- Email: [email protected]
MP52-05 MOUSE PERIPROSTATIC ADIPOSE (PPA) TISSUE MIMICS HUMAN PPA ACTIVITY ON PROSTATE CANCER CELLS: A MODEL SYSTEM TO STUDY PPA – PROSTATE CANCER INTERACTIONS
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Monday, May 19, 2014
it to develop novel therapies for inhibiting prostate cancer growth in the bone-niche. Source of Funding: Phi Beta Psi Sorority Grant. UCSD Dept of Surgery and Moores Cancer Center Start Up Funding.
MP52-03 DISRUPTION OF CHD8-CTCF CHROMATIN COMPLEX IN PROSTATE CANCER ALTERS DNA METHYLATION PATTERNS Nathan Damaschke, Wei Huang, Chee Paul Lin, Jin Hee Lee, David Jarrard*, Madison, WI INTRODUCTION AND OBJECTIVES: Epigenetic features drive cancer progression, but little is known regarding the targeting of DNA methylation. CCCTC-binding factor (CTCF), Chromodomain helicase DNA-binding protein 8 (CHD8), and Brother of the regulator of imprinted sites (BORIS) are interrelated chromatin proteins involved in the regulation of epigenetic marks. BORIS and CTCF are paralogues which compete for CTCF binding sites, exhibit opposing functions, and require CHD8. We evaluated the role that this complex plays in protecting methylated regions, and investigated their expression in prostate cancer (PCa). METHODS: Doxocycline-inducible CTCF targeting shRNAs were stably introduced into PCa lines PPC1, LNCaP and HPV16 immortalized prostate epithelial cells. Proliferation and cell cycle was assessed using flow cytometry. DNA methylation was tested at CTCF binding sites using quantitative pyrosequencing. To evaluate gene expression in PCa, tissue microarrays consisting of benign (N¼96), localized (N¼146), metastatic PCa (N¼44) and HGPIN (N¼50) were chromogenically stained for CTCF, CHD8, and BORIS. A novel, automated quantitative imaging system VECTRAÔ was used to evaluate epithelial staining in both the nucleus and cytoplasm. RESULTS: Knockdown of CTCF was performed to disrupt this chromatin complex. It had no effect on cell proliferation or apoptosis. However, CTCF loss leads to an increase in methylation across CTCF binding sites within the Cav1 gene and the Igf2-H19 imprinted region. Quantitative protein expression in clinical samples demonstrated a marked downregulation of CHD8 expression in HGPIN (P<0.01), localized (P < 0.001), and metastatic PCa (P < 0.0001) compared to benign. CHD8 expression was downregulated in 52% of primary cancer samples indicating a common alteration. Decreased nuclear CHD8 expression was associated with positive margins (P ¼ 0.047), extracapsular extension (P<0.01), and presence of metastases (P¼0.025). BORIS expression, as was the BORIS/CTCF ratio, was increased in localized PCa (P¼0.03 and 0.03) compared to benign. Heterogeneity of staining was assessed using the Simpson’s coefficient which revealed CTCF expression to be more heterogeneous in cancerous tissue (both P < 0.001), especially with higher Gleason grade (p<0.01). CONCLUSIONS: In the first detailed analysis in cancer, a marked loss of CHD8 expression and increased BORIS/CTCF ratio indicate disruption of this chromatin complex. Loss of this complex results in DNA hypermethylation potentially explaining the targeting of DNA methylation changes in cancer. Source of Funding: NIH
MP52-04 COLOCALIZATION OF ANDROGEN RECEPTOR AND ESTROGEN RECEPTOR ALPHA IN THE STROMAL MICROENVIRONMENT SUPPORTS A ROLE FOR ESTROGENS IN EARLY PROSTATE CANCER PROGRESSION Tristan Nicholson*, Priyanka Sehgal, Sally Drew, Wei Huang, William Ricke, Madison, WI INTRODUCTION AND OBJECTIVES: Androgens, acting via androgen receptor (AR), are known to be important in prostate cancer (PRCA) progression; there is also an emerging role for estrogens,
e581
acting via estrogen receptors (ERs), in PRCA. We evaluated expression and localization of AR and ERa (considered the key estrogenic mediator of prostate carcinogenesis) to better understand the role of these sex steroid receptors in PRCA progression. We asked two questions: (1) what is the prevalence of cells expressing AR, ERa and both receptors during different stages of PRCA progression, and (2) what does the relative prevalence of double positive cells imply about the underlying biology of the stromal microenvironment during PRCA progression? METHODS: Multiplexed immunohistochemistry was performed to detect AR, ERa and smooth muscle alpha-actin (ACTA2) utilizing a tissue microarray consisting of benign prostate tissue (BPT), high-grade prostatic intraepithelial neoplasia (HGPIN), primary tumor from PRCA, and metastasis (MET). To assess the number of sex steroid receptor positive cells and marker staining intensity, we used an automated pathology platform. RESULTS: Expression and staining intensity of AR increased with PRCA progression in prostate epithelium, carcinoma cells and the stromal microenvironment. Epithelial and stromal ERa expression and staining intensity was highest in HGPIN and decreased in PRCA and MET, relative to BPT. However, among stromal cells negative for ACTA2, AR positive cells were less prevalent in PRCA compared to BPT, and there was no significant difference in the prevalence of ERa positive cells. In the epithelium and the stromal microenvironment, double positive (for AR & ERa) cells were most prevalent in HGPIN, relative to BPT. CONCLUSIONS: Taken together, the present results underscore the importance of androgens and estrogens in the biology of PRCA progression. We found an increased prevalence of cells expressing both AR and ERa in early, but not late stages of PRCA progression, suggesting that while the androgens are important in all stages of progression, the role for estrogens and sex steroid synergy may be most prominent during early prostate carcinogenesis. Source of Funding: F30DK093173
R01CA123199,
T32GM07356,
MP52-05 MOUSE PERIPROSTATIC ADIPOSE (PPA) TISSUE MIMICS HUMAN PPA ACTIVITY ON PROSTATE CANCER CELLS: A MODEL SYSTEM TO STUDY PPA e PROSTATE CANCER INTERACTIONS Palamadai N. Venkatasubramanian, Evanston, IL; Massa Mafi, Milwaukee, WI; Charles B. Brendler, Beth A. Plunkett, Evanston, IL; Jennifer Doll*, Milwaukee, WI INTRODUCTION AND OBJECTIVES: Visceral obesity promotes prostate cancer (PCa) progression; yet, the molecular mechanisms remain unclear. We recently showed that periprostatic adipose (PPA) tissues from obese PCa patients stimulate higher proliferative rates in both PCa and endothelial cells compared to that of PPA from lean or subcutaneous adipose (SQA) tissues from lean or obese patients. We also demonstrated that obesity shifts the fatty acid (FA) profile in the PPA and increases angiogenesis. Here, we examined mouse tissues to determine if their adipose depots had similar characteristics to that of human in order to develop a model system in which to study the obese fat e PCa interaction. METHODS: Adipose (SQA, PPA and the gonadal fat pat) were harvested from wildtype (WT) and obese (ob/ob) mice (3-4 mo.; n¼3-5/ group). Serum-free conditioned media from adipose tissue explant culture were collected at 48 h and used in MTT proliferation assays on PC-3 cells. Data were normalized to gram adipose tissue weight. Lipolytic activity of tissues was assessed using the free glycerol assay (Sigma). FA content was determined by magnetic resonance spectroscopy (MRS). RESULTS: Similarly to our previous human tissue data, obese mouse PPA tissues had an increased monounsaturated to saturated FA ratio compared to WT mice (1.983+/-0.36 vs. 1.157+/-0.19; P<0.002). Basal lipolytic activity was 52% lower in obese visceral adipose tissue compared to that of WT, while no difference was observed with obesity
THE JOURNAL OF UROLOGYâ
e582
in SQA tissues. Interestingly, obese mice develop prostatic hyperplasia, which is characterized by increased intraepithelial lipid accumulation (Doll et al., in revision). In initial functional tests using lean mice, PPA secretions increased PC-3 cell proliferation 1.8 and 2.3 times that of the visceral fat pad and SQA tissues, respectively (P<0.001), again consistent with our human tissue data. CONCLUSIONS: From these initial studies, it is evident that the PPA has pro-tumorigenic characteristics resembling that of human adipose tissues in PCa patients. Thus, the obese mouse can be used in future studies to elucidate how the obese PPA tissue e PCa interaction alters each tissue in combination with PCa mouse models. Such studies will provide unique insight into the molecular mechanisms of obesitydriven PCa. Source of Funding: This study was funded in parts by a Creativity Award from the Prostate Cancer Foundation, a Pilot grant and RCDA award from NorthShore University HealthSystem, the Northwestern University Prostate SPORE P50-CA090386, and philanthropic support secured secured through the Division of Urology and the John and Carol Walter Center for Urological Health, NorthShore University HealthSystem.
MP52-06 IN VITRO CO-CULTURE MODEL TO STUDY ADRENAL STIMULUS OF CRPC Jan Matthijs Moll*, Wilma Teubel, Rotterdam, Netherlands; Ian Hickson, Ralph Graeser, Beerse, Belgium; Guido Jenster, Wytske van Weerden, Rotterdam, Netherlands INTRODUCTION AND OBJECTIVES: Numerous studies have demonstrated the androgen receptor (AR) is still active in CPRC despite low serum testosterone levels. Among other mechanisms of AR-activation, intratumoural androgen synthesis, either via conversion of adrenal steroids or de novo synthesis from cholesterol, is a mechanism of castration resistance. Our group has previously demonstrated that clinical CRPC samples expressed markers for conversion of adrenal androgens and not for de novo synthesis. In this study, we compared growth stimulation of castration-na€ive and CRPC cell lines by adrenal androgens (conversion) or androgen precursors (de novo synthesis) and subsequently tested a novel coculture model to mimic adrenal stimulus of (CR)PC. METHODS: VCaP and DuCaP CRPC lines were generated by long-term culturing in androgen-depleted medium with or without the anti-androgens bicalutamide or flutamide. To test cell growth, VCaP and DuCaP cells and their CRPC sublines were cultured in the presence of androgen precursors (pregnenolone and progesterone) or adrenal androgens (DHEA and androstenedione) at levels found in men. Cultures were treated with vehicle, the CYP17A1-inhibitor Abiraterone (0.1 mM) or the anti-androgen MDV3100 (1 mM). Cell proliferation was assessed by MTT-assay on day 9 with each experiment performed in triplicate. For the co-culture system, VCaP cells were cultured with human adrenal (H295R) cells in separate compartments between which only medium, but not cells, could diffuse freely, and treated with vehicle, Abiraterone or R1881. RESULTS: In VCaP and DuCaP and their CRPC derivatives, androgen precursors pregnenolone and progesterone did not induce cell growth. Physiological levels of adrenal androgens stimulated cell growth, which was similar to optimal stimulus by DHT. Treatment with Abiraterone could not block adrenal androgen induced growth, while MDV3100 blocked steroid-induced growth in all conditions. Co-culturing of VCaP cells with H295R cells induced optimal cell growth, which could be inhibited by treatment with Abiraterone. CONCLUSIONS: These data support our previous clinical observation that (CR)PC seems to rely more on conversion than on de novo androgen synthesis. Co-culturing PC cells with human adrenal cells using a 2-compartment system is a relevant model to test CRPC stimulation in vitro.
Vol. 191, No. 4S, Supplement, Monday, May 19, 2014
Source of Funding: The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n 115188, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/ 2007-2013) and EFPIA companies in kind contribution.
MP52-07 DIFFERENTIAL MICRORNA EXPRESSION LEVELS IN GLEASON 6 PROSTATE BIOPSIES: A POTENTIAL TEST FOR GUIDANCE IN DETERMINING WHICH PATIENTS SHOULD UNDERGO TREATMENT VERSUS ACTIVE SURVEILLANCE Christopher Lebeis*, Boston, MA; Kari Bailey, Benjamin Waldorf, Casey Kowalik, Travjs Sullivan, David Canes, Alireza Moinzadeh, John Libertino, Kimberly Christ, Burlington, MA INTRODUCTION AND OBJECTIVES: Pathologic upgrading from biopsy to prostatectomy occurs in approximately 30% of prostate cancer cases. Biomarkers that more accurately assess risk of aggressive prostate cancer at the time of screening are critical for the future management of this disease. The aim of this study was to evaluate microRNA (miRNA) expression levels associated with Gleason 6 TRUS prostate biopsy specimens as an adjunct tool to better characterize the pathologic findings and predict which patients would be upgraded at the time of prostatectomy. METHODS: Total RNA was isolated from paraffin embedded TRUS biopsies from patients with Gleason Sum (GS) 6 that remained GS 6 (same grade) at prostatectomy and those that were upgraded to GS7 (upgrade) at the time of prostatectomy. For the discovery phase, pools consisting of ten samples each were created for the same grade and upgraded groups and miRNAs were profiled via microarray. Validation of miRNA expression levels was performed on 52 same grade and 55 upstaged samples by qRT-PCR. RESULTS: Microarray analysis identified 17 miRNAs with 003F1.6-fold differential expression between patients who had Gleason upgrading between biopsies and prostatectomy compared to patients who were same grade. Of these, twelve miRNAs were down-regulated and five were up-regulated in the upgraded group. Eight miRNAs were further validated by qRT-PCR on individual samples (n¼77) which confirmed differential expression (p<0.05) of seven miRNAs in the two study groups. Expanding the study to include 30 additional biopsy specimens with <20% tumor resulted in six miRNAs that remained significant. ROC curve analyses indicated that a combination of two miRNAs may be useful for discriminating patients with higher grade prostate cancer when all samples were included (AUC¼0.73) or excluding the less than 20% samples (AUC¼.78). CONCLUSIONS: GS6 TRUS biopsy samples demonstrated different miRNA expression levels between samples that were same grade or upgraded at prostatectomy. These differences in expression levels could provide the clinician with additional information to allow them to better stratify patients to active surveillance versus those that need to proceed to treatment. Future efforts will include developing a model to predict the GS at prostatectomy based on these miRNAs. Validation in a larger cohort is warranted. Source of Funding: none
MP52-08 TARGETING IGFBP-2 AND IGFBP-5 IN CASTRATION AND ENZALUTAMIDE RESISTANT PROSTATE CANCER Joseph Ischia*, Vancouver, Canada; Mototsugu Muramaki, Kobe, Japan; Yi Xia, Eliana Beraldi, Vancouver, Canada; Hideaki Miyake, Kobe, Japan; Amina Zoubeidi, Michael Cox, Martin Gleave, Vancouver, Canada INTRODUCTION AND OBJECTIVES: Castration and the next generation androgen receptor (AR) antagonist enzalutamide have been shown to improve survival in men with advanced prostate cancer.
Vol. 191, No. 4S, Supplement, Monday, May 19, 2014
it to develop novel therapies for inhibiting prostate cancer growth in the bone-niche. Source of Funding: Phi Beta Psi Sorority Grant. UCSD Dept of Surgery and Moores Cancer Center Start Up Funding.
MP52-03 DISRUPTION OF CHD8-CTCF CHROMATIN COMPLEX IN PROSTATE CANCER ALTERS DNA METHYLATION PATTERNS Nathan Damaschke, Wei Huang, Chee Paul Lin, Jin Hee Lee, David Jarrard*, Madison, WI INTRODUCTION AND OBJECTIVES: Epigenetic features drive cancer progression, but little is known regarding the targeting of DNA methylation. CCCTC-binding factor (CTCF), Chromodomain helicase DNA-binding protein 8 (CHD8), and Brother of the regulator of imprinted sites (BORIS) are interrelated chromatin proteins involved in the regulation of epigenetic marks. BORIS and CTCF are paralogues which compete for CTCF binding sites, exhibit opposing functions, and require CHD8. We evaluated the role that this complex plays in protecting methylated regions, and investigated their expression in prostate cancer (PCa). METHODS: Doxocycline-inducible CTCF targeting shRNAs were stably introduced into PCa lines PPC1, LNCaP and HPV16 immortalized prostate epithelial cells. Proliferation and cell cycle was assessed using flow cytometry. DNA methylation was tested at CTCF binding sites using quantitative pyrosequencing. To evaluate gene expression in PCa, tissue microarrays consisting of benign (N¼96), localized (N¼146), metastatic PCa (N¼44) and HGPIN (N¼50) were chromogenically stained for CTCF, CHD8, and BORIS. A novel, automated quantitative imaging system VECTRAÔ was used to evaluate epithelial staining in both the nucleus and cytoplasm. RESULTS: Knockdown of CTCF was performed to disrupt this chromatin complex. It had no effect on cell proliferation or apoptosis. However, CTCF loss leads to an increase in methylation across CTCF binding sites within the Cav1 gene and the Igf2-H19 imprinted region. Quantitative protein expression in clinical samples demonstrated a marked downregulation of CHD8 expression in HGPIN (P<0.01), localized (P < 0.001), and metastatic PCa (P < 0.0001) compared to benign. CHD8 expression was downregulated in 52% of primary cancer samples indicating a common alteration. Decreased nuclear CHD8 expression was associated with positive margins (P ¼ 0.047), extracapsular extension (P<0.01), and presence of metastases (P¼0.025). BORIS expression, as was the BORIS/CTCF ratio, was increased in localized PCa (P¼0.03 and 0.03) compared to benign. Heterogeneity of staining was assessed using the Simpson’s coefficient which revealed CTCF expression to be more heterogeneous in cancerous tissue (both P < 0.001), especially with higher Gleason grade (p<0.01). CONCLUSIONS: In the first detailed analysis in cancer, a marked loss of CHD8 expression and increased BORIS/CTCF ratio indicate disruption of this chromatin complex. Loss of this complex results in DNA hypermethylation potentially explaining the targeting of DNA methylation changes in cancer. Source of Funding: NIH
MP52-04 COLOCALIZATION OF ANDROGEN RECEPTOR AND ESTROGEN RECEPTOR ALPHA IN THE STROMAL MICROENVIRONMENT SUPPORTS A ROLE FOR ESTROGENS IN EARLY PROSTATE CANCER PROGRESSION Tristan Nicholson*, Priyanka Sehgal, Sally Drew, Wei Huang, William Ricke, Madison, WI INTRODUCTION AND OBJECTIVES: Androgens, acting via androgen receptor (AR), are known to be important in prostate cancer (PRCA) progression; there is also an emerging role for estrogens,
e581
acting via estrogen receptors (ERs), in PRCA. We evaluated expression and localization of AR and ERa (considered the key estrogenic mediator of prostate carcinogenesis) to better understand the role of these sex steroid receptors in PRCA progression. We asked two questions: (1) what is the prevalence of cells expressing AR, ERa and both receptors during different stages of PRCA progression, and (2) what does the relative prevalence of double positive cells imply about the underlying biology of the stromal microenvironment during PRCA progression? METHODS: Multiplexed immunohistochemistry was performed to detect AR, ERa and smooth muscle alpha-actin (ACTA2) utilizing a tissue microarray consisting of benign prostate tissue (BPT), high-grade prostatic intraepithelial neoplasia (HGPIN), primary tumor from PRCA, and metastasis (MET). To assess the number of sex steroid receptor positive cells and marker staining intensity, we used an automated pathology platform. RESULTS: Expression and staining intensity of AR increased with PRCA progression in prostate epithelium, carcinoma cells and the stromal microenvironment. Epithelial and stromal ERa expression and staining intensity was highest in HGPIN and decreased in PRCA and MET, relative to BPT. However, among stromal cells negative for ACTA2, AR positive cells were less prevalent in PRCA compared to BPT, and there was no significant difference in the prevalence of ERa positive cells. In the epithelium and the stromal microenvironment, double positive (for AR & ERa) cells were most prevalent in HGPIN, relative to BPT. CONCLUSIONS: Taken together, the present results underscore the importance of androgens and estrogens in the biology of PRCA progression. We found an increased prevalence of cells expressing both AR and ERa in early, but not late stages of PRCA progression, suggesting that while the androgens are important in all stages of progression, the role for estrogens and sex steroid synergy may be most prominent during early prostate carcinogenesis. Source of Funding: F30DK093173
R01CA123199,
T32GM07356,
MP52-05 MOUSE PERIPROSTATIC ADIPOSE (PPA) TISSUE MIMICS HUMAN PPA ACTIVITY ON PROSTATE CANCER CELLS: A MODEL SYSTEM TO STUDY PPA e PROSTATE CANCER INTERACTIONS Palamadai N. Venkatasubramanian, Evanston, IL; Massa Mafi, Milwaukee, WI; Charles B. Brendler, Beth A. Plunkett, Evanston, IL; Jennifer Doll*, Milwaukee, WI INTRODUCTION AND OBJECTIVES: Visceral obesity promotes prostate cancer (PCa) progression; yet, the molecular mechanisms remain unclear. We recently showed that periprostatic adipose (PPA) tissues from obese PCa patients stimulate higher proliferative rates in both PCa and endothelial cells compared to that of PPA from lean or subcutaneous adipose (SQA) tissues from lean or obese patients. We also demonstrated that obesity shifts the fatty acid (FA) profile in the PPA and increases angiogenesis. Here, we examined mouse tissues to determine if their adipose depots had similar characteristics to that of human in order to develop a model system in which to study the obese fat e PCa interaction. METHODS: Adipose (SQA, PPA and the gonadal fat pat) were harvested from wildtype (WT) and obese (ob/ob) mice (3-4 mo.; n¼3-5/ group). Serum-free conditioned media from adipose tissue explant culture were collected at 48 h and used in MTT proliferation assays on PC-3 cells. Data were normalized to gram adipose tissue weight. Lipolytic activity of tissues was assessed using the free glycerol assay (Sigma). FA content was determined by magnetic resonance spectroscopy (MRS). RESULTS: Similarly to our previous human tissue data, obese mouse PPA tissues had an increased monounsaturated to saturated FA ratio compared to WT mice (1.983+/-0.36 vs. 1.157+/-0.19; P<0.002). Basal lipolytic activity was 52% lower in obese visceral adipose tissue compared to that of WT, while no difference was observed with obesity
THE JOURNAL OF UROLOGYâ
e582
in SQA tissues. Interestingly, obese mice develop prostatic hyperplasia, which is characterized by increased intraepithelial lipid accumulation (Doll et al., in revision). In initial functional tests using lean mice, PPA secretions increased PC-3 cell proliferation 1.8 and 2.3 times that of the visceral fat pad and SQA tissues, respectively (P<0.001), again consistent with our human tissue data. CONCLUSIONS: From these initial studies, it is evident that the PPA has pro-tumorigenic characteristics resembling that of human adipose tissues in PCa patients. Thus, the obese mouse can be used in future studies to elucidate how the obese PPA tissue e PCa interaction alters each tissue in combination with PCa mouse models. Such studies will provide unique insight into the molecular mechanisms of obesitydriven PCa. Source of Funding: This study was funded in parts by a Creativity Award from the Prostate Cancer Foundation, a Pilot grant and RCDA award from NorthShore University HealthSystem, the Northwestern University Prostate SPORE P50-CA090386, and philanthropic support secured secured through the Division of Urology and the John and Carol Walter Center for Urological Health, NorthShore University HealthSystem.
MP52-06 IN VITRO CO-CULTURE MODEL TO STUDY ADRENAL STIMULUS OF CRPC Jan Matthijs Moll*, Wilma Teubel, Rotterdam, Netherlands; Ian Hickson, Ralph Graeser, Beerse, Belgium; Guido Jenster, Wytske van Weerden, Rotterdam, Netherlands INTRODUCTION AND OBJECTIVES: Numerous studies have demonstrated the androgen receptor (AR) is still active in CPRC despite low serum testosterone levels. Among other mechanisms of AR-activation, intratumoural androgen synthesis, either via conversion of adrenal steroids or de novo synthesis from cholesterol, is a mechanism of castration resistance. Our group has previously demonstrated that clinical CRPC samples expressed markers for conversion of adrenal androgens and not for de novo synthesis. In this study, we compared growth stimulation of castration-na€ive and CRPC cell lines by adrenal androgens (conversion) or androgen precursors (de novo synthesis) and subsequently tested a novel coculture model to mimic adrenal stimulus of (CR)PC. METHODS: VCaP and DuCaP CRPC lines were generated by long-term culturing in androgen-depleted medium with or without the anti-androgens bicalutamide or flutamide. To test cell growth, VCaP and DuCaP cells and their CRPC sublines were cultured in the presence of androgen precursors (pregnenolone and progesterone) or adrenal androgens (DHEA and androstenedione) at levels found in men. Cultures were treated with vehicle, the CYP17A1-inhibitor Abiraterone (0.1 mM) or the anti-androgen MDV3100 (1 mM). Cell proliferation was assessed by MTT-assay on day 9 with each experiment performed in triplicate. For the co-culture system, VCaP cells were cultured with human adrenal (H295R) cells in separate compartments between which only medium, but not cells, could diffuse freely, and treated with vehicle, Abiraterone or R1881. RESULTS: In VCaP and DuCaP and their CRPC derivatives, androgen precursors pregnenolone and progesterone did not induce cell growth. Physiological levels of adrenal androgens stimulated cell growth, which was similar to optimal stimulus by DHT. Treatment with Abiraterone could not block adrenal androgen induced growth, while MDV3100 blocked steroid-induced growth in all conditions. Co-culturing of VCaP cells with H295R cells induced optimal cell growth, which could be inhibited by treatment with Abiraterone. CONCLUSIONS: These data support our previous clinical observation that (CR)PC seems to rely more on conversion than on de novo androgen synthesis. Co-culturing PC cells with human adrenal cells using a 2-compartment system is a relevant model to test CRPC stimulation in vitro.
Vol. 191, No. 4S, Supplement, Monday, May 19, 2014
Source of Funding: The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n 115188, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/ 2007-2013) and EFPIA companies in kind contribution.
MP52-07 DIFFERENTIAL MICRORNA EXPRESSION LEVELS IN GLEASON 6 PROSTATE BIOPSIES: A POTENTIAL TEST FOR GUIDANCE IN DETERMINING WHICH PATIENTS SHOULD UNDERGO TREATMENT VERSUS ACTIVE SURVEILLANCE Christopher Lebeis*, Boston, MA; Kari Bailey, Benjamin Waldorf, Casey Kowalik, Travjs Sullivan, David Canes, Alireza Moinzadeh, John Libertino, Kimberly Christ, Burlington, MA INTRODUCTION AND OBJECTIVES: Pathologic upgrading from biopsy to prostatectomy occurs in approximately 30% of prostate cancer cases. Biomarkers that more accurately assess risk of aggressive prostate cancer at the time of screening are critical for the future management of this disease. The aim of this study was to evaluate microRNA (miRNA) expression levels associated with Gleason 6 TRUS prostate biopsy specimens as an adjunct tool to better characterize the pathologic findings and predict which patients would be upgraded at the time of prostatectomy. METHODS: Total RNA was isolated from paraffin embedded TRUS biopsies from patients with Gleason Sum (GS) 6 that remained GS 6 (same grade) at prostatectomy and those that were upgraded to GS7 (upgrade) at the time of prostatectomy. For the discovery phase, pools consisting of ten samples each were created for the same grade and upgraded groups and miRNAs were profiled via microarray. Validation of miRNA expression levels was performed on 52 same grade and 55 upstaged samples by qRT-PCR. RESULTS: Microarray analysis identified 17 miRNAs with 003F1.6-fold differential expression between patients who had Gleason upgrading between biopsies and prostatectomy compared to patients who were same grade. Of these, twelve miRNAs were down-regulated and five were up-regulated in the upgraded group. Eight miRNAs were further validated by qRT-PCR on individual samples (n¼77) which confirmed differential expression (p<0.05) of seven miRNAs in the two study groups. Expanding the study to include 30 additional biopsy specimens with <20% tumor resulted in six miRNAs that remained significant. ROC curve analyses indicated that a combination of two miRNAs may be useful for discriminating patients with higher grade prostate cancer when all samples were included (AUC¼0.73) or excluding the less than 20% samples (AUC¼.78). CONCLUSIONS: GS6 TRUS biopsy samples demonstrated different miRNA expression levels between samples that were same grade or upgraded at prostatectomy. These differences in expression levels could provide the clinician with additional information to allow them to better stratify patients to active surveillance versus those that need to proceed to treatment. Future efforts will include developing a model to predict the GS at prostatectomy based on these miRNAs. Validation in a larger cohort is warranted. Source of Funding: none
MP52-08 TARGETING IGFBP-2 AND IGFBP-5 IN CASTRATION AND ENZALUTAMIDE RESISTANT PROSTATE CANCER Joseph Ischia*, Vancouver, Canada; Mototsugu Muramaki, Kobe, Japan; Yi Xia, Eliana Beraldi, Vancouver, Canada; Hideaki Miyake, Kobe, Japan; Amina Zoubeidi, Michael Cox, Martin Gleave, Vancouver, Canada INTRODUCTION AND OBJECTIVES: Castration and the next generation androgen receptor (AR) antagonist enzalutamide have been shown to improve survival in men with advanced prostate cancer.