Abstracts from the New England Section of the AUA 2020: A Virtual Experience

NE AUA 2020 Abstracts Utilization of Capillary Electrophoresis Mass Spectrometry in the Development of a Metabolomic Signature for Prostate Cancer Andrew Gusev, BA 1 , Leo L. Cheng, PhD 1 , Alex Buko, PhD 2 , Takushi Oga, PhD 2 , Adam S. Feldman, MD MPH 1 1 Massachusetts General Hospital, Boston, MA, USA; 2 HumanMetabolome Technologies, Boston, MA, USA Introduction: Prostate cancer (PCa) pathogenesis is influenced by alterations in cellular metabolism. Metabolomics measures these biochemical changes to create global tissue metabolite profiles. Urinary studies are noninvasive and can potentially identify biomarkers for PCa. We used Capillary Electrophoresis Mass Spectrometry (CE-MS) to analyze urine from men undergoing prostate biopsy for suspicion of PCa to investigate their metabolomic profiles. Materials&Methods: 150urinespecimenswereprospectivelycollectedfrommenundergoing prostate biopsy.After histopathologic evaluation of all biopsy cores was completed, 40 urine samples were selected for metabolomic investigation. 20 samples were taken frommen with entirely benign prostate biopsies, and 20 from men with biopsy-proven PCa. An analysis of charged metabolites by CE-MS was performed as described (J Proteome Res. 2:488; 2003). Urinary metabolites were extracted from 100 μL urine by mixing with methanol containing 20μMofinternalstandards.CE-MSexperimentswereperformedwiththeAgilentCEsystem. Screening of potential biomarkers was performed with statistical protocols and pathway analyses. Metabolites with levels below the detection limit in all samples were excluded. Relative abundances of metabolites were normalized to levels of creatinine. Results : CE-MS analysis produced thousands of features in the combined anionic and cationic modes. A volcano plot comparing p values against fold change identified 60 metabolites that were statistically different between urine samples of men with PCa and those of men without PCa. Pathway analysis of these using MetaboAnalyst (Metabolites 9:57; 2019) showed high activity in ceramide, short chain fatty acid (SCFA), branched chain amino acid, serine, threonine, and tryptophan metabolism. Figure 1 demonstrates the pathway analysis with fold change represented by color and vertical scale, and number of significant metabolites with size. Figure 2 graphically contrasts metabolomic profiles. Conclusions : CE-MSanalysis identifiedseveralmetabolicpathwaysthatwereupregulated inurineofmenwithPCa.Thesemetabolitesare involved insteroid,aromatic,microorganism andSCFAprocessesandwarranttargetedstudieswhichareunderwayinourlab.Ifvalidated, theyhavepotentialtoserveasnon-invasivebiomarkersforPCadiagnosisandtherapeutics. P12 Estrogen Receptors Regulate Prostate Growth in Benign Prostatic Hyperplasia upon SRD5A2 Promoter Methylation Christina Sharkey, BS, Sijun Liu, MD, Zongwei Wang, PhD, Aria F. Olumi, MD BIDMC, Boston, MA, USA Introduction: Steroid5 α -reductasetypeII(SRD5A2) isthepredominantenzymeresponsible for prostatic development and growth. We found that expression of SRD5A2 in the prostate is variable, and that one-third of prostate tissue samples from BPH patients do not express SRD5A2.WedemonstratedthattheabsenceofSRD5A2expressionisassociatedwith SRD5A2 methylation in the promoter region. We have demonstrated that there is an “androgenic to estrogenicswitch”whenSRD5A2 isabsent intheprostategland.Herewewishedto identify if SRD5A2 promoter methylation regulates estrogen receptor (ER) expression. Materials & Methods: We used human prostatic stromal and epithelium cells, and prostate specimens that collected from patients who underwent transurethral resection of the prostate (TURP). ER α and ER β histological expression was determined by immunohistochemistry. Genome DNAwas extracted and SRD5A2 promoter methylation was determined with DNA methylation-specific PCR. The level of SRD5A2 promoter methylation was correlated to ER expression. Results: ER β was expressed in both stroma and epithelial compartments, whereas ER α was mainly expressed in the basal epithelium. In cultured prostatic stromal and epithelium cells, ER β only expressed in the nucleus. Six out of twenty-three patients were SRD5A2 hypermethylation at the promoter region and had higher ER β expression. Conclusions: ER α and ER β co-localize in human benign prostatic tissue. Specifically, ER β is expressed in the nucleus of both the prostatic stroma and epithelial. SRD5A2 promoter methylation is associated with the expression of ER. Targeting the estrogenic signaling may serve as an effective treatment strategy in subset of ER-sensitive BPH patients. P11 Modulation of TRPV4 Channel Activity by ATP in Rat and Human Urinary Bladder Seneca D. Hutson, BSc 1 , Katarina Zvarova, MD, PhD 2 , Peter Zvara, MD, PhD 2 1 UniversityofVermont,Burlington,VT,USA; 2 UniversityofSouthernDenmark,Odense,Denmark Introduction: Transient receptor potential V4 (TRPV4) channels involved in bladder sensory signaling represent a promising target for the treatment of bladder overactivity. Stretch-inducedATPreleasefromtheurotheliumstimulatessensorynerveendings.Existing experimental evidence suggests that TRPV4 channels are likely involved in this process through activation ofATPrelease, although the exact mechanismof this effect needs further elucidation. In this study we investigated functional interaction between selective TRPV4 modulating drugs and ATP in rat and human bladder. Materials &Methods: Bladder tissue was functionally assessed using in vitro myography. Experiments used full thickness strips from healthy rat bladder and partial thickness strips containing detrusor muscle and urothelium from the bladder of adult female patients undergoing radical cystectomy. Contractile response of the bladder tissue to TRPV4-modulating drugs (agonist - GSK1016790A, antagonist - HC067047) and their interaction with P2X 2/3 receptor agonist adenosine triphosphate (ATP) were evaluated in Ca-containing solution. Ca 2+ -free solution was then used to demonstrate the reliance of these events on calcium. Results: TRPV4 agonist (GSK1016790A) evoked dose-dependent increases in the baseline tone and amplitude of phasic contractions of muscle strips fromboth rat (n = 6) and human (n = 3) bladders. The response first developed at GSK concentration of 1nM and reached a peak at 100nM. This effect was significantly reduced by pretreatment with 1µM TRPV4 antagonist (HC067047). ATP (1mM) administered before GSK completely blocked the GSK effect. Adding 1-100µm of ATP at the peak of GSK-evoked response increased the amplitude of phasic activity. A 1mM dose of ATP, at the peak of GSK-evoked response, reversed the effect of GSK, returning the tone to baseline and completely abolishing phasic activity. In Ca 2+ -free solution, both tonic and phasic responses to GSK were reduced by > 90%. These effects were observed in both rat and human tissue. Conclusions: Our results show that TRPV4 increases tone and amplitude of phasic activity in bladder strips in both human and rat bladder, supporting its likely role in bladder overactivity. Lower concentrations of ATP potentiate TRPV4-induced phasic activity and higherconcentrationsofATPcompletelyinhibitallTRPV4bladdereffects.Theseobservations point to the possibility that ATP may modulate TRPV4 channel activity during bladder filling via negative feedback, thus contributing to the control of bladder sensory signaling. P10 Poster Session I 35