- Email: [email protected]
MP87-18 ASSOCIATION OF GERMLINE GENETIC VARIANTS WITH TMPRSS2-ERG FUSION STATUS IN PROSTATE CANCER
THE JOURNAL OF UROLOGYâ
e1172
MP87-16 INTERFERON-INDUCED MICRORNA TURNOVER LEADING TO EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN CANCER U-Ging Lo*, Rey-Chen Pong, Elizabeth Hernandez, Shi-Hong Ma, Jer-Tsong Hsieh, Dallas, TX INTRODUCTION AND OBJECTIVES: Despite the role of interferon-g (IFNg) in tumor immune surveillance; studies have implicated the dark side of IFNg based on its pro-tumorigenic activity. IFNg can induce transcriptional activation of IFN-stimulated genes (ISGs) via JAK-STAT signaling pathway. The most highly induced ISGs are interferon-induced tetratricopeptide repeat (IFIT) family members. By studying a differential regulation on a unique tumor suppressor miR-363 from its polycistronic miR-106a-363 cluster, we unveiled a new microRNA (miRNA) turnover machinery composed of IFIT5, which is first described as a viral RNA binding protein. Up to date, the impact of IFIT5 on cancer metastasis is unclear. METHODS: Luciferase reporter gene assay was for examining the IFNg-induced IFIT5 gene activation. Transwell migration assay was for demonstrating the function of IFIT5 with cancer metastasis. Sitedirected mutagenesis, in vitro transcription, RNA pull down and in vitro RNA degradation assay were for determining the specificity of miRNA species regulated by IFIT5-mediated turnover machinery. RESULTS: IFIT5 gene promoter activity and protein level were significantly elevated by IFNg. IFIT5 complex represents unique posttranscriptional machinery for turnover of a specific population of tumor suppressor miRNAs. We examined several IFIT5-regulated miRNA candidates, and found IFNg can suppress miR-101, miR-335, miR-203, and miR-128, and phenocopied the miRNA expression profile of IFIT5overexpressing cells. Seed regions of miR-101 and miR-128 have sequence-matched target sites at ZEB1 mRNA 30 UTR, and indeed can suppress ZEB1. Meanwhile, IFIT5 is elevated in higher grade prostatic tumors, and positively correlated with ZEB1, ZEB2 and Slug in prostate cancer. Knockdown of IFIT5 lead to suppression of ZEB1 and Slug, along with decreased migration mobility in cancer cells. On the contrary, IFNg treatment enhanced cell migration, and this effect is diminished by the loss of IFIT5. We also modified the 50 end structure of precursor miR101 and miR-128, and examined its regulation by IFIT5-mediated miRNA turnover machinery. Both pre-miR-101 and pre-miR-128 with mutated blunt end are resistant to degradation in an IFIT5-expressing PC3 cell and show greater impact on suppressing cell migration, compared to the mutant precursor with single stranded overhang. CONCLUSIONS: We demonstrated that IFNg potentiate prostate cancer metastasis via IFIT5-mediated miRNA turnover machinery, which regulates specific tumor suppressor miRNAs that target critical EMT factors including ZEB1 and Slug. Source of Funding: DoD postdoc training award (W81XWH14-1-0249)Urology Care Foundation Research Scholar Award
Vol. 197, No. 4S, Supplement, Monday, May 15, 2017
suspicious regions (MSR), and MSR < 0.25 mL. The MRI targeted biopsy specimen (MRI+) and a systematic biopsy specimen geometrically distant from the MSR (MRI-) were stained for leukocytes (CD45). Blinded to the MRI result, leukocyte density (LD) was measured manually (number per mm) and presence of PCa was noted. The groups were compared using the unpaired t test. RESULTS: For the overall study cohort, the mean LD for MRI+ biopsy cores was significantly higher than for MRI- biopsy cores (71.9+25.4 versus 42.1+10.6, p¼0.04). Within the subset of only MRI+ biopsy cores, mean LD was not different based on the presence of PCa (71.8+28.0 versus 72.0+53.7, p¼0.99). Within the subset of only MRIbiopsy cores, mean LD was not different based on the presence of PCa (39.3+11.2 versus 52.2+28.3, p¼0.34). An example of similar LD in two MRI+ biopsy cores, despite PCa in one but not the other is provided. CONCLUSIONS: The LD was significantly higher for biopsy cores from MRI suspicious areas, irrespective of the presence of PCa. The MRI appearance may be representative of leukocytes (benign inflammation of tumor infiltrating lymphocytes) rather than PCa.
Source of Funding: Washington University School of Medicine, Division of Urology
MP87-17
MP87-18
PROSTATE MRI REPRESENTS LEUKOCYTE DENSITY AND NOT THE PRESENCE OF CANCER ON BIOPSY
ASSOCIATION OF GERMLINE GENETIC VARIANTS WITH TMPRSS2-ERG FUSION STATUS IN PROSTATE CANCER
Eric Kim*, Dengfeng Cao, Russell Pachynski, Robert Grubb III, Gerald Andriole, St. Louis, MO
Indu Kohaar*, Lakshmi Ravindranath, Denise Young, Amina Ali, Rockville, MD; Qiyuan Li, Fujian Sheng, China, People’s Republic of; Albert Dobi, David McLeod, Rockville, MD; Inger Rosner, Bethesda, MD; Isabell Sesterhenn, Silver Spring, MD; Matthew Freedman, Cambridge, MA; Shiv Srivastava, Gyorgy Petrovics, Rockville, MD
INTRODUCTION AND OBJECTIVES: Suspicious lesions on prostate MRI have been correlated to prostate cancer (PCa) on prostatectomy specimens. However, targeted biopsies of even highly suspicious prostate MRI lesions (i.e. PIRADS 4 or 5) are found to be benign in multiple reported series. One potential factor leading to this discrepancy is the presence of leukocytes, as they may be present in both a small focus of benign inflammation or present as tumor-infiltrating lymphocytes. METHODS: Reviewing patients with PIRADS 4 or 5 lesions that received MRI targeted as well as systematic biopsy from December 2014 to December 2015, we developed a study cohort for additional pathologic review. We excluded patients with a clinical history of prostatitis, prior prostate biopsy within 12 months, multiple MRI
INTRODUCTION AND OBJECTIVES: Oncogenic activation of ERG resulting from prevalent gene fusions is present in two thirds of prostate cancer (CaP) patients of European Ancestry including Caucasian Americans (CA). Our laboratory and others have recently reported that major cancer driver genes, including ERG, show significant racial/ethnic differences in CaP with lower frequencies in African Americans (AA), Africans and Asians. Racial differences of CaP associated SNPs have also been extensively described. However, there is limited data on germline association with ERG fusion status.
THE JOURNAL OF UROLOGYâ
Vol. 197, No. 4S, Supplement, Monday, May 15, 2017
The goal of this study is to identify germline molecular determinants associating with ERG status of CaP. METHODS: Blood derived genomic DNA samples were prepared from 270 AA men and 129 CA men treated by radical prostatectomy at Walter Reed National Military Medical Center. ERG status was determined in whole mounted prostate specimens by immunohistochemistry (IHC) for ERG protein expression as a surrogate for the TMPRSS2-ERG fusion. Blinded blood samples were genotyped for SNPs on the Illumina Golden Gate platform using Infinium Oncoarray, a 500K genome wide BeadChip kit from Illumina. Data analysis approaches included association analyses based on logistic regression, Principal Component Analysis (PCA) and Efficient Mixed-Model Association eXpedited (EMMAX) analysis. Genotype imputation analysis is being performed by IMPUTE2 program. RESULTS: After applying rigorous sample and SNP QC steps on the datasets, SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. Logistic regression, principal component analysis by EIGENSTRAT and a variance component approach, EMMAX analysis were performed to account for population structure. By EMMAX we identified SNPs associated with ERG status. The SNPs most significantly (<10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G proteincoupled receptor B3 (ADGRB3). Fine-mapping of SNPs is underway by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, followed by independent validation. CONCLUSIONS: This study identified SNPs differentially associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options. Source of Funding: Work is supported by DoD/PCRP Health Disparity Award; W81XWH-13-2-0096
MP87-19 ETS RELATED GENE (ERG) DRIVEN ANDROGEN RECEPTOR AGGREGATION IS A KEY REGULATOR OF ENDOPLASMIC STRESS AND CELL SURVIVAL DURING PROSTATE CARCINOGENESIS Taduru Sreenath*, Natallia Mikhalkevich, Shashwat Sharad, Rishita Gupta, Oluwatosin Diaro, Kevin Babcock, Charles Xavier, Ahmed Mohamed, Muhammad Jamal, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Rockville, MD; Isabell Sesterhenn, Silver Spring, MD; David McLeod, Rockville, MD; Inger Rosner, Bethesda, MD; Shiv Srivastava, Rockville, MD INTRODUCTION AND OBJECTIVES: Deregulated androgen receptor (AR) signaling due to either mutations or altered expression of the AR and its cofactors (activators or suppressors) has been identified as critical in prostate cancer development and progression. AR regulated oncogenic activation of Ets Related Gene (ERG) represents one of the most common and validated prostate cancer driver gene. In our recent studies using prostate specific ERG transgenic mouse prostate glands, we a observed novel morphological phenotypes of endoplasmic reticulum (ER) stress. Since AR was the critical regulator of ERG expression through TMPRSS2 promoter in human prostate cancer, the present study was aimed towards understanding the post-translational interactions between ERG and AR in ER stress and subsequent cell survival mechanisms in mouse and cell culture models. Understanding such mechanistic insights will potentially have major therapeutic implications. METHODS: Histological phenotype in the mouse prostate glands were examined by light and electron microscopy. Cell culture models of LNCaP, HEK293 and COS7 cells were utilized to examine the AR aggregations, Co-IP and Proximal Ligation Assay in the presence and
e1173
absence of ERG. Various domain deletions of AR were utilized to identify specific AR domain interactions with ERG and its contribution in AR aggregation. Luminal cell surface markers on the isolated mouse prostate glands and spontaneously immortalized mouse prostate epithelial cells from ERG transgenic mouse (MoE1) were analyzed by FACS analysis. RESULTS: Co-expression of ERG and AR in LNCaP and COS7 cells showed significant aggregation of AR in filter assays. Co-IP experiments and PLA assays in VCaP, LNCaP and HEK 293 cell revealed that ERG physically interacts with AR. Epithelial cells of ERGTg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to radiation induced cell death. Both epithelial cells grown into spheres and established MoE1 cells displayed increased CD49f (low) and significant increase in the EpCAM negative population. CONCLUSIONS: Overall, our experiments demonstrate the mechanistic link that the physical interactions between ERG and AR initiate the ER stress in prostate epithelium through AR misfolding/aggregation. Our observation of ERG induced AR aggregation is one of the initial events that lead to ER stress to cell survival indicate a critical function for ERG in the etiology of prostate cancer initiation and progression. Source of Funding: This research in part was supported by the National Cancer Institute R01CA162383 (S. S.) and HU000110-2-0002 funds.
MP87-20 THERAPEUTIC TARGETING OF TRANSCRIPTIONAL REPRESSOR BCL-6 IN ENZALUTAMIDE-RESISTANT CASTRATION-RESISTANT PROSTATE CANCER Hiroshi Hongo*, Takeo Kosaka, Yota Yasumizu, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Recently, several new drugs have been approved for castration-resistant prostate cancer (CRPC) patients, including the next generation anti-androgen enzalutamide (ENZ). However, survival benefits with ENZ are limited, and progression on ENZ is inevitable. We analyzed the gene expression profile of an ENZ-resistant CRPC cell line and identified BCL-6 as a potential therapeutic target. METHODS: Three cell lines were used: LNCaP, a human prostate cancer cell line that exhibits androgen-dependent proliferation; C4-2, an ENZ-sensitive CRPC cell line; and C4-2AT6, an ENZ-resistant CRPC cell line which had been established by incubating C4-2 in androgen ablation conditions. We performed whole genome expression analysis and explored gene profile changes among the three cell lines. RESULTS: Whole genome expression analysis by CGH array and Exome demonstrated that BCL-6 expression was higher in the order C42AT6 > C4-2 > LNCaP. We validated these results using western blotting. The protein expression level of BCL-6 was also higher in C4-2AT6 cells. We evaluated the cytotoxic effect of a BCL-6 inhibitor, 79-6, on C4-2AT6 cells. Relative cell viability when treated with 10 mM 79-6 was 65.7 0.8%. Western blotting revealed that 79-6 promoted p53 expression and PARP cleavage in C4-2AT6 cells. We also examined the effect of BCL-6 inhibition by gene knockdown with siRNA. Knockdown of BCL-6 significantly reduces the cell viability of C4-2AT6 cells (78.4 1.4%). Next, we examined the sensitivity of LNCaP and C4-2AT6 cells to ENZ. Relative cell viability when treated with 1 mM ENZ was 73.6 1.2% and 93.3 2.7%, respectively. C42AT6 cells exhibited ENZ resistance. We examined the synergistic effect of ENZ and 79-6 on C4-2AT6 cells. The relative cell viability when treated with 1 mM ENZ and 10 mM 79-6 was 43.5 2.1%. CONCLUSIONS: BCL-6 inhibition was able to overcome ENZ resistance in CRPC cell lines. Source of Funding: This work was supported by JSPS KAKENHI Grant Number 15K20109 and 26861299.
e1172
MP87-16 INTERFERON-INDUCED MICRORNA TURNOVER LEADING TO EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN CANCER U-Ging Lo*, Rey-Chen Pong, Elizabeth Hernandez, Shi-Hong Ma, Jer-Tsong Hsieh, Dallas, TX INTRODUCTION AND OBJECTIVES: Despite the role of interferon-g (IFNg) in tumor immune surveillance; studies have implicated the dark side of IFNg based on its pro-tumorigenic activity. IFNg can induce transcriptional activation of IFN-stimulated genes (ISGs) via JAK-STAT signaling pathway. The most highly induced ISGs are interferon-induced tetratricopeptide repeat (IFIT) family members. By studying a differential regulation on a unique tumor suppressor miR-363 from its polycistronic miR-106a-363 cluster, we unveiled a new microRNA (miRNA) turnover machinery composed of IFIT5, which is first described as a viral RNA binding protein. Up to date, the impact of IFIT5 on cancer metastasis is unclear. METHODS: Luciferase reporter gene assay was for examining the IFNg-induced IFIT5 gene activation. Transwell migration assay was for demonstrating the function of IFIT5 with cancer metastasis. Sitedirected mutagenesis, in vitro transcription, RNA pull down and in vitro RNA degradation assay were for determining the specificity of miRNA species regulated by IFIT5-mediated turnover machinery. RESULTS: IFIT5 gene promoter activity and protein level were significantly elevated by IFNg. IFIT5 complex represents unique posttranscriptional machinery for turnover of a specific population of tumor suppressor miRNAs. We examined several IFIT5-regulated miRNA candidates, and found IFNg can suppress miR-101, miR-335, miR-203, and miR-128, and phenocopied the miRNA expression profile of IFIT5overexpressing cells. Seed regions of miR-101 and miR-128 have sequence-matched target sites at ZEB1 mRNA 30 UTR, and indeed can suppress ZEB1. Meanwhile, IFIT5 is elevated in higher grade prostatic tumors, and positively correlated with ZEB1, ZEB2 and Slug in prostate cancer. Knockdown of IFIT5 lead to suppression of ZEB1 and Slug, along with decreased migration mobility in cancer cells. On the contrary, IFNg treatment enhanced cell migration, and this effect is diminished by the loss of IFIT5. We also modified the 50 end structure of precursor miR101 and miR-128, and examined its regulation by IFIT5-mediated miRNA turnover machinery. Both pre-miR-101 and pre-miR-128 with mutated blunt end are resistant to degradation in an IFIT5-expressing PC3 cell and show greater impact on suppressing cell migration, compared to the mutant precursor with single stranded overhang. CONCLUSIONS: We demonstrated that IFNg potentiate prostate cancer metastasis via IFIT5-mediated miRNA turnover machinery, which regulates specific tumor suppressor miRNAs that target critical EMT factors including ZEB1 and Slug. Source of Funding: DoD postdoc training award (W81XWH14-1-0249)Urology Care Foundation Research Scholar Award
Vol. 197, No. 4S, Supplement, Monday, May 15, 2017
suspicious regions (MSR), and MSR < 0.25 mL. The MRI targeted biopsy specimen (MRI+) and a systematic biopsy specimen geometrically distant from the MSR (MRI-) were stained for leukocytes (CD45). Blinded to the MRI result, leukocyte density (LD) was measured manually (number per mm) and presence of PCa was noted. The groups were compared using the unpaired t test. RESULTS: For the overall study cohort, the mean LD for MRI+ biopsy cores was significantly higher than for MRI- biopsy cores (71.9+25.4 versus 42.1+10.6, p¼0.04). Within the subset of only MRI+ biopsy cores, mean LD was not different based on the presence of PCa (71.8+28.0 versus 72.0+53.7, p¼0.99). Within the subset of only MRIbiopsy cores, mean LD was not different based on the presence of PCa (39.3+11.2 versus 52.2+28.3, p¼0.34). An example of similar LD in two MRI+ biopsy cores, despite PCa in one but not the other is provided. CONCLUSIONS: The LD was significantly higher for biopsy cores from MRI suspicious areas, irrespective of the presence of PCa. The MRI appearance may be representative of leukocytes (benign inflammation of tumor infiltrating lymphocytes) rather than PCa.
Source of Funding: Washington University School of Medicine, Division of Urology
MP87-17
MP87-18
PROSTATE MRI REPRESENTS LEUKOCYTE DENSITY AND NOT THE PRESENCE OF CANCER ON BIOPSY
ASSOCIATION OF GERMLINE GENETIC VARIANTS WITH TMPRSS2-ERG FUSION STATUS IN PROSTATE CANCER
Eric Kim*, Dengfeng Cao, Russell Pachynski, Robert Grubb III, Gerald Andriole, St. Louis, MO
Indu Kohaar*, Lakshmi Ravindranath, Denise Young, Amina Ali, Rockville, MD; Qiyuan Li, Fujian Sheng, China, People’s Republic of; Albert Dobi, David McLeod, Rockville, MD; Inger Rosner, Bethesda, MD; Isabell Sesterhenn, Silver Spring, MD; Matthew Freedman, Cambridge, MA; Shiv Srivastava, Gyorgy Petrovics, Rockville, MD
INTRODUCTION AND OBJECTIVES: Suspicious lesions on prostate MRI have been correlated to prostate cancer (PCa) on prostatectomy specimens. However, targeted biopsies of even highly suspicious prostate MRI lesions (i.e. PIRADS 4 or 5) are found to be benign in multiple reported series. One potential factor leading to this discrepancy is the presence of leukocytes, as they may be present in both a small focus of benign inflammation or present as tumor-infiltrating lymphocytes. METHODS: Reviewing patients with PIRADS 4 or 5 lesions that received MRI targeted as well as systematic biopsy from December 2014 to December 2015, we developed a study cohort for additional pathologic review. We excluded patients with a clinical history of prostatitis, prior prostate biopsy within 12 months, multiple MRI
INTRODUCTION AND OBJECTIVES: Oncogenic activation of ERG resulting from prevalent gene fusions is present in two thirds of prostate cancer (CaP) patients of European Ancestry including Caucasian Americans (CA). Our laboratory and others have recently reported that major cancer driver genes, including ERG, show significant racial/ethnic differences in CaP with lower frequencies in African Americans (AA), Africans and Asians. Racial differences of CaP associated SNPs have also been extensively described. However, there is limited data on germline association with ERG fusion status.
THE JOURNAL OF UROLOGYâ
Vol. 197, No. 4S, Supplement, Monday, May 15, 2017
The goal of this study is to identify germline molecular determinants associating with ERG status of CaP. METHODS: Blood derived genomic DNA samples were prepared from 270 AA men and 129 CA men treated by radical prostatectomy at Walter Reed National Military Medical Center. ERG status was determined in whole mounted prostate specimens by immunohistochemistry (IHC) for ERG protein expression as a surrogate for the TMPRSS2-ERG fusion. Blinded blood samples were genotyped for SNPs on the Illumina Golden Gate platform using Infinium Oncoarray, a 500K genome wide BeadChip kit from Illumina. Data analysis approaches included association analyses based on logistic regression, Principal Component Analysis (PCA) and Efficient Mixed-Model Association eXpedited (EMMAX) analysis. Genotype imputation analysis is being performed by IMPUTE2 program. RESULTS: After applying rigorous sample and SNP QC steps on the datasets, SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. Logistic regression, principal component analysis by EIGENSTRAT and a variance component approach, EMMAX analysis were performed to account for population structure. By EMMAX we identified SNPs associated with ERG status. The SNPs most significantly (<10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G proteincoupled receptor B3 (ADGRB3). Fine-mapping of SNPs is underway by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, followed by independent validation. CONCLUSIONS: This study identified SNPs differentially associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options. Source of Funding: Work is supported by DoD/PCRP Health Disparity Award; W81XWH-13-2-0096
MP87-19 ETS RELATED GENE (ERG) DRIVEN ANDROGEN RECEPTOR AGGREGATION IS A KEY REGULATOR OF ENDOPLASMIC STRESS AND CELL SURVIVAL DURING PROSTATE CARCINOGENESIS Taduru Sreenath*, Natallia Mikhalkevich, Shashwat Sharad, Rishita Gupta, Oluwatosin Diaro, Kevin Babcock, Charles Xavier, Ahmed Mohamed, Muhammad Jamal, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Rockville, MD; Isabell Sesterhenn, Silver Spring, MD; David McLeod, Rockville, MD; Inger Rosner, Bethesda, MD; Shiv Srivastava, Rockville, MD INTRODUCTION AND OBJECTIVES: Deregulated androgen receptor (AR) signaling due to either mutations or altered expression of the AR and its cofactors (activators or suppressors) has been identified as critical in prostate cancer development and progression. AR regulated oncogenic activation of Ets Related Gene (ERG) represents one of the most common and validated prostate cancer driver gene. In our recent studies using prostate specific ERG transgenic mouse prostate glands, we a observed novel morphological phenotypes of endoplasmic reticulum (ER) stress. Since AR was the critical regulator of ERG expression through TMPRSS2 promoter in human prostate cancer, the present study was aimed towards understanding the post-translational interactions between ERG and AR in ER stress and subsequent cell survival mechanisms in mouse and cell culture models. Understanding such mechanistic insights will potentially have major therapeutic implications. METHODS: Histological phenotype in the mouse prostate glands were examined by light and electron microscopy. Cell culture models of LNCaP, HEK293 and COS7 cells were utilized to examine the AR aggregations, Co-IP and Proximal Ligation Assay in the presence and
e1173
absence of ERG. Various domain deletions of AR were utilized to identify specific AR domain interactions with ERG and its contribution in AR aggregation. Luminal cell surface markers on the isolated mouse prostate glands and spontaneously immortalized mouse prostate epithelial cells from ERG transgenic mouse (MoE1) were analyzed by FACS analysis. RESULTS: Co-expression of ERG and AR in LNCaP and COS7 cells showed significant aggregation of AR in filter assays. Co-IP experiments and PLA assays in VCaP, LNCaP and HEK 293 cell revealed that ERG physically interacts with AR. Epithelial cells of ERGTg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to radiation induced cell death. Both epithelial cells grown into spheres and established MoE1 cells displayed increased CD49f (low) and significant increase in the EpCAM negative population. CONCLUSIONS: Overall, our experiments demonstrate the mechanistic link that the physical interactions between ERG and AR initiate the ER stress in prostate epithelium through AR misfolding/aggregation. Our observation of ERG induced AR aggregation is one of the initial events that lead to ER stress to cell survival indicate a critical function for ERG in the etiology of prostate cancer initiation and progression. Source of Funding: This research in part was supported by the National Cancer Institute R01CA162383 (S. S.) and HU000110-2-0002 funds.
MP87-20 THERAPEUTIC TARGETING OF TRANSCRIPTIONAL REPRESSOR BCL-6 IN ENZALUTAMIDE-RESISTANT CASTRATION-RESISTANT PROSTATE CANCER Hiroshi Hongo*, Takeo Kosaka, Yota Yasumizu, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Recently, several new drugs have been approved for castration-resistant prostate cancer (CRPC) patients, including the next generation anti-androgen enzalutamide (ENZ). However, survival benefits with ENZ are limited, and progression on ENZ is inevitable. We analyzed the gene expression profile of an ENZ-resistant CRPC cell line and identified BCL-6 as a potential therapeutic target. METHODS: Three cell lines were used: LNCaP, a human prostate cancer cell line that exhibits androgen-dependent proliferation; C4-2, an ENZ-sensitive CRPC cell line; and C4-2AT6, an ENZ-resistant CRPC cell line which had been established by incubating C4-2 in androgen ablation conditions. We performed whole genome expression analysis and explored gene profile changes among the three cell lines. RESULTS: Whole genome expression analysis by CGH array and Exome demonstrated that BCL-6 expression was higher in the order C42AT6 > C4-2 > LNCaP. We validated these results using western blotting. The protein expression level of BCL-6 was also higher in C4-2AT6 cells. We evaluated the cytotoxic effect of a BCL-6 inhibitor, 79-6, on C4-2AT6 cells. Relative cell viability when treated with 10 mM 79-6 was 65.7 0.8%. Western blotting revealed that 79-6 promoted p53 expression and PARP cleavage in C4-2AT6 cells. We also examined the effect of BCL-6 inhibition by gene knockdown with siRNA. Knockdown of BCL-6 significantly reduces the cell viability of C4-2AT6 cells (78.4 1.4%). Next, we examined the sensitivity of LNCaP and C4-2AT6 cells to ENZ. Relative cell viability when treated with 1 mM ENZ was 73.6 1.2% and 93.3 2.7%, respectively. C42AT6 cells exhibited ENZ resistance. We examined the synergistic effect of ENZ and 79-6 on C4-2AT6 cells. The relative cell viability when treated with 1 mM ENZ and 10 mM 79-6 was 43.5 2.1%. CONCLUSIONS: BCL-6 inhibition was able to overcome ENZ resistance in CRPC cell lines. Source of Funding: This work was supported by JSPS KAKENHI Grant Number 15K20109 and 26861299.