cell carcinomas
Omar Al-Janabi
Clinic of Urology, University of Erlangen
P1.2
HIF-2α is highly expressed in papillary renal cell carcinoma type II: a potential
reason for the worse prognosis?
Carl Ludwig Behnes
Institute of Pathology, University of Goettingen
P1.3
Deregulation of the CSN-CRL pathway during urological tumorigenesis?
Linda Gummlich
Clinic of Surgery, Charité Berlin
P1.4
Quantitative imaging of intratumoral heterogeneity in clear cell renal
carcinoma
Rouven Höfflin
Dept. of Molecular Urooncology, University of Heidelberg
P1.5
Prediction of second-line therapy response in metastatic renal cell carcinoma
patients by blood plasma marker proteins
Sebastian Hölters
Clinic of Urology, University of Homburg/Saar
P1.6
Targets and function of dysregulated microRNAs in clear cell renal cell cancer
Julia Liep
P1.7
Integrated microRNA and mRNA signature associated with the transition
from the locally confined to the metastasized renal cell carcinoma
Zofia Wotschofsky
P1.8
Molecular cytogenetic differences between Collecting Duct Carcinomas and
upper urinary tract urothelial carcinomas
Volker Jung
Taubert
12:1513:15
P1.9
Novel antiangiogenic compounds with antitumor activity for innovative
approaches in cisplatin resistant testicular germ cell cancer treatment
Bianca Nitzsche
Institute of Physiology, Charité Berlin
P1.10
Epigenetic regulation of genes involved in epithelial mesenchymal transition
in prostate cancer
Ulrike Brandt
P1.11
Epigenetic intervention counteracts resistance towards temsirolimus in
prostate cancer
Jasmina Makarevic
Clinic of Urology, University of Frankfurt a.M.
P1.12
Prostate cancer: An integrated evaluation of metabolomics, transcriptomics,
and proteomics expression data
Regina Reszka
Metanomics, Berlin
P1.13
Cytostatic drug WIST-C overcomes docetaxel induced resistance
Martina Rottach
Clinic of Urology, University of Greifswald
P1.14
Absolute quantification by qRT-PCR - a new and sensitive method to measure
absolute amounts of miRNA in high risk prostate cancer tissue
Maria Schubert
Clinic of Urology, University of Wuerzburg
P1.15
Evaluation of Patient Derived Molecular Neuroendocrine Signatures in PC3
using RNAseq data
Jost von Hardenberg
Clinic of Urology, University of Mannheim
Lunch
15
3rd
Symposium
Focus: Immunology / Infectiology of the genito-urinary tract
Moderation: Florian Wagenlehner & Hans Krause
13:1513:35
V3.1
Bacterial virulence factors
Ulrich Dobrindt
Institute for Hygiene, University of Muenster
V3.2
Host-response in urogenital infections
Hamid Hossain
Institute of Medical Microbiology, University of Giessen
V3.3
Interstitial cystitis
Lars-Christian Horn
Institute of Pathology, University of Leipzig
V3.4
Immunologie of testes / epididymides
Andreas Meinhardt
Short lectures
13:3513:55
13:5514:10
14:1014:25
14:2514:45
V3.5
Infection of the prostate caused by Propionibacterium acnes
Behnham Sayanjali
Max-Planck-Institute for Infection Biology, Berlin
V3.6
Investigation of Escherichia coli urinary tract isolates with characteristics of
small colony variants
Johannes Putze
Institute for Hygiene, University of Muenster
14:4515:15
16
Coffee Break / Poster Exhibition
Poster II
2nd Poster Presentation
Moderation: Susanne Füssel, Frederik Roos & Katharina Braun
15:1516:30
Füssel
15 Posters
Roos
P2.1
OASIS/CREB3L1 is downregulated in human bladder tumors and mediates
suppression of cancer cell spreading and migration in vitro
Laura Dierichs
P2.2
Responses of bladder cancer cells towards tyrosine-kinase inhibition by
dovitinib (TKI-258) in relation to the eptithelial mesenchymal transition
status
Jörg Hänze
Clinic of Urology, University of Marburg
P2.3
HDAC inhibition suppresses bladder cancer cell adhesion to collagen under
flow conditions
Eva Juengel
Clinic of Urology, University of Frankfurt a.M.
P2.4
Invasion of urothelial carcinoma of the bladder: MMP7 is associated with
increased cell invasion in urothelial cancer: evidence from functional models
Daniel Knauf
Clinic of Urology, University of Mannheim
P2.5
Analysis of microRNA expression in urothelial carcinoma of the upper urinary
tract
Stephanie Kriebel
Clinic of Urology, University of Bonn
P2.6
Acute epididymitis induces alterations in sperm protein composition
Adrian Pilatz
P2.7
Investigations on the localization and significance of the prolactin-inducible
protein (PIP) in the male urogenital tract
Srikanth Karnati
P2.8
Role of TET3 and 5-hmC in establishment of sperm epigenome and in
production of fertile sperm
Undraga Schagdarsurengin
Clinic of Urology, Dept. of Molecular Andrology, University of Giessen
17
Braun
18
P2.9
Mixed testicular atrophy is related to atherosclerosis in the ApoE(-/-) / LDL
receptor(-/-) double knockout mouse model: New data of the arterial supply
of the tubuli
Kai Steinfeld
P2.10
Polysialylation of NCAM correlates with onset and termination of seasonal
spermatogenesis
Sebastian Galuska
Institute of Biochemistry, University of Giessen
P2.11
Bacterial epididymitis induces fibrotic transformation and regional changes in
the activin/follistatin ratio
Vera Michel
P2.12
Necrosis is the dominant cell dead pathway in UPEC induced epididymoorchitis model and is responsible for structural and functional damage of rat
testis
Sudhanshu Bhushan
P2.13
Identification of mouse sperm glycoprofile to evaluate possible alterations
after urogenital tract infection: possible implications for immune privilege
and sperm function
Ferhad Khosravi
P2.14
Uropathogenic E. coli inactivate host survival AKT signalling pathway in
Sertoli cells
Zhengguo Zhang
P2.15
Urethral brush cells are polymodal cholinergic chemosensors
Klaus Deckmann
4th
Symposium
Focus: Cellular biology of the lower urinary tract
Moderation: Roman Nawroth & Karl-Dietrich Sievert
16:3016:50
V4.1
Cell-test systems for stem cell-based tissue engineering
Sabine Neuss-Stein
V4.2
Sphinkter regeneration using MSCs – options and limitations
Karl-Dietrich Sievert
Clinic of Urology, University of Tuebingen
V4.3
Afferent system: non-neuronal cholinergic system
Wolfgang Kummer
Institutr of Anatomy und Cell Biology, University of Giessen
Short lectures
16:5017:10
17:1017:30
17:3018:00
V4.4
Cellular changes during BPS
Christian Gratzke
Clinic of Urology, LMU Muenchen
V4.5
Expression of Aquaporin water channels by human urothelium: contribution
to transurothelial permeability in vitro and modulation by osmolality
Peter Rubenwolf
Clinic of Urology, University of Mainz
V4.6
3D-ultrastructure of the human urinary bladder revealed by FIB-SEM and
3View SEM
Jochen Neuhaus
Clinic of Urology, University of Leipzig
Keynote
Lecture
Hans Lilja
Moderation: Roman Nawroth & Karl-Dietrich Sievert
18:0018:30
K2
Relationship between the biology of PSA and related kallikrein-markers and
prostate cancer in man and mice (models)
Hans Lilja
Memorial Sloan-Kettering Cancer Center, New York, NY, USA
from 19:00
Experimental Evening at Museum Mathematikum
Liebigstraße 8, Gießen; http://www.mathematikum.de
19
SATURDAY, 16.11.2013
EisenbergerSession
Crosstalks Tutor & Fellow
Moderation: Gerhard Unteregger & Max Burger
09:0009:30
E1.1
The Department of Clinical Laboratories, Surgery and Medicine at Memorial
Sloan-Kettering Cancer Center as host for Eisenberger-fellows of the DGU
Hans Lilja
Memorial Sloan-Kettering Cancer Center, New York, NY, USA
09:3010:00
E1.2
MSP, PSA und hK2 in der Pathogenese des Prostatakarzinoms
Katharina Braun
Clinic of Urology, University of Bochum/Marienhospital Herne
E2.1
The Department of Transplant and Infection Immunology as host for
Eisenberger-fellows of the DGU
Martina Sester
Dept. of Transplantation and Infection Immunology, University of Homburg/Saar
E2.2
T-regs in metastasised renal cell carcinoma
Martin Janssen
10:0010:30
20
Coffee Break / Poster Exhibition
Poster III
3rd Poster Presentation
Moderation: Peter Olbert, Sven Perner & Martin Janssen
10:3012:00
Olbert
15 Posters
Perner
P3.1
GDNF-family neurotrophic factors in urinary bladder and expression of their
receptors (GFRα) in bladder sensory neurons
Rajender Nandigama
P3.2
Treatment strategy dependent tissue injury produced by a new acoustic lens
design for electromagnetic lithotripters
Andreas Neisius
Clinic of Urology, University of Mainz
P3.3
Coming closer to endoscopic “real time target identification” - Technical
improvements of an experimental lithotripsy laser system with spec-tral realtime object analysis
Arkadiusz Miernik
Clinic of Urology, University of Freiburg
P3.4
Overexpression of Drosha is associated with outcomes in patients with
urothelial carcinomas
Nadine Ratert
P3.5
Alternative therapy strategies for the non-muscle invasive bladder cancer on
the basis of carbon nanomaterials
Christiane Rieger
Clinic of Urology, University of Dresden
P3.6
The function of Kruppel-like factor 4 (KLF4) as a transcriptional repressor of
IGF2-promoter during prostate carcinogenesis
Temuujin Dansranjavin
P3.7
Resveratrol and prostate a very complicated “dangerous liaison”
Marina Di Domenico
Department of Biochemistry, Biophysics and General Pathology, Second
University of Naples, I
P3.8
Cardiolipin species composition differs in benign prostate epithelia and
prostate cancer and may be associated with tumor progression
Sebastian Fussek
Clinic of Urology, University of Greifswald
21
Janssen
22
P3.9
MiR-205 is progressively down-regulated in lymph node metastasis but fails
as a prognostic biomarker in high-risk prostate cancer
Charis Kalogirou
Clinic of Urology, University of Wuerzburg
P3.10
Re-expression of an epigenetically repressed microRNA inhibits prostate
cancer cell growth
Nina Korzeniewski
Dept. of Molecular Urooncology, University of Heidelberg
P3.11
HMGB1 modulates immune responses in a cell-specific manner in the rat
experimental autoimmune orchitis
Ferial Aslani
P3.12
Epigenetic dysregulation of inflammatory factors in prostatitis and prostate
cancer
Siva Reddy Velagala
P3.13
Androgen receptor regulates CD4+CD25+Foxp3+ T cell differentiation by
binding to foxp3 locus
Magdalena Walecki
P3.14
Functional analysis of specific T cells in patients under BCG-therapy
Julia Elsässer
Dept. of Transplantation and Infection Immunology, University of Homburg/Saar
P3.15
Urine protein profiling identified Alpha-1-microglobulin and Haptoglobin as
biomarkers for early diagnosis of acute allograft rejection following kidney
transplantation
Beatrice Stubendorff
5th
Symposium
Focus: Epithelial-mesenchymal transition / Tumour-stroma-interaction
Moderation: Ruth Knüchel-Clarke & Kerstin Junker
12:0012:20
V5.1
Stromal regulation and metastasis
Jonathan Sleeman
Center of Biomedicine and Medical Technology, University of Heidelberg
V5.2
Tumour-stroma-interactions
Gerhard Unteregger
V5.3
2-Photon microscopy of protective immunity in vivo
Stephan Halle
Institute of Immunology, University of Hannover
12:2012:40
12:4013:00
13:0013:15
Awards
Resumee
Outlook 2014
from 13:15
Short Lunch
Bernd Wullich, Chairman of AuF
Florian Wagenlehner & Wolfgang Schulz, AuF
Gerhard Unteregger & Frederik Roos, AuF
23
24
SOCIAL
PROGRAM
25
26
BEGRÜßUNGSABEND
Donnerstag, 14.11.2013, ab 19:45 Uhr
im Restaurant „Dach Café“
In gewohnt legerer Atmosphäre möchten
wir Sie am ersten Abend des Symposiums
zu einem Get Together in der Gaststätte
Dach Café in Gießen begrüßen.
Der Kostenbeitrag für diese Veranstaltung
beträgt 20 €, für Studenten 10 €.
Kontakt:
Restaurant Dach Café
Ludwigsplatz 11, 35390 Gießen
Tel. 0641 – 686 91 000
Wegbeschreibung siehe Lageplan auf S. 5
EXPERIMENTELLER ABEND
Freitag, 15.11.2013, ab 19:00 Uhr
im Museum „Mathematikum“
Zum Festabend laden wir Sie herzlich in das
besondere
Ambiente
des
Museum
Mathematikum ein. Während der zwischenzeitlich angebotenen Führungen erleben Sie
Mathematik zum Anfassen.
Der Kostenbeitrag für diese Veranstaltung ist
in den Tagungsgebühren enthalten.
Kontakt:
Museum Mathematikum
Liebigstraße 8, 35390 Gießen
Tel. 0641 – 969 79 70
Wegbeschreibung siehe Lageplan auf S. 5
27
28
ABSTRACTS
29
30
V1.3
Targeting a cancer stem cell population in renal cell cancer using small molecule
inhibitors
Fendler A1,2,3, Busch J2, Jung K2,3, Birchmeier W1
1
2
3
Department of Signal Transduction, Invasion and Metastasis of Epithelial Cells, Max Delbrück
Center of Molecular Medicine, Berlin
Department of Urology, Charité – University Hospital Berlin
Berlin Institute of Urologic Research, Charité – University Hospital Berlin
Tumors frequently exhibit extensive intratumoral heterogeneity and it has been hypothesized that
cancers are hierarchically organized and sustained by cancer stem cells (CSC) that are able to self
renew and recapitulate tumor heterogeneity. CSC’s have been linked to therapy resistance and the
specific inhibition of the CSC population might help to develop novel and more efficient treatment
strategies of cancer. Current therapies of metastatic renal cell carcinoma are not curative,
highlighting the ongoing need for novel and more efficient strategies. Thus, we aimed to identify a
cancer stem cell population in clear cell renal cell cancer (ccRCC) and to investigate the potential of
small molecule inhibitors to specifically target the CSC population.
To identify a population with CSC properties in ccRCC, primary renal cancer cells were isolated from
patients undergoing nephrectomy. Fluorescence activated cell sorting (FACS) identified a rare
population of CXCR4+/c-Met+/CD44+ cells in primary renal cancer cells. These cells displayed higher
sphere formation in vitro and their frequency increased with higher tumor stage. Xenograft assays in
immune deficient NSG mice support the idea of a tumor-initiating capacity of this population. To
assess treatment strategies that specifically target the cancer stem cell population in ccRCC, small
molecule inhibitors directed against c-Met (Crizotinib), CXCR4 (AMD 3100), Wnt/-Catenin (ICG001, XAV939, LF3), GSK3 (CHIR 99021), Notch (DAPT), and MAPK signaling (U0126) were tested on
their effect on sphere formation and cell proliferation of adherent cultures. Additionally small
molecule inhibitors that have been approved for patient therapy (Sorafenib, Rapamycin) were tested.
Small molecule inhibitors showed mixed effect on the proliferation of adherent cells and sphere
formation and we could distinguish different classed of inhibition: (i) inhibition of both spheroid
formation and growth of adherent cells, (ii) inhibition of spheroid formation but no effect on growth
of adherent cells, (iii) no influence on sphere formation but inhibition of adherent cell growth, (iv) no
effect.
In conclusion, we identified small molecule inhibitors that can specifically target a population with
cancer stem-like properties in ccRCC, while others appear to only target more differentiated cells.
Targeting the CSC population might have a beneficial effect in treatment of ccRCC in the future.
Contact:
[email protected]
31
V1.4
Epigenetic silencing of ITIH5 is associated with bladder cancer progression and early
relapse of pT1 high grade urothelial tumors
Rose M1, Gaisa NT1, Antony P1, Fiedler D1, Heidenreich A2, Otto W3, Denzinger S3, Bertz S4, Hartmann
A4, Karl A5, Knüchel R1, Dahl E1
1
2
3
4
5
Molecular Oncology Group, Institute of Pathology, Medical Faculty of the RWTH Aachen
University
Department of Urology, Medical Faculty of the RWTH Aachen University
Department of Urology, Caritas St. Josef Medical Centre, University of Regensburg
Institute of Pathology, University Hospital Erlangen
Department of Urology, LMU Munich
Background: Invasive bladder cancer is known to cause unfavorable prognosis. Disease management
of this urothelial cancer subtype is still poor due to lack of prognostic and predictive markers, and
most of these patients die in consequence of metastatic disease. Accumulating indications have
shown that inter- -trypsin inhibitor heavy chain 5 (ITIH5) is associated with tumor suppression,
especially metastasis, in various cancers such as breast cancer. However, its putative role in bladder
cancer is completely unknown. Therefore, we aimed to study ITIH5 expression as well as its
prognostic and biological impact on human bladder cancers.
Methods: Epigenetic ITIH5 gene regulation was studied in bladder cell lines and primary bladder
tumors by real-time PCR, immunohistochemistry, methylation-specific PCR (MSP) and
pyrosequencing. In-vitro chromatin remodeling was approached by 5-aza-2’-deoxycytidine/
trichostatin A (DAC/TSA) treatment of bladder cancer cell lines. Statistical evaluation of ITIH5
methylation and ITIH5 expression with patient characteristics and clinical outcome was performed
using SPSS15.0. The biological function of ITIH5 was examined by re-expression of a full-length ITIH5
cDNA in the urothelial cancer cell line RT112 that served as in vitro model for proliferation, colony
spreading and tumor cell migration studies.
Results: Real-time analyses showed downregulation of ITIH5 mRNA in 61% (n=45) of urothelial
cancer samples, particularly in muscle-invasive tumors (p<0.001). ITIH5 loss in bladder cancer was
further pronounced on protein level. DNA methylation analysis verified tumor-specific ITIH5
promoter methylation in 50% of pTa low grade and 68% of invasive bladder tumors. This epigenetic
promoter modification was tightly linked (p<0.001) with the inactivation of ITIH5 mRNA synthesis in
bladder tumor samples. A direct correlation between ITIH5 DNA methylation and its expression was
functionally confirmed by in vitro demethylation experiments. Moreover, pyrosequencing analysis
revealed that ITIH5 hypermethylation frequency was closely associated with progressive bladder
cancers. In light of that, a cohort (n=120) of clinically challenging pT1 high grade bladder tumors was
analyzed for ITIH5 expression. We found an association between ITIH5 protein loss and unfavorable
prognosis of bladder cancer patients (recurrence-free survival; hazard ratio: 4.35, p=0.048). Finally,
stable ITIH5 re-expression in human RT112 bladder cancer cells mediated suppression of both cell
migration and colony spreading.
Conclusions: We provide for the first time evidence that ITIH5 loss by aberrant ITIH5 promoter
methylation during bladder cancer development may promote bladder cancer progression. Moreover,
ITIH5 protein might become a prognostic biomarker for relapse risk stratification in high grade
bladder cancer patients.
Contact:
[email protected]
32
V1.5
Expression changes of long noncoding RNA HOTAIR have tissue specific effects on
HOX gene expression and phenotype in urothelial carcinomas
Heubach J, Niegisch G, Schulz WA, Hoffmann MJ
Urologische Klinik, Heinrich-Heine-Universität, Medizinische Fakultät
Objective: Tumor heterogeneity in urothelial carcinoma (UC) which complicates diagnosis and
therapy may be caused by aberrant differentiation. In many cancers, aberrant differentiation may be a
consequence of deregulated homeotic HOX gene expression. However, expression and regulation of
HOX genes is poorly studied in UC. We have therefore determined the expression patterns of HOXC
and HOXD genes and the long noncoding RNA (lncRNA) HOTAIR. We studied its function in UC as it
has been reported to regulate posterior HOXD genes and induce a more aggressive phenotype in
breast cancer.
Materials and Methods: Expression of HOX genes and HOTAIR was determined by qRT-PCR in UC
tissues and cell lines. HOTAIR expression was modulated by ectopic expression and siRNA in selected
UC cell lines. Cell migration was analyzed by means of transwell Boyden chambers coated with
Collagen IV.
Results: HOXC5-6 and C11-13 genes were generally overexpressed in tumor tissues and many cell
lines. Adjacent HOTAIR was strongly overexpressed in a subset of tumors and in 7/12 cell lines. We
did not find the expected inverse correlation between HOTAIR and HOXD10, but rather a strong
positive correlation between HOTAIR and HOXD12 (r=0.93). Neither ectopic expression nor siRNA
knockdown of HOTAIR resulted in HOXD10 expression changes, except in a single cell line, 5637.
Functional assays with cell lines stable transfected with HOTAIR revealed cell type dependent effects
on cell proliferation and migration.
Conclusion: Our data confirm that major changes in HOX expression take place in urothelial
carcinoma contributing to altered differentiation. Although we also found HOTAIR to be often
overexpressed in UC its effects differ from those described in other tumors demonstrating that
effects of lncRNAs can be tissue or even cell type specific.
Supported by Forschungskommission der Medizinischen Fakultät der Heinrich-Heine-Universität
Düsseldorf
Contact:
[email protected]
33
V1.6
Prevalence and clinical features of HOXB13 mutation carriers in German prostate
cancer patients
Lüdeke M1,2, Xu J3, Zheng SL3, Sonntag P4, Schulwitz H4, Rinckleb AE1,2, Schrader AJ1, Schrader M1, Vogel
W2, Hoegel J2, Herkommer K4, Maier C1,2
1
2
3
4
Department of Urology, University Hospital Ulm
Institut of Human Genetics, University Hospital Ulm
Center for Cancer Genomics, Wake Forest University School of Medicine, Winston-Salem, USA
Department of Urology, Klinikum rechts der Isar, Technische Universität München
Introduction: Recently, a germline missense mutation in the homeobox gene HOXB13 (G84E) has
been proposed as high risk variant for prostate cancer (PrCa) susceptibility in a cohort of European
Americans. In order to assess the relevance of the G84E mutation in the German population, we
performed a case control study on familial and sporadic PrCa patients.
Methods: The study cohort consisted of 379 unrelated familial and 367 sporadic PrCa cases, as well
1015 controls. Additionally, 646 affected relatives from familial cases were included for associations
with clinical features (stratification by Gleason score 8, advanced tumor stage or PSA at diagnosis
>20ng/ml). Genotyping of HOXB13 G84E was conducted in part by an iPLEX massarray and by
TaqMan method.
Results: Carriers of G84E were rare in controls (0.4 %) and showed increased frequencies in both
sporadic (1.6 %) and familial PrCa cases (3.2 %). Estimated risks were OR = 4.2 (p = 0.026) and OR =
8.3 (p = 0.0003) respectively. The risk effect size increased with the number of affected individuals
per pedigree: OR = 12.6 (p < 0.0001) for 3 or more, OR = 14.4 (p < 0.0001) for 4 or more affected. The
strongest association with clinical features was observed between G84E and advanced tumor stage
(OR = 9.2; p < 0.0001).
Conclusion: The frequency of HOXB13 mutation carriers in our study cohort was comparable to the
observations in European Americans, establishing HOXB13 as the first high risk gene for PrCa in the
German population. The association between G84E mutations and advanced tumor stage may be of
greater interest for clinical practice, but needs further validation. Also the penetrance of the HOXB13
G84E mutation should be investigated in further studies in order to elucidate its suitability as a
genetic predictor for PrCa.
Contact:
[email protected]
34
V1.7
Immunohistochemical study on invasive tumor cells of clear cell renal cell carcinoma
Rabien A , Blauhut S1, Ergün B1, Jung K1,2, Kilic E3
1
1
2
3
Department of Urology, Charite-Universitätsmedizin Berlin
Berlin Institute for Urologic Research, Berlin
Institute of Pathology, Charité – Universitätsmedizin Berlin
Background: The molecular mechanisms that lead to invasion of renal cell carcinoma (RCC) are not
clear until now. As about 30% of the patients have metastases even at the time of diagnosis and the
five-year survival rate is just about 70%, new insights in early metastatic processes are needed to
better understand the conditions for invasion and to find suitable biomarkers for renal cancer. We
investigated the molecular characteristics of invasive tumor cells of the major tumor subtype of clear
cell RCC, which were currently invading blood vessels or fat tissue in the environment of the tumor.
Material and Methods: In a first approach nine cases of clear cell RCC with respective invasive
properties were selected by an experienced pathologist (E.K.) using hematoxylin / eosin stained
sections of formalin-fixed paraffin-embedded (FFPE) tissue. Immunohistochemical staining of FFPE
tissue containing blood vessels or fat tissue with invading tumor cells was done using primary
antibodies for molecules driving / inhibiting invasion (matrix metalloproteinases MMP-2, -9, -14,
EMMPRIN, RECK), for markers of the epithelial / mesenchymal phenotype (cytokeratin-19, Ecadherin, N-cadherin, S100A4, ZEB1, beta-Catenin) and for stem cells (CD105, in progress: CD44, CMET, CXCR4).
Results: Depending on the marker, staining of the invading tumor cells was heterogenous. Some
single cells were positive for the epithelial marker cytokeratin-19. The epithelial E-Cadherin as well as
the mesenchymal N-Cadherin congruently stained (often larger) areas of the tissue. S100A4 and
ZEB1 showed nuclear staining in many cells which accumulated in distinct areas at the edge of the
blood vessels. MMP-2 was expressed at the whole edges of blood vessels, but less in the invading
cells. CD105 stained blood vessels and rarely single cells which need to be differentiated from
microcapillaries. The MMP inhibitor RECK appeared in occasional granular spots spread in the tumor
cells. Beta-Catenin as well as EMMPRIN were widely expressed at the plasma membrane of tumor
cells with extremely rare beta-catenin staining in the cytoplasm. Staining of the tumor and invading
tumor cells seemed to be equal. In contrary, MMP-9 and MMP-14 levels were higher at the edge of
the tumor mass and in the invading tumor cells.
Conclusions: Heterogenous expression of the majority of markers investigated points to different cell
types within the population of invading tumor cells, apart from tumor-supporting cells. Epithelial-tomesenchymal transition does not seem to play a role in invading clear cell RCC, or it is a temporally
short phenomenon which could not be recognized in these samples. Elevated expression of MMP-9
and MMP-14, however, could indicate an important regulation which will be further examined.
Contact:
[email protected]
35
V2.3
Transcriptional control of tubular lumen size control in normal and diseased kidneys
Yurtdas Y1,2,3,*, Aue A1,*, Ruffert J1,*, Hinze C1,3,*, Kilic E4, Schmidt-Ott KM1,3
Max-Delbrueck Center for Molecular Medicine
Department of Urology, Charité Universitätsmedizin Berlin
3
Department of Nephrology, Charité Universitätsmedizin Berlin
4
Department of Pathology, Charité Universitätsmedizin Berlin
* equal contribution
1
2
Introduction: The establishment of lumen structure of defined size requires highly regulated genetic
and cellular interactions for the maintenance of physiological function of the kidney. Under
pathological conditions, perturbed lumen size formation can be found in cystic kidney diseases and
also in renal tumors. The transcription factor Grainyhead-like2 (Grhl2) is an epithelium specific
mammalian homolog of Drosophila grainyhead, which is specifically expressed in epithelial cells of
the distal nephron and in the collecting duct of the kidney. Understanding the mechanisms governing
renal lumen size regulation can provide useful insights into the pathogenesis of cystic kidney diseases
and renal tumors. In this study, we show that Grhl2 regulates collecting duct lumen size and identify
a zinc finger transcription factor Ovol2 as a novel relevant target gene downstream of Grhl2.
Methods: Formalin fixed, paraffin embedded and frozen tissue specimens of healthy human kidney
and mouse kidney sections were evaluated immunohistochemically. The role of Grhl2 in kidney
development was investigated in vivo by the generation of conditional Grhl2 knockout mouse model.
In parallel, in vitro analysis by using an inner medullary collecting duct cell line (IMCD-3) in 3D
culture was performed to analyze the role of Grhl2 in epithelial morphogenesis on a cellular level.
Grhl2 loss of function and gain of function experiments were carried out on IMCD-3 cells via
lentiviral gene transfer and an integration of Grhl2 rescue construct, respectively. Chromatin
immunoprecipitation followed by next generation sequencing (ChIP-seq) from IMDC-3 cells were
performed to determine the individual target genes downstream of Grhl2. To test the significance of
Ovol2 downstream of Grhl2, Ovol2 was overexpressed in Grhl2-deficient IMDC-3 cells.
Results: Immunohistochemical analysis of kidney sections from both mice and humans showed that
the expression of Grhl2 was restricted in the nuclei of collecting duct and distal tubule cells of the
kidney. The measurement of collecting duct lumen area showed a remarkably reduced lumen size in
vivo and cyst lumen area in 3D culture in vitro in the Grhl2 knockout and knockdown scenario,
respectively. Furthermore, the new Grhl2 key targets were found via gene expression analysis and
ChIP-seq experiments. We focused on Ovol2 as the target gene of Grhl2 in the establishment of
lumen formation. Our analysis showed that the re-expression of Ovol2 in Grhl2 knockdown IMCD-3
cells partially rescued a lumen phenotype, compared to Grhl2 knockdown IMDC-3 cells.
Conclusion: Our results reveal the role of Grhl2 and its target gene Ovol2 in the regulation of lumen
size and show that Grhl2/Ovol2 axis crucially influences lumen size in collecting duct.
For further studies, we aim to characterize the molecular and cellular function of Grhl2/Ovol2
cascade in kidney diseases.
Contact:
[email protected]
36
V2.4
Influence of testosterone on the regulation of inflammatory responses in testicular and
immune cells
Fijak M1, Sauber L-J1, Walecki M1, Eisel F1, Aslani F1, Wahle E1, Bhushan S1, Hackstein H2, Schuler G3,
Meinhardt A1
1
2
3
Department of Anatomy and Cell Biology, Justus-Liebig-University Giessen
Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University Giessen
Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-LiebigUniversity Giessen
Beside their spermatogenic function androgens also play also a role in the modulation of
autoimmune disease and contribute to suppression of inflammatory/autoimmune response.
Previously, we have shown that substitution of reduced testosterone levels in a rat model of chronic
testicular inflammation - experimental autoimmune orchitis (EAO) - has led to a significant
amelioration of disease characteristics by inhibition of inflammatory responses in the testes. This was
documented by a strong decrease of elevated macrophage and CD4+T cells numbers in the
interstitial space concomitant with significant increase of “anti-inflammatory“ regulatory T cells
(CD4+CD25+Foxp3+) as well as significantly lower mRNA levels of TNF-, IL-6 and MCP-1 as
compared to EAO controls. Anti-inflammatory effects of testosterone supplementation during
chronic testicular inflammation prompted us to investigate a putative direct influence of androgens
on the generation of regulatory T cells and immune response in Sertoli (SC) and peritubular cells
(PTC), both important contributors to immunological balance in the testis.
Collected conditioned media from isolated Leydig cells were used for cultivation of isolated splenic T
cells. Differentiation of regulatory T cells was estimated by measurement of expression of Foxp3
transcription factor by FACS and secretion of IL-10 and TGF- by ELISA. In another approach, isolated
primary SC and PTC were pre-incubated with different concentrations of testosterone before
induction of inflammatory response by LPS. IL-6, IL-10, TNF- and MCP-1 mRNA expression was
investigated by quantitative real-time RT-PCR.
Testosterone in conditioned media collected from Leydig cells stimulated the expression of Foxp3
transcription factor in splenic CD4+ T cells as well secretion of IL-10 and TGF- by these cells. The
specificity of testosterone effect was proven by addition of androgen antagonist flutamide.
In SC and PTC inflammatory response induced by LPS was inhibited by pre-incubation with increasing
concentrations of testosterone. Significant reduction of TNF- mRNA levels were achieved in the
presence of 1000 nM and 100 nM of testosterone in SC and PTC, respectively. The anti-inflammatory
effect of testosterone in SC was abolished by use of flutamide.
In view of the high intratesticular testosterone levels under normal conditions and their decrease
under inflammatory conditions, our data point to a role of androgens in the immune homeostasis of
the testis. Androgen action could be mediated by direct effect on SC and PTC as well as on the de
novo generation and functional differentiation of regulatory T cells.
Contact:
[email protected]
37
V2.5
New biomarkers to differentiate malignant germ cell tumours of the testis established
by the SILAC-method (stable isotope labelling by/with amino acids in cell culture) in
combination with high-resolution mass spectrometry
Bremmer F1, Bohnenberger H1, Oellerich T2, Kueffer S1, Strauss A3, Urlaub H4, Serve H2, Radzun HJ1,
Ströbel P1, Maatoug Y1 and Behnes CL1
1
2
3
4
Institute of Pathology, University Medical Center, Göttingen
Department of Hematology/Oncology, Johann Wolfgang Goethe University, Frankfurt
Clinic for Urology, University Medical Center, Göttingen
Bioanalytical Mass Spectrometry, MPI Biophysical Chemistry, Göttingen
Background: Malignant germ cell tumours of the testis are the most common malignant tumours in
young men between 18 to 35 years. Differentiation of the histological subtypes is essential for the
therapeutic management. Thus it is important to find new biomarkers for the various histological
subtypes. Furthermore, biomarkers may help to understand pathophysiological processes in these
tumour types.
Methods: In this study we carried out quantitative proteomic studies using high-resolution mass
spectrometry in combination with the SILAC method (stable isotope labeling by amino acid in cell
culture). For this purpose, the two germ cell tumour cell lines NTERA-2 and TCAM-2 were cultured in
the presence of amino acids of different mass and subsequently analyzed by mass spectrometry. The
detected proteins were further investigated by western blot and immunohistochemical analysis.
Results: The SILAC and high-resolution mass spectrometry of the investigated cell lines revealed a
total number of 342 differential expressed proteins. After intensive research in literature- and
protein-data bases we chose antibodies against Destrin, CD81, and PHF6 for western blot analysis.
The results confirmed the findings of mass spectrometry analysis. We further verified these findings
on immunohistochemical analysis of 248 formalin-fixed and paraffin-embedded testis tumour tissue
samples (seminomas, embryonic carcinomas and mixed germ cell tumours)allowing to distinguish
different germ cell tumours subtypes, especially seminomas and embryonic carcinomas.
Conclusion: (I) High-resolution mass spectrometry in combination with the SILAC method is suitable
to differentiate between different tumour cell lines on the protein level. (II) The results of SILAC
measures are reproducible in western blot analysis. (III) The method is helpful to establish new
biomarkers differentiating histological subtypes of tumours. (IV) The detected proteins can be
applied for immunohistochemical analysis on formalin-fixed and paraffin-embedded tumour tissue
samples especially to distinguish seminomas from embryonic carcinomas as shown in this study.
Contact:
[email protected]
38
P1.1
Transcript levels of Piwi-like 1-4 genes are associated with clinicopathological
parameters in renal cell carcinomas
Al-Janabi O1, Wach S1, Nolte E1, Weigelt K1, Rau TT2, Stöhr C2, Legal W1, Schick S3, Greither T4,
Hartmann A2, Wullich B1, Taubert H1
1
2
3
4
Urologische Klinik, Universitätsklinikum Erlangen, Friedrich-Alexander University Erlangen
Institute of Pathology, Universitätsklinikum Erlangen, Friedrich-Alexander University Erlangen
Tumorcenter, Friedrich-Alexander University Erlangen
Center for Reproductive Medicine and Andrology, Martin Luther University Halle
Background: Piwi-like gene family members (Piwil 1-4) are considered as stem-cell associated
genes/proteins. They are expressed predominantly in the germ line but with a re-expression in
different tumors. The expression of Piwil 1-4 genes has not been studied and correlated with clinicopathological parameters in renal cell carcinomas (RCC) yet.
Material and methods: Transcript levels of Piwil 1-4 were analyzed by quantitative real time PCR in 74
clear cell RCC (ccRCC) tissues and corresponding normal tissues.
Results: A strong and significant correlation of transcript levels of Piwil 1, 2, and 4 but not with Piwil 3
could be detected in the tumor tissues and in the normal tissues (P < 0.001; Spearman’s rank test).
Piwil 4 gene expression was significantly higher in ccRCC than in corresponding normal renal tissue
(P < 0.001; Mann-Whitney U-test). When separating the ccRCC patient cohort according to the
median of Piwil 1-4 expression in a low and a high expression group and according to age in younger
(≤ 64 yrs) and older patients (>64yrs); the younger patients displayed significantly higher mRNA
expression levels of Piwil 1 in comparison to the older patients (P = 0.01; Fisher’s exact test).
Interestingly, the expression of Piwil 1 showed a left-right polarity in the normal tissues but not in the
tumor tissues (P = 0.0005; Fisher’s exact test).
Conclusion: Associations between the expression levels of the Piwi-like family members and
clinicopathological parameters could be detected for ccRCC suggesting a potential role in diagnostics
and tumorigenesis of ccRCC and in embryology at least for renal tissue.
Contact:
[email protected]
39
P1.2
HIF-2 is highly expressed in papillary renal cell carcinoma type II: a potential reason
for the worse prognosis?
Behnes CL1, Thelen P2, Strauss A2, Radzun H-J1 Ströbel P1, Bremmer F1
1
2
Department of Pathology, University of Göttingen, Göttingen
Department of Urology, University of Göttingen, Göttingen
Background: Hypoxia inducible factors (HIFs) are widely expressed in different cell types and human
cancers. HIFs are heterodimeric complexes composed of an alpha subunit (HIF-1, HIF-2 and HIF3) and a beta subunit (HIF-1) and promote cancer progression and neoangiogenesis by inducing
VEGF secretion. Whereas HIF-1 and its functions are well known especially in renal cell cancer, HIF2 has not been investigated so far.
We analyzed the expression of HIF-1 and HIF-2 in comparison to the expression of VEGF and
capillary density in papillary renal cell carcinoma (RCC), which represents a rare tumor and is divided,
based on histological criteria, into two subtypes, of which type II papillary RCC shows a worse
prognosis.
Methods: In the present study the expression of HIF-2, HIF-1, VEGF, CD31 and Ki67 were
examined in 42 papillary RCC of histological type I and 32 papillary RCC of histological type II (n = 74)
by immunohistochemistry. To demonstrate significant differences or correlations the data were
subsequently statistically analysed.
Results: Subtype II papillary RCC showed a significant higher expression of HIF2, whereas papillary
RCC subtype I demonstrated a significant higher expression of HIF-1. The VEGF expression, the
capillary density (CD31), and the proliferation rate (Ki67) were significant higher in papillary RCC
subtype II in comparison to subtype I. Further analysis showed a significant correlation between
VEGF expression and capillary density only in papillary RCC subtype II. A correlation between HIF-2
and the proliferation rate could not be demonstrated in both subtypes of papillary RCC.
Conclusion: Papillary RCC types II demonstrate a significant increased expression of HIF-2 and
VEGF in comparison to papillary RCC type I, which shows a significant higher expression of HIF-1.
The HIF-2 and VEGF could be the reason for the higher capillary density as well as for the worse
prognosis of subtype II compared to subtype I.
Contact:
[email protected]
40
P1.3
Deregulation of the CSN-CRL pathway during urological tumorigenesis?
Gummlich L1,2, Kilic E3, Jung J2,4, Dubiel W1
1
2
3
4
Department of General, Visceral, Vascular and Thoracic Surgery, Division of Molecular Biology,
Charité – Universitätsmedizin Berlin
Berlin Institut for Urological Research, BFIU, Charité – Universitätsmedizin Berlin
Department of Pathology, Charité – Universitätsmedizin Berlin
Department of Urology, Charité – Universitätsmedizin Berlin
Urological cancers belong to the most frequently occurring tumors. Renal cell carcinoma (RCC), for
example, accounts for approximately 2% of all cancers worldwide. Despite a lot of effort spent to
personalize therapeutical approaches, a group of RCC patients still appears to be therapy-resistant.
New applicable targets are desperately needed for the treatment of urological cancer. The COP9
signalosome (CSN)-cullin-RING ubiquitin (Ub)-ligase (CRL) pathway is a prominent segment of the
Ub proteasome system (UPS). It specifically ubiquitinates regulatory proteins and is often
deregulated in cancer. Altered expression of the CRL components called F-box proteins in RCC
influences the presence of key tumor promoting proteins in urological tissues. The exact mechanism
of how deregulated CRL components are integrated in urological tumorigenesis is however unknown.
Recently, our group revealed a post-transcriptional fine-tuning of COP9 signalosome (CSN)
biosynthesis regulated by the c-myc/Lin28b/Let-7 pathway. Interestingly, analysis of RCC patient
samples revealed down regulated let-7 miRNAs depending on metastatic state. Based on these
observations we think that the CSN-CRL pathway is an attractive subject for urological cancer
research.
CSN-CRL pathway components were immunohistochemically stained in a subset of RCC samples to
determine their expression pattern in urological tumor tissue. Selected CSN-CRL pathway
components are expressed partly cytosolic and in the nucleus of the tubule, ureter, as well as the
tumor cells. CSN subunit expression levels varied only slightly, whereas CAND1 staining revealed a
deregulation in the intensity in urological cancer tissue nuclei. CSN subunit 8 and CAND1 are
currently further examined in a tissue microarray with an appropriate cohort focusing on nucleus
staining patterns. Interestingly, no subunit of the CSN complex was expressed irregularly in 4 RCC
cell lines, implying a deregulation of the whole complex rather than a single subunit. The CAND1Skp2-p27 axis was also observed deregulated in these cell lines. In 786-O cells Skp2 is overexpressed
whereas p27 appears with an atypical double band in immunoblots. In order to study the interplay of
particular CSN-CRL pathway components and the possible impact of the p27 double band during
renal tumorigenesis further investigations are in progress.
Contact:
[email protected]
41
P1.4
Quantitative imaging of intratumoral heterogeneity in clear cell renal carcinoma
Höfflin R , Lahrmann B2, Grabe N2, Roth W3, Hadaschik B4, Pahernik S4, Hohenfellner M4, Duensing S1,4
1
1
2
3
4
Section of Molecular Urooncology, University Hospital Heidelberg
Hamamatsu Tissue Imaging and Analysis Center (TIGA), BIOQUANT, University of Heidelberg
Department of Pathology, University Hospital Heidelberg
Department of Urology, University Hospital Heidelberg
Background: Recent results from next generation sequencing analyses show a high degree of
intratumoral genomic heterogeneity in advanced clear cell renal cell carcinoma (ccRCC). Moreover,
reconstruction of tumor phylogeny based on mutational data suggests a branched pattern of clonal
evolution of tumor cell populations. The two most commonly mutated genes with therapeutic
relevance in ccRCC are VHL and mTOR. It is possible to indirectly determine the mutational status of
these genes by analyzing the abundance of their respective downstream targets or changes in protein
phosphorylation using routine immunohistochemistry (IHC). This proof-of-concept study was
designed to test whether intratumoral heterogeneity centered on the VHL and mTOR pathway can be
analyzed on the protein level in a quantitative manner and exploited as prognostic biomarker and
tool for clinical decision-making.
Methods: Formalin-fixed, paraffin-embedded (FFPE) specimens from a total of 34 primary tumors and
metastatic lesions were processed for routine IHC analysis and analyzed for markers of pVHL loss
(upregulation of HIF1- and/or HIF2-), activation of mTOR (phospho-mTOR S2448, phospho-S6
ribosomal protein S235/236) and expression of the proliferation marker Ki-67. Stained sections were
scanned using a Nanozoomer 2.0 HT Scansystem at 20x magnification. The quantitative evaluation
was performed with Visiopharm Software using two different algorithms: 1. positive pixel count to
quantify cytoplasmic staining for pTOR S2448 and pS6RP S235/236. 2. positive nuclear count to
quantify staining for HIF1-, HIF2- and Ki-67. In order to compare identical regions, adjacent
sections were aligned and virtually segmented into squares of 1 mm². After exclusion of nontumorous tissue, scores were computed for individual squares and later combined into 3D maps to
reflect abundance of protein expressin in a given tumor area.
Results: We were able to obtain quantitative protein expression data for all markers and tumors. We
found that intratumoral heterogeneity of protein expression is extensive. One of our key findings is
that an activation of the mTOR signaling pathway occurs preferentially in the tumor
periphery/invasion front. In addition, we detected strong focal pTOR S2448 immunostaining in two
out of ten small ccRCCs with synchronous distant metastases. These focal lesions were suggestive of
epithelial-mesenchymal-transition (EMT) and are currently being further investigated. Based on
quantitative IHC image analyses we were able to create 3D maps representing differences in protein
expression for all tumors analyzed.
Conclusion: We have established an image analysis method for IHC/brightfield microscopy which
makes it possible to visualize intratumoral heterogeneity of biomarkers in 3D. As this method can be
fully automated and is applicable to other biomarkers, it can potentially be integrated into routine
pathology and clinical decision-making.
Contact:
[email protected]
42
P1.5
Prediction of second-line therapy response in metastatic renal cell carcinoma patients
by blood plasma marker proteins
Hölters S1, Bergmann L2, Grünwald V3,Keilholz U4, Ohlmann C1 ,Staehler M5, Schmerler D6, Junker K1
1
2
3
4
5
6
Clinic of Urology and Pediatric Urology, Saarland University Medical Center, Homburg
Medical Clinic II,University Hospital Frankfurt
Department of Haematology, Haemostaseology, Oncology and Stem Cell Transplantation,
Hannover Medical School
Medical Clinic, Charité - Universitätsmedizin Berlin
Department of Urology, University Hospital Munich
Department of Clinical Chemistry and Laboratory Medicine, University Hospital Jena
Introduction & Objectives: For patients with metastatic renal cell carcinoma (mRCC) vascular
endothelial growth factor (VEGF)-targeted therapy (e.g. Sunitinib or Sorafenib) represents a
promising first-line therapy after tumournephrectomy. For patients who progress during first-line
therapy, inhibitors of mammalian target of rapamycin (mTOR) (e.g. Temsirolimus or Everolimus)
represent an option for second-line therapy. Within the framework of the MARC-2 clinical trial, the
aim of our study is the identification and validation of marker proteins associated with second-line
therapy (Everolimus) response.
Material & Methods: We included blood plasma samples from 19 mRCC patients receiving
Everolimus. Therapy response was defined as stable disease and partial remission. We analysed the
samples by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDITOF MS) using CM10 and Q10 arrays. For the identification of potential marker proteins, 2dimensional gelelectrophoresis and matrix-assisted laser desorption/ioniziation time-of-flight mass
spectrometry (MALDI-TOF MS) will be conducted.
Results: We identified 20 mass peaks by SELDI-TOF MS with Q10 and 16 with CM10 chips for the
discrimination between responders (defined as stable disease or partial remission after two month of
therapy) and non-responders (defined as progressive disease after 2 month of therapy). With the
Q10, but not with the CM10 chip, we obtained five peaks for the discrimination between both patient
groups before the first drug application. These peaks could be useable to preselect patients for the
second-line therapy with Everolimus. For therapy monitoring, we found seven mass peaks (four with
Q10 and three with CM10 chips) after two weeks and 24 mass peaks (11 with Q10 and 13 with CM10
chips) after four weeks of therapy to differentiate between responders and non-responders. These
mass peaks could be useful to estimate the therapy response for the patients.
Conclusion/Outlook: Our preliminary results argue for the presence of blood plasma proteins which
potentially predict therapy response to Everolimus before as well as during therapy. The confirmation
and identification of the SELDI-TOF MS outcomes by 2-dimensional gel electrophoresis and MALDITOF MS is on-going as well as the screening of an extended sample set.
Sponsored by: iOMEDICO AG
Contact:
[email protected]
43
P1.6
Targets and function of dysregulated microRNAs in clear cell renal cell cancer
Liep J , Wotschofsky Z1,2, Jung K1,2
1,2
1
2
Department of Urology, Charité – Universitätsmedizin Berlin
Berlin Institute for Urologic Research, Charité – Universitätsmedizin Berlin
Background: MicroRNAs (miRNAs) play a pivotal role in various types of tumors. Literature data
reveal also the role of miRNAs in carcinogenesis and tumor progression of clear cell renal cell cancer
and its metastasis. In a previous study (doi:10.7150/ijbs.5106) using microarray analysis and RT-PCR
techniques, we could identify a specific miRNA expression pattern with several dysregulated miRNAs
whereby most of them were down-regulated. Epigenetic mechanism could be revealed to be a key
player in this downregulation of some of the found miRNAs. The altered miRNA-profile together with
predicted miRNA-target interactions affords a solid basis for further functional analyses of individual
miRNAs in RCC metastatic progression. The aim of our study was to identify targets and function of
some selected miRNAs of our profiling (miR-127 and miR-145).
Materials and Methods: We started with a comprehensive target research using different search
machines (miRWalk and targetscan) to get a list of predicted targets for each miRNA. Based on
literature research we chose some potential targets out of this list, which could be interesting for
carcinogenesis. To test, if these potential targets are really regulated by the miRNAs, renal cell lines
(786-O and ACHN) were transfected with the miRNAs. The impact of the miRNA overexpression
towards the appropriate target expression on mRNA level by RT-qPCR was measured. Furthermore,
we wanted to investigate the effect of the miRNAs by means of scratch assay.
Results: By this approach, we could identify some potential targets of our miRNAs of interest. The
two genes BIRC2 and TNFRSF10B were downregulated after increasing miR-145 concentration, and
the polycomb group protein EZH2 seems to be regulated by miR-127. The functional analysis reveals
that miR-127 seems to have an influence on migration of tumor cells.
Conclusions: Our study identifies some new miRNA-target interactions. Furthermore, the functional
analysis gives hint that the found miRNA-target interaction plays an important role in tumor
progression and metastasis. These interesting findings are a good starting point for further analysis
to investigate the detailed role of miRNAs in progression of clear cell renal cell cancer and its
metastasis.
Contact:
[email protected]
44
P1.7
Integrated microRNA and mRNA signature associated with the transition from the
locally confined to the metastasized renal cell carcinoma
Wotschofsky Z1,2, Billaud J-N3, Jung M1,2, Meyer H-A1,4
1
2
3
4
Department of Urology, Charité – Universitätmedizin Berlin
Ingenuity Systems, Redwood City, USA
Institute of Physiology, Charité – Universitätmedizin Berlin
Background and Objective: MicroRNAs (miRNAs) are endogenous small non-coding RNAs that
regulate gene expression by interfering translation or stability of target transcripts. One miRNA can
interact with several hundred mRNAs, while one mRNA can be regulated by several miRNAs. This
interplay between miRNA and their mRNA has been proposed as an important process in cancer
development and progression. We have investigated molecular networks impacted by predicted
mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma
(ccRCC) diagnosed with or without metastasis.
Material and Methods: miRNA and mRNA microarray expression profiles derived from primary clear
cell renal cell carcinomas from patients with (in total 16 samples) or without diagnosed metastasis (in
total 22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this
purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of
experimentally validated and predicted mRNA targets was used. By applying an expression pairing
tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs
and their target genes with opposite or same expression. The resulting identified interactions were
revalidated by RT-qPCR in another cohort of RCC patients. The predicted miRNA-mRNA interactions
were also tested by functional analyses using miRNA knock-down and over expression experiments in
renal cancer cell lines.
Results: Among the significantly differentially expressed miRNAs, we have identified 3 miRNAs (miR146a, miR-128a and miR-17-5p) that were upregulated in primary tumors from patients without
metastasis and down regulated in primary tumors from patients with metastasis. We have further
identified the mRNA targets which expression were inversely correlated to these 3 miRNAs, and have
been previously experimentally demonstrated in cancer setting in humans. Specifically we showed
that BRAC1, MCM10, CDKN3, UHRF1, IL8 were downregulated and targeted by miR-146a-5p. The
relation between these identifies target genes and miRNA-146a was validated in cell culture
experiments.
Conclusions: We identified novel target genes of dysregulated miRNA which are involved in the
transition from primary RCC without metastases into tumors generating distant metastasis.
Contact:
[email protected]
45
P1.8
Molecular cytogenetic differences between Collecting Duct Carcinomas and upper
urinary tract urothelial carcinomas
Jung V1,13, Becker F1,11,13, Parr M1,13, Hartmann A2,13, Füssel S3,13, Toma M4,13, Grobholz R5, Pflugmann T6,
Wullich B7,13, Strauss A8,13, Behnes CL9,13, Otto W10,13, Stöckle M1,13, Junker K1,12,13
1
2
3
4
5
6
7
8
9
10
11
12
13
Department of Urology, University of the Saarland, Homburg/Saar
Department of Pathology, University Hospital Erlangen
Department of Urology, Technical University of Dresden
Department of Pathology, Technical University of Dresden
Department of Pathology, Kantonsspital Aarau, Aarau, CH
Department of Urology, St.Franziskus Clinics, Mönchengladbach
Department of Urology, University Hospital Erlangen
Department of Urology, Georg-August University Göttingen
Department of Pathology, Georg-August University Göttingen
Department of Urology, Caritas Clinics St. Joseph, University Regensburg
Urological Group Practice & Clinic Derouet/Pönicke/Becker, Boxberg Center, Neunkirchen
Department of Urology, University Hospital Jena
German Network of Renal Cell Tumors
Collecting duct carcinoma (CDC) is a rare renal neoplasm that is associated with poor prognosis due
to its highly aggressive course and limited response to immuno- or chemotherapy. Histologically,
CDC is defined as a subtype of renal cell carcinomas, but in some cases, it is difficult to differentiate
from urothelial carcinomas (UC). Therefore the aim of this study was to determine genetic alterations
of CDC in comparison to that of urothelial carcinomas of the upper urinary tract (UUT-UC) to clarify
the histological origin of this rare tumor entity.
Twenty-nine CDC samples were obtained from seven different German centers and compared with
twenty-six urothelial carcinomas of the upper urinary tract. Comparative genomic hybridization
(CGH) was used to investigate the genetic composition of patients’ tumors and allowed the detection
of losses and gains of DNA copy numbers throughout the entire genome. The clinical data were
correlated with CGH results.
CGH analysis of CDC revealed DNA aberrations in many chromosomes. DNA losses were more
frequently observed than gains, while high-level amplifications were not detected. The mean
frequency of CDC chromosomal aberrations (4.9/case) was slightly lower than that in UUT-UC
(5.4/case). Recurrent CDC DNA losses occurred at 8p (n=9/29), 16p (9/29), 1p (n=7/29) and 9p
(n=7/29), and gains occurred in 13q (n=9/29). In contrast to CDC, the most frequently detected UUTUC DNA aberration was a loss at 9q (n=13/26). DNA losses at 9q, 13q and 8q as well as gains at 8p
showed significant variations in UUT-UC compared to CDC. There was no correlation between the
patients’ clinical course and the presence or absence of these recurrent genetic alterations.
CDCs are characterized by a different genetic pattern compared to UUT-UC. Regarding the published
data on renal cell carcinoma, we conclude that CDC appears to be a unique entity among kidney
carcinomas.
Contact:
[email protected]
46
P1.9
Novel antiangiogenic compounds with antitumor activity for innovative approaches in
cisplatin resistant testicular germ cell cancer treatment
Nitzsche B1,2, Pries A1, Schrader M3, Preissner R1, Honecker F4, Höpfner M1
1
2
3
4
Institute of Physiology, Charité – Universitätsmedizin Berlin
Berlin Institute for Urologic Research, Charité – Universitätsmedizin Berlin
Department of Urology, University of Ulm
Department of Oncology and Hematology, Hubertus Wald Tumor Center - University Cancer
Center Hamburg
Objective: Effective treatment of testicular germ cell tumors (TGCT) resistant to conventional
chemotherapy is still insufficient. As angiogenesis is essential for the development, growth and
progression of tumors we hypothesised that targeting angiogenic growth factor receptor signalling
pathways may be a promising approach for novel treatment of therapy resistant TGCTs.
Design and method: Two recently identified antiangiogenic compounds, HP-2 and HP-14, blocking
the VEGFR-2 and related signalling pathways of endothelial and VEGFR-2 expressing cancer cells
were investigated for their suitability to inhibit the growth and vascularisation of normal and
cisplatin-resistant testicular germ cell tumors (TGCT) in vitro and in vivo. Performing proliferation
assays (crystal violet method), the antineoplastic effects of HP-2 and HP-14 alone or in combination
with platinum compounds were evaluated in both platinum sensitive and –resistant testicular germ
cell tumor cells (2102EP, 2102EP-R).
For in vivo evaluations TGCT cells (2102EP, 2102EP-R, Tera-1, Tera-2) were inoculated onto the
chorioallantoic membrane of fertilized chicken eggs (CAM assay). The developing tumors were
treated with the HP-compounds and the inhibition of angiogenesis and tumor growth was
documented.
Results and conclusions: HP-2 and HP-14 effectively suppressed the growth of TGCTs, both in vitro
and in vivo. We could show that the growth of testicular germ cell cancer cells, resistant to
conventional platinum-based chemotherapy can be potently inhibited by the novel HP-compounds
alone or in combination with cisplatin. Together, these data suggest that HP-2 and HP-14 may be
interesting new drugs for targeted therapy of urologic cancers, particularly for those being resistant
to the conventionally successful cisplatin-based interventions.
Contact:
[email protected]
47
P1.10
Epigenetic regulation of genes involved in epithelial mesenchymal transition in
prostate cancer
Brandt U1, Wagenlehner F1, Waliszewski P1, Steger K1, Weidner W1, Gattenloehner S2,
Schagdarsurengin U1, Dansranjavin T1
1
2
Clinic of Urology, Pediatric Urology and Andrology; Justus-Liebig-University Giessen
Institute of Pathology, Justus-Liebig-University Giessen
Aim: Epithelial to mesenchymal transition (EMT) plays a pivotal role in molecular mechanisms of
prostate cancer (PCa) metastasis. Hypoxia-inducible factor 1-alpha (Hif-1) is a key transcriptional
factor controlling hypoxia induced EMT. In our study, we have analyzed the epigenetic regulation of
EMT associated genes EPO, FBLN2, SLUG and SNAIL1 in PCa cell lines after stabilization of Hif-1
protein.
Methods: The endogenous Hif-1 protein was stabilized and enriched by treatment with 1 mM HIF1 prolyl hydroxylase inhibitor dimethyloxaloylglycine (DMOG). We investigated the effect of Hif1-stabilisation and 5´-Aza-2´-deoxycitidine (Aza) 5 μM on relative expression of EPO, FBLN2, SLUG
and SNAIL1 in prostate cancer cell lines LNCaP, Du145 and PC3 by qRT-PCR. The methylation of the
promoter regions were analyzed by COBRA (combined bisulfit restriction analysis) and methylation
specific PCR.
Results: Untreated PCa cell lines exhibited no expression of all analyzed genes, except SLUG showing
a weak relative expression (RE) in PC3 cells. The promoter regions of EPO, FBLN2, SLUG and SNAIL1
were partially methylated in Du145 and PC3 cells. In LNCaP cells all candidate genes, except SLUG,
were unmethylated. The stabilization of Hif-1 protein with DMOG led to an overexpression of only
EPO (RE 11) in LNCaP and of SNAIL1 (RE 10) in PC3 cells. Aza treatment resulted in the activation of
EPO (RE 1,5), FBLN2 (RE 1,3), SLUG (RE 1,7) and SNAIL1 (RE 3,2) in LNCaP cells. In PC3 cells we
observed the activation of FBLN2 (RE 4,3) and 5-fold upregulation of SLUG (from RE 0,6 up to RE 3,4).
The combined treatment of Aza with DMOG resulted in the activation of SLUG (RE 1,8) in LNCaP
cells and of SLUG (RE 3,3) and SNAIL1 (RE 0,6) in PC3 cells.
Conclusion: These data provide evidence that the CpG-methylation status of genes regulating EMT
modulates their inducibility by Hif-1. Therefore we suppose that the methylation status of Hif-1
target genes could be useful for characterization of EMT and of metastatic potential of PCa.
Contact:
[email protected]
48
P1.11
Epigenetic intervention counteracts resistance towards temsirolimus in prostate
cancer
Makarevic J, Tsaur I, Jüngel E, Haferkamp A, Blaheta R
Department of Urology, University of Frankfurt
Introduction: Specific blocking of the “mammalian target of rapamycin” (mTOR)-kinase by the mTORinhibitor temsirolimus (Torisel ®) have led to encouraging clinical results. However, chronic use of
temsirolimus may lead to resistance, which counteracts the antitumoral effect of this drug. In this
study, we evaluated growth and invasion of prostate cancer cells with acquired resistance to the
mTOR-inhibitor temsirolimus. Further investigation was designed to determine whether additionally
targeting histone deacetylase (HDAC) by an HDAC-inhibitor (valproic acid, VPA) might counteract
undesired feedback mechanism caused by chronic use of temsirolimus.
Material and Methods: Prostate cancer cells were either treated with temsirolimus over 12 months,
starting at 1 nM and increasing stepwise to 5 μM (PC3TEM), or only treated with medium (PC3).
Growth, proliferation, adhesion and invasion of PC3 versus PC3TEM were investigated. Expression of
cell cycle regulating proteins, mTOR related intracellular signaling and the expression profile of alpha
and beta integrin subtypes were also evaluated. Additionally, HDAC was blocked in PC3TEM cells and
the consequences on invasive growth investigated.
Results: PC3 TEM accumulated in the G2/M-phase, accompanied by cdk1, cdk2, cyclin B elevation
and reduction of p21 and p27. The target proteins Akt, mTOR, rictor and p70S6k were strongly
activated in PC3TEM compared to PC3 cells. Distinct modifications were also seen with respect to
alpha and beta integrin expression. Additional application of VPA blocked growth and adhesion of
PC3TEM cells, reverted integrin modulation and deactivated proteins of the mTOR signaling pathway.
Discussion: Chronic use of the mTOR inhibitor temsirolimus causes prostate cancer cell resistance.
HDAC-inhibition counteracts this process, possibly by cross-communicating with the mTOR signaling
axis. Specific targeting of HDAC may, therefore, enhance the benefit of an mTOR-inhibitor based
regimen.
Contact:
[email protected]
49
P1.12
Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and
proteomics expression data
Meyer H-A1, Kamlage B2, Reszka R3, SchatzP3, Stephan C1,4, Dietrich D5, Kristiansen G5, Jung K1,4
1
2
3
4
5
6
Institute of Physiology, Charité – Universitätsmedizin Berlin
Department of Urology, Charité - Universitätsmedizin Berlin
Metanomics GmbH, Berlin
Metanomics Health GmbH, Berlin
Institute of Pathology, University Hospital of Bonn
Background: Metabolite profiling research offers a deeper insight into biochemical changes in cancer
metabolism. Moreover the integrated analysis of transcription, metabolomics and proteomics data
can improve the understanding of the underlying biological processes.
Material and Methods: A set of 254 metabolites was determined by gas chromatography/liquid
chromatography-mass spectrometry in matched malignant and non-malignant prostatectomy
samples from 95 prostate cancer (PCa) patients. Transcription profiling data were obtained from own
micro array experiments (matched malignant and non-malignant prostatectomy samples from 15 PCa
patients using Affymetrix U133 arrays) as well as public GEO expression data. Expression levels of
selected proteins were determined by tissue micro array in 41 matched frozen tissue samples.
The data were related to clinicopathological variables and disease recurrence in the follow-up.
Transcription and metabolomics data were statistical analysed (ANOVA, Mann–Whitney U test) and
significant regulated metabolites/genes/proteins were selected.
Results: Significant regulated metabolites/genes between malignant and non-malignant samples
were used for network analysis. Enriched pathways which are involved in PCa progression or
recurrence such as carbohydrate and fatty acid metabolism were identified. The role of fatty acid
metabolism in PCa was analysed in more detail. Several fatty acids such as cerebronic acid, 2hydroxybehenic acid, tricosanoic acid showed higher concentrations in malignant than in nonmalignant samples which correspond to the observed higher mRNA and protein expression level of
fatty acid synthase (FASN) in PCa. In contrast to normal prostate tissue, where protein expression
level of FASN was correlated to the level of measured metabolites we found in malignant samples a
deregulation of the corresponding pathway.
Conclusion: Our integrated analysis of transcription, metabolite and proteomics data confirm and
extent the role of several biological pathways which are involved in PCa progression.
Contact:
[email protected]
50
P1.13
Cytostatic drug WIST-C overcomes docetaxel induced resistance
Rottach M1, Peter T1, Mandelkow R1, Weiss M1, Walther R2, Burchardt M1, Stope MB1
1
2
Department of Urology, University Medicine Greifswald
Department of Medical Biochemistry and Molecular Biology, University Medicine Greifswald
Background: Induction of cytoprotective pathways during docetaxel treatment of advanced prostate
cancer (PCa) frequently confers secondary resistance to therapy. The alternative taxane compound
WIST-C given as second-line therapy restores pro-therapeutical effects followed by overcoming
docetaxel-induced resistance mechanisms. The aim of this study was to identify and characterize
WIST-C-mediated cytostatic mechanisms in PCa cells, particularly compared to docetaxel-induced
molecular effects.
Methods: Prostate cancer cell lines were treated with the taxane WIST-C and analyzed by
proliferation assay (CASY TT cell analyser, Roche). Drug-dependent modulation of resistanceassociated heat shock proteins HSP27, HSP70, HSP90, as well as androgen receptor (AR) and
prostate specific antigene (PSA) expression levels were monitored by Western blotting and
quantitative RT-PCR. Furthermore, the induction and activation of apoptotic factors was assessed.
Results: Prior to incubation experiments WIST-C IC50 concentrations were determined indicating the
cytostatic efficacy of the drug. Analysis of putative factors of chemoresistance revealed decreased
levels of HSP27 and AR accompanied by an attenuation of transcriptional activity of AR as shown by
reduced levels of PSA mRNA. Moreover, WIST-C treatment led to an induction of the pro-apoptotic
factor p53.
Conclusion: These results indicate WIST-C efficacy counter-acting docetaxel-induced cytoprotective
HSP27 machinery in PCa cells pointing to differential cellular responses to both taxanes in PCa cells.
Contact:
[email protected]
51
P1.14
Absolute quantification by qRT-PCR - a new and sensitive method to measure absolute
amounts of miRNA in high risk prostate cancer tissue
Schubert M1*, Kneitz B1, Spahn M2, Riedmiller H1, Kneitz S3
1
2
3
Department of Urology and Pediatric Urology, University Hospital, Wuerzburg; Comprehensive
Cancer Center Mainfranken
Department of Urology, Inselspital Bern, CH
Department of Physiological Chemistry, University Wuerzburg
Introduction: We previously showed miRNA-221 as potential prognostic biomarker whose downregulation is associated with clinical recurrence in high risk prostate cancer (PCa). To become a
reliable biomarker expressional analyses from independent study centres are necessary for validation.
The Δct-method (relative qRT-PCR) is widely used for this. Due to high error rates and only a relative
quantification statement, comparison of PCR results of material from different study collectives is
limited. Often statistical features (e.g. Z-Score) are necessary to adapt expression levels of different
study groups to make results comparable.
There is urgent need to develop a method that is: more exact in determination of expression levels,
applicable in daily routine and allows for exact comparison of results. Based on these conditions we
demonstrate the method of absolute miRNA-quantification.
Material and Methods: A universal reference (miRXplore™) with predefined amounts of miRs was
used and serial dilution performed for qRT-PCR. This allowed for determination of absolute amounts
of miR (in attomol) within a sample. For validation of miR-221 we used the same collective (A: n=99)
with clinical data as in our previous analyses. Absolute quantification for miR-221 and the
housekeeper were performed, followed by Cox regression and Kaplan Meier (KM) analyses. Absolute
quantification was performed in a second independent cohort (B: n=108) to compare expression
levels.
Results: The new method was well reproducible and revealed precise results. The expressional
analysis of cohort A reached comparable significance levels as in relative qRT-PCR: KM estimates
predicted sign. difference (p< 0.01) in groups with high and low miR-221 expression regarding clinical
failure and cancer related death (CRD). Dichotomized miR-221 expression was multivariately sign. for
prediction of CRD (p< 0.01; CI 0.004-0.3). Absolute quantification revealed approximated expression
levels in both PCa cohorts.
Conclusion: We demonstrate a new precise and reproducible method to quantitate absolute amounts
of miRs. We confirm miR-221 as potential prognosticator in high risk PCa. By absolute quantification
expressional analyses from different study centres are comparable more precisely. In the future this
method might therefore be used for validation of potential biomarkers in independent study centres
and prospective settings.
*2011-2012: Supported by a Ferdinand Eisenberger grant of the Deutsche Gesellschaft für Urologie
(German Society of Urology), grant ID ScM1/FE-11
Contact:
[email protected]
52
P1.15
Evaluation of Patient Derived Molecular Neuroendocrine Signatures in PC3 using
RNAseq data
von Hardenberg J1,2, Kerr G1, Voloshanenko O1, Worst TS1,2, Michel MS2, Boutros M1
1
2
German Cancer Research Center (DKFZ), Div. Signaling and Functional Genomics, Heidelberg
Department of Urology, University Medical Center Mannheim, University of Heidelberg
Introduction: Neuroendocrine prostate cancer (NPCA) represents an aggressive subtype of prostate
cancer. Apart from NCI-H660 there are no cell based model systems available to study this aggressive
prostate cancer subtyp. Recently the established prostate cancer cell line PC3 was suggested to serve
as a NPCA model system. In western blot analysis we observed the expression of chromogranin A
(CGA) and neuron specific enolase (NSE) in PC3 but also in Du-145. This questioned the approach to
characterize only according to two neuroendocrine markers. The aim of this study was to test if we
could better estimate the NPCA signature in PC3 and other prostate cancer cell lines. We therefore
evaluated patient derived molecular NPCA signatures in these cell lines.
Methods: Western blot analysis were used to identify the protein expression of CGA and NSE in PC3,
Du-145 and LNCaP. Subsequently, gene expression information from 17 prostate cancer cell lines
was used to evaluate known molecular signatures of NPCA. Alignment data from RNA Sequencing
(RNAseq) experiments from different labs were compared and integrated by estimating expression
using a standardized workflow. Expression information of known basal, luminal and neuroendocrine
genes was then used to profile these 17 cell lines. Additionally, recently identified molecular
signatures of NPCA in patient samples were used for unsupervised clustering.
Results: CGA was highly expressed in PC3 and Du-145 but not in LNCaP cells. The profiling of
RNAseq expression data according to known prostate markers proofed the feasiblity of this approach.
The NPCA cell line NCI-H660 grouped by itself. PC3 and Du-145 did not show high expressions of
neuroendocrine markers in the RNAseq data. Using the patient derived NPCA signature NCI-H660
showed a unique expression profile not clustering with other cell lines. We could not observe a NPCA
signature in other established cell lines.
Conclusion: Identifying molecular signatures using RNAseq helps to more precisely characterize
prostate cancer cell lines. A molecular NPCA signature could only be observed in the established
neuroendocrine cell line NCI-H660 although PC3 and Du-145 showed expressions of CGA and NSE
on the protein level.
Contact:
[email protected]
53
V3.6
Investigation of Escherichia coli urinary tract isolates with characteristics of small
colony variants
Putze J1, Greune L2, Schmidt MA2, Svanborg C3, Dobrindt U1
1
2
3
Institut für Hygiene, Universitätsklinikum Münster
Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung - ZMBE, Münster
Division of Microbiology, Immunology and Glycobiology - MIG, Lund University, Lund, S
Introduction: Urinary tract infections (UTIs) are a worldwide occurring disease with an estimate of
more than 10 million cases per year in Western Europe. The most prevalent causative agent of UTIs is
Escherichia coli. Besides symptomatic UTI, E. coli also causes asymptomatic bacteriuria (ABU). ABU
strains cause only mild or no symptoms during carriage. Bacterial small colony variants (SCVs) grow
slow and form tiny colonies compared to other strains of the same species. The appearance of SCVs
has mainly been described for Staphylococcus aureus and Pseudomonas aeruginosa. SCVs are known
to cause chronic-persistent infections, being more resistant to antibiotic treatment, occurring
intracellularly and may display several other virulence- associated traits. On the other hand, SCVs
frequently exhibit deficiencies like auxotrophy and electron-transport-defects also accounting for the
slow growth behavior.
Material and Methods: We focused on traits of UPEC which contribute to persistent and recurrent
infection and characterized two reisolates of ABU E. coli strain 83972 obtained from deliberately
colonized patients showing an SCV and SCV-like phenotype, respectively as well as additional isolates
with decreased growth ability from asymptomatic carriage. We analyzed growth kinetics as well as
bacterial cell morphology. In addition, phenotypic features like curli and cellulose expression, biofilm
formation and resistance against antibiotics were assessed. We compared the adhesion
characteristics to human bladder epithelial cells and human kidney epithelial cells. Furthermore the
invasion capacity, uptake and survival in non-professional and professional phagocytes were
determined. As a key aspect we sequenced the reisolates of E. coli ABU strain 83972.
Results: UPEC and ABU isolates with decreased growth rate from cases of extended bladder
colonization differed in their phenotypic traits. SCV characteristics could not be determined for all
slow-growing urinary isolates. The SCV and the SCV-like reisolates of ABU E. coli 83972 differ in their
phenotypes from their progenitor 83972.
Conclusion: Our results demonstrate that SCV formation is also one strategy of asymptomatically
colonizing E. coli to adapt to adverse and changing growth conditions in the urinary tract. We discuss
our findings against the background of bacterial traits which distinguish UTI and ABU strains or
contribute to adaptation to prolonged urinary tract colonization.
Contact:
[email protected]
54
P2.1
OASIS/CREB3L1 is downregulated in human bladder tumors and mediates suppression
of cancer cell spreading and migration in vitro
Dierichs L1, Rose M1, Schubert C1, Gaisa NT1, Heer M1, Heidenreich A2, Knüchel R1, Dahl E1
1
2
Molecular Oncology Group, Institute of Pathology, Medical Faculty of the RWTH Aachen
University
Department of Urology, Medical Faculty of the RWTH Aachen University
Background: Invasive bladder cancers are associated with an unfavorable clinical outcome.
Understanding the underlying biological mechanisms of this cancer subtype may help to approach
novel therapeutic strategies. Previously, DNA array expression analysis identified the bZIP
transcription factor OASIS (old astrocyte specifically induced substance), also known as CREB3L1, to
be downregulated during bladder cancer development. We furthermore showed that aberrant
promoter methylation of OASIS in bladder cancer tumors is associated with invasive high grade
tumors. In this study, we aimed to decipher the functional role of OASIS loss in bladder cancer by
generating a bladder cancer cell model overexpressing OASIS. Furthermore, the putative OASIS
target gene HTRA3 was analyzed in this cellular system.
Methods: OASIS and HTRA3 mRNA expression was analyzed in a large cohort of bladder cancer
samples (n=64), comprising carcinoma in situ (CIS), papillary and invasive bladder tumor samples by
using real-time PCR. Normal urothelium tissues (n=14) served as control. Using
immunohistochemistry OASIS protein expression was determined. In order to characterize a putative
tumor suppressive role OASIS, we established a stable gain-of-function in vitro model using the
invasive J82 urothelial cancer cell line. Based on this, we analyzed colony growth (colony formation
assay) and tumor cell motility (wound healing assay) of both independent J82-mock and J82-OASIS
clones. Correlation of the OASIS expression and HTRA3 expression was performed by calculating a
Spearman correlation coefficient.
Results: Real-time PCR analysis showed a downregulation of OASIS mRNA in bladder cancer tissues
(Δ fold change: -3.8) that was also evident on protein level. OASIS re-expression in the invasive
bladder cancer cell line J82 led to a significant (p<0.001) suppression of colony growth. Moreover, we
functionally demonstrated a reduction of tumor cell migration in vitro that is potentially mediated by
an OASIS-induced HTRA3 expression. A positive correlation of OASIS and HTRA3 expression was
also confirmed in bladder cancer tissues (n=48) (Spearman test; r=0.6649, p<0.001).
Conclusion: In the current study we provide for the first time evidence that OASIS expression may
represses bladder cancer invasion. By identifying HTRA3 as a potential target gene of OASIS in
invasive bladder cancer cells a putative impact of OASIS on TGF- signaling could be suggested but
have to further analyzed in future studies.
Contact:
[email protected]
55
P2.2
Responses of bladder cancer cells towards tyrosine-kinase inhibition by dovitinib (TKI258) in relation to the eptithelial mesenchymal transition status
Hänze J, Henrici M, Hegele A, Hofmann R, Olbert P
Klinik für Urologie und Kinderurologie, Philipps Universität Marburg
Dovitinib (TKI-258) is a receptor tyrosine kinase (RTK) inhibitor targeting fibroblast growth factor
receptor (FGFR), and structurally related RTKs. Dovitinib is investigated as anticancer drug in various
cancers including bladder cancer with aberrant RTK signalling. Here, we analyzed the dovitinib
response in relation to the epithelial mesenchymal transition (EMT) status as a possible predictor of
therapy response. EMT status was determined in human bladder cancer cell lines based on
mesenchymal marker N-cadherin and epithelial marker E-cadherin. Dovitinib dependent responses
were analyzed by determination of IC50 values in viability/proliferation dose response curves and by
survival fractions in colony formation experiments. We observed significant correlations of these
parameters with the EMT status. Thus, the EMT status in bladder cancer cells may be exploited to
predict therapy responses towards dovitinib treatment.
Contact:
[email protected]
56
P2.3
HDAC inhibition suppresses bladder cancer cell adhesion to collagen under flow
conditions
Juengel E1, Santos SM2, Schneider T1, Makarevic J1, Hudak L1, Bartsch G1, Haferkamp A1, Blaheta RA1
1
2
Klinik für Urologie und Kinderurologie, Universitätsklinikum, Goethe-University, Frankfurt a.M.
Institut für Klinische Pharmakologie, Universitätsklinikum, Goethe-University, Frankfurt a.M.
Introduction: The influence of the histone deacetylase (HDAC)-inhibitor, valproic acid (VPA), on
bladder cancer cell adhesion in vitro was investigated in this paper.
Materials & Methods: TCCSUP and RT-112 bladder cancer cells were treated with VPA (0.5 or 1 mM)
twice or thrice weekly for 14 days. Controls remained untreated. Tumour cell interaction with
immobilized collagen was evaluated by a flow-based adhesion assay using a shear force of 2 or 4
dyne/cm2. The effects of VPA on the integrin adhesion receptors 3, 5, ß1, ß3 and ß4 were
assessed by flow cytometry to determine integrin surface expression and by western blotting to
determine the cytoplasmic integrin level.
Results: VPA, 0.5 mM and 1 mM, significantly prevented binding of both RT-112 and TCCSUP cells to
collagen, compared with the untreated controls. Adhesion was reduced to a higher extent when RT112 (subjected to 2 dyne/cm2) or TCCSUP (subjected to 2 or 4 dyne/cm2) tumour cells were treated
with VPA three times a week, compared to the two times a week protocol. VPA caused a significant
up-regulation of the integrin 3, 5, ß1, ß3 and ß4 subtypes on the TCCSUP cell surface membrane.
In RT-112 cells, only integrin 5 was elevated on the cell surface following VPA exposure. Western
blotting revealed an up-regulation of 3, 5, ß3 and ß4 integrins and down-regulation of the integrin
ß1 protein by VPA in TCCSUP. VPA also up-regulated 5 and down-regulated ß1 integrin in RT-112
cells, but also reduced 3 and ß3 in TCCSUP. VPA exerted adhesion-blocking properties on bladder
cancer cells under physiologic flow conditions. The effects were accompanied by distinct
modifications of the integrin expression profile, which differ depending on the cell lines used.
Conclusion: Application of VPA might be an innovative option to prevent bladder cancer
dissemination.
Contact:
[email protected]
57
P2.4
Invasion of urothelial carcinoma of the bladder: MMP7 is associated with increased cell
invasion in urothelial cancer: evidence from functional models
Knauf D¹, Gorzelanny C², Erben P¹, Schneider SW², Steidler A¹, Bolenz C¹
¹ Department of Urology, Mannheim Medical Center, University of Heidelberg, Mannheim
² Department of Experimental Dermatology, Mannheim Medical Center, University of Heidelberg,
Mannheim
Introduction: Matrix metalloproteinases (MMPs) play a crucial role in the lymphovascular invasion
(LVI) of malignant cells and metastatic spread of cancer. Elevated levels of MMP7 have been reported
to correlate positively with invasiveness of urothelial carcinoma (UC) cells and are associated with a
poorer oncological outcome. Therefore we aimed to functional evaluate MMP7 using in vitro assays
in UC.
Material & Methods: MMP7 expression was characterized in human UC cells (UMUC-3, RT112,
HT1197, T24/83 and RT4) and in a benign urothelial cell line (Urotsa) by quantitative RT-PCR and
Western Blot. The invasive potency of the different cell types where determined using an in vitro
invasion assay based on electrophysiological resistance breakdown across a MDCK-C7 monolayer. A
coefficient close to zero reflects high invasive potency. MMP7 expression was modulated using RNAi
interference before seeding in the invasion assay.
Results: Urothelial T24/83, HT1197, RT112, RT4 and UroTsa cells expressed the inactive pro-form of
MMP7, whereas the active form was only detected in HT1197 and RT112 possessing the highest
invasive potency (coefficients of 0.073 and 0.076, respectively). Optimiced transfection of UC
HT1197 cells with MMP7 siRNA inhibited MMP7 expression and reduced significantly the invasive
behaviour in the invasion assay (p<0.005, Mann-Whitney U-test).
Conclusions: Our results indicate an invasive potency of UC cells dependent on the expression of the
active form of MMP7. Therapeutical targeting of MMP7 could be an option in the prevention of
lymphovascular invasion and metastatic spread of UC cancer cells.
Contact:
[email protected]
58
P2.5
Analyse der microRNA Expression im Urothelkarzinom des oberen Harntrakts
Kriebel S , Schmidt D1, Kristiansen G2, Müller SC1, Ellinger J1
1
1
2
Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum Bonn
Institut für Pathologie, Universitätsklinikum Bonn
Background: MicroRNAs play a major role in the cancerogenesis of multiple malignant tumours and
are also relevant for the urothelial Carcinoma of the urinary bladder. So far, the microRNA expression
of the urothelial carcinoma of the upper urinary tract (UUT-UC) has not been investigated.
Methodology: RNA was extracted out of 47 UUT-UC samples as well as 36 corresponding ureter
samples. The expression of the microRNAs was analyzed using quantitative Real-Time PCR. As target
genes 11 microRNAs were selected, which had been described in previous studies as dysregulated in
the urothelial cancer of the urinary bladder. A statistical analysis was carried out using the MannWhitney Test including a Bonfferoni correction for multiple testing (significance level p<0.0045).
Results: MicroRNAs miR-21, miR-96, miR-135, miR-141, miR-182, miR-205, miR-429, miR-520b (all
p<0,001) were significantly upregulated in UUT-UC; miR-10a (p=0,012), miR-200b (p=0,006) and miR1244 (p=0,600) were similary expressed as in the normal tissue. Especially miR-96 and miR-182
allowed a precise differentiation (area under curve >0.92) between the two groups. The microRNA
miR-205 (p=0,002) was upregulated in the poorly differentiated UUT-UC (G3 vs. G2/G1).
Conclusion: MicroRNA expression profiling allows a differentiation of normal urothelium and UUTUC; microRNAs (especially miR-96 and miR-182) could be used as non-invasive Biomarkers in urine or
blood. There also exists a potential prognostic relevance for miR-205.
Contact:
[email protected]
59
P2.6
Acute epididymitis induces alterations in sperm protein composition
Pilatz A , Karnati S2, Lochnit G3, Paradowska-Dogan A1, Lang T4, Schultheiss D5, Schuppe H-C1, Hossain
H6, Baumgart-Vogt E2, Weidner W1, Wagenlehner F1
1
1
2
3
4
5
6
Klinik für Urologie, Kinderurologie und Andrologie, Justus-Liebig-Universität, Gießen
Institut für Anatomie und Zellbiologie, AG Medizinische Zellbiologie, Justus-Liebig-Universität,
Gießen
Institut für Biochemie, Justus-Liebig-Universität, Gießen
Institut für Anatomie und Zellbiologie, AG Reproduktionsbiologie, Justus-Liebig-Universität,
Gießen
Urologische Belegabteilung, Evangelischen Krankenhaus Mittelhessen, Gießen
Institut für medizinische Mikrobiologie, Justus-Liebig-Universität, Gießen
Introduction: Infection and inflammation in the urogenital tract are important etiological factors
implicated in male infertility. Here, the acute epididymitis is the only ascending infection with direct
impact on epididymis and testis. In spite of epididymitis occurring frequently in patients within
reproductive years, the impact on fertility has not been systematically investigated. To date, it is
unknown if urogenital infections cause changes in protein composition of sperm.
Materials and Methods: Between 2010 and 2012, 8 patients with acute epididymitis, gave written
informed consent and provided a semen sample three months after initial presentation and
treatment according to the guidelines. As a control group, 10 healthy men were included in the study.
Semen analysis was performed within 1 h of collection according to WHO 2010 recommendations. To
select the motile/viable sperm for proteome analysis, direct swim-up technique was performed from
1 ml of total semen. Samples of patients and controls were separately pooled due to low protein
content. Proteome analysis was performed by 2D electrophoresis and protein identification by
MALDI-TOF mass spectroscopy. The sub-cellular localization and biological function of the identified
proteins was determined using the information of the UniProt Knowledgebase. In addition
representative sperm preparations were examined by immunofluorescence for the presence of the subunit of the mitochondrial ATP synthase (ATP5B).
Results: Proteome analysis identified 35 proteins in sperm from epididymitis patients were downregulated, irrespective of subcellular localization and biological functions. According to the literature,
13 of these have been reported to be differentially expressed after capacitation, 10 showed an
impaired expression in infertile males, and 13 proteins have been found in the epididymal fluid. Most
of the proteins were part of the cytoskeleton, followed by the cytoplasm, nucleus and mitochondria.
Most of the proteins were classified to the following categories: “organization and cell motility",
"transcription, translation, and protein folding" and "energy and metabolism". Immunofluorescence
analysis confirmed ATP5B is less abundant in epididymitis samples compared to controls.
Conclusions: Even when normal semen parameters are observed in patients following epididymitis by
conventional semen analysis, significant changes in the sperm protein composition occurs. These
changes may be implicated as additional factors contributing to postinflammatory
subfertility/infertility.
Contact:
[email protected]
60
P2.7
Investigations on the localization and significance of the prolactin-inducible protein
(PIP) in the male urogenital tract
Karnati S1, Xiao Y1, Janga H1, Jongik D2, Izykowsky N2, Schuppe H-C3, Baumgart-Vogt E1, Weidner W3,
Wagenlehner F3, Pilatz A3
1
2
3
Institut für Anatomie und Zellbiologie, AG Medizinische Zellbiologie, Justus-Liebig-Universität,
Gießen
Institut für Pathologie, Medizinische Hochschule Hannover
Klinik für Urologie, Kinderurologie und Andrologie, Justus-Liebig-Universität, Gießen
Introduction: The prolactin-inducible protein (PIP) is a glycoprotein that is found in both the salivary
glands and the ejaculate. The localization of PIP in the genital tract is contradictory in the literature.
Several publications describe PIP as a biomarker for metastatic breast cancer, carcinoma of the
prostate as well as for azoospermia. Further, proteomic studies performed in various diseases of the
urogenital tract showed that PIP is differentially regulated; however, the exact biological function of
this protein in the ejaculate (seminal plasma, sperm) is unknown.
Materials und Methods: To investigate the localization of PIP in the urogenital tract,
immunohistochemistry was performed in mouse and human tissues of testis, epididymis, seminal
vesicles and prostate. In addition, human sperms were stained for PIP by immunofluorescence.
Finally, with a commercial ELISA, the quantification of PIP in seminal plasma as well as in sonicated
sperms purified by the swim-up technique was performed. A total of 95 seminal plasma samples were
analyzed for PIP (all with complete semen analysis according to WHO 2010): 10 samples before and
after vasectomy of identical men, 10 samples from patients with acute epididymitis and 65 samples
from the andrological routine were used in this study.
Results: In murine and human genital organs PIP was localized exclusively in the cytoplasm of
epithelial cells of the seminal vesicle. The PIP concentration in seminal plasma in men before
vasectomy was 480 ± 63 pg / ml and thus comparable to the situation after vasectomy with 458 ± 37
pg / ml (p = 0.2). Patients with completed antimicrobial treatment for acute epididymitis had
significantly lower PIP levels with concentrations of 431 ± 68 pg / ml (p <0.01). Correlation analysis in
95 seminal samples revealed PIP concentrations to be significantly associated with the fructose
concentrations (classic seminal vesicle marker) in semen (r = 0.3, p <0.01), while no significant
correlations were evident with zinc (prostate marker) and alpha-glucosidase (epididymis marker) as
well as the sperm parameters (concentration, motility, morphology). PIP was not detectable with
ELISA in swim-up purified and sonicated sperm samples. Immunofluorescence for PIP on human
sperms showed no staining, suggesting that PIP is not expressed in human sperms.
Conclusions: PIP is exclusively expressed in the seminal vesicle of the urogenital system and
constitutes a component of the seminal plasma. The observed down regulation of PIP in patients with
acute epididymitis might suggest a possible accompanying inflammation of the seminal vesicles.
Contact:
[email protected]
61
P2.8
Role of TET3 and 5-hmC in establishment of sperm epigenome and in production of
fertile sperm
Dansranjavin T1, Deuker J1, Steger K1, Bergmann M2, Weidner W1, Spiess A3, Schorsch M4,
Schagdarsurengin U1
1
2
3
4
Abteilung Molekulare Andrologie, Klinik und Poliklinik für Urologie, Kinderurologie und
Andrologie, JLU Giessen
Institut für Veterinär-Anatomie, -Histologie und Embryologie, JLU Giessen
Abteilung für Andrologie, Universitätsklinikum Hamburg-Eppendorf
Kinderwunschzentrum Wiesbaden
Objectives: Ten eleven translocation dioxygenase 3 (TET3) promotes DNA-demethylation by
converting 5-methylcytosine to 5-hydroxymethylcytosine and is, therefore, a key player of postfertilization chromatin remodeling in paternal pronucleus. The aim of our study was to analyze,
whether TET3 and 5-hmC are involved in heterochromatization during spermiogenesis, and whether
TET3 has an impact on male fertility status. Methods: TET3-expression in spermatogenesis was
examined by in-situ hybridization and by immunohistochemistry assays on human and bovine testis
sections exhibiting normal spermatogenesis. Presence of 5-hmC was analyzed by
immunofluorescence. TET3-mRNA-level in spermatozoa was analyzed by qRT-PCR in ejaculates from
healthy normozoospermic donors (n=28) and patients, who underwent intracytoplasmic sperm
injection (ICSI, n=48) and in vitro fertilization (IVF, n=19). Post-fertilization TET3 expression was
analyzed in bovine early embryos. Results: TET3-mRNA was detectable in human as well as in bovine
testis in spermatocytes up to round spermatids, whereas TET3-protein was expressed in round up to
elongated spermatids. We could detect the presence of 5-hmC in round up to elongating spermatids.
Sperm of healthy donors and work-up sperm of IVF-patients showed the highest TET3-mRNA in
comparison to ICSI-patients (p<0.001). Spermatozoa from younger men (≤35y.) exhibited significant
higher TET3-mRNA in comparison to elders (p=0.016). Among patients, elevated TET3-mRNA
associated significantly to higher progressive motility (>50% vs. ≤50%, p<0.001) and to pregnancy
(pregnancy vs. no pregnancy, p=0.02). Conclusions: The present study reveals for the first time the
function of TET3 in chromatin remodeling process during spermiogenesis. Moreover, we show that
the level of TET3-mRNA in spermatozoa is associated to fertility parameters.
Contact:
[email protected]
62
P2.9
Mixed testicular atrophy is related to atherosclerosis in the ApoE(-/-) / LDL receptor(/-) double knockout mouse model: New data of the arterial supply of the tubuli
Steinfeld K1, Middendorff R2, Kampschulte M3, Mietens A2, Langheinrich A3, Krombach GA3, Linn T4,
Mühlfeld C5, Wudy S6, Hartmann M6, Pilatz A1, Paradowska-Dogan A1, Altinkilic B1, Bergmann M7,
Weidner W1
1
2
3
4
5
6
7
Department of Urology, Pediatric Urology and Andrology, University of Giessen
Department of Anatomy and Cell Biology, University of Giessen
Department of Radiology, University of Giessen
Department of Internal Medicine, University of Giessen
Department of Functional and Applied Anatomy, Medical School Hannover
Department of General Pediatrics and Neonatology, University of Giessen
Department of Veterinary Anatomy, Histology and Embryology, University of Giessen
Aims/Objectives: We wanted to investigate the possible relationship between atherosclerotic lesions
and unexplained infertility in men using the mouse model mentioned below and clinical studies with
special focus on the arterial supply of the tubuli.
Material and Methods: The ApoE /LDL receptor double knockout (KO) mouse model is an ideal tool
to investigate altheroslerosis related spermatogenetic alterations comparable to humans. In testes
from KO- and wild-type mice at the age of 20, 40, 60 and 80 weeks testis volume and total vascular
volume fraction were quantified by micro-CT. In semithin sections total length, volume and surface
area of capillaries were estimated by newCAST. Spermatogenesis was analyzed by spermatogenetic
scores. Sperm counts were quantified in the epididymis. Testosterone levels were determined in
serum.
Results: KO mice exhibit diminished testis and total vascular volume fraction, changes of total length
(P=0.0001), volume and surface area (P=0.0046) of capillaries, mixed atrophy in various seminiferous
tubules and a reduction of testosterone levels.
Conclusions: Mixed testicular atrophy in KO mice is linked to reduced testis volume, vascular volume
fraction and low testosterone serum levels, suggesting a direct relation between atherosclerosis and
disturbed spermatogenesis.
(DFG, KFO 181/2-Project 8)
Contact:
[email protected]
63
P2.10
Polysialylation of NCAM correlates with onset and termination of seasonal
spermatogenesis
Hänsch M1, Simon P1, Schön J2, Geyer R1, Middendorff R3, Müller K4, Galuska SP1
1
2
3
4
Institute of Biochemistry, Justus-Liebig-University, Giessen
Berlin-Brandenburg School for Regenerative Therapies, (BSRT), Charité, Berlin
Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen
Leibniz Institute for Zoo and Wildlife Research, Berlin
Roe deer (Capreolus capreolus) are seasonal breeders and cyclic structural changes of roe bucks’
testis come along with a totally arrested (winter) and a highly activated spermatogenesis (summer).
For this reason, roe buck represents an interesting model to study general mechanisms of initiation
and termination of spermatogenesis. We investigated if polysialic acid (polySia) - a linear
homopolymer of 2,8-linked sialic acids, which could act as a negative regulator of cell-cell adhesion might be involved in the activation and/or inactivation of spermatogenesis. To address this point,
testis samples of adult male roe deer were collected after castration at different time point of the
year (February, April, June, August, October, December). Intriguingly, we observed that polySia
attached to the neural cell adhesion molecule NCAM was enhanced during the onset of
spermatogenesis in April. In addition, polySia was highly expressed in December. Predominantly,
polySia was detectable between Sertoli cells and spermatogonia in the basal regions of testicular
tubules as well as in the adluminal part of Sertoli cells.
Thus, polySia is expressed during key steps of the “on/off mechanisms” of seasonal spermatogenesis
and might represent one mediator of the interaction as well as communication between Sertoli cells
and spermatogonia.
Contact:
[email protected]
64
P2.11
Bacterial epididymitis induces fibrotic transformation and regional changes in the
activin/follistatin ratio
Michel V, Bhushan S, Middendorff R, Meinhardt A
Institute for Anatomy and Cell Biology, Department of Reproduction Biology, Justus-Liebig-University
Giessen
Uropathogenic E.coli (UPEC) are identified in >80% of urinary tract infections, with 40% of acute
epididymitis patients showing persistent impaired semen parameters despite antibiotic treatment.
This clinical observation suggests a link between urogenital infection and male infertility. Activin A, a
critical mediator of inflammation and fibrosis, increases dramatically during acute infection in many
tissues, and inhibition of activin by its binding protein, follistatin, may reduce the severity of disease
and damage.
The aim of this study was to investigate the activin-to-follistatin ratio in epididymitis, and its
potential correlation with epididymal inflammation and fibrosis. We established an acute
experimental epididymitis mouse model by infection of C57BL/6 mice with the uropathogenic isolate
UPEC CFT073. Sham-operated C57BL/6 mice injected with PBS served as the control group.
Following 3 days of infection, preliminary immunohistochemical and qRT-PCR experiments indicated
regional changes in the epididymal activin-follistatin ratio. Fibrosis development in the caput, corpus
and cauda epididymis was visualized histologically by Masson-Goldner staining. Acute UPEC
infection resulted in an increase in collagen fibers and severe fibrotic transformation of the
epididymal tissue architecture particularly around tubule cross-sections.
These findings point to a putative link between the fibrotic damage following UPEC-induced acute
epididymitis and the activin-follistatin signalling axis. Future studies will investigate the chronic
progression of epididymal fibrosis and elucidate the potential for targeting these pathways in the
treatment and fertility preservation of epididymitis patients.
Contact:
[email protected]
65
P2.12
Necrosis is the dominant cell dead pathway in UPEC induced epididymo-orchitis model
and is responsible for structural and functional damage of rat testis
Bhushan S1, Lu Y1, Tchatalbachev S2, Marconi M3, Bergman M4, Weidner W3, Chakraborty T2, Meinhardt
A1
1
2
3
4
Institute for Anatomy and Cell Biology, Depratment of Reproductive Biology, Giessen
Institute for Medical Microbiology, Giessen
Institute of Pediatric Urology and Andrology, Clinic and Policlinic of Urology, Giessen
Institute for Veterinary Anatomy, Department of Histology, and Embryology, Giessen
Introduction: Bacterial infections of the male genital tract result from ascending canalicular
infections of the male excurrent ducts and thus could cause male infertility. Uropathogenic
Escherichia coli (UPEC) is a relevant pathogen in urogenital tract infection.
Method and materials: To explore how testicular defenses react against UPEC infection, epididymoorchitis model was established by injecting UPEC CFT073 into vas deference in close proximity to the
epididymis. Mimicking an infection ascending from the urinary tract, in the testis UPEC bacteria were
found exclusively in the interstitial space 7 days post infection. Tracer experiments revealed that the
integrity of the blood-testis and blood epididymis barrier was intact 7 days post-infection indicating
that evasion of bacteria occurs probably intracellularly. In the testis, UPEC infection resulted in
impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in
sperm numbers and a significant increase in TUNEL (+) cells. To investigate potential mechanism of
germ cell death, hall mark steps of apoptosis were investigated. Activation of caspase-8 (extrinsic
apoptotic pathway), caspase-3/-6 (intrinsic apoptotic pathway), caspase-1 (pyroptosis pathway) and
the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic
pathway and pyroptosis, were not observed in infected testis. Notably, electron microscopical
examination revealed degenerative features of Sertoli cells (SC) in UPEC infected testis. Furthermore,
the passive release of high mobility group protein B1 (HMGB1), as an indication of the necrosis, was
observed in infected testis.
Conclusion: In summary, the findings indicate that UPEC infection causes the induction of an
organized self-destruction cascade termed programmed necrosis in the testis, which may ultimately
be the major mechanism contributing to impairment.
Contact:
[email protected]
66
P2.13
Identification of mouse sperm glycoprofile to evaluate possible alterations after
urogenital tract infection: possible implications for immune privilege and sperm function
Khosravi F¹, Lang T¹, Mink W², Kühnhardt S², Galuska SP², Meinhardt A¹
¹ Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen
² Institute of Biochemistry, Justus-Liebig-University, Giessen
Introduction: Uropathogenic E. coli (UPEC) are a common etiological cause of genito-urinary tract
infections such as prostatitis, epididymitis or epididymo-orchitis affecting in total approximately 1015% of all infertile men. Eradication of pathogens occurs via antibiotic treatment, but in 50-60% of
acute epididymitis, patient impairment of spermatogenesis remains. The sperm glycocalix is
generated during spermatogenesis as well as during epididymal maturation and O-linked and Nlinked glycans are essential for fertility, e.g. induction of sperm acrosome reaction and sperm-zona
pellucida binding and interaction. Changes in the glycoprofile may predispose damaged spermatozoa
for recognition by immune cells. There are three types O- glycan transferases: Nacetylgalactosaminyltransferase, O-mannosyltransferase and O-fucosyltransferase are initiating
enzymes for O-glycan biosynthesis. These enzymes have tissue and/or cell specific expression.
Aim: We aimed to determine the N- and O-glycan profile of mouse sperm under normal and
infectious conditions. We hypothesise that UPEC or secreted virulence factors may impair male
reproductive potential by possible modification of sperm glycoproteins.
Results: In first step expression levels of 21 different O-glycan transferases were analysed in the
mouse epididymis. Then we have established chromatography systems for determination and
quantification of monosaccharide moieties of sperm. This system is functional to evaluate sugar
alternations during sperm maturation.
Conclusions: Expression of O-glycan transferse genes in epididymis indicates that epididymis plays an
important role in synthesis of sperm glycocalyx. Glycoprofile analysis of mouse sperm indicates the
alterations in sperm glycocalyx during sperm maturation in epididymis.
Outlook: We are trying to identify the O- and N-linked saccharides of sperm by mass spectrometry
analysis. We plan to evaluate the alterations of sperm glycoprofile and expression level of glycan
transferases in mouse epididymis after UPEC infection and their consequences in male infertility.
Contact:
[email protected]
67
P2.14
Uropathogenic E. coli inactivate host survival AKT signalling pathway in Sertoli cells
Zhang Z , Bhushan S1, Tchatalbachev S2, Chakraborty T2, Meinhardt A1
1
1
2
Department of Anatomy and Cell Biology, Unit of Reproductive Biology, Justus-Liebig-University
Giessen
Department of Medical Microbiology, Justus-Liebig-University Giessen
Uropathogenic Escherichia coli (UPEC) are the major cause of acute and chronic ascending bacterial
genital tract infections in men which ultimately can result in impaired spermatogenesis. UPEC can
modulate host survival pathways to attenuate host inflammatory responses and escape from host
immune response. Here, we have shown that in isolated rat Sertoli cells (SC) UPEC virulence factor
alpha-hemolysin can inactivate AKT, also known as Protein Kinase B (PKB), by dephosphorylation. The
inactivation of AKT leads to activation of several downstream targets by dephosphorylation, one of
which is FOXO (Forkhead box class of transcription factors). We have observed the activation of
FOXO1 and FOXO3 by dephosphorylation at serine 256 and serine 253 in SC. Following
dephosphorylation, FOXOs can remain in the nucleus and execute diverse cellular functions such as
cell cycle arrest, apoptosis, reactive oxygen species detoxification and DNA repair. We have
demonstrated that the FOXO localize in the nucleus consequently FOXO DNA-binding activity
significantly increased after UPEC infection in SC. Although FOXO translocate to the nucleus with
high DNA binding activity, we have not observed any significant change in the expression of cyclin
D1 (a regulator of cell cycle progression), p27Kip1 and p15INK4B (both of which can inhibit cell cycle
progression). Similarly we have not observed any visible change in the expression of SOD2 (Sodium
dismutase, a detoxification enzyme of reactive oxygen species) and Catalase. The underlying
mechanism of FOXO targeted gene silencing still remains unclear. Taken together these results
suggest that UPEC can evade host immune cell response by manipulating host surviving signalling
pathway. Moreover suppression of AKT signalling pathway may lead to damage of Sertoli cells which
could impair spermatogenesis and germ cell death.
Contact:
[email protected]
68
P2.15
Urethral brush cells are polymodal cholinergic chemosensors
Deckmann K1, Filipski K1, Krasteva-Christ G1, Rafiq A1, Althaus M2, Fronius M2, Bschleipfer T3, Kummer
W1
1
2
3
Institute for Anatomy and Cell Biology, JLU Giessen
Institute of Animal Physiology, JLU Giessen
Department of Urology, JLU Giessen
Epithelial cell with a tuft of stiff microvilli on the cell surface are named brush cells. They have been
reported in the gastrointestinal tract and the respiratory tract. For the respiratory tract it is know that
brush cell are chemosensors. Recently, we identified them in the urethra as well. They are cholinergic
(expressing the acetylcholine synthesizing enzyme choline acetyltransferase=ChAT) and express the
brush cell marker protein, villin, and components of the canonical taste transduction signaling
cascade (-gustducin, PLC2, TRPM5).
In this work, we investigated the functional similiarity of urethral brush cells to type II taste cells of
the tongue described in literature. Type II taste cells detect bitter, sweet and umami gustatory stimuli
via taste receptors of the Tas1R and Tas2R families and the canonical taste transduction signaling
cascade to generate signals to activate afferent nerve fibers. We used an antibody directed against an
extracellular domain of the cation channel TPRM5 to isolate the presumptive urethral brush cells for
RT-PCR analysis. This revealed mRNA expression of several taste receptors. In whole cell patch-clamp
recordings, the bitter substance denatonium benzoate caused activation of a delayed and long-lasting
inward current in 15/21 cells. In CLSM recording of free intracellular calcium concentration ([Ca2+]i)
we investigated the responses to various stimuli. Candidate chemoreceptor cells from two different
strains of choline acetyltransferase (ChAT)-eGFP mice were identified by their eGFP fluorescence,
and from wild-type mice by fluorescent (FITC) antibody labeling of TPRM5. 91% (43/47) of the cells
responded to ATP (0.5 mM) with an increase in [Ca2+]i, 92% (45/49) to denatonium benzoate (25
mM; bitter compound), and 90% (38/42) to L-glutamate (25 mM; “umami”). The response to
denatonium benzoate was dose-dependent (2.5-25 mM).The effect could be blocked by a specific
TRPM5 blocker (TPPO; 0.25 mM) as well as by specific PLC 2 Inhibitor U73122 (10 μM).
Interestingly, urethral brush cells act as polymodal sensors in contrast to unimodal type II taste cells.
Like taste cells, they communicate to neighboring cells. We noted [Ca2+]i rise in response to
denatonium in non-eGFP expressing cells when situated in the vicinity of ChAT-eGFP positive cells. In
the presence of cholinergic blockers, denatonium evoked [Ca2+]i increase only in ChAT-eGFP cells
but not in eGFP negative cells, demonstrating stimulus evoked cholinergic signaling between
chemosensory and surrounding cells.
We interpret this cell type as a sentinel at the entrance to the urogenital tract initiating protective
mechanisms by acetylcholine release.
Funding: LOEWE Schwerpunkt “Non-neuronale cholinerge Systeme”
Contact:
[email protected]
69
V4.5
Expression of Aquaporin water channels by human urothelium: contribution to
transurothelial permeability in vitro and modulation by osmolality
Rubenwolf P1,3, Georgopoulos NT2, Baker S3, Southgate J3
1
2
3
Department of Urology, Johannes Gutenberg University Medical School Mainz, Mainz
Department of Chemical and Biological Sciences, School of Applied Sciences, University of
Huddersfield, UK
Jack Birch Unit for Molecular Carcinogenesis, University of York, UK
Background: It is generally assumed that human urothelium is impermeable to water and to the
constituents of the urine. However, recent evidence indicates that human urothelium expresses a
network of water- and urea-transporting channels, the aquaporins, which may constitute a molecular
basis for transurothelial water and solute transfer. This finding challenges the traditional concept of
the impermeable urothelial barrier, as it suggests that urothelium may be able to mediate water and
solute transport and thus modify the composition and final concentration of the urine. The aim of the
study was to study the functional aspects of urothelial AQPs.
Methods: In an in vitro model of human urothelium, we have investigated how changes in osmolality
affect AQP expression and have examined the role of AQP channels in water and urea transport. The
effect of exposing normal human urothelial cell cultures to osmotic stress was examined
immunochemically. Barrier strength of differentiated cultures was assessed by measuring
transepithelial electrical resistance (TER) and determining permeability coefficients for water and
urea. Mercuric chloride was used as an AQP channel blocker.
Results: AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A
small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium
revealed a significant barrier function (mean TER 3862 Ω.cm²), with mean diffusive water and urea
permeability coefficients PD of 6.3 and 2.4 cm/s, respectively. AQP blockade with mercuric chloride
resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets
remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase
in water and urea flux across an osmotic gradient.
Conclusion: When considered in toto with the emerging evidence from other studies, our results
support an active role for human urothelium in sensing and responding to hypertonic salt
concentrations through alterations in AQP protein expression, with AQP channels as a potential
mechanism for modifying urine composition. The contribution of these mechanisms to normal
urinary physiology has yet to be realised, but may be of particular significance in disorders where the
urothelial barrier is compromised, such as dysfunctional bladder syndromes and infectious or
interstitial cystitis.
Contact:
[email protected]
70
V4.6
3D-ultrastructure of the human urinary bladder revealed by FIB-SEM and 3View SEM
Neuhaus J1, Schröppel B2, Wolburg H3, Fallier-Becker P3, Dass M4, Zimmermann H4, Schwalenberg T1,
Stolzenburg J-U1
1
2
3
4
Department of Urology, University of Leipzig
Natural and Medical Sciences Institute at the University of Tuebingen
Department of Pathology, University of Tuebingen
Zeiss Microscopy Labs, Training, Application and Support Center (TASC), Munich
Objective: Urinary bladder function depends on the structural integrity of its cellular components.
Pathological conditions, e.g. bladder pain syndrome / interstitial cystitis (BPS/IC), are characterized
by macroscopic, microscopic and ultrastructural alterations of the bladder wall, including
suburothelial myofibroblasts. Ultrastructural features of those cells are well known. However, their
three-dimensional appearance has not been addressed so far at ultrastructural level. We here present
the first 3D-reconstruction of suburothelial myofibroblasts of the human bladder at ultrastructural
level using two different electron microscopic approaches.
Material and Methods: Bladder biopsies from two male patients aged 79 yrs and 83 yrs undergoing
urothelial tumor transurothelial resection were included in this pilot study. The tissue was prepared
for electron microscopy using either standard protocol or the enhanced heavy metal block staining
protocol (Deerinck et al. 2010). We used a Crossbeam® Auriga 40, a Crossbeam® Auriga 60 (Zeiss),
and a Sigma™VP 3View (Zeiss) to produce serial section electron microscopic image stacks of up to
1000 images. The minimum voxel resolution was 4.5 x 4.5 x 10 nm. Image stacks were pre-processed
using ImageJ and 3D-reconstructions were done with Amira® 3D Image Analysis software (vsg,
Düsseldorf, Germany). For comparison we did confocal immunofluorescence imaging using a LSM5
Pascal laserscanning microscope (Zeiss).
Results: 3D-ultrastructural analysis of suburothelial myofibroblasts revealed the delicate
membraneous structure of the cells, characterized by a network of cytoplasmic protrusions and spots
of vesicle production, resembling exosomes, supported by CD9 confocal immunofluorescence.
Interestingly, we could identify gap junctions between the protrusions of one single cell, i.e.
establishing „short circuits“ within the cell. This feature was only revealed by careful examination of
the three-dimensional structure of the cells. The sponge-like structure of the plasma membrane leads
to an immense increase of the surface area. Suburothelial myofibroblasts were characterized by
myofilaments, partial coverage by a basal lamina, fibronexus junctions and plasmalemmal attachment
plaques. No classical adhesion junctions were found between those cells.
Conclusions: We here show for the first time the delicate 3D-ultrastructure of suburothelial
myofibroblasts in the human bladder. Quite surprising was the finding of gap junction formation
between protrusions of the one and the same cell, the functional relevance of which has to be
explored. Our results demonstrate the potency of 3D-ultrastructure analysis to gain further insights
into the structure-function relationship of cells in the human bladder and might reveal new aspects of
bladder pathology when extended to pathologically altered tissues in future studies.
Contact:
[email protected]
71
P3.1
GDNF-family neurotrophic factors in urinary bladder and expression of their receptors
(GFR) in bladder sensory neurons
Nandigama R1, Bschleipfer T2, Kummer W1, Nassenstein C1
1 Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany,
2 Department of Urology, Pediatric Urology and Andrology, Justus-Liebig-University, Giessen,
Germany
Bladder sensory C-fibre neurons express transient receptor potential (TRP) channels. TRPV1 plays a
role in bladder pain and bladder hyperreflexia in inflammation. Neurotrophic factors like nerve
growth factor (NGF) are usually associated with development of the peripheral nervous system.
Recent studies, however, reported their role in inflammation dependent hypersensitivity of sensory
neurons and their effects in TRP channel mediated excitability of sensory neurons. Although NGF has
been associated in bladder inflammation, the role of glial cell line-derived neurotrophic factors
(GDNF) has not been studied yet. Here we investigated expression of GDNF-family ligands (GFLs) in
urinary bladder and of their receptors (GFR) in bladder sensory neurons. Quantitative real-time PCR
showed expression of all GFLs in urinary bladder. Real-time PCR analysis revealed the following rank
order of GFL expression: artemin (Ct value 29) > neurturin (Ct value 32) > GDNF (Ct value 36) >
persephin (Ct value 38). RT-PCR analysis for receptors for GDNF-neurotrophic factors (GFR)
showed expression of all GFR (1-4) and its co-receptor RET in L5-S2 DRG where cell bodies of
bladder afferent neurons are located. Single-cell RT-PCR studies in TRPV1 positive isolated
retrogradely labelled bladder sensory neurons showed expression of receptors GFR1 (receptor for
GDNF; 1/7 cells), GFR3 (receptor for artemin; 6/7 cells) and its co-receptor RET (co-receptor for all
GFLs; 6/7 cells), whereas expression of GFR2 (receptor for neurturin) was absent in all 7 single cells.
Taken together, these data indicate that GDNF-family neurotrophic factors and their receptors
(GFR) may play a role in bladder sensory pathways.
Contact:
[email protected]
72
P3.2
Treatment strategy dependent tissue injury produced by a new acoustic lens design for
electromagnetic lithotripters
Neisius A1,2, Smith N3, Kuntz NJ1, Schykowski T1, Astroza GM1, Lipkin ME1, Gustafson MR3, Simmons
WN3, Preminger GM1, Zhong P1,3
1
2
3
Division of Urologic Surgery, Duke Comprehensive Kidney Stone Center, Durham, NC, USA
University Medical Center Mainz, Department of Urology, Mainz
Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA
Purpose: The acoustic lens of a Siemens Modularis electromagnetic shock wave lithotripter has been
further modified to reduce pre-focal cavitation while generating a pressure waveform and broad focal
zone mimicking the Dornier HM3 electrohydraulic device. We sought to determine the threshold for
the maximum acoustic energy which can be safely applied to a kidney under clinically relevant
treatment protocols, and the dependency of tissue injury on pulse repetition frequency (PRF).
Materials and Methods Tissue injury (TI) produced by the original and modified lenses in a swine
model were first evaluated starting from the highest output level to determine the threshold energy
for safe lithotripsy. Thereafter, TI was assessed under an effective acoustic pulse energy for the
leading compressive wave (E+) of 44 mJ. A clinical protocol with a soft ramping scheme was used to
deliver 3000 shock waves to each kidney using a PRF of 1.0 and 1.5 Hz, leading to a total accumulated
energy of 112.84 J. Following lithotripsy, kidneys were perfused, harvested, dehydrated, cast in
paraffin wax, and sectioned. Photographic images were taken every 120 μm and analyzed to
determine the functional renal volume (FRV) damage.
Results: Gross subcapsular hematomas were produced by both the original and modified lenses at E+
of 51 mJ. Using E+ of 44 mJ, the modified lens showed quantatively macroscopic tissue injury
(subcapsular hematoma) in 1/6 renal units (17%) in the 1.5 Hz group. No macroscopic tissue injury
was detected in the 1.0 Hz group (0/6 renal units). After processing of the digitalized images TI was
detected in 0.432 % (±0.51%) of the FRV (with a maximum level of 1.324% in the kidney with the
gross hematoma) in the 1.5 Hz group and in 0.009% (±0.015%) of the FRV in the 1 Hz group
(p=0.025), respectively.
Conclusions: This study demonstrates that the energy threshold for gross TI of the modified lens is
comparable to the original lens. Our data further confirms that the initiation of TI depends on
acoustic pulse energy, and the extent of TI depends on the total accumulated acoustic energy
delivered to a kidney, as well as on PRF. Overall, our results suggest that a rational design of
treatment strategies to minimize tissue injury in SWL is warranted.
Supported by a Ferdinand Eisenberger grant of the Deutsche Gesellschaft für Urologie (German
Society of Urology), grant ID NeA1/FE-11
Contact:
[email protected]
73
P3.3
Coming closer to endoscopic “real time target identification” - Technical improvements
of an experimental lithotripsy laser system with spec-tral real-time object analysis
Miernik A1, Nuese C2, Knabe B2, Eilers Y2, Bolwien C2, Brandenburg A2, Lambrecht A2, Wetterauer U1,
Schoenthaler M1
1
2
University Medical Centre Freiburg, Department of Urology, Freiburg,
Fraunhofer Institute for Physical Measurement Techniques IPM, Freiburg
Introduction: Photonic technologies are gaining increasing attention in different fields of medicine.
Almost every application of laser energy induces a specimen spe-cific spectral response. Analysis of
reflected spectra may be used for acquisition of additional diagnostic information. Future laser
systems with integrated spectroscopic real-time analysis of targeted structures will provide a new
therapeutic quality in dif-ferent fields of medicine.
In urology, lasers are used for various applications such as the treatment of benign prostate
hyperplasia or tumour ablation of the bladder. Concerning interventional uri-nary stone treatment,
the Holmium-YAG laser is the gold standard for endoscopic disintegration. In this setting, a
differentiation of organic and non-organic structures (tissue vs. stone vs. endoscope) might
significantly increase safety and accuracy of the treatment. We hereby present constructional
improvements of a previously de-scribed experimental laser system for endoscopic stone lithotripsy
with real-time ob-ject identification.
Methods: Pure samples of urinary stones of different compositions, native human caliculi, endoscope
components and organic samples (porcine renal parenchyma, collecting system, blood) were analysed
by an experimental fibre-coupled fluores-cence spectroscopy setup in a dry and a wet model. After
determining the optimum laser excitation wavelength and acquisition of the spectral information
with consecu-tive data analysis, an experimental setup including a photo diode controlled by a micro-computer was constructed to improve data processing. The system was evaluat-ed for its ability
of real-time object identification.
Results: With the experimental setup as described, it was possible to obtain specific spectra for all
specimens. Mathematical post processing data revealed narrow band areas of the light spectrum
required for discriminatory specimen analysis. Signal in-tensity levels were sufficient for a reliable
discrimination of stone material, endoscope surfaces and organic samples. By optimizing the system,
measurement (integration) times could be reduced significantly.
Conclusions: Implementation of a computer-controlled photo diode into a fibre-coupled fluorescence
spectroscopy laser system improved and accelerated data pro-cessing of acquired spectral signals
significantly. This will improve the efficiency of real-time object analysis during endoscopic
interventions. The hereby acquired data will promote the development of future medical laser
systems with real time target identification.
(Supported by the Ferdinand Eisenberger-Grand MiA1/FE-12)
Contact:
[email protected]
74
P3.4
Overexpression of Drosha is associated with outcomes in patients with urothelial
carcinomas
Ratert N1,2, Ecke T3, Jung K1,2, Erbersdobler A4, Kilic E5, Rabien A1
1
2
3
4
5
Department of Urology, Charite-Universitätsmedizin, Berlin
Department of Urology, Helios Klinikum Bad Saarow
Institute of Pathology, Universität Rostock
Institute of Pathology, Charité – Universitätsmedizin Berlin
Background: It is already known that abnormal miRNA expression is associated with tumorigenesis.
Aberrant microRNA (miRNA) expressions are also associated with different clinical outcomes in
cancer patients. Consequently, it is postulated that proteins which are needed for miRNA maturation
may also play a key role in tumor behavior. The biogenesis of miRNAs is a multistep process involving
a couple of protein complexes. Briefly, miRNA genes are transcribed by RNA-Polymerase (RNAP)II/III
into a long single or multiple primary miRNA (pri miRNA). Afterwards pri miRNA is processed by
RNase III enzyme Drosha and its cofactor DiGeorge syndrome critical region gene 8 (DGCR8) into
hairpin precursor miRNA (pre miRNA). In cytoplasm pre miRNA is converted by RNase III enzyme
Dicer into double stranded miRNA duplex containing the mature as well as the complementary
strand. Finally, mature miRNA is loaded onto the miRNA Induced Silencing Complex (miRISC).
Argonaute proteins are the central components of miRISC and are needed for the repression of
mRNA translation. The aim of this study was to investigate the expression of the miRNA-related
proteins Argonaute1 (Ago1), Argonaute 2 (Ago2), and Drosha by immunohistochemical staining in
bladder cancer and to evaluate the expression data in relation to clinicopathological parameters and
as prognostic tools.
Material and Methods: A total of 112 urothelial carcinomas and 30 normal tissue samples of the
bladder collected by transurethral resection of the bladder (TURB) or cystectomy were used for the
tissue microarray (TMA) study. The cases were not stratified in any way but selected in accordance to
availability of tissue as well as of follow up data. Immunostaining was done with primary antibodies
against Ago1, Ago2, and Drosha. Fast-Red TR/Naphthol AS-MX was used as chromogen. Negative
controls were performed by staining with missing respective primary antibody. Immunostainings
were evaluated as 0 (no immunoreactivity) or 1 (positive immunoreactivity).
Results: Ago1, Ago2, and Drosha expression levels were up-regulated in malignant compared to
normal bladder tissue samples. Performing separate Kaplan-Meier analysis for G1/G2 and G3 tumors,
Drosha expression exhibited possible prognostic potential for G1/G2 (log rank test=10.875; P=0.001)
but not for G3 tumors concerning overall survival. In order to avoid bias results for the prognostic
analysis due to starting point of analysis we separated the patient collective in TURB and cystectomy
treated patients. Drosha exhibited possible prognostic potential for TURB (log rank test=6.636;
P=0.010) as well as for cystectomy patients (log rank test=4.139; P=0.042) with regard to the overall
survival, whereas results for Ago1 and Ago2 remained insignificant.
Conclusions: In this study, we investigated Ago1, Ago2, and Drosha expression in an
immunohistochemical manner for the first time in bladder cancer. Drosha appears to be a potential
prognosticator of overall survival time with respect to G1/G2 tumors.
Contact:
[email protected]
75
P3.5
Alternative therapy strategies for the non-muscle invasive bladder cancer on the basis
of carbon nanomaterials
Rieger C1, Kunze D1, Temme A2, Fuessel S1, Wirth MP1
1
2
Department of Urology, University of Technology, Dresden
Department of Neurosurgery2, University of Technology, Dresden
Introduction: An improvement of the intravesical treatment for recurrence and progression
prophylaxis of the non-muscle invasive bladder cancer is needed. Therefore the use of siRNAs might
represent a promising treatment option but also a multifunctional transporter for conventional
chemotherapeutic (CT) and therapeutic nucleic acids (AS-ODN) for chemosensitization of the tumor
cells will be an option. This novel carrier system is based on carbon nanotubes (CNTs) which can be
functionalized for different purposes like increased mucoadhesive properties. To analyze such
innovative cancer treatments suitable orthotopic BCa models are needed. Following instillation of
human BCa cells labeled with a luciferase gene to the mouse bladder tumor growth can be monitored
noninvasively in such in vivo models.
Material and methods: UM-UC-3 BCa cells were stably transduced with the gene of the Fireflyluciferase. Different UM-UC-3LUC clones were then selected and characterized. Cells were
transfected with siRNAs against Bcl-xL and survivin, either one siRNA alone (40 nM) or with
combinations of two siRNAs (20 nM each). Cell counts, apoptosis induction as well as targets mRNA
and protein levels were examined after 48h. Cells were also transfected with AS-ODNs against Bcl-xL
(250nM). mRNA expression and cell counts were examined after 24h. The mucoadhesive properties
of CNTs to pig or mouse bladder urothelium were assessed using Franz diffusion cells (Gauer Glas).
Bladder tumor implantation was achieved by intravesical instillation of 2 x 106 tumor cells in nude
mice. The bladder was pretreated with poly-L-lysine (0.1 mg/ml) or trypsin (0.5%).
Results: Two UM-UC-3LUC clones were selected. Single and combined siRNA treatment strongly
decreased the mRNA and protein expression of the targets in both UM-UC-3LUC clones. For
example, siRNA treatment against survivin led to a 95% reduction of survivin mRNA level. Different
Bcl-xL AS-ODNs were selected by mRNA structure prediction algorithms. BX2100 showed the
strongest effect on UM-UC-3LUC cells. Different types of CNTs (varying in length and diameter) were
tested in first ex vivo mucoadhesive studies on pig and mice bladders to show the attachment to
urothelium. After inoculation of UM-UC-3LUC bladder cancer cells through a urethral catheter into
the murine bladder an in vivo signal could be monitored using bioluminescence imaging.
Conclusions: Two UM-UC-3LUC clones were successfully generated and characterized, which is an
important requirement for the subsequent establishment of an orthotopic BCa model. The
knockdown of antiapoptotic Bcl-xL by using siRNAs as well as AS-ODNs and the use of CNTs as
multifunctional transporter represent a promising treatment option for BCa and will be further
investigated in an in vivo BCa model.
Funding provided by German Cancer Aid
Contact:
[email protected]
76
P3.6
The function of Kruppel-like factor 4 (KLF4) as a transcriptional repressor of IGF2promoter during prostate carcinogenesis
Dansranjavin T1, Lammert A1, Wagenlehner F1, Waliszewski P1, Brandt U1, Steger K1, Weidner W1,
Gattenloehner S2, Schagdarsurengin U1
1
2
Clinic of Urology, Pediatric Urology and Andrology; Justus-Liebig-University Giessen
Institute of Pathology, Justus-Liebig-University Giessen
Objective: KLF4 is a potent transcriptional repressor involved in stem cell maintenance and in
tumorigenesis. The present study reveals KLF4-bonding in differentially methylated region of IGF2promoter 0 (IGF2-DMR0) and elucidates the role of KLF4 in the regulation of IGF2/H19-imprinting
cluster in prostate carcinogenesis.
Methods: PCa-cell lines (LnCAP and Du145), 57 PCa (prostate carcinoma) and 71 BPH (benign
prostatic hyperplasia) were subjected to DNA-methylation analyses in IGF2-DMR0 and in imprinting
control region (ICR) by bisulfite pyrosequencing, and to IGF2- and H19-mRNA-quantification by
qRTPCR. Loss of imprinting (LOI) was determined by Apa I and Rsa I polymorphic sites within IGF2
and H19, respectively. KLF4-bonding on IGF2-DMR0 was analyzed by chromatin
immunoprecipitation (ChIP).
Results: KLF4 was significantly enriched in unmethylated IGF2-DMR0 of LnCAP and depleted in
hypermethylated IGF2-DMR0 of Du145 cells (relative bond: 265 vs. 8). DNA-demethylation in Du145
with 5-azacytidine led to a 14-fold increase of KLF4-bond in IGF2-DMR0. In tissue specimen, DMR0hypermethylation was significantly associated to elevated IGF2-mRNA (p< 0.001) emphasizing the
repressor role of KLF4 in prostate tumors. BPH exhibited more often IGF2-overexpression (33.8%)
compared to PCa (15.8%) (p=0.025). In contrast to hypermethylation of IGF2-DMR0, ICRhypermethylation showed a significant association to H19- as well as to IGF2-LOI (p=0.005 and
p=0.014, respectively).
Summary: Our study demonstrates that detachment of the transcriptional repressor KLF4 from IGF2DMR0 by its hypermethylation contributes to IGF2-overexpression. IGF2-overexpression could be
examined in BPH as well as in PCa. ICR-hypermethylation in PCa and BPH was correlated significantly
to IGF2- and H19-LOI.
Contact:
[email protected]
77
P3.7
Resveratrol and prostate a very complicated “dangerous liaison”
Di Domenico M1,6, Mancini FP2, Kisslinger A3, Iannelli P2, Iorio R1, Feola A1, Saar M4, Pierantoni GM5,
Tramontano D5
1
2
3
4
5
6
Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, I
Department of Sciences and Technologies, University of Sannio, Benevento, I
Institute of Experimental Oncology and Endocrinology, CNR, Naples, I
Department of Urology and Pediatric Urology, University of Saarland, Homburg/Saar
Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico
II”, Naples, I
Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple
University, Philadelphia, PA, USA
Background: Several lines of evidence support a strong connection between nutrition and prostate
cancer. One plausible link between diet and prostate cancer is oxidative stress which triggers a host
of pro-carcinogenic processes. Dietary antioxidants have been demonstrated to promote protection
against several human disorders and dietary chemoprevention is attracting increasing interest.
Resveratrol, a phytoalexin from grapes, exerts a variety of biological protective effects that are
antioxidative, pro-apoptotic/anti-cancer, and at least calorie-restrictive. P66Shc, a redox enzyme
member of the Shc family, shows a striking convergence of activities with those of resveratrol.
Therefore, we tested the effect of resveratrol on the activation of p66Shc in a prostate cell culture
system.
Methods The non-transformed human prostate cell line EPN and the EPN-PKM3 cells bearing PKM, a
dead kinase mutant of the non-receptor kinase Pyk2, were used in this study. A soft-agar assay was
performed on both cell lines and cell proliferation was analyzed in the presence of resveratrol by
direct counting. Western blot analysis and immunoprecipitation served to study various molecular
targets.
Results: Resveratrol reduces cell proliferation of both cell lines and induces a dose- and timedependent increase of p66Shc Ser36 phosphorylation. Hereby the effect of resveratrol on p66Shc
Ser36 phosphorylation is not ERK dependent.
Conclusion: This is the first evidence linking resveratrol and p66Shc in the prostate and may provide
insight into the mechanisms underlying the effect of resveratrol on prostate cell regulation.
Key words: prostate cancer, resveratrol, p66shc
Contact:
[email protected]
78
P3.8
Cardiolipin species composition differs in benign prostate epithelia and prostate cancer
and may be associated with tumor progression
Fussek S¹, Evert K², Schild L³, Streitbörger A¹, Grossebrummel H¹, Pechoel M¹, Zimmermann U¹, Evert
M², Burchardt M¹, Stope MB¹
¹ Department of Urology, University Medicine Greifswald
² Department of Pathology, University Medicine Greifswald
³ Department of Pathobiochemistry, Otto-von-Guericke University of Magdeburg
Background: Cardiolipin (CL) is a mitochondrial phospholipid serving an important role in membrane
structure and mitochondrial function. The composition of CL acyl-chain residues has been found to
be highly variable. Previously, fatty-acid residues of CL were shown to differ between tumor and nontumor cells in rat liver and brain cancer. Furthermore, a functional link between CL acyl-chain
distribution and cell proliferation was reported in lymphocytes of patients with leukemia. In this
study, the acyl-chain patterns of CLs in benign prostate epithelial cells and prostate cancer cells were
investigated.
Methods: CLs were extracted from established PCa cell lines (LNCaP, PC-3 and 22Rv1) and ten
human prostate specimen by a modified Folch extraction procedure and analyzed using highperformance liquid chromatography tandem mass spectrometry (HPLC-MS / MS). Tissue purity was
evaluated by two independent pathologists. Differences in CL expression were evaluated by SPSS
software.
Results: CL acyl-chain composition showed differences between benign prostate epithelial cells and
prostate cancer cells. 11 of 19 analyzed CL species differed statistically significant. Remarkably,
among the benign samples the same CL pattern was found while the prostate cancer specimen
showed a high variability. Moreover all three prostate cancer cell lines presented significant different
CL fatty-acid residues contents.
Conclusion: This study demonstrated the different acyl-chain composition of CL in benign prostate
epithelial and prostate cancer cells. The differences of CL patterns within prostate cancer specimen
and the cell culture cells suggest a role in prostate cancer progression.
Contact:
[email protected]
79
P3.9
MiR-205 is progressively down-regulated in lymph node metastasis but fails as a
prognostic biomarker in high-risk prostate cancer
Kalogirou C1, Spahn M1,2, Krebs M1,Joniau S3, Lerut E4, Burger M1, Scholz C-J5, Kneitz S6, Riedmiller H1,
Kneitz B1
1
2
3
4
5
6
Klinik und Poliklinik für Urologie und Kinderurologie, Universtitätsklinik Würzburg
Universitätsklinik für Urologie, Inselspital Bern, CH
Universitätsklinik für Urologie, Leuven, B
Universitätsklinik für Urologie, Leuven, B
Interdisziplinäres Zentrum für klinische Forschungs, Universität Würzburg
Biozentrum Würzburg, Institut für Physiologische Chemie I, Würzburg
Abstract: The treatment of high-risk prostate cancer (HRPCa) is a tremendous challenge for urooncologists. The identification of predictive moleculobiological markers allowing risk assessment of
lymph-node metastasis and systemic progression is essential to establish effective treatment. In the
current study we investigate the prognostic potential of miR-205 in HRPCa study- and validation
cohorts setting defined clinical endpoints for both. We demonstrate miR-205 to be significantly
down-regulated in over 70 % of the HRPCa samples analysed and that reconstitution of miR-205
causes inhibition of proliferation and invasiveness in prostate cancer (PCa) cell lines. Additionally,
miR-205 is increasingly down-regulated in lymph-node metastases compared to the primary tumour
indicating that miR-205 plays a role in migration of PCa cells from the original location into
extraprostatic tissue. Nevertheless down-regulation of miR-205 in primary PCa was not correlated to
the synchronous presence of metastasis and failed to predict the outcome for HRPCa patients.
Moreover, we found a tendency for miR-205 up-regulation to correlate with an adverse outcome of
PCa patients suggesting a pivotal role of miR-205 in tumourigenesis. Overall we showed that miR205 is involved in the development and metastasis of PCa, but failed to work as a useful clinical
biomarker in HRPCa. These findings might have implications for the use of miR-205 as a prognostic
or therapeutic target in HRPCa.
Keywords: high-risk prostate cancer; microRNA; miR-205; prognosis; biomarker
Contact:
[email protected]
80
P3.10
Re-expression of an epigenetically repressed microRNA inhibits prostate cancer cell
growth
Korzeniewski N1, Hohenfellner M2, Duensing S1,2
1
2
Section of Molecular Urooncology, University Hospital Heidelberg
Department of Urology, University Hospital Heidelberg
Background: The cause of prostate cancer is largely unknown, although compelling evidence exists
that extensive chromatin remodeling, which also affects microRNA (miRNA) loci, occurs at early
stages of prostate carcinogenesis. MiRNAs are small non-coding RNA molecules that play a crucial
role in a plethora of regulatory cellular processes by controlling mRNA stability and protein
expression. There is mounting evidence that miRNAs also play an important role in prostate cancer
development and progression. Determining changes in the miRNA profile that occur following
prostate carcinogenesis may reveal novel targeting of signaling pathways important for either the
initiation of carcinogenesis or the continued propagation of prostate tumor cells. Here, we surveyed
the expression of miRNAs after DNA hypomethylation in order to determine which miRNAs are
affected by methylation-driven chromatin modification in prostate cancer cells.
Methods: Hormone-sensitive LNCaP cells were treated for seven days with the DNA
methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) which is known to promote re-expression
of tumor suppressor genes through promoter hypomethylation compared to control treated LNCaP
cells. A miRNA array consisting of approximately 1400 capture probes (miRCURY; Exiqon) was then
used to identify dysregulation of miRNAs in treated versus untreated LNCaP cells.
Results: We identified a novel miRNA, mir-555, to be re-expressed following 5-aza-dC treatment of
LNCaP cells and found that ectopic expression of this miRNA negatively affects cell cycle
progression. We further discovered that the cell cycle arrest phenotype observed following ectopic
miR-555 expression is dependent on AR expression. We next performed an oligonucleotide gene
expression microarray analysis on the same 5-aza-dC treated samples and found that miR-555
potentially targets several genes important for mitotic progression, suggesting new targets for novel
therapeutic interventions.
Conclusion: Taken together, our results lend additional support to the notion that miRNAs play an
important role in prostate cancer. We show that miRNAs are dynamically regulated during chromatin
remodeling processes and we are currently utilizing these results to identify mitotic signaling
pathways that could be novel therapeutic targets to prevent prostate cancer cell progression.
Contact:
[email protected]
81
P3.11
HMGB1 modulates immune responses in a cell-specific manner in the rat experimental
autoimmune orchitis
Aslani F1, Meinhardt A1, Elsässer H-P2, Schuppe H-C3, Bhushan S1, Fijak M1
1
2
3
Department of Anatomy and Cell Biology, Justus-Liebig-University, Giessen
Institute of Cytobiology and Cytopathology, Philipps-Universität, Marburg
Department of Urology, Pediatric Urology and Andrology, Justus-Liebig University of Giessen
High mobility group box protein 1 (HMGB1), the non-histone chromosomal protein, plays an
important role in onset and chronification of autoimmune diseases once released from the nuclei. In
this study, we analyzed how HMGB1 can regulate inflammatory reactions in vivo in a rat model of
experimental autoimmune orchitis (EAO) and in vitro in primary testicular cells. HMGB1 was
translocated from the nuclei in EAO testis and in the testis of infertile men with leukocytic infiltrates.
HMGB1 levels in EAO testis were elevated at late chronic phase of disease as compared to early
proinflammatory cytokines such as IL-6 and TNF-. We found that testicular somatic cells show a cellspecific expression profile of HMGB1 receptors TLR4 and receptor for advanced glycation end
products (RAGE). The highly sensitive and specific proximity ligation assay was used to analyze
HMGB1 receptor binding in testicular cells. HMGB1-TLR4 binding was dominant in testicular
macrophages. However, Sertoli and peritubular cells showed higher levels of HMGB1-RAGE
interaction. In support, HMGB1 triggered RAGE-dependent ERK1/2 MAPK and CREB activation in
Sertoli and peritubular cells, whilst in testicular macrophages HMGB1 induced TLR4-signaling as
evidenced by p38 MAPK and p65 NF-B phosphorylation which stimulated an increase in mRNA
levels of TNF- and IL-6. Recent data shows that RAGE induced ERK activation leads to enhanced
autophagy levels. In line with these data, extracellular HMGB1 triggered formation of LC3 positive
punctae and double membrane autophagosomes in Sertoli cells. Increased autophagy levels in Sertoli
cells might explain how these cells survive the inflammatory environment in EAO testis. Considering
HMGB1’s late phase of action, inhibition of HMGB1 may be a putative target for therapeutic
intervention in treatment of chronic testicular inflammation.
Contact:
[email protected]
82
P3.12
Epigenetic dysregulation of inflammatory factors in prostatitis and prostate cancer
Velagala SR1, Seifer P1, Dansranjavin T1, Steger K1, Weidner W2, Wagenlehner F2, Schagdarsurengin U1
1
2
Dept. of Molecular Andrology, JLU Giessen
Clinic of Urology, Pediatric Urology and Andrology, UKGM, Standort Giessen
Objective: Recently, a new hypothesis has been proposed for prostate carcinogenesis that exposure
to environmental factors such as infectious agents and dietary carcinogens, and hormonal imbalances
lead to injury of the prostate and to the development of chronic inflammation and regenerative ‘risk
factor’ lesions, referred to as proliferative inflammatory atrophy (PIA). This preliminary study aimed
to analyze the epigenetic status of different inflammatory factors in prostate tumors in order to
reveal the link between prostate inflammation and carcinogenesis. Material and Methods: Three
prostate cancer cell lines PC-3, DU145 and LnCAP, and primary matching tissue samples from 14 PCa
(prostate carcinoma) and 14 BPH (benign prostatic hyperplasia) were subjected to epigenetic studies,
including CpG-promoter methylation analyses by COBRA (combined bisulfite restriction analysis)
and bisulfite sequencing, 5-aza-CdR-treatment and qRTPCR. Additionally, we analyzed the mRNA of
cytokines in ejaculate (spermatozoa) and in urine samples before and after prostate massage from 5
patients with prostatitis-syndrome (chronic pelvic pain syndrome, n=4; asymptomatic inflammatory
prostatitis, n=1). Results: Based on available literature 44 inflammatory factors could be selected as
involved in prostatitis and/or prostate cancer. In-silico analyses revealed that 12/44 have CpG-island
promoters (C3, CCL5, CXCL5, CXCL12, ERß, HIF1, IL4, IL6, IL11, IL12A, IL12B, IL13 and NGF),
among which 6 (IL6, CCL5, C3, IL4, IL13 and CXCL12) exhibited in PCa cell lines hypermethylated
promoters and down regulated expression, respectively. Four latter genes could be reexpressed via 5aza-CdR-treatment. Analyses on primary tumor probes revealed that cytokines, especially IL4, IL13
and CXCL12, are frequently hypermethylated in PCa as well as in BPH. Interestingly, in comparison to
IL4 and IL13, whose mRNA was frequently down regulated in the majority of PCa and BPH, CXCL12
exhibited an intense mRNA-expression in PCa and BPH. Studies on urine samples from prostatitis
patients revealed that mRNA level of cytokines in first and exprimate urine is nearly the same, except
some cases, where a prostate massage led to a massive (up to 5-fold) increase of cytokine-mRNA in
urine (e.g. IL4, IL13 and CXCL12). Remarkably, spermatozoa from prostatitis patients, especially from
asymptomatic inflammatory prostatitis, exhibited high levels of cytokine-mRNA (e.g. IL6, IL13 and
CCL5). Conclusion: Our study provides indications that cytokines, particularly IL4, IL13 and CXCL12,
are frequently epigenetically affected in prostate tumors and that these cytokines are present in high
levels in the urine of prostatitis patients. Therefore, we suggest that these cytokines might play a
crucial role in leading prostatitis to prostate tumor.
Contact:
[email protected]
83
P3.13
Androgen receptor regulates CD4+CD25+Foxp3+ T cell differentiation by binding to
foxp3 locus
Walecki M1, Eisel F1, Hackstein H2, Baal N2, Steger K3, Paradowska-Dogan A3, Meinhardt A1, Fijak M1
1
2
3
Department of Anatomy and Cell Biology, Giessen
Institute for Clinical Immunology and Transfusion Medicine, Giessen
Department of Urology, Pediatric Urology and Adrology, Giessen
Regulatory T cells (Tregs) are able to inhibit proliferation and cytokine production in the effector T
cells and play a major role in immune responses and prevention of development of autoimmune
diseases. The experimental autoimmune orchitis (EAO) in the rodent is a model of chronic testicular
inflammation leading to infertility. Our previous results have shown supplementation of EAO rats as
well as rat splenic T cells in vitro with testosterone causes differentiation of Tregs characterized by
increased expression of the transcription factor Foxp3. Foxp3 is a master regulator in the function of
Treg cells and its expression is under epigenetic control. Many common transcription factors are
involved in Foxp3 regulation as well as in the methylation status by binding the foxp3 locus.
Peripheral naïve CD4+CD25-Foxp3- T cells can differentiate into Tregs by stimulation and induction
of Foxp3 by several factors and interaction proteins. In the present study, we aim to investigate the
effect of androgens on the expression, regulation and methylation status of human Foxp3 and
putative interaction with androgen receptor (AR). We analyzed the effect of androgens on the
differentiation of human male and female Treg cells. Our results showed that the Foxp3 expression
significantly increased in T cells from human with lower testosterone level. We found an interaction
of androgen receptor with foxp3 locus in isolated primary human CD4+CD25+CD127- regulatory T
cells from male and female peripheral blood after the stimulation with androgens. No binding was
detectable in CD4+CD25-CD127+ T cells (Foxp3 negative). The AR-foxp3 interaction changes the
acetylation status of histones within the binding region but not the DNA methylation of tested CpG
within foxp3 gene.
Contact:
[email protected]
84
P3.14
Functional analyses of specific T cells in patients under BCG-therapy
Elsäßer J , Janssen M1,2, Becker F3, Suttmann H4, Ohlmann C-H1, Schmitt K5, Sester U2,6, Stöckle M1,
Sester M2
1,2
1
2
3
4
5
6
Department of Urology, Saarland University, Homburg
Department of Transplant and Infection Immunology, Institute of Virology, Saarland University,
Homburg
Boxberg Centre, Urological Group and Clinic Derout/Pönicke/Becker, Neunkirchen
Urologicum Hamburg
Department of Pathology, Saarland University, Homburg
Department of Internal Medicine IV, Saarland University, Homburg
Introduction: Immunotherapy with Bacille Calmette Guérin (BCG) is the standard-therapy for
prevention of progress and relapse of non-muscle invasive urothelial carcinoma. However, knowledge
on the induction and functionality of systemic T cell immunity is limited.
Method: In 18 bladder cancer patients, blood was drawn longitudinally before each instillation during
the induction course. Multiparameter flow cytometry is applied to quantify and characterize specific
T cells from whole blood using intracellular cytokine staining after stimulation with purified protein
derivative (PPD). PPD is used as a commercially available mycobacterial antigen preparation for
tuberculin skin test and it allows detection of therapy-induced immunity due to its cross reactivity
towards BCG. 54 age-matched healthy subjects served as controls.
Results: Before the first instillation, 9/18 patients showed a pre-existing immunity toward PPD, which
could be caused by BCG-vaccination or prior contact to mycobacteria. However, baseline levels of
IFN- producing PPD-specific T cells were comparable to controls and showed a significant increase
after completion of the 2nd instillation (p<0.0001). Systemic immunity was induced in all patients,
although the increase was less pronounced in patients with pre-existing immunity. Consistent with
repeated antigen-challenge, PPD-specific cytokine-profiling after instillation revealed a lower
percentage of multifunctional IFN-γ/IL-2 double-positive T cells compared to healthy controls
(p=0.0003). Of note, unlike in patients with pre-existing immunity, cytokine profiles in patients with
primary immunity was shifted towards IL-2 single producing T cells (35.9% vs. 13.4%, p=0.02).
Conclusion: Interestingly we found out, that patients under BCG-therapy shows similar cytokine
profiles as patients with active tuberculosis. Decreased functionality is a typical feature of specific
immunity in both patients with active TB and BCG therapy. Considering this in the context of
tuberculosis diagnosing, among patients with active infection, the percentage of IL2 single positive
cells may allow distinction between patients with primary infection and cases with boosted immunity
after prior contact.
Contact:
[email protected]
85
P3.15
Urine protein profiling identified Alpha-1-microglobulin and Haptoglobin as
biomarkers for early diagnosis of acute allograft rejection following kidney transplantation
Stubendorff B1,2, Finke S1, Walter M1, Kniemeyer O3, von Eggeling F4, Gruschwitz T1, Steiner T1,5, Ott
U6,7, Wolf G6,7, Wunderlich H1,8, Junker K1,2
1
2
3
4
5
6
7
8
Department of Urology, University Hospital, Jena
Clinic of Urology and Pediatric Urology, Saarland University Medical Center, Homburg/Saar
Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product
Research and Infection Biology - Hans-Knöll-Institute and Integrated Research and Treatment
Center – Center for Sepsis Control and Care (CSCC), University Hospital, Jena
Institute of Human Genetics, Core Unit Chip Application (CUCA), University Hospital, Jena
Department of Urology, Helios Hospital Erfurt
KfH Kuratorium für Dialyse und Nierentransplantation e.V., KfH Nierenzentrum, Jena
Department of Internal Medicine III, University Hospital, Jena
Clinic of Urology and Pediatric Urology, St. Georg Hospital, Eisenach
Introduction: Early diagnosis of acute allograft rejection and effective immunosuppressive therapy
lead to improvements in graft survival following kidney transplantation. However, there are no noninvasive diagnostic parameters available that enable early and reliable detection of acute rejection.
The objective of our study was to determine whether acute allograft rejection can be predicted at an
early postoperative stage and to identify potential biomarkers for clinical practice.
Materials and Methods: Urine samples of 116 kidney recipients were included. Rejection was proven
by biopsy (n=58) and stable transplant function was monitored for at least 2 years (n=58).
Postoperative urine samples were collected between 3rd and 10th day following transplantation.
Urinary protein profiles were analyzed by SELDI-TOF-MS. Multiplex-Fluorescence-2DE and Peptide
Mass Fingerprinting were used for identification of candidate proteins. Urinary concentration of
candidate proteins was validated by ELISA.
Results: A protein signature including 4 masses differentiated acute rejection from stable transplant
patients at the postoperative stage with 73% sensitivity and 88% specificity. Alpha-1-microglobulin
(A1MG) and Haptoglobin (Hp) were identified as putative biomarkers for acute rejection. Protein
levels were significantly higher in postoperative urine samples from patients with upcoming rejection
than in stable transplant patients (A1MG: 29.13 μg/ml vs. 22.06 μg/ml, p=0.001; Hp: 628.34 ng/ml vs.
248.57 ng/ml, p=0.003). The combination of both proteins enabled the diagnosis of early rejection
with 85% sensitivity and 80% specificity.
Conclusion: The specific urine protein pattern enables the prediction of early acute allograft rejection
already few days after kidney transplantation. A1MG and Hp appear to be reliable rejection
biomarkers.
Further analyses have to show if early diagnosis is possible for patients with allograft rejection that
occurs several month after transplantation by analyzing changes in urine protein pattern in regular
intervals.
Contact:
[email protected]
86
ANHANG
87
88
AuF-Preisträger
2009:
Dipl.-Biol. Annika Schäfer (verh. Fendler), Berlin
Dr. med. Matthias Saar, Homburg/Saar
2010:
Dr. rer. nat. Natalie Sampson, Innsbruck
Dr. med. Friedemann Zengerling, Ulm
2011:
Dipl.-Biol. Elke Nolte, Erlangen
Dipl.-Biol. Elke Schneider, Mainz
Rudi Ascherl & Dr. rer. nat. Kerstin Boll, Leipzig
Dr. med. Matthias Heck, München
2012:
PD Dr. med. Carsten Stephan, Berlin
Dr. rer. nat. Bettina Schlick, Innsbruck
Dr. med. Carl Ludwig Behnes, Göttingen
Dr. rer. nat. Nina Korzeniewski, Heidelberg
89
90
Wir bedanken uns für die finanzielle Unterstützung bei
unseren Förderern und Sponsoren:
91
Die DGU bedankt sich bei den genannten Firmen für die nachstehend aufgeführten Beträge
als Gegenleistung für Werbezwecke/Standkosten:
Astellas
10.000 €
Farco-Pharma
7.500 €
Pierre-Fabre
1.500 €
Janssen
1.000 €
Pfizer
1.000 €
Takeda
1.000 €
Apogepha
1.000 €
Bionorica
1.000 €
GlaxoSmithKline
800 €
Lilly
700 €
Bayer
500 €
AMS
500 €
Serag-Wiessner
500 €
Ausblick: 5. AuF-Symposium 2013 in Gießen
92
6. Symposium
CME
Urologische Forschung
der Deutschen Gesellschaft für Urologie
Interdisziplinäre Forschung in der Urologie:
Mehrwert durch Vernetzung
Homburg 2014
In Kooperation mit
der Arbeitsgemeinschaft Uropathologie
der Deutschen Gesellschaft für Pathologie
13. bis 15. November 2014
Kulturzentrum Saalbau
Homburg/Saar
http://auf-symposium.dgu.de

PROGRAMM 5. Symposium Urologische Forschung

Transcription

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