2014 AUA Research Forum: “Showcasing Early Career  Investigators”  Sunday, May 18, 2014    3:00 – 5:30 pm 

 2014 AUA Research Forum: “Showcasing Early Career Investigators” Sunday, May 18, 2014 3:00 – 5:30 pm Orange County Convention Center, Room W230AB 3:00 3:30 3:35 3:45 3:55 4:05 4:15 4:25 4:35 4:45 4:55 5:05 5:20 Early Career Investigators Poster Viewing Program Introduction Leo Giambarresi, PhD Bridging the Research and Clinical Realms in Prostate Cancer Health‐Related Quality of Life Measurement The Relationship Between Male Fertility and Somatic Health Peter Chang, MD Acellular Bi‐layer Silk Biomaterial for Augmentation Cystoplasty Carlos Estrada, MD Michael Eisenberg, MD The Role of Rho‐kinase in the Neuropathy of Erectile Dysfunction Joanna Hannan, PhD Determine Whether Vaginal Delivery‐Associated IR Injury Can Be Mitigated by Cytoprotection and Whether Cytoprotection Accelerates Recovery from Vaginal Delivery‐Induced SUI Una Lee, MD Does Active Surveillance Miss the Window for Cure? Matched Comparison of Immediate Versus Delayed Prostatectomy in a Nationwide Population ‐Based Cohort Stacy Loeb, MD Hydrogen Sulphide Supplementation in Renal Transplantation: A Potential Solution for Ischemia Reperfusion Injury Alp Sener, MD Cortisol Awakening Response and Salivary Cortisol Response to Stress in Women with Overactive Bladder and Controls Ariana Smith, MD Prostate Epithelial Lineage Hierarchy AUA/Urology Care Foundation Research Activities Leo Giambarresi, PhD Presentation of Awards Li Xin, PhD The American Urological Association (AUA) is honored to support urology research, especially the work of our early career investigators. The annual Research Forum is the only event at the Annual Meeting that is dedicated to promoting their work. It is truly about “Showcasing Early Career Investigators.” The presenters for the Research Forum were selected from nominations made by AUA sections, affiliated societies, and members of the Research Council. All nominees are invited to display posters. The top nine ranked submissions will present from the podium during the Forum program. Each presenter will have eight minutes to present their work. A panel of judges will score and rank the presentations. The top three presenters will receive mounted achievement certificates from the AUA Office of Research. The presenters’ abstracts are listed first in this syllabus. Then you will find poster abstracts from nominees who are not presenting from the podium and wish to exhibits posters on their work. In addition, current Urology Care Foundation Scholars were invited to display posters. CME Disclosure The American Urological Association (AUA) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The AUA designates this live activity for a maximum of 2.5 AMA PRA Category 1 Credits™. Physicians should claim only the credit commensurate with the extent of their participation in the activity. All speaker disclosures have been submitted and meet AUA CME requirements. “Showcase” Presenter Abstracts Peter Chang, MD Harvard Medical School / Beth Israel Deaconess Medical Center Bridging the Research and Clinical Realms in Prostate Cancer Health‐Related Quality of Life Measurement Introduction and Objective: Patient reported outcome (PRO) questionnaires such as the Expanded Prostate Cancer Index Composite (EPIC)1,2 assess prostate cancer (PCa) specific health related quality of life (HRQOL) from the patient perspective, and their use in research has clarified PCa treatment effects.3 However, 10‐15 years after their development, there still remains a large discrepancy between practitioner‐ documented and patient‐reported HRQOL.4 This represents a disconnect between research efforts and clinical practice – existing instruments are too burdensome to use clinically; they are time consuming and difficult to score. Therefore, practitioners have not only been underestimating patients’ symptoms, but have also been unable to apply models that can predict post‐treatment HRQOL outcomes based on baseline HRQOL,5 limiting the ability to individualize treatment recommendations. Our objective was to bridge the research and clinical realms by developing and validating a PRO questionnaire that retained the properties of EPIC and could be administered and scored at the point‐of‐care. We then sought to demonstrate its practicality of use in clinical practice, show its responsiveness to HRQOL changes over time, and expand its clinical applicability by calculating minimally important differences (MID) in domain score changes and recalibrating an existing multivariable predictive model for clinical use. Methods: We developed the EPIC for Clinical Practice (EPIC‐CP) questionnaire by eliminating conceptually overlapping items from the 3 page EPIC‐262 and revising the format to mirror the AUA Symptom Index6, thereby enabling domain score calculation at the point of care. We administered EPIC‐CP to a new validation cohort of PCa patients in community‐based and academic oncology, radiation, and urology practices to evaluate EPIC‐CP’s validity and ease of use in clinical practice.7We then modeled each treatment group’s EPIC‐CP domain scores at pre‐
treatment, short‐term, and long‐term follow‐ up in 1,201 subjects from a previously described PROST‐QA cohort.3 We considered post‐treatment domain score changes from pre‐treatment ≥ 0.5 standard deviations (SD) clinically significant and with a p‐value ≤ 0.01 as statistically significant. We determined domain MIDs using the 0.5 pooled SD of the 2, 6, 12, and 24 month post‐treatment changes from pre‐treatment. Lastly, we recalibrated an EPIC‐26‐based nomogram predicting 2‐year post‐prostatectomy functional erections, enabling its use in clinical practice using EPIC CP.8 Peter Chang, MD (cont’d) Results: EPIC‐CP is a 16‐item, 1 page questionnaire that retains all five EPIC HRQOL domains: urinary incontinence, urinary irritation/obstruction, bowel, sexual, and vitality/hormonal.7 Each domain has three items, and item answers are added to calculate a domain score (lower score = better HRQOL). In the EPIC‐ CP validation cohort (N = 307), 175 treated and 132 untreated PCa subjects completed EPIC‐CP efficiently (96% in <10 minutes, and 11% missing items). It was deemed ‘very convenient’ by clinicians in 87% of routine clinical encounters, and clinicians accurately scored completed questionnaires 94% of the time. EPIC‐CP shows convergent validity: domain scores correlate highly with respective domain scores from longer versions of EPIC (r ≥ 0.93 for all domains). EPIC‐CP has high internal consistency (Cronbach’s α = 0.64‐0.84) and discrimitive validity – sensitivity to PCa treatment‐related effects (p < 0.05 for each domain). For every domain, EPIC‐CP is responsive to similar post‐treatment HRQOL changes over time as had been observed using EPIC‐26 in the PROST‐QA study.3 The EPIC‐CP MIDs for changes in the urinary incontinence, urinary irritation/obstruction, bowel, sexual, and vitality/hormonal domains are 1.0, 1.3, 1.2, 1.6, and 1.0, respectively. The EPIC‐CP‐based sexual prediction model performs well (AUC=0.76) and shows robust agreement with its EPIC‐26 based counterpart, with predicted probability differences between models of ≤10% for 95% of individuals and a mean difference of 0.0 (SD=0.05) across all individuals.8 Conclusions: EPIC‐CP bridges the gap between the research and clinical realms in the management of PCa‐ related HRQOL changes. We demonstrated its ease of use, validity (convergent, discriminative), and responsiveness, increased its clinical applicability by defining the minimally important differences in score changes, and showed how it can predict post‐treatment sexual outcomes after radical prostatectomy. Its use in clinical practice can help practitioners individualize treatment decisions and recognize and treat post‐ treatment HRQOL deficits, all of which contribute to patient‐centered care, and improvement of care quality. Areas of future study include identifying the barriers to EPIC‐CP (and other PRO) use in clinical practice, as well as assessing the effect of EPIC‐CP use on symptom distress, HRQOL outcome, and patient satisfaction. REFERENCES: 1. Wei JT, Dunn RL, Litwin MS, et al. Development and validation of the Expanded Prostate Cancer Index Composite (EPIC) for comprehensive assessment of health‐related quality of life in men with prostate cancer. Urology. 2000; 56:899‐905. 2. Szymanski KM, Wei JT, Dunn RL, et al. Development and validation of an abbreviated version of the Expanded Prostate Cancer Index Composite instrument for measuring health‐related quality of life among prostate cancer survivors. Urology. 2010 Nov;76(5): 1245‐50. Epub 2010 Mar 28. Peter Chang, MD (cont’d) 3. Sanda, MG, Dunn RL, Michalski J, et al. Q uality of life and satisfaction with outcome among prostate‐ cancer survivors. N Engl J Med, 2008. 358(12):1250‐61. 4. Sonn GA, Sadetsky N, Presti JC, et al. Differing perceptions of quality of life in patients with prostate cancer and their doctors. J Urol. 2013 Jan;189(1 Suppl):S59‐65; discussion S65. doi: 10.1016/j.juro.2012.11.032. 5. Alemozaffar M, Regan MM, Cooperberg MR, et al. Personalized prediction of erection recovery after primary prostate cancer treatment: validation in the community‐based CaPSURE cohort of a predictive model developed in PROST‐QA. JAMA. 2011 Sep 21;306(11):1205‐14. 6. Barry MJ, Fowler FJ Jr, O’Leary MP, et al.. The American Urological Association symptom index for benign prostatic hyperplasia. The Measurement Committee of the American Urological Association. J Urol 1992; 148(5): 1549‐57. 7. Chang P, Szymanski KM, Dunn RL, et al. Expanded prostate cancer index composite for clinical practice: development and validation of a practical health related quality of life instrument for use in the routine clinical care of patients with prostate cancer. J Urol. 2011 Sep;186(3):865‐72. Epub 2011 Jul 23. 8. Chipman JJ, Sanda MG, Dunn RL, Wei JT, Litwin MS, Crociani C, Regan MM, Cha ng P. Measuring and Predicting Prostate Cancer Quality of Life Changes using the Expanded Prostate Cancer Index Composite for Clinical Practice (EPIC‐CP). J Urol. 2013 Sep 24 (Epub ahead of print). Michael Eisenberg, MD Stanford University School of Medicine Exploring the Diagnosis, Treatment, and Consequences of Male Infertility Upon Both the Patient and His Offspring Background: Aberrations in reproductive fitness may be a harbinger of medical diseases in men. As such, diminished sperm production may indicate men at risk for poor health outcomes. We sought to determine the association between impaired semen parameters and mortality in the years after an infertility evaluation. Methods: We performed a multi‐center retrospective analysis of a cohort of 11,935 men evaluated for infertility at fertility clinics in Texas and California from 1989 to 2011. Men accumulated time at risk from the date of the initial infertility evaluation until the time of final follow‐up or death. Men were stratified based on abnormal semen parameters as determined by the WHO 5th edition criteria. Medical comorbidities were quantified using the Charlson Comorbidity Index (CCI) as well as the Centers for Medicare & Medicaid Services‐Hierarchical Condition Categories (CMS‐HCC) Model developed for ambulatory patients. All cause mortality was determined by data linkage to the National Death Index or Social Security Death Index. Age adjusted standardized mortality rates were calculated using population‐based mortality data from the National Center for Health Statistics. In addition, Cox regression analysis was used to compare infertile men based on semen parameters. Results: During 92,104 person years of follow‐up, 69 of 11,935 men died (0.58%). The mean age at infertility evaluation was 36.6 years with a mean follow up of 7.7 years. Compared to the general population, men evaluated for infertility had a lower risk of death with 69 deaths observed compared with 176.7 expected (SMR 0.39, 95% CI 0.30‐ 0.49). When stratified by semen parameters, however, men with impaired semen parameters (i.e. male factor infertility) had significantly higher mortality rates compared with men with normal parameters (i.e. no male factor infertility). Low semen volume, sperm concentration, sperm motility, total sperm count, and total motile sperm count were associated with higher risk of death. In contrast, abnormal sperm morphology was not associated with mortality. While adjusting for current health status attenuated the association between semen parameters and mortality, men with two or more abnormal semen parameters still 2.3‐fold higher risk of death compared to men with normal semen (95% CI 1.12‐4.65). Conclusions: The current report found that a man’s semen quality was inversely associated with mortality. Men with impaired sperm counts, sperm motility, or semen volume (i.e. male factor infertility) had higher mortality rates compared to men with normal semen quality. Moreover, the risk of death increased with an increasing number of semen parameter in the subfertile range. While reassuring that the absolute risk of death remains low for men evaluated for infertility in the decade after an infertility evaluation, the current report suggests continued health monitoring of these men is warranted after reproductive efforts have ceased. Carlos Estrada, MD Boston Children’s Hospital/Harvard Medical School Acellular Bi‐layer Silk Biomaterial for Augmentation Cystoplasty Background: In several different urological conditions, the urinary tract is anatomically or functionally obstructed, which can result in renal damage from elevated urinary storage pressures. These diseases include posterior urethral valves, neurogenic bladder secondary to spina bifida or spinal cord injury, and bladder and cloacal exstrophy. These conditions often require bladder augmentation, and currently, enterocystoplasty is the gold standard. However, the use of intestinal segments can result in significant complications including chronic urinary tract infection, metabolic abnormalities, urinary stone formation, and risk of malignancy in the transposed bowel. Given these limitations, tissue engineered alternatives have been vigorously studied for decades. The main successes in this field have come from the use of primary autologous cells used in conjunction with PGA scaffolds. This strategy has limitations, primarily its reliance on diseased autologous cells that require a separate biopsy to procure and are cumbersome to expand and seed onto the scaffold in vitro prior to implantation. We have studied biomaterials derived from Bombyx mori silkworms as an alternative for bladder tissue engineering given their mechanical robustness, processing plasticity, and biodegradability. Our published results have shown that a novel acellular bi‐layer silk scaffold supports robust tissue regeneration in murine and porcine models of bladder augmentation. In addition, it was superior to our comparison groups, SIS and PGA, in tissue organization, lack of fibrosis, and lack of inflammation. The goal of the current project that is in process is to evaluate the ability of this scaffold to regenerate bladder tissue in large animal models of bladder disease ‐ bladder exstrophy and bladder outlet obstruction ‐ in order to demonstrate potential clinical viability. Project Outline: The two aims of our project are: 1) Evaluate the histological and functional performance of silk scaffolds for bladder augmentation in a fetal sheep model of bladder exstrophy, and 2) Determine the feasibility of silk matrices to support tissue regeneration and functional voiding following augmentation cystoplasty in a porcine model of bladder outlet obstruction. For Aim 1, a fetal sheep model of bladder exstrophy will be created. Briefly, fetal lambs at ~95 d gestation will undergo open surgical creation of a bladder exstrophy defect by marsupializing the anterior portion of the bladder to the abdominal wall in utero. In this model, prolonged luminal exposure of the bladder to amniotic fluid and lack of mechanical cycling causes chronic inflammatory and maturational changes to the bladder wall including fibrosis, ulceration and squamous metaplasia, as well as tissue atrophy which ultimately leads to low capacity, poorly compliant bladder phenotypes at birth. At 1‐4 d following delivery (~140 d, full term), animals will be divided into two experimental groups. In Group 1 (N=2), the bladder exstrophy defect will be Carlos Estrada, MD (cont’d) primarily closed with surrounding host bladder tissue while in Group 2 (N=2), silk scaffolds (4x4cm2) will be implanted for bladder augmentation. In addition, a control group of normal lambs (Group 3, N=2), receiving no surgical intervention, will be assessed as a benchmark for native bladder function. All groups will be maintained for a total of 3 months and will be subjected to outcome analyses, including ability to voluntary void, urodynamic parameters, renal function, and histological, immunohistochemical, mechanical and contractility outcome analyses. For Aim 2, a porcine model of bladder outlet obstruction will be created. Briefly, juvenile male swine will be subjected to urethral ligation and urinary diversion will be accomplished with a suprapubic catheter in line with a calibrated resistance valve (5 cm H2O pressure) in order to produce controlled bladder outlet obstruction for a period of 4 weeks. This model has been shown to promote fibrotic tissue remodeling and lead to reduced bladder compliance and attenuated contractility. After 4 weeks, the obstruction will be removed and catheters will be left to free drainage and animals will be divided into two experimental groups. Group 1 (N=2) will serve as negative controls receiving cystotomy alone, while augmentation cystoplasty will be performed in Group 2 (N=2) with silk scaffolds (6x6cm2). Both groups will then be maintained for a total of 3 m and will be subjected to outcome analyses as in Aim 1. Conclusion: We aim to produce an “off the shelf” scaffold for bladder augmentation that would overcome the substantial limitations of previous biomaterial options and tissue engineering strategies. Our acellular bi‐layer silk construct may offer advantages over conventional gastrointestinal segments and previously described cellularized biomaterials for augmentation cystoplasty. Johanna Hannan, PhD Johns Hopkins School of Medicine The Role of Rho‐kinase in the Neuropathy of Erectile Dysfunction Introduction and Objectives: RhoA and Rho‐kinase (ROCK) are implicated in the inhibition axonal growth/neurite sprouting, and neurodegeneration via regulation of inflammation and apoptosis following peripheral nerve injury. We hypothesize that degeneration of the cavernous nerve (CN) after injury is due to increased RhoA/ROCK signaling in the major pelvic ganglion (MPG) leading to nNOS uncoupling, inflammation, apoptosis and nerve degeneration. This study aimed to characterize the role of ROCK inhibition in preserving erectile responses, penile contractions, nNOS uncoupling, markers of nerve injury and apoptosis, and neurite outgrowth following bilateral cavernous nerve injury (BCNI). Methods: Male Sprague‐Dawleys rats (12 wks) were separated into sham, BCNI and BCNI treated with Y‐27632 (ROCK inhibitor, 5 mg/kg twice daily; n=16/group). 14 days after BCNI, groups underwent CN stimulation to determine erectile function. Penes were dissected and contractile responses to phenylephrine (PE) and electrical field stimulation (EFS) were assessed. MPGs were excised and protein (Western blots) and/or gene expression (q‐PCR) of ROCK1, ROCK2, RhoA, neuronal nitric oxide synthase (nNOS), caspase1, caspase3, transcription factor 3 (ATF‐3), glial fibrillary acidic protein (GFAP), β‐tubulin3 (BT3), tyrosine hydroxylase (TH), and vesicular acetylcholine transporter (VAChT) were assessed. Additional MPGs (n=3/group) were cultured in reduced growth factor matrigel for 48h and neurite growth was measured. Results: While erectile function was severely decreased in rats with BCNI, daily administration of Y‐
27632 improved erectile responses 2‐fold compared to BCNI (p<0.05). Protein and gene expression of membrane bound RhoA, ROCK1 and ROCK2 were increased in rat MPG after BCNI and were normalized with ROCK inhibition (p<0.05). nNOS MPG gene expression was decreased by 50% after BCNI and recovered to sham levels with Y‐27632 (p<0.05). nNOS protein uncoupling was increased in BCNI MPGs and completely prevented in y‐27632 treated rats (p<0.05). Apoptotic markers caspase1 and caspase3 were significantly elevated after BCNI and were significantly reduced with ROCK inhibition (p<0.05). Nerve injury marker ATF‐3 and Schwann cell activation marker GFAP were significantly elevated after BCNI which corresponded with a decrease in neuronal markers BT3, TH, and VACht (p<0.05). Treatment with Y‐27632 prevented the increase of injury markers and the decrease in neuronal markers. Cavernous contractile responses to PE were unchanged. EFS‐mediated penile contractions were significantly lower following BCNI and treatment with Y‐27632 increased EFS‐mediated contractions 2‐fold compared to sham and BCNI penes (p<0.05). MPG neurite outgrowth was unchanged in sham and BCNI; however, BCNI+Y MPGs had significantly increased growth (S:328±9μm; BCNI:349±14μm; BCNI+Y:405±11μm, p<0.05). GAP43, a growth protein highly expressed during axonal regeneration, was increased 25% following BCNI and 30% with Y‐ 27632 treatment. Johanna Hannan, PhD (cont’d) Conclusion: Inhibition of ROCK preserved erections, prevented nNOS uncoupling, prevented neuronal injury and apoptosis, mediated increased neurite growth and increased nerve‐mediated penile contractions in BCNI rats. Preventing the activation of RhoA/ROCK signaling in the MPG and CN after injury may inhibit neurodegeneration and post‐radical prostatectomy erectile dysfunction. Una J. Lee, MD Virginia Mason Medical Center Determine Whether Vaginal Delivery‐Associated IR Injury Can Be Mitigated By Cytoprotection and Whether Cytoprotection Accelerates Recovery from Vaginal Delivery‐induced SUI Stress urinary incontinence (SUI) affects more than half of women aged 40 to 60, has a greater impact on quality of life than diabetes or arthritis, and costs more than $20 billion annually to treat or manage. The etiology of urinary incontinence is complex and multifactorial. One of the strongest risk factors for SUI is vaginal delivery. Vaginal delivery damages the pelvic floor muscles, nerves, vasculature, and connective tissues, contributing to the development of both urinary and fecal incontinence. One‐third of women develop SUI immediately following vaginal delivery, but in most women this resolves within a year. Importantly, postpartum SUI is a significant risk factor for the development of SUI later in life, suggesting that impaired recovery from childbirth injury contributes to the pathophysiology of SUI. In order to better understand and define the complex pathophysiology of SUI, several animal models have been developed, including simulating birth trauma by vaginal distension in rodents. While hypoxic injury has been demonstrated in the urethra, vagina, and bladder in a rodent model of simulated childbirth injury, the contribution of hypoxia and reperfusion injury to the development of SUI has yet to be investigated. Ischemia‐reperfusion (IR) injury is a significant biologic process demonstrated in multiple organ systems exposed to hypoxic conditions. Upon reperfusion of tissues, a severe inflammatory response is initiated and oxidative stress‐related cellular injury occurs, leading to cell and tissue damage and loss of function. In other organ systems (cardiac, bowel, liver, and kidney), tissue damage can be attenuated by cytoprotective drugs that upregulate heme‐oxygenase‐1 (HO‐1), which is an anti‐inflammatory, anti‐apoptotic, and anti‐proliferative factor involved in the cellular response to IR injury. We propose to investigate the effects of cytoprotective drugs on IR injury in the tissues damaged during childbirth, which may lead to novel therapies for preventing parturition‐induced SUI. Our long‐term goal is to reduce the risk of SUI associated with vaginal delivery. The objective of this project is to determine whether vaginal delivery‐associated IR injury can be mitigated by cytoprotection. The central hypothesis is that IR injury during vaginal delivery causes damage to the urethra, external urethral sphincter, and the connective tissue supporting the urethra, leading to SUI, and the administration of cytoprotective agents at the time of childbirth may mitigate the development of SUI. Our hypothesis has been formulated on the basis of studies of human pregnancy and animal models of urinary incontinence. For instance, in humans, vaginal delivery and longer second stages of labor are associated with increased levels of IR injury markers in serum. This project consists of two aims: Una Lee, MD (cont’d) 1) Determine whether vaginal delivery‐associated IR injury can be mitigated by cytoprotection. 2) Determine whether cytoprotection accelerates recovery from vaginal delivery‐induced SUI. Under the first aim, HO‐1 expression will be pharmacologically upregulated in a rodent model of vaginal distension‐induced SUI to determine whether accelerating the cellular response to oxidative stress protects the tissues from IR injury. Under the second aim, a cytoprotective agent for IR injury will be administered in a rodent model of vaginal distension‐induced SUI to determine whether HO‐1 upregulation accelerates functional recovery from the effects of simulated childbirth. This project is innovative because it represents a paradigm shift in the field by intervening, pharmacologically, at the time of greatest risk to prevent the onset of SUI. The proposed research is significant because elucidating the injury process during vaginal delivery will reveal how SUI may be prevented. Ultimately, intervening at the time of vaginal delivery may address SUI not only in the postpartum period, but also prevent its development in older parous women. Stacy Loeb, MD New York University‐Langone Medical College Does Active Surveillance Miss the Window for Cure? Matched Comparison of Immediate Versus Delayed Prostatectomy in a Nationwide Population‐Based Cohort INTRODUCTION AND OBJECTIVES: Active surveillance is an important option to delay or avoid aggressive treatment in men with localized prostate cancer. However, there is relatively little long term data to confirm that this strategy does not miss the window of opportunity for cure. The objective of our study was to perform a comparison of immediate versus delayed radical prostatectomy (RP) in matched cases from a nationwide population‐based registry. METHODS: From the National Prostate Cancer Register of Sweden, we identified 367 men with low to intermediate risk prostate cancer who underwent delayed RP after a period of surveillance from 1997‐2005. Using year of diagnosis, age at diagnosis, PSA, clinical stage, and Gleason score, these men were matched 1:1 to 367 men who underwent immediate RP (time from diagnosis to RP: 20.7 vs. 3.6 months, p<0.0001). Conditional logistic regression and Cox proportional hazards models were used to compare RP pathology features, biochemical progression, and prostate cancer mortality between groups. RESULTS: Despite similar demographics and clinical characteristics, men undergoing delayed versus immediate RP were significantly less likely to have organ‐confined (51% vs. 64%, p<0.001) and Gleason 6 disease (56% vs. 72%, p<0.0001) at RP. On multivariable analysis adjusting for clinical risk category, Charlson comorbidity index, socioeconomic status, marital status, and educational level, delayed RP was associated with a greater risk of Gleason >=7 at RP (OR 2.14, 95% Cl 1.54‐
2.97, p<0.0001). There were no significant differences in other pathologic features on multivariable analysis. At five years, there were no significant differences in the rate of PSA relapse. There were also no significant differences in prostate cancer‐specific mortality between the two groups at a median follow‐up of 9.5 years. CONCLUSIONS: Men who underwent delayed RP after a period of surveillance had more high‐
grade disease at RP compared to matched men who had immediate RP. However, there was no significant difference in biochemical recurrence or prostate cancer mortality, although the number of deaths from prostate cancer was small. Overall, our data suggest that a strategy of initial active surveillance with selective delayed RP is a viable strategy to reduce overtreatment that does not compromise the opportunity for cure during intermediate follow‐up. SOURSE OF FUNDING: Funding was provided from The Swedish Research Council 825‐2012‐5047 and The Swedish Cancer Alp Sener, MD, PhD Western University Hydrogen Sulphide Supplementation in Renal Transplantation: A Potential Solution for Ischemia Reperfusion Injury The disparity between the number of patients on the renal transplant waiting list and available organ donors have led to an increase in the use of organs procured from more “marginal” donors whose organs are more susceptible to ischemia‐reperfusion injury (IRI) during transplantation. IRI is unavoidable during transplantation and is associated with acute tubular necrosis, increased rates of delayed graft function as well as impaired early and long‐term graft function and survival. Recently, treatment of ischemic tissues with gasotransmitters has become a promising avenue in minimizing IRI. Gasotransmitters, such as nitric oxide and carbon monoxide, are a family of small endogenous gaseous molecules that, while initially thought to only be toxic, are now known to possess many important physiological properties. Hydrogen sulfide (H2S) is the newest member of this family and is produced endogenously at low concentrations in virtually all tissue and is implicated in many physiological functions such as cellular signaling and vasodilation as well as exhibiting a host of anti‐inflammatory, anti‐apoptotic and anti‐oxidant effects. The theme of our research has been to investigate the utility of H2S in mitigating renal IRI during transplantation with the ultimate goal of creating a novel supplement to the standard organ perfusion solutions in order to improve renal graft function and survival following the warm and/or cold IRI experienced by grafts in the peri‐transplant period. To achieve this aim, our two main objectives were to characterize the real‐time protective properties of supplemental H2S against prolonged periods of warm renal IRI as well as determining its protective role in organ preservation during extremes of cold storage and transplantation. In our first series of experiments mimicking warm renal injury, uninephrectomized rats underwent a traumatic clamping of the renal pedicles for 1 hour followed by and 2 hours of reperfusion in the presence of either phosphate buffered saline (PBS; control) or PBS plus 150 μM of the H2S donor, NaHS. Reperfusion of the organs was captured in real time using intravital microscopy, where H2S treated kidneys had significantly improved renal capillary perfusion as well as decreased inflammatory infiltrate compared to control animals. In addition, H2S treated animals exhibited improved serum creatinine levels as well as improved serum markers of systemic inflammation. Histopathologic analysis revealed that H2S treated kidneys exhibited less ATN and tubular apoptosis as well as decreased expression of pro‐inflammatory mediators compared to control kidneys. Overall, we were able to demonstrate, for the first time, that H2S treatment improved real‐time renal vascular flow and function and decreased the renal injury and inflammation associated with prolonged warm renal IRI. Alp Sener, MD, PhD (cont’d) To achieve our second objective, evaluating the protective effects of H2S against renal IRI in the context of prolonged cold IRI associated with renal transplantation, we utilized a murine model of syngeneic renal transplantation. Bilaterally nephrectomized recipient rats received kidneys from genetically identical donors following 24 hours of cold (4°C) storage in standard University of Wisconsin (UW) solution or UW solution plus 150 μM NaHS. Animals receiving UW treated grafts exhibited extremely poor survival after this extreme period of cold storage (0% survival at 5 days post‐op) whereas H2S treatment dramatically improved recipient survival (80% at 14 days post‐
op). H2S treatment also decreased post‐transplant serum creatinine levels to normal levels by post‐operative day 4 and improved graft function, whereas UW treated animals died in an anuric state. Histopathologic analysis revealed that H2S treated kidneys exhibited decreased levels of apoptosis, necrosis and inflammatory infiltrates. In addition, these kidneys showed decreased expression of pro‐inflammatory and pro‐apoptotic genes and increased expression of antiapoptotic genes compared to UW animals. These experiments clearly showed that supplemental H2S treatment during prolonged cold ischemia associated with transplantation dramatically improves renal graft survival and function and mitigates graft injury and inflammation compared to standard UW solution. Taken together our studies have established that exogenous H2S treatment is capable of protecting kidneys from both prolonged warm and cold IRI associated with clinical renal transplant. Considering the increasing use of marginal donors and the increased sharing of organs which has led many centers to utilize grafts at limits of cold storage times in order to accommodate expanding waiting lists, supplemental H2S could represent a novel, potent and cost‐
effective solution for the future success of renal transplantation. Ariana Smith, MD University of Pennsylvania Cortisol Awakening Response and Salivary Cortisol Response to Stress in Women With Overactive Bladder and Controls Introduction: Overactive bladder (OAB) is characterized by frequency, urgency, nocturia and urgency incontinence, and OAB urinary symptoms have been associated with greater subjective psychologic stress. Physiologic measures of stress response modulated by the hypothylamic pituitary axis may contribute to lower urinary tract symptoms in women with overactive bladder (OAB). Methods: Postmenopausal women age 45‐75 diagnosed with OAB were recruited from a tertiary care Urology practice and age matched healthy controls were recruited from a women’s health center. Participants were excluded if they were undergoing treatment for stress reduction, using anxiolytic or hormonal medications, reported pain, urinary infection, bladder cancer, or psychiatric disease. Physiologic measures including salivary cortisol levels, heart rate, and urinary markers as well as validated symptom assessment for stress, anxiety, depression, and bladder dysfunction were obtained at baseline and at the time of induced psychosocial stress. The Trier Social Stress Test (TSST) was used as a motivated performance task for the induction of psychological stress. A two‐day cortisol awakening response (CAR) and baseline salivary cortisol samples were obtained in all participants prior to TSST (T‐30, T‐15 minutes )and at the TSST visit (T=0, T+30, T+40, T+50 minutes). CAR, mean cortisol levels and cortisol area under the curve (AUC) were calculated. Independent sample T‐tests compared CAR in OABs and controls, and analysis of variance (ANOVA) compared cortisol AUC pre‐ and post‐ TSST in OABs and controls. Validated questionnaires, visual analog scales (VAS) for urgency, voiding diaries, and urinary samples were obtained pre and post‐TSST. Independent T‐tests compared OABs and controls. Results: Eleven patients with OAB and 7 controls completed the performance task and provided all requested questionnaire, salivary and urinary samples. Voiding diary confirmed OAB symptoms in patients and lack of symptoms in controls. Heart rate(HR) monitoring confirmed physiologic response to TSST in both OAB patients and controls. No difference in HR was detected between groups. No difference in CAR was detected between groups (p=0.97). Regarding psychosocial stress response, controls showed a 2.2% mean cortisol decrease from pre‐ to post‐TSST, while OABs showed a 72.2% increase (Figure 1). The TSST produced a significant increase in cortisol AUC (p = 0.001) with no significant effect of OAB status (p = 0.26), although controls showed a 3.8‐fold cortisol AUC increase and OABs showed a 6.5‐fold cortisol AUC increase. VAS revealed an increase in pressure in the bladder in OAB patients post TSST (p=0.04). State Trait Anxiety Inventory showed that patients with OAB are more anxious than controls around the time of TSST, (p=0.05). Ariana Smith, MD (cont’d) Conclusion: Women with OAB have similar baseline physiologic measures of stress response as controls, reflected in CAR. Women with OAB may have greater physiologic responsiveness to stress as measured by salivary cortisol following TSST, greater anxiety, and exacerbation of bladder pressure. Evaluation of markers of stress response may provide targets for potential diagnostic and therapeutic interventions. Funding: This work was funded by NIH/NIDDK O’Brien Urology Center Grant P50 DK052620‐15 Li Xin, PhD Baylor College of Medicine Prostate Epithelial Lineage Hierarchy A major therapeutic challenge for prostate cancer is to distinguish indolent cancer from aggressive diseases that need immediate therapeutic intervention. One of the prevailing hypotheses is that the identity of cells of origin for prostate cancers can determine their malignant potentials. A major project in my laboratory is to understand the lineage hierarchy in the prostate, which serves as a prerequisite to reveal the identities of the cells of origin for prostate cancer. When I worked at UCLA with Owen Witte, I established a dissociated prostate cell regeneration assay by modifying a classic tissue fragment recombination assay, making it a reliable assay to monitor the capacities of stem cells in the prostate for multi‐lineage differentiation and self‐
renewal. In this assay, dissociated single cells from adult murine prostate tissues are mixed with urogenital sinus mesenchymal (UGSM) cells derived from midgestation embryos and transplanted under the renal capsules of immunodeficient mice. I showed that only a fraction of prostate cells possessed the stem cell capacity to regenerate prostate tissues de novo in this assay. I, along with my colleagues, managed to use flow cytometry to fractionate individual cell lineages (basal epithelial cells, luminal epithelial cells, and stromal cells) in the prostate based on their surface antigen expression profiles, and demonstrated that FACS‐sorted prostate basal cells carry the stem cell activity. A major limitation of the dissociated prostate regeneration assay is that prostate cells were taken out of their native microenvironment and stimulated by embryonically derived UGSM cells that have strong reprogramming capacity. It was not clear if the regenerative capacity that basal cells displayed in this assay contributes to the maintenance of prostate epithelia substantially in vivo. In addition, luminal cells do not proliferate in this assay. Therefore, it was not clear if they are also capable of serving as one cell‐of‐origin for prostate cancer. To overcome these limitations, my laboratory obtained and generated mouse models through which we can specifically label basal cells or luminal cells with fluorescence reporters so that we can track their fates. In this way, we assayed the biology of the cells in their native microenvironment. In contrast to the observation in the prostate regeneration assay, we found unexpectedly that prostate basal and luminal cell lineages are self‐sustained independently under physiological conditions. I reasoned that the distinct behaviors of prostate basal cells in these studies reflect the different experimental conditions of the two assays. The experimental conditions in the regeneration assay resemble some processes that also occur during tissue inflammation, which is an important factor that promotes the initiation of prostate‐related disorders. To determine whether inflammation affects the maintenance of prostate lineage hierarchy and the initiation of prostate cancer, my Li Xin, PhD (cont’d) laboratory further performed the lineage tracing study in the context of a uropathogenic bacteria induced mouse model for prostatitis. We showed that prostate inflammation drives differentiation of basal cells into luminal cells. Collectively, these studies reveal that prostate tissues use different mechanisms to maintain tissue homeostasis under physiological and pathological conditions, and unify the seemingly contradictory previous studies from several independent labs in this field.
Research Forum Poster Abstracts Eugene K. Lee, MD University of Kansas KU675, a Concomitant Heat Shock Protein Inhibitor of HSC70 and Hsp90 that Manifests Isoform Selectivity for Hsp90α Introduction: Prostate cancer is the most common cancer in men and second leading cause of cancer death in the United States. Since 1952, when the susceptibility of prostate cancer to androgen (AR) withdrawal was first reported by Huggins and Hodges, patients with advanced prostate cancer have traditionally been treated with inhibitors of the androgen receptor pathway. Despite dramatic initial clinical response, androgen withdrawal is inefficient at the treatment of metastatic disease and many patients fail these androgen‐
targeted therapies. Although recent trials demonstrate a modest survival advantage with novel agents, the long‐term effectiveness remains limited, and a critical need for innovative therapies to treat prostate cancer remains. The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N‐terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, we report the characterization of a C‐terminal Hsp90 inhibitor, KU675, which demonstrates anti‐cancer activity in prostate cancer cells in the absence of a HSR and displays relative selective inhibition of the Hsp90α isoform and Hsc70. Methods: Hormone dependent and refractory prostate cancer cells were used in both direct and indirect in vitro Hsp90 inhibition assays (antiproliferative, luciferase refolding, and Western blot for client protein degradation) to characterize the effects of KU675 in PC3‐
MM2 and LNCaP‐LN3 cell lines. Direct binding studies were conducted using fluorescence spectroscopy and Hsp90α, Hsp90β and Hsc70 recombinant proteins. Results: KU675 exhibits anti‐proliferative and cytotoxic activity along with client protein degradation without induction of the heat shock response (HSR) in both prostate cancer cell lines. Furthermore, KU675 demonstrates direct inhibition of Hsp90 complexes as measured by the inhibition of luciferase refolding in cancer cells. In direct binding studies, the internal Eugene K. Lee, MD (cont’d) fluorescence signal of KU675 was used with fluorescence spectroscopy to measure the binding constant (KD) of KU675 to recombinant Hsp90α, Hsp90β and Hsc70. The KD for Hsp90α was determined to be 191μM, while the KD for Hsp90β is 726 μM demonstrating the relative isoform selectivity of KU675. Western blot experiments with cell lines treated with KU675 or shRNA knockdown of Hsp90α or Hsp90β support the isoform selectivity binding data by revealing the degradation of Hsp90α specific client proteins. KU675 also displays binding to HSC70 with a KD value at 76.3uM, which is supported by decreased expression of HSC70 specific client proteins in Western blot studies. Conclusion: Overall, these findings suggest the possibility of designing isoform selective inhibitors of Hsp90 as well as dual inhibitors of other heat shock proteins for the treatment of cancer. Luis Braga, MD McMaster University Time to Febrile Urinary Tract Infection (fUTI) in Children with Antenatal Hydronephrosis (AHN): A Prospective Study INTRODUCTION AND OBJECTIVES: According to our previous AHN study, female gender, uncircumcised boys and those with hydroureteronephrosis (HUN) without vesicoureteral reflux (VUR) seemed to be at a higher risk of developing fUTI. Due to intrinsic retrospective data limitations, a time to event analysis was not possible and the role of CAP and VUR could not be properly evaluated. Therefore, we sought to verify the impact of those factors on fUTI rates in infants with AHN, in a prospective fashion. METHODS: Pts seen at a tertiary children’s hospital for postnatal management of AHN from Jun 10‐Oct 13 were prospectively followed. Those with postnatally detected hydronephrosis (HN), posterior urethral valves, duplication anomalies and neurogenic bladders were excluded. The primary outcome was fUTI, defined as a catheter specimen, >100000 single organism urine culture, with temp >38°C. Seven a priori risk factors were explored: age, AHN grade [low (I‐II) vs. high (III‐IV)], etiology (isolated HN vs. HUN), CAP use, VUR grade, gender, and circumcision. Univariate analysis was conducted to identify potential fUTI risk factors. Time to fUTI curves analyzed by Cox proportional regression (Hazard ratio ‐ HR) were generated to adjust for confounders. RESULTS: Data on 328 pts (77% male) were collected, of whom 61 (19%) had a fUTI. Median [IQR] age at fUTI was 6 [2,11] mos. High grade AHN was present in 195 (60%) infants. CAP was prescribed for 122 (37%) pts. Of pts on CAP, 68% had high grade AHN. Of 217 pts who had a VCUG, 63 (29%) had VUR (44: IV‐V, 17:I‐III), 2/3 of VUR pts were on CAP. Circumcision was performed in 82 (32%) boys. Table shows univariate analysis results. In the Cox proportional regression model, female gender (HR=2.7, p=0.03), uncircumcised males (HR=2.8, p=0.03) (Fig), VUR (HR=11, p<0.01) (Fig) and lack of CAP (HR=6.3, p<0.01) remained significantly associated with fUTI. A sensitivity analysis excluding VUR pts showed that HUN was a significant risk factor as well (HR=3.8, p<0.01). HN grade was not found to be associated with fUTI once all covariates were adjusted for. CONLUSIONS: Females and uncircumcised boys with HUN without VUR had significantly higher fUTI rates and should probably benefit from CAP use. SOURSE OF FUNDING: none Brian Eisner, MD Massachusetts General Hospital, Harvard Medical School The Epidemiology of Stone Disease Nephrolithaisis is a common cause of morbidity, with a lifetime prevalence in the United States estimated at nearly 10%.1 Historically, nephrolithaisis was considered a disorder of urine chemistry, but within the last 2 decades, epidemiologic studies have demonstrated associations between nephrolithaisis and various systemic diseases, including hypertension, diabetes mellitus, and cardiovascular disease.2‐5 Recent epidemiologic studies have demonstrated that kidney stone history is a risk factor for future end‐stage renal disease (ESRD). 6, 7 Oxalate nephropathy has been implicated in the pathogenesis of ESRD for patients with the rare genetic disorders known as primary hyperoxaluria as well as in patients who have undergone bariatric surgery.8‐10 However, little is known about the mechanisms underlying risk of ESRD in the common stone‐ former. The goal of the my current research on this topic was to examine the relationship between kidney stone disease and risk of ESRD in a large cohort representative of the US population as well as to better understand the potential mechanisms governing such relationships. Study # 1 (data collection completed, submitted to 2014 AUA): The National Health and Nutrition Examination Survey (NHANES 2008‐2010) database was interrogated for patients with a history of kidney stones. Those with and without stones were evaluated in a multivariate analysis. Kidney stone disease was associated with a significant increase in risk of developing chronic kidney disease (OR 1.42, 95% CI 1.04 to 1.93) and the need for dialysis (OR 2.25, 95 CI 1.09 to 4.62). These data confirm findings of prior studies using other patient cohorts using a cohort (NHANES) which was designed to be representative of the US population. Study #2 (data collection in progress): One plausible mechanism for the findings described in Study #1 is oxalate nephropathy. Although oxalate has been implicated in patients with primary hyperoxaluria and bariatric surgery, urine oxalate has not been shown to cause renal dysfunction in populations of “common stone‐formers.” To examine this hypothesis, we have developed a database of over 500 stone‐formers seen in our clinic in the past 4 years and will examine abnormalities of 24‐hour urine composition and estimated glomerular filtration rate while adjusting for co‐variates associated with renal dysfunction. Brian Eisner, MD (cont’d) Based on the findings from the aforementioned studies, prospective patient studies as well as animal model studies could be used to further evaluate the relationship between kidney stones and renal dysfunction. REFERENCES 1. Scales CD,Jr, Smith AC, Hanley JM et al: Prevalence of kidney stones in the United States. Eur Urol 2012; 62: 160. 2. Reiner AP, Kahn A, Eisner BH et al: Kidney Stones and Subclinical Atherosclerosis in Young Adults: The CARDIA Study. J Urol 2011; 185: 920. 3. Madore F, Stampfer MJ, Rimm EB et al: Nephrolithiasis and risk of hypertension. Am J Hypertens 1998; 11: 46. 4. Taylor EN, Stampfer MJ, Curhan GC: Diabetes mellitus and the risk of nephrolithiasis. Kidney Int 2005; 68: 1230. 5. Sakhaee K: Nephrolithiasis as a systemic disorder. Curr Opin Nephrol Hypertens 2008; 17: 304. 6. Rule AD, Bergstralh EJ, Melton LJ,3rd et al: Kidney stones and the risk for chronic kidney disease. Clin J Am Soc Nephrol 2009; 4: 804. 7. Rule AD, Krambeck AE, Lieske JC: Chronic kidney disease in kidney stone formers. Clin J Am Soc Nephrol 2011; 6: 2069. 8. Nelson WK, Houghton SG, Milliner DS et al: Enteric hyperoxaluria, nephrolithiasis, and oxalate nephropathy: potentially serious and unappreciated complications of Roux‐en‐
Y gastric bypass. Surg Obes Relat Dis 2005; 1: 481. 9. Lorenz EC, Michet CJ, Milliner DS et al: Update on oxalate crystal disease. Curr Rheumatol Rep 2013; 15: 340. 10. Kurts C: A crystal‐clear mechanism of chronic kidney disease. Kidney Int 2013; 84: 859. Dirk Lange, PhD University of British Columbia A Role for Developmental Pathways in Mediating Ureteral Smooth Muscle Dysfunction and Aperistalsis in the Stented Ureter Background: Ureteral dysfunction is associated with serious complications including severe hydronephrosis, renal damage, bladder dysfunction and infection. Ureteral peristalsis is a physiologic mechanism ensuring the unidirectional transport of urine from the kidney to the bladder, mediated by coordinated smooth muscle (SM) contractions initiated via pacemaker cells in the renal pelvis and subsequently transmitted distally along the ureter. Ureteral dysfunction is related to changes in the contractility of ureteral SM and research in this area is focused on studying morphological and physiological changes involving SM contractile machinery. While such studies have provided valuable insight into functional abnormalities in ureteral SM, the specific mechanisms leading to these changes remain unknown. We believe that a better understanding of specific signaling mechanisms that are the underlying cause of the mechanistic and physiological abnormalities in ureteral SM is important for the identification of potential targets for future therapeutic agents. The Sonic Hedgehog (Shh) signaling pathway plays a significant role in the development of the urinary system during embryogenesis. Specifically, it maintains ureteral function during development, by regulating the development of pacemaker and ureteral SM cells, both of which are required for coordinated ureteral peristalsis. Inactivation of this pathway results in the loss of ureteral peristalsis and promotes the formation of hydroureter and hydronephrosis. While the role of Hedgehog signaling in the developing ureter has been well established, its role in the maintenance of ureteral peristalsis in the developed urinary system remains unknown. Study Overview: In a recent study identifying molecular mechanisms that drive stent‐associated ureteral aperistalsis we have identified a possible role for Hh signaling in the maintenance of ureteral SM function in the developed ureter. For this study, eight farm pigs underwent unilateral Double J stent insertion, while four additional animals served as unstented controls. At 48 hours and 7 days post stent insertion, ureteral peristalsis was assessed macroscopically pre‐ and post stent removal and SM function was assessed ex vivo using tissue baths. Overall, peristalsis was found to be absent and SM function was significantly decreased stented versus non‐stented contralateral ureters in each animal and unstented control animals. Interestingly, ureteral aperistalsis coincided with the loss of Gli1 expression in ureteral SMCs at 7 days but not 48 hours suggesting additional mechanisms at early stages of ureteral stenting. Epithelial Shh expression did not change. Dirk Lange, PhD (cont’d) To determine whether the loss of SM Gli1 expression was also present in long‐ term stented animals, we assessed the expression of Shh and Gli 1, in proximal ureteral tissue samples from pigs stented for 2, 4, 7 and 10 weeks from a previous study. The 10‐ week animals had their stents removed at week 7, allowing for the study of Shh and Gli1 expression following 3 weeks of recovery. Overall we found a decrease in Gli1 expression in the ureteral SM layers of stented ureters as the degree of inflammation increased. Over time, as the degree of inflammation decreased, Gli‐1 expression in the SM layers of the stented ureters increased. Stent removal at week 7 followed by a 3‐week recovery period resulted in the recovery ofGli1 expression towards unstented levels. Interestingly, epithelial Shh expression was unaffected. Conclusions: Our work suggests a role for developmental pathways in mediating ureteral SM dysfunction and aperistalsis. While the current findings pertain to stent‐induced ureteral dysfunction, they do raise the possibility that developmental pathways also mediate SM dysfunction associated with other urologic disorders. Jay Raman, MD Penn State‐ Milton S. Hershey Medical Center Povidone Iodine Rectal Preparation to Reduce Infectious Complications Following Prostate Needle Biopsy Transrectal ultrasound guided prostate needle biopsy (TRUS PNB) is the referent method for obtaining prostate tissue for histologic assessment of prostate cancer. Each biopsy core requires piercing through the rectal mucosa into the highly vascular prostate which can inherently contribute to infection. Fluoroquinolones antibiotics have historically provided optimal prophylaxis prior to TRUS PNB. The emergence of fluoroquinolone resistant organisms has risen to almost 20‐25%. Not surprisingly, data on infectious complications following TRUS PNB highlight an increasing rate of infection and sepsis. At our medical center, the sepsis rate rose precipitously in 2011 to 4.2%. Given the scope of the problem, innovative strategies became necessary. Our approach involved use of topical agents as opposed to broadening systemic coverage. Povidone‐iodine is a commonly used topical antiseptic that dramatically reduces microorganism colonies when applied to a surgical site. Rectal cleansing with povidone‐iodine has been demonstrated to be safe and effective in reducing rectal flora counts prior to colon procedures. Within our institution, a 1‐year quality improvement measure implementing povidone iodine rectal preparation (PIRP) prior to TRUS PNB demonstrated a 2% reduction in culture positive febrile infections. Based upon these preliminary observations, we designed a prospective study investigating PIRP versus standard of care prophylaxis prior to TRUS PNB (Figure). Our specific project objectives (with data acquired to date) were as follows: Objective 1 ‐ Characterize the magnitude of decrease in rectal vault bacterial counts before and after povidone iodine treatment. Rectal cultures before and after PIRP administration noted a 97% reduction in microorganism colonies (2.1 x 105 CFU/ml vs. 6.3 x 103 CFU/ml, p < 0.001). Objective 2 ‐ Quantify urine and blood bacterial counts after biopsy in patients receiving povidone iodine rectal preparation versus current standard of care. Urine bacterial counts following TRUS PNB were 1 CFU/mL PIRP vs. 6 CFU/ml for standard. Blood bacterial counts following TRUS PNB were 0 CFU/mL PIRP vs. 1 CFU/mL for standard of care. Objective 3 ‐ Determine differences in clinical sepsis events between patients receiving povidone iodine preparation versus standard of care. Initial data indicates a 0% sepsis rate in PIRP patients versus a 5.5% rate in standard of care TRUS PNB patients. Jay Raman, MD (cont’d) Objective 4 ‐ Assess patient tolerability and side effects from PIRP. No side effects from mucosal exposure of PIRP were noted on phone questionnaire 7 days following treatment. While our data collection continues, we believe that our strategy is effective with strong scientific merit as highlighted by the bacteriology data shown above. Furthermore, the approach is cheap, easily reproducible, and does not subject the patients to additional systemic antibiotics which may further foster resistant organisms. Fubo Wang, MD, PhD Second Military Medical University‐ Shanghai Changhai Hospital, China Urine Long Noncoding RNA Panel to Predict Diagnosis of Prostate Cancer Introduction: It is urgently needed to develop novel biomarkers to supplement PSA testing. Urine long noncoding RNA (lncRNA) emerged as a potent biomarker in detecting cancer and other human disease. However, the role of lncRNAs in urine to diagnose prostate cancer (PCa) was not studied systemically in the large Chinese cohorts. Objective: We sought to investigate lncRNA expression profiles by RNA‐Seq and aimed to identify a urine lncRNA panel to predict diagnosis of PCa with PSA=4‐20 ng/ml. Methods: lncRNA expression profiles were investigated with 66 pairs of PCa and matched adjacent normal tissues by RNA‐Seq. Quantitative reverse‐transcriptase polymerase chain reaction (qRT‐
PCR) assay was then applied to evaluate the expression of selected lncRNAs in 120 paired PCa tissues and 90 benign prostatic hyperplasia (BPH). A logistic regression model was constructed using an independent cohort (n=345) and then validated using an independent cohort (n=300). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. Results: RNA‐Seq revealed several novel differentially expressed lncRNAs, which could be good candidates for PCa diagnosis. Post‐DRE urine sediment provided informative RNA in 645 patients. Then, we identified a lncRNA panel (include 5 novel lncRNAs) that provided a high diagnostic accuracy of discriminating positive biopsies from negative biopsies (AUC‐ROC = 0.842 and 0.854 for training and validation data set, respectively). The lncRNA panel is more effective for detecting positive biopsies from negative biopsies than PCA3 score alone (p < 0.01). In the diagnostic “gray zone”, the lncRNA panel also show a good diagnostic performance to discriminate positive biopsies from negative biopsies (AUC‐ROC = 0.837 for the panel versus AUC‐ROC = 0.673 for PCA3 score). Conclusions: The lncRNA panel in urine sediments after DRE was found to be a good predictor of PCa in the initial prostate biopsy in Chinese population. Further large‐scale multicenter studies in China are needed to confirm our findings. Urology Care Foundation Scholar Abstracts Philip Abbosh, MD, PhD Fox Chase Cancer Center The Role of PBRM1 Mutation in Renal Cell Carcinoma Clear cell renal cell carcinoma (ccRCC) is known to derive from a series of mutations, most notably the Von Hippel Lindau tumor suppressor gene (VHL). However, VHL mutations alone are insufficient for carcinogenesis. Recently, multiple large‐scale exomic sequencing of ccRCC specimens and cell lines has uncovered a cornucopia of mutations. Interestingly, many of these mutations are in chromatin‐modifying genes and occur concomitantly with VHL mutation, suggesting that mutation of these genes occur in a stepwise fashion and together cooperate to cause cancer. The most commonly mutated chromatin‐modifying gene, PBRM1, is mutated in about 40% of specimens. PBRM1 encodes a subunit of the highly conserved Switch/Sucrose Non‐
Fermenting (SWI/SNF) complex. SWI/SNF’s best understood role is to remodel chromatin and primarily activate and but at times to repress genes which it targets. In multiple developmental and cancer models, the SWI/SNF and Polycomb group (PcG) activities antagonize each other, whereby loss of one activity promotes expansion of the action of the other complex. PcG is an evolutionarily conserved complex which mediates gene silencing via methylation of histone tails. Expansion of PcG activity has been demonstrated repeatedly in RCC; no mechanism as to why its activity is expanded has been proposed. If the same antagonistic relationship which is present in multiple SWI/SNF and PcG model systems is present in ccRCC, then exploration of this relationship might provide novel platforms on which to base prevention, early detection, and treatment algorithms for patients. The hypothesis of this project is that PBRM1 loss allows PcG activity to expand and that this will have significant implications into the biology of RCC. I plan to explore and exploit the effect of the loss of SWI/SNF activity via loss of PBRM1 by mutation in ccRCC. This project addresses multiple research items in the National Urology Research Agenda including understanding the mechanism of carcinogenesis, functional epigenetics and DNA hypermethylation in RCC, and development of animal models of disease. Jennifer Bishop, PhD Vancouver Prostate Centre Androgen Receptor Regulated Emergence of Tumor Cell Heterogeneity during Enzalutamide Resistance Jennifer L. Bishop1, Kirsi Ketola1, Barinder Sangha1, Arkijimal Angeles1, Alexander Sio1, Ka Mun Nip1 and Amina Zoubeidi1,2 1. Vancouver Prostate Centre, Vancouver BC 2. Department of Urologic Sciences, University of British Columbia, Vancouver BC Background: The rapid recurrence of Enzalutamide (ENZ) resistant tumors in castration‐resistant prostate cancer (CRPC) patients underscores the need for improved therapies. Recent evidence suggests that continued androgen‐deprivation therapy may promote the emergence of more aggressive tumor cell types, including highly metastatic cells, stem cells (CSC) or neuroendocrine (NE)‐like cells. Our data indicates that the level of AR activity in ENZ resistant cells is associated with phenotypic plasticity of these cells; thus, we hypothesize that ENZ initiates distinct, AR‐
controlled, differentiation programs in subsets of cancer cells that promote progression of resistant disease. Methods: ENZ resistant cells were established from castration‐resistant LNCaP xenografts that recurred under ENZ treatment in vivo, characterized for expression of AR and PSA, and genetically profiled in an unbiased manner using Illumina Platform gene expression analysis. Increased expression of select genes and cellular phenotypes were confirmed by qRT‐PCR, flow cytometric analysis, protein expression levels and in vitro and in vivo assays for metastatic capacity. Results: Genes involved in pluripotency, NE differentiation and immune regulation, were upregulated in PSA‐low cells and CSC, NE and immune‐evasive cell phenotypes were validated by flow cytometry and qPCR analysis. Increased frequency in cell surface expression of CSC markers CD44, CD133 and CD117, as well as immune checkpoint molecules PD‐L1 and 2, ICOSL and B7H3, were observed in PSA‐low resistant cells compared to CRPC, as were terminal markers of NE differentiation including NSE, SYP and CgA. In contrast, PSA‐high ENZ resistant cells did not display CSC, NE or immune‐evasive signatures, but displayed epithelial‐to‐mesenchymal (EMT) characteristics in vivo and in vitro compared to CRPC, suggesting AR activity regulates phenotypic plasticity in resistant cells. Genes involved in the GLI and FOXM1developmental pathways, which govern stemness as well as EMT, were overexpressed in PSA‐low and high cell lines, suggesting common pathways may regulate phenotypic heterogeneity that emerges during treatment resistance. Conclusions: Our data indicates that level of AR activity in ENZ resistant cell lines may govern their phenotypic plasticity. Thus, elucidating the molecular mechanisms that govern aggressive cell phenotypes, and establishing their importance in prostate disease progression, may guide clinical evaluation of compounds targeting cellular heterogeneity associated with ENZ resistant CRPC to improve current standard of care. Saunders Ching, PhD University of California, San Francisco Coordinated Activity of Spry1 and Spry2 is Required for Normal Development of the External Genitalia Development of the mammalian external genitalia is controlled by a complex network of signaling molecules and transcription factors. Because FGF signaling plays a central role in this complicated morphogenetic process, we investigated the role of Sprouty genes, which are important intracellular modulators of FGF signaling, during embryonic development of the external genitalia in mice. We found that Sprouty genes are expressed by the urethral epithelium during embryogenesis, and that they have a critical function during urethral canalization and fusion. Development of the genital tubercle (GT), the anlage to the prepuce and glans penis in males and glans clitoris in females, was severely affected in male embryos carrying null alleles of both Spry1 and Spry2. In Spry1–/–;Spry2–/– embryos, the internal tubular urethra was absent, and urothelial morphology and organization was abnormal. These effects were due, in part, to elevated levels of epithelial cell proliferation in Spry1–/–;Spry2–/– embryos. Despite changes in overall organization, terminal differentiation of the urothelium was not significantly affected. Characterization of the molecular pathways that regulate normal GT development confirmed that deletion of Sprouty genes leads to elevated FGF signaling. However, levels of signaling in other cascades were largely preserved. Together, these results show that levels of FGF signaling must be tightly regulated during embryonic development of the external genitalia in mice, and that Sprouty gene products play an important role in this regulation. Irina Debnath, PhD New York University School of Medicine Regulation of Proteus mirabilis virulence by MrpJ INTRODUCTION AND OBJECTIVES: Proteus mirabilis is a major cause of catheter‐associated urinary tract infections (UTIs), which affect a large portion of our aging population in long‐term care facilities and hospitalized patients with indwelling urinary catheters. In addition to severe bladder and kidney infections, this bacterium manifests increasing levels of antibiotic resistance and causes urolithiasis, further complicating disease progression and treatment. Genomic sequencing of P. mirabilis clinical isolate HI4320 established the presence of 17 chaperone‐usher fimbriae. The majority of these operons encode a transcriptional regulator that represses flagella. Expression of mrpJ, the prototypical member of these regulators, is highly upregulated in vivo; furthermore, a mutation of this gene renders P. mirabilis unable to compete in a mouse model of UTI. The objective of this work is to define the targets and mechanism behind MrpJ‐mediated virulence. METHODS: To determine the targets of MrpJ‐mediated transcriptional regulation, we have adopted two complementary approaches: 1) A cDNA microarray analysis of wild‐type HI4320, HI4320ΔmrpJ and HI4320 with mrpJ in trans (mimics expression level during experimental UTI). 2) A ChIP‐seq study (chromatin immunoprecipitation with massively parallel DNA sequencing) of MrpJ using a strain expressing Histagged MrpJ in trans. RESULTS: Our microarray analysis discovered evidence for MrpJ regulation of several known and predicted virulence factors including its own mrp fimbrial operon, other fimbriae, swarmer cell differentiation factors, Pta toxin, ZapA protease, and a type VI secretion system (T6SS). This microarray analysis was followed by quantitative RT‐PCR verification of candidate genes. Our ChIP results further demonstrate the direct regulation by MrpJ of flagellar regulator flhDC and the mrp operon. ChIP‐seq is currently underway to determine the direct genome‐wide targets of MrpJ regulation. CONCLUSIONS: Our findings indicate that MrpJ is a master regulator of virulence for P. mirabilis. This study will lead to molecular level identification of virulence genes governed by transcriptional regulator MrpJ, an essential virulence factor during P. mirabilis‐mediated UTI. Comparison of the microarray and ChIP‐seq data will enable us to pinpoint the direct and indirect gene regulatory network resulting in the discovery of novel virulence genes and set the stage for future therapeutic targeting. Source of Funding: NIH/NIAID K22 AI083743 (M.M.P.) Arindam Ghosh, PhD University of Alabama at Birmingham mTOR Regulation of Renal Cancer Kidney cancer is one of the 10 most common types of cancer in men and women affecting over 300,000 people in the US. The PI3K/Akt/mTOR pathway is critical for tumorigenesis and genetic alterations of components in this pathway have been reported in a variety of tumors. An established downstream target of PI3K signaling is the mammalian target of rapamycin (mTOR), an atypical serine/threonine protein that assimilates into two distinct multiprotein complexes mTORC1 and mTORC2. mTOR has emerged as a biologically significant pathway in RCC as several studies have reported an increase in mTOR signaling in renal cancers in comparison to normal renal tissue. Recent deep sequencing efforts of RCC samples have identified mutations in mTOR. However, these mutations have not been examined. In this study, we aim to characterize the functional significance of these point mutations with regard to tumorigenic phenotypes and their effects on mTOR regulation and signaling. In addition, our preliminary data suggests that mTORC2 signaling is activated in RCC cell lines and tissue samples, and we have recently reported a role for mTORC2 in the regulation of S‐phase kinase‐associated protein 2 (SKP‐2) in RCC cells. In thus study we aim to assess the role of the mTOR/SKP‐2 axis in modulating levels of FOXO1/3a, well known tumor suppressors that are down regulated in RCC. The overall goal of this project is to define the mechanisms by which mTOR is activated in RCC and the relevant tumorigenic pathways modulated by this signaling pathway. Catherine A. Gordon, PhD Stanford University NuSAP is Regulated by RB1 and Modulates Prostate Cancer Progression We have recently shown that nucleolar and spindle‐associated protein (NUSAP1; encoding NuSAP) is overexpressed in recurrent prostate cancer tumors, and validated this finding in independent prostate cancer datasets. Although NuSAP is essential for cell cycle progression, binding to and stabilizing microtubules during mitosis, little is known about its role in prostate cancer progression. Hence, in this study, we performed an extensive analysis to understand the role of NuSAP in aggressive prostate cancer. We first aimed to identify a mechanism leading to NuSAP upregulation. In our previous study, we found that E2F1 (E2F transcription factor‐1) directly binds to the promoter of NUSAP1, enhancing NUSAP1 expression. Since E2F1 is known to be negatively regulated by the retinoblastoma protein (RB1), and RB1 is frequently lost in aggressive prostate cancer, we hypothesized that the expression of NuSAP is regulated, at least in part, by the RB1/E2F1 axis. We confirmed that NUSAP1 transcripts are anti‐correlated with RB1 transcripts and correlated with E2F1 transcripts in prostate cancer microarray datasets, and then used lentiviral‐
based technology to knock down RB1 in human prostate cancer cell lines. RT‐qPCR and western blot revealed that NuSAP expression increases upon knockdown of RB1, and decreases upon further knock down of E2F1, supporting the notion that NuSAP is regulated by the RB1/E2F1 axis. We next aimed to determine the function of NuSAP in prostate cancer progression. Using lentiviral‐based technology, we knocked down and overexpressed NuSAP in prostate cancer cell lines, and examined proliferation, invasion, apoptosis, cell cycle stages, migration, and global gene expression. Proliferation, invasion, and scratch assays revealed that knock down of NuSAP reduces proliferation, invasion, and migration of prostate cancer cell lines, while overexpression of NuSAP increases invasion and migration of prostate cancer cell lines. Flow cytometry analyses revealed that knockdown of NuSAP leads to an increase in the number of apoptotic cells and number of cells in the G2/M phase of the cell cycle. RNA‐Seq analysis further revealed genes significantly differentially expressed upon knockdown or overexpression of NuSAP. Of note, genes involved in invasion, cellular movement, angiogenesis, and metastasis are activated upon overexpression of NuSAP. Examination of microarray datasets additionally revealed that NUSAP1 transcripts are increased in metastatic prostate tumors compared to primary prostate tumors and normal prostate tissue. Taken together, our work provides a mechanism accounting for NuSAP overexpression in prostate cancer, and identifies novel functions of NuSAP in aggressive prostate cancer. Deciphering the role of NuSAP in prostate cancer progression may lead to new ways to prognosticate and treat the aggressive forms of prostate cancer. Chunming Guo, PhD Boston Children’s Hospital and Harvard Medical School A Mouse Model of Urinary Incontinence Abstract: Embryonic and juvenile disorders often predispose adult onset diseases such as urinary incontinence (UI). UI is one of the most common chronic health problems especially in women and senior citizens. Despite the importance, molecular pathophysiology of this disease is poorly understood. The human Urofacial Syndrome (UFS) is a rare autosomal recessive disease characterized by dysfunctional urinations, including UI, frequency and urgency. These conditions are often coupled with repeated episodes of urinary tract infections, and could gradually progress to upper tract damage and eventually to life threatening renal failure. Several independent group have identified HPSE2 is a UFS disease gene. We have successfully generated a mouse Hpse2 mutant line. This is the first and the only mouse model for studying UFS. We reasoned that genetically defined mouse model, such as Hpse2 mutant, would allow us to study specific aspects of molecular and cellular basis of complex urological disorder including UI, which would otherwise be prohibitively difficulty to study because of the etiological complexity. Hpse2 mutants die at the weaning age. The exact cause of lethality is not further investigated but is likely due to malnutrition and renal damage. Hpse2 mutant bladders exhibit increased levels of fibrosis. Ongoing studies are designed to determine whether and how Hpse2 regulates signal pathways related to bladder remodeling and hyperactivity. Andrew Hau, PhD University of California, San Diego uPAR and mTORC2: Coupled Targets for Therapeutics Development in Bladder Cancer Bladder cancer currently ranks as the fifth most common and the single most expensive cancer in the United States. The primary treatment for patients with muscle‐invasive bladder cancer includes radical cystectomy and bilateral regional lymph node dissection. However, for patients with bladder cancer, it is well established that invasive behavior is a major predictor for diminished outcomes due to high recurrence and distant metastasis. Therefore, new targeted therapies for bladder cancer and a better understanding of the molecular mechanisms governing invasive behavior are urgently needed. We have previously demonstrated that mammalian target of rapamycin complex 2 (mTORC2) signaling is a critical determinant of bladder cancer invasion and its activity is correlated with an invasive phenotype in patient specimens. Presently, we have identified a potential link between mTORC2 and the urokinase receptor (uPAR), a signaling receptor that is frequently found to be upregulated in many invasive tumor types but has not been characterized in detail in bladder cancer. The objective of this study is to further delineate the signaling interplay between mTORC2 and uPAR in bladder cancer, and to investigate the efficacy of recently developed uPAR‐specific human antibodies, with and without mTORC2 inhibition to test for synergistic or additive effects, on tumor invasion in orthotopic xenograft mouse models. Mark Hsu, MD Stanford University Endoscopic Molecular Imaging of Bladder Cancer Introduction: The current clinical paradigm of bladder cancer diagnosis centers around white light cystoscopy (WLC) for identification of suspicious bladder tumors, transurethral resection of bladder tumors, and cystoscopic surveillance after complete resection of non‐muscle invasive cancer. Use of WLC for visualization of bladder tumors is highly user‐dependent in being able to identify all target sites for resection, and can be challenging in the setting of differentiating nonpapillary/flat tumors from coexisting inflammatory lesions. Endoscopic molecular imaging offers a potentially transformative approach for bladder cancer diagnosis by combining cancer selective, fluorescently labeled binding agents (e.g. antibodies, peptides, small molecules) with complementary optical imaging technology. CD47 is a surface molecule highly expressed by bladder cancer cells and may serve as a promising imaging and therapeutic target for bladder cancer. In feasibility experiments, we have demonstrated targeted imaging of bladder cancer in human cystectomy specimens using fluorescently labeled anti‐CD47 and a clinical grade photodynamic diagnosis (PDD) fluorescent cystoscope. In addition, we are developing a mouse orthotopic bladder cancer model using primary human bladder cancer cells. Taking advantage of these clinically relevant ex vivo and in vivo model systems, we propose to develop a strategy for endoscopic molecular. Shu Lin, PhD University of California, Los Angeles Roles of EMT on Stem Cell Properties of Prostate Stem and Cancer Cells During Castration‐resistant Prostate Cancer Progression Androgen ablation remains the mainstay of treatment for men with advanced and metastatic prostate cancer. However, despite the introduction of new generation anti‐androgens, a majority of men succumb to castration resistant prostate cancer (CRPC). The molecular mechanisms governing the emergence of treatment resistance in CRPC patients are not well understood. Recent experience suggests that tumor regeneration from castration‐resistant stem‐like cells induce resistance to hormonal therapy. Therefore, elucidating novel targets essential for driving stem‐like activity is critical to prevent and defeat CRPC. It has been shown that normal and cancer stem cells exploit normal development process of epithelial‐mesenchymal transition (EMT) to survive and metastasize, and that EMT confer stem cell properties to more differentiated cancer cell progeny in breast and other cancers. However, it is unclear if EMT is linked with stem cells in normal and malignant prostate. Our lab has reported that N‐cadherin, a marker of EMT, is upregulated after neoadjuvant hormone ablation and in CRPC and is sufficient to cause metastasis and CRPC. Therapeutic targeting of N‐cadherin by novel N‐cadherin antibody inhibited metastatic and CRPC progression. The cell population displaying N‐cadherin co‐expressed a number of stem cell‐associated genes in CRPC models. Here, we verified EMT linked to stem cells in both normal prostate and CRPC. We found in LAPC‐9 CRPC tumors, the cell population expressing N‐cadherin behaved like stem cells with enhanced sphere‐forming ability, which could be specifically inhibited by novel N‐cadherin antibody 2A9. We isolated stem‐like CD49fhi/Trop2hi cells from prostatectomy specimens and found that forced N‐cadherin expression promoted sphere formation of those cells. Our evaluation of gene expression in N‐cadherin‐positive prostate cancer cell lines and CRPC tumors demonstrated that N‐cadherin expression activated common EMT transcriptional regulators including Zeb1. We asked if Zeb1 regulated stem cell properties in normal and malignant prostate. We found that forced Zeb1 expression induced EMT with enhanced cell invasiveness in LNCaP human prostate cancer cells. However, Zeb1 overexpression inhibited cell proliferation and CRPC tumor growth of LNCaP. Zeb1 overexpression also surprisingly inhibited sphere formation of normal stem/progenitor cells from prostatectomy specimens. Our data suggest that Zeb1‐regulated EMT promotes both quiescence and invasiveness in normal and malignant prostate in which the quiescent cells may survive and play a role in treatment resistance, while N‐cadherin mediates stem cell proliferation and self‐renewal. Our research will likely provide useful information of EMT‐related biomarkers for preventing and developing efficient therapeutics to combat the treatment resistance to new generation anti‐androgens. Tomasz Owczarek, PhD Columbia University Cell of Origin of Bladder Cancer Bladder cancer manifests as two different subtypes: noninvasive papillary carcinoma, which has an excellent prognosis, and muscle invasive carcinoma, which has a particularly high mortality rate. Bladder carcinoma arises in a specific epithelium, called the urothelium, which consists of at least three cell types: basal, intermediate and umbrella cells. Reciprocal relationships between the cell types that form the urothelium have not been fully elucidated, but evidence suggests that at least the basal and umbrella cells arise from distinct cell populations. My proposal is based on the hypothesis that these distinct cell populations give rise to distinct forms of bladder cancer. I propose to take advantage of a unique genetically engineered mouse model of invasive bladder cancer developed in the Abate‐Shen laboratory. I will adapt this model to direct genetic alterations to distinct cell types within the urothelium to assess phenotypic outcome. I will combine these studies with analysis of genomic changes and gene expression signatures specific of tumors that have originated from different cell types. My ability to utilize predictive genes signatures based on this study may lead to identification of new molecular targets and should improve the estimation of patient survival, therefore significantly enhance bladder cancer management. Bharathi Rajaganapathy, PhD Beaumont Health System Research Institute Exploring the Molecular Mechanism of Inflammatory Response in a New Rat Model of Radiation Cystitis Most therapeutic effects for cancer are associated with a variety of late side effects for cancer survivor patients. Conventionally, cancer patients undergo radiation therapy as part of their treatment plan for pelvic malignancies. As result of increased use of radiation therapy, there are a growing number of cancer survivors who are experiencing adverse effects of radiation therapy. Radiation cystitis is one such common condition that is caused by radiation therapy; hence, there is a need to monitor cancer survivors for the late physical and biological side effects of radiation therapy. In this proposal, we attempt to develop tacrolimus (well‐ known immunosuppressant) as a novel drug with a new mechanism to treat Radiation cystitis by targeting the bladder locally using intravesical bladder targeted delivery. Fewer side effects can be anticipated, and, therefore, it will be beneficial for radiation cystitis patients who suffer unwanted side effects. Inflammation is believed to have an important role in radiation cystitis, so the role of tacrolimus in inflammatory mechanism will be studied using biotechnological techniques. We will utilize state of the art facilities including, Small Animal Radiation Research Platform (SARRP), Magnetic Resonance Imaging (MRI) and various molecular techniques in this study to develop a successful therapy for Radiation Cystitis. Sungyong You, PhD Cedars−Sinai Medical Center An Epigenomic Pathway From Cholesterol to Intracrine Androgen Prostate cancer kills about 28,000 men in the United States each year. Although progress has been made in understanding the molecular features of different forms of the disease, therapeutic options when primary therapy fails are limited. Recent studies on humans and laboratory models have provided evidence that high circulating cholesterol is a risk factor for aggressive prostate cancer. A variety of studies have also shown that cholesterol reduction, using drugs that are safe and which have minimal side effects, appears to prevent the emergence of aggressive disease in some patients. If we had a better understanding of the molecular role(s) played by cholesterol in prostate cancer we would be able to identify patients, possibly even prior to diagnosis, who would benefit from cholesterol reduction. Epidemiologic evidence suggests that such interventions may prevent prostate cancer from advancing to lethal disease and may also improve prognosis after therapy. Our laboratory recently made a discovery that for the first time appears to connect cholesterol metabolism to epigenetic mechanisms of gene regulation and aggressive prostate cancer. Our preliminary findings indicate that this discovery is relevant to how prostate cancer evades hormonal therapy (androgen deprivation therapy, ADT). We have identified a protein, called scaffold attachment factor B1 (SAFB1), which regulates or is associated with key proteins known to drive prostate cancer toward greater virulence. Our data indicate that expression of SAFB1 declines as prostate cancer progresses to a metastatic state. This suggests that SAFB1 may act to inhibit disease progression. Molecular data we have assembled are consistent with this conclusion. We have also discovered that SAFB1 appears to regulate a metabolic pathway that controls the level of androgen present in the tumor cell. This is a very important finding because it suggests that mechanisms controlling the prostate cancer cell’s response to cholesterol may directly impact the levels of androgen present in the tumor cell, even when circulating androgen is eliminated by hormonal therapy. Recent studies from several groups have shown that androgen synthesized and retained within the tumor cell may be an important mechanism of resistance to hormonal therapy (a process called “intracrine” signaling). The goals of our study are to characterize all of the gene regulatory events controlled by SAFB1 in human prostate cancer cells. We will also test the hypothesis that SAFB1 controls a family of genes (UGTB2s) that are involved in the inactivation of androgen and export of androgen from tumor cells. Our data indicate that when SAFB1 levels decline, expression of the UGT2B genes also declines, suggesting that the tumor cells lose their ability to inactivate androgen. This metabolic Sungyong You, PhD (cont’d) change should promote the activity of the androgen receptor, a critical protein thought to mediate prostate cancer progression to lethal disease. Finally, we will test whether silencing of the SAFB1 gene promotes progression toward a castration‐resistant phenotype, a hypothesis consistent with our preliminary data. We will also use a novel method of raising and lowering cholesterol in mice, a method we developed, to determine whether SAFB1 loss collaborates with high cholesterol to promote prostate cancer growth and whether cholesterol reduction can inhibit tumor growth under these circumstances. Our genome‐wide analyses will reveal a molecular network that will for the first time integrate cholesterol metabolism with intracrine androgen signaling. We anticipate that a number of outcomes from this study may be clinically relevant: (1) we may identify targets that will help select patients for active surveillance; (2) we will learn about how tumor cells retain androgen in the hormone‐repressed state; and (3) we will learn how cholesterol and lipid metabolism affects the entire prostate cancer genome. My long‐term scientific interests are directed toward the identification of novel pathogenic drivers and useful biomarkers that arise during the natural history of prostate cancer, especially with respect to the development of novel systems biology approaches toward these questions. I believe that in the future, the old domains of “wet lab” approaches and “statistics,” which frequently were occupied by scientists with non‐overlapping specializations and training, is obsolete. It is now critical to conduct biochemistry or molecular biology experiments coupled with state‐of‐the‐art technology and interpret the results using computational methods in an interdisciplinary manner. With this objective in mind, as an intensively trained bioinformatician and translational scientist, I am committed to prostate cancer research, in particular to, the systems biology of prostate cancer and the application of new discoveries to clinical practice.