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MP99-01 PROTEOMIC ANALYSIS OF URINARY EXTRACELLULAR VESICLES FROM HIGH GLEASON SCORE PROSTATE CANCER
THE JOURNAL OF UROLOGYâ
e1320
Vol. 197, No. 4S, Supplement, Tuesday, May 16, 2017
Prostate Cancer: Basic Research & Pathophysiology IV
OVEREXPRESSION OF HSD3B1 CONFERS RESISTANCE TO ENZALUTAMIDE IN PROSTATE CANCER Cameron Armstrong*, Chengfei Liu, Wei Lou, Christopher Evans, Allen Gao, Sacramento, CA
Moderated Poster 99 Tuesday, May 16, 2017
MP99-02
9:30 AM-11:30 AM
MP99-01 PROTEOMIC ANALYSIS OF URINARY EXTRACELLULAR VESICLES FROM HIGH GLEASON SCORE PROSTATE CANCER Kazutoshi Fujita*, Suita, Japan; Hideaki Kume, Ibaraki, Japan; Kyosuke Matsuzaki, Atsunari Kawashima, Takeshi Ujike, Akira Nagahara, Motohide Uemura, Yasushi Miyagawa, Suita, Japan; Takeshi Tomonaga, Ibaraki, Japan; Norio Nonomura, Suita, Japan INTRODUCTION AND OBJECTIVES: Extracellular vesicles (EVs) are microvesicles secreted from various cell types. EVs contain microRNAs, proteins, and mRNAs and play a role in intercellular communications via the mechanisms of exocytosis and endocytosis. We aimed to discover a new biomarker for high Gleason score (GS) prostate cancer (PCa) in urinary EVs via quantitative proteomics. METHODS: EVs were isolated from urine after massage from 18 men (negative biopsy [n ¼ 6], GS 6 PCa [n ¼ 6], or GS 8-9 PCa [n ¼ 6]). EV proteins were labeled with iTRAQ and analyzed by LC-MS/MS. Candidate proteins were further analyzed by selected reaction monitoring/multiple reaction monitoring (SRM/MRM). RESULTS: Proteins extracted from EVs were enriched with CD9 protein, which is a marker of EVs, compared with unprocessed urinary proteins. EVs labeled with anti-CD9 antibody conjugated with Au colloids were also confirmed by electron microscopy. We identified and quantified 3530 proteins in the urinary EVs by LC-MS/MS. Thirty-six proteins increased in patients with PCa compared to those with negative biopsy (ratio > 2.0, p < 0.1). Four proteins increased in patients with GS 8-9 PCa compared to those with negative biopsy or GS 6 PCa (ratio > 2.0, p < 0.1). Twenty-seven proteins were chosen for further analysis and verified in 29 independent urine samples (negative [n ¼ 11], PCa [n ¼ 18]) using SRM/MRM. Among these candidate markers, fatty acid binding protein 5 (FABP5) was higher in the cancer group than in the negative group (p ¼ 0.009) and was significantly associated with GS (p for trend ¼ 0.011). Univariate logistic analysis showed that FABP5 was significantly associated with prostate cancers with GS 7 or more (p < 0.001). Even after adjusting for age, PSA, and PSA density, FABP5 was significantly associated with prostate cancer with GS 7 or more (p ¼ 0.003). The receiver-operator characteristics curve analysis showed that the area under the curve (AUC) for the prediction of GS ¼ 7 by FABP5 was 0.856 (95% CI 0.708e1.00, p ¼ 0.002), whereas the AUC value for prediction by serum PSA was 0.511 (95% CI 0.280e0.757, p ¼ 0.87). CONCLUSIONS: We applied the proteomic analysis to discover biomarkers in EVs in urine collected after prostate massage. FABP5 in urinary EVs could be a potential biomarker of high GS prostate cancer. Additional large-scale studies are warranted to confirm this finding.
INTRODUCTION AND OBJECTIVES: Most prostate cancer (PCa) patients receiving enzalutamide (Enza) develop drug resistance within 24 months of exposure. This creates a need to better understand the underlying causes of Enza resistance so that improved treatment methods can be developed. Previous studies demonstrate that uncontrolled intraprostatic androgen synthesis promotes Enza resistance. HSD3B1 is a key enzyme contributing to androgen synthesis and its expression is associated with PCa progression. The aim of this study is to determine the contribution of HSD3B1 to Enza resistance in PCa. METHODS: Enza resistant C4-2B PCa cells (C4-2B MDVR) were generated by chronically exposing parental C4-2B cells to increasing Enza concentrations (5-40 mM) for >12 months and maintained in 20 mM Enza. Differences in gene expression between C4-2B MDVR and parental cells was determined by microarray and RNA-seq. HSD3B1 expression was knocked down in C4-2B MDVR cells using shRNA and cell number was determined in media containing FBS, charcoal dextran stripped FBS (CD-FBS), or CD-FBS supplemented with 100 nM pregnenolone (P5), 100 nM DHEA, or 10 nM DHT in the presence and absence of 20 mM Enza. PSA secretion was determined by ELISA and PSA-luciferase activity was measured by reporter assay. C4-2B MDVR cells were also treated with apigenin, which has been shown to reduce HSD3B1 expression, and cell number and PSA outcomes were determined under the previously mentioned treatment conditions. RESULTS: HSD3B1 expression is higher in C4-2B MDVR cells compared to parental C4-2B cells as measured by Microarray and RNA-seq. This correlates to increased intracrine androgens in C42B MDVR cells compared to C4-2B cells as examined by LC-MS. Knockdown of HSD3B1 in C4-2B MDVR resensitized cells to Enza in FBS, CD-FBS+DHT and CD-FBS+P5 conditions as determined by a reduction in cell number and PSA secretion and/or luciferase activity in response to Enza. No response was seen in CD-FBS or CDFBS+DHEA. Supplementation of parental C4-2B cells with 100 nM P5, but not DHEA, improved resistivity to Enza. Apigenin, a potential inhibitor of HSD3B1 expression, resensitized C4-2B MDVR cells to Enza. CONCLUSIONS: HSD3B1 overexpression in C4-2B MDVR cells contributes to Enza resistance and modulation of this enzyme could be a viable strategy to improve Enza treatment response in PCa cells. HSD3B1 activity appears to be reliant on select androgen precursors, such as pregnenolone, indicating preference towards a specific androgen synthesis pathway by HSD3B1 in mediating Enza resistance. Source of Funding: This work is supported in part by grants NIH/NCI CA140468, CA168601, CA179970, DOD PC130062, and US Department of Veterans Affairs, ORD VA Merits I01BX0002653.
MP99-03 RIOK2 IS A MEDIATOR OF OBESITY ENHANCED PROSTATE CANCER GROWTH Everardo Macias*, Los Angeles, CA; David Corcoran, Jen-Tsan Chi, Durham, NC; Stephen Freedland, Los Angeles, CA
Source of Funding: This study was supported by grants from the Princess Takamatsu Cancer Research Fund, JSPS KAKENHI Grant Number JP15K10588, and Osaka University project MEET.
INTRODUCTION AND OBJECTIVES: Obesity is a growing global and U.S. health problem. For 2012, an estimated 117,000 cancer cases in the U.S. were deemed preventable by achieving and maintaining a healthy weight, including 11% of all advanced prostate cancers (PC). Obesity is associated with greater risk of high-grade PC, recurrence after therapy, metastases, and PC specific death. We
e1320
Vol. 197, No. 4S, Supplement, Tuesday, May 16, 2017
Prostate Cancer: Basic Research & Pathophysiology IV
OVEREXPRESSION OF HSD3B1 CONFERS RESISTANCE TO ENZALUTAMIDE IN PROSTATE CANCER Cameron Armstrong*, Chengfei Liu, Wei Lou, Christopher Evans, Allen Gao, Sacramento, CA
Moderated Poster 99 Tuesday, May 16, 2017
MP99-02
9:30 AM-11:30 AM
MP99-01 PROTEOMIC ANALYSIS OF URINARY EXTRACELLULAR VESICLES FROM HIGH GLEASON SCORE PROSTATE CANCER Kazutoshi Fujita*, Suita, Japan; Hideaki Kume, Ibaraki, Japan; Kyosuke Matsuzaki, Atsunari Kawashima, Takeshi Ujike, Akira Nagahara, Motohide Uemura, Yasushi Miyagawa, Suita, Japan; Takeshi Tomonaga, Ibaraki, Japan; Norio Nonomura, Suita, Japan INTRODUCTION AND OBJECTIVES: Extracellular vesicles (EVs) are microvesicles secreted from various cell types. EVs contain microRNAs, proteins, and mRNAs and play a role in intercellular communications via the mechanisms of exocytosis and endocytosis. We aimed to discover a new biomarker for high Gleason score (GS) prostate cancer (PCa) in urinary EVs via quantitative proteomics. METHODS: EVs were isolated from urine after massage from 18 men (negative biopsy [n ¼ 6], GS 6 PCa [n ¼ 6], or GS 8-9 PCa [n ¼ 6]). EV proteins were labeled with iTRAQ and analyzed by LC-MS/MS. Candidate proteins were further analyzed by selected reaction monitoring/multiple reaction monitoring (SRM/MRM). RESULTS: Proteins extracted from EVs were enriched with CD9 protein, which is a marker of EVs, compared with unprocessed urinary proteins. EVs labeled with anti-CD9 antibody conjugated with Au colloids were also confirmed by electron microscopy. We identified and quantified 3530 proteins in the urinary EVs by LC-MS/MS. Thirty-six proteins increased in patients with PCa compared to those with negative biopsy (ratio > 2.0, p < 0.1). Four proteins increased in patients with GS 8-9 PCa compared to those with negative biopsy or GS 6 PCa (ratio > 2.0, p < 0.1). Twenty-seven proteins were chosen for further analysis and verified in 29 independent urine samples (negative [n ¼ 11], PCa [n ¼ 18]) using SRM/MRM. Among these candidate markers, fatty acid binding protein 5 (FABP5) was higher in the cancer group than in the negative group (p ¼ 0.009) and was significantly associated with GS (p for trend ¼ 0.011). Univariate logistic analysis showed that FABP5 was significantly associated with prostate cancers with GS 7 or more (p < 0.001). Even after adjusting for age, PSA, and PSA density, FABP5 was significantly associated with prostate cancer with GS 7 or more (p ¼ 0.003). The receiver-operator characteristics curve analysis showed that the area under the curve (AUC) for the prediction of GS ¼ 7 by FABP5 was 0.856 (95% CI 0.708e1.00, p ¼ 0.002), whereas the AUC value for prediction by serum PSA was 0.511 (95% CI 0.280e0.757, p ¼ 0.87). CONCLUSIONS: We applied the proteomic analysis to discover biomarkers in EVs in urine collected after prostate massage. FABP5 in urinary EVs could be a potential biomarker of high GS prostate cancer. Additional large-scale studies are warranted to confirm this finding.
INTRODUCTION AND OBJECTIVES: Most prostate cancer (PCa) patients receiving enzalutamide (Enza) develop drug resistance within 24 months of exposure. This creates a need to better understand the underlying causes of Enza resistance so that improved treatment methods can be developed. Previous studies demonstrate that uncontrolled intraprostatic androgen synthesis promotes Enza resistance. HSD3B1 is a key enzyme contributing to androgen synthesis and its expression is associated with PCa progression. The aim of this study is to determine the contribution of HSD3B1 to Enza resistance in PCa. METHODS: Enza resistant C4-2B PCa cells (C4-2B MDVR) were generated by chronically exposing parental C4-2B cells to increasing Enza concentrations (5-40 mM) for >12 months and maintained in 20 mM Enza. Differences in gene expression between C4-2B MDVR and parental cells was determined by microarray and RNA-seq. HSD3B1 expression was knocked down in C4-2B MDVR cells using shRNA and cell number was determined in media containing FBS, charcoal dextran stripped FBS (CD-FBS), or CD-FBS supplemented with 100 nM pregnenolone (P5), 100 nM DHEA, or 10 nM DHT in the presence and absence of 20 mM Enza. PSA secretion was determined by ELISA and PSA-luciferase activity was measured by reporter assay. C4-2B MDVR cells were also treated with apigenin, which has been shown to reduce HSD3B1 expression, and cell number and PSA outcomes were determined under the previously mentioned treatment conditions. RESULTS: HSD3B1 expression is higher in C4-2B MDVR cells compared to parental C4-2B cells as measured by Microarray and RNA-seq. This correlates to increased intracrine androgens in C42B MDVR cells compared to C4-2B cells as examined by LC-MS. Knockdown of HSD3B1 in C4-2B MDVR resensitized cells to Enza in FBS, CD-FBS+DHT and CD-FBS+P5 conditions as determined by a reduction in cell number and PSA secretion and/or luciferase activity in response to Enza. No response was seen in CD-FBS or CDFBS+DHEA. Supplementation of parental C4-2B cells with 100 nM P5, but not DHEA, improved resistivity to Enza. Apigenin, a potential inhibitor of HSD3B1 expression, resensitized C4-2B MDVR cells to Enza. CONCLUSIONS: HSD3B1 overexpression in C4-2B MDVR cells contributes to Enza resistance and modulation of this enzyme could be a viable strategy to improve Enza treatment response in PCa cells. HSD3B1 activity appears to be reliant on select androgen precursors, such as pregnenolone, indicating preference towards a specific androgen synthesis pathway by HSD3B1 in mediating Enza resistance. Source of Funding: This work is supported in part by grants NIH/NCI CA140468, CA168601, CA179970, DOD PC130062, and US Department of Veterans Affairs, ORD VA Merits I01BX0002653.
MP99-03 RIOK2 IS A MEDIATOR OF OBESITY ENHANCED PROSTATE CANCER GROWTH Everardo Macias*, Los Angeles, CA; David Corcoran, Jen-Tsan Chi, Durham, NC; Stephen Freedland, Los Angeles, CA
Source of Funding: This study was supported by grants from the Princess Takamatsu Cancer Research Fund, JSPS KAKENHI Grant Number JP15K10588, and Osaka University project MEET.
INTRODUCTION AND OBJECTIVES: Obesity is a growing global and U.S. health problem. For 2012, an estimated 117,000 cancer cases in the U.S. were deemed preventable by achieving and maintaining a healthy weight, including 11% of all advanced prostate cancers (PC). Obesity is associated with greater risk of high-grade PC, recurrence after therapy, metastases, and PC specific death. We