Yingjie Song

MP70-13 DEPLETION OF PMEPA1 GENE CONFERS INCREASED CELL PROLIFERATION IN MOUSE PROSTATE EPITHELIUM

expression profiles of PC3CR by microarray to investigate the mechanisms of CBZ resistance. RESULTS: We incubated PC3 cells with gradually increasing concentrations of CBZ for 1.5 years, and established a CBZ-resistant cell line, PC3CR. PC3CR cells underwent cell division with 3 nM CBZ. We compared the cytotoxic effect of CBZ on PC3 and PC3CR cells using a cell viability assay. The half maximal inhibitory concentration (IC50) of CBZ in PC3 and PC3CR cells were 11.0 nM and 3.7 nM, respectively. Mice PC3CR xenograft tumors also had CBZ resistance (Fig. A) Functional annotation clustering (FAC) analysis using microarray data demonstrated cell division (GO: 0051301) and mitotic nuclear division (GO: 0007067) were the most enhanced clusters in PC3CR compared to PC3. These results suggested that enhancement of cell cycle progression signaling was related with CBZ resistance in CRPC. We perform in silico screening for compounds overcoming CBZ resistance by Connectivity Map (CMAP) analysis. Etoposide (VP16) was one of the candidate drugs which reverted gene expression pattern of CBZ-resistant cells into CBZ-sensitive cells. Topoisomerase IIa (TOP2A) is a main target of VP16, so we examined TOP2A expression in PC3CR. Quantitative PCR showed up-regulation of TOP2A in PC3CR. In cell viability assay, 1mM etoposide significantly inhibited PC3CR proliferation (80.3 3.2%). VP16 is ordinary used with platinum-based agents for neuroendocrine tumor. We tested cytotoxic effect of CDDP for CBZ-resistant CPRC. The relative cell viabilities of PC3CR treated with 3mM CDDP was 86.4 1.6%. CDDP was also effective for CBZ-resistant CRPC cells. Next, we tested anti-tumor effect of VP16 and CDDP using PC3CR xenograft tumor model. Both singleagent treatments with VP16 and CDDP significantly inhibited PC3CR xenograft tumors (Fig. B). Moreover, VP16 and CDDP in combination use had a synergic effect for the xenograft tumors. CONCLUSIONS: Etoposide based chemotherapy may be an optimal treatment for CPRC in the post-cabazitaxel setting.

Abstract A35: Increased expression of PCGEM1 lncRNA in prostate cancer of African American men

Introduction: Prostate cancer (CaP) affects 1 in 7 men throughout their life time. One of the major risk factors for the development of CaP is race/ethnicity. African American (AA) men have significantly higher incidence and mortality from CaP compared to Caucasian American (CA). Our laboratory and others have recently described that cancer driver genes, including ERG and PTEN, have significantly different frequencies in AA versus CA CaP. In recent years progress has been made in evaluating the expression and role of long noncoding RNAs (lncRNAs) in prostate tumorigenesis, but variations in lncRNAs expression across race/ethnicity (AA vs CA) remains poorly understood. The objective of this study is to compare the expression of CaP related lncRNAs among AA and CA men. Methods: Whole-mounted prostate sections from 30 patients, 15 AA and 15 CA matched for age, Gleason score and pathologic stage, were evaluated for the expression of lncRNAs that have been reported to be associated with CaP or other cancers. Probe sets were designed for the following 20 lncRNAs: CTBP1-AS, PCA3, PRNCR1, Hoxa11as, H19, HOTTIP, MEG3, PCGEM1, ST7OT1, SChLAP1, ANRIL/p15AS/CDK2BAS, HOTAIR, SRA-1/SRA, MALAT1/NEAT2, GAS5/SNHG2, PlncRNA-1/CBR3-AS1, Xist, hY1/RNY1, hY3/RNY3, PCAT-1. Tumor and benign epithelium was dissected under microscope from the slides, and the isolated RNA, passing BioAnalyzer quality control, was evaluated by using Nanostring platform. PCGEM1 expression analysis was updated in an existing QRT-PCR dataset of 88 patients (24 AA and 64 CA). Results: Copy numbers of each gene, normalized to ten housekeeping genes, was compared between tumor and matched normal prostate epithelium and across ethnic groups. We have identified a panel of lncRNAs (CTBP1-AS, PCA3, PCAT-1, PRNCR1) overexpressed in tumor versus matched normal samples in 95% of patients when combined. Three genes were significantly downregulated in CaP (MEG3, HOTTIP, H19). Most significantly, PCGEM1 had robust overexpression in prostate tumors (over 10-fold upregulation) only in AA patients (7 of 15) but not in CAs (0 of 15), which was in concordance with evaluation of PCGEM1 in existing dataset, underscoring selective overexpression in AA CaP. Conclusions: PCGEM1 was robustly overexpressed only in AA patients, whereas several lncRNAs were overexpressed (CTBP1-AS, PCA3, PCAT-1, PRNCR1) or downregulated (MEG3, HOTTIP, H19) in tumors of both AA and CA patients. Biomarker potential and biological functions of PCGEM1 are under further investigation. Citation Format: Jocelyn Lee, Muhammad Jamal, Wusheng Yan, Denise young, Yingjie Song, Yongmei Chen, Shilpa Katta, Lakshmi Ravindranath, Alagarsamy Srinivasan, Jennifer Cullen, Jacob Kagan, Sudhir Srivastava, Albert Dobi, Inger L. Rosner, David G. McLeod, Isabell A. Sesterhenn, Shiv Srivastava, Gyorgy Petrovics. Increased expression of PCGEM1 lncRNA in prostate cancer of African American men. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A35.

Abstract 2016: Evaluation of prostate cancer biomarkers and therapeutic targets in FFPE specimens using the NanoString platform

Introduction: Identification of prognostic indicators of disease progression is one of the highest priorities in prostate cancer (CaP) research. The majority of CaP tissue specimens available for biomarker discovery and validation are formalin fixed paraffin embedded (FFPE) specimens. The RNA quality of these tissues for traditional gene expression analysis approaches is not adequate, especially of cases with long term- follow up necessary for prognostic marker studies. We systematically evaluated and optimized the NanoString platform for CaP gene expression analysis in FFPE whole mounted prostate specimens with up to 15 years follow up. Methods: NanoString readouts were compared between a series of input total RNA prepared through different tissue dissection methods. We then performed NanoString analysis of 151-CaP probe set on 63 archived CaP samples to evaluate this modified protocol. ERG gene expression was validated by immunohistochemistry (IHC) assay using CPDR ERG-MAb (9FY). Results: We have performed NanoString analysis of CPDR designed 151- CaP gene panel on whole-mounted prostate tissue specimens using our optimized protocol. The prostate epithelial cell markers KLK3, MSMB and PAP had the highest signals in all samples reflecting the prostate epithelial origin of the specimens. Established CaP specific genes, such as AMACR, PCA3 and PSGR were most highly over-expressed in tumors compared to matched benign prostate epithelium. ERG positive tumors had strong expression of both TMPRSS2-ERG fusion probes and probes for several ERG splice forms (ERG 1,2,3 and 8), with no signals in the matched benign samples. The ERG gene expression status was consistent with blinded ERG IHC results. Conclusions: The feasibility of NanoString profiling in archived whole mounted FFPE prostate specimens was demonstrated. This approach is well suited for a medium throughput evaluation and validation of prognostic biomarker and therapeutic target candidates in CaP. Citation Format: Jocelyn Lee, Wusheng Yan, Denise Young, Yingjie Song, Yongmei Chen, Shilpa Katta, Ahmed Mohamed, Lakshmi Ravindranath, Alagarsamy Srinivasan, Jennifer Cullen, Jacob Kagan, Sudhir Srivastava, Albert Dobi, Inger Rosner, David G. McLeod, Isabell A. Sesterhenn, Shiv Srivastava, Gyorgy Petrovics. Evaluation of prostate cancer biomarkers and therapeutic targets in FFPE specimens using the NanoString platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2016. doi:10.1158/1538-7445.AM2015-2016