Ramez Andrawis

Identification of Differentially Methylated Genes in Normal Prostate Tissues from African American and Caucasian Men

Purpose: Aberrant DNA methylation changes are common somatic alterations in prostate carcinogenesis. We examined the methylation status of six genes in prostate tissue specimens from African American (AA) and Caucasian (Cau) males. Experimental Design: We used pyrosequencing to quantitatively measure the methylation status of GSTP1, AR, RARβ2, SPARC, TIMP3, and NKX2-5. Real-time PCR was used to determine gene expression, and gene reactivation was analyzed by 5-aza-2′-deoxycytidine and/or trichostatin A treatment. Results: Statistical analysis showed significantly higher methylation in the prostate cancer tissue samples in comparison with matched normal samples for GSTP1 (P = 0.0001 for AA; P = 0.0008 for Cau), RARβ2 (P < 0.001 for AA and Cau), SPARC (P < 0.0001 for AA and Cau), TIMP3 (P < 0.0001 for AA and Cau), and NKX2-5 (P < 0.0001 for AA; P = 0.003 for Cau). Overall, we observed significant differences (P < 0.05) in the methylation level for all genes, except GSTP1, in the AA samples in comparison with the Cau samples. Furthermore, regression analysis revealed significantly higher methylation for NKX2-5 (P = 0.008) and TIMP3 (P = 0.039) in normal prostate tissue samples from AA in comparison with Cau, and a statistically significant association of methylation with age for NKX2-5 (P = 0.03) after adjusting for race. Conclusion: Our findings show higher methylation of several genes in prostate tissue samples from AA in comparison with Cau and may potentially contribute to the racial differences that are observed in prostate cancer pathogenesis. Clin Cancer Res; 16(14); 3539–47. ©2010 AACR.

Identification and Functional Validation of Reciprocal microRNA-mRNA Pairings in African American Prostate Cancer Disparities

Purpose: African Americans (AA) exhibit higher rates of prostate cancer incidence and mortality compared with European American (EA) men. In addition to socioeconomic influences, biologic factors are believed to play a critical role in prostate cancer disparities. We investigated whether population-specific and -enriched miRNA–mRNA interactions might contribute to prostate cancer disparities. Experimental Design: Integrative genomics was used, combining miRNA and mRNA profiling, miRNA target prediction, pathway analysis, and functional validation, to map miRNA–mRNA interactions associated with prostate cancer disparities. Results: We identified 22 AA-specific and 18 EA-specific miRNAs in prostate cancer versus patient-matched normal prostate, and 10 “AA-enriched/-depleted” miRNAs in AA prostate cancer versus EA prostate cancer comparisons. Many of these population-specific/-enriched miRNAs could be paired with target mRNAs that exhibited an inverse pattern of differential expression. Pathway analysis revealed EGFR (or ERBB) signaling as a critical pathway significantly regulated by AA-specific/-enriched mRNAs and miRNA–mRNA pairings. Novel miRNA–mRNA pairings were validated by qRT-PCR, Western blot, and/or IHC analyses in prostate cancer specimens. Loss/gain of function assays performed in population-specific prostate cancer cell lines confirmed miR-133a/MCL1, miR-513c/STAT1, miR-96/FOXO3A, miR-145/ITPR2, and miR-34a/PPP2R2A as critical miRNA–mRNA pairings driving oncogenesis. Manipulating the balance of these pairings resulted in decreased proliferation and invasion, and enhanced sensitization to docetaxel-induced cytotoxicity in AA prostate cancer cells. Conclusions: Our data suggest that AA-specific/-enriched miRNA–mRNA pairings may play a critical role in the activation of oncogenic pathways in AA prostate cancer. Our findings also suggest that miR-133a/MCL1, miR-513c/STAT1, and miR-96/FOXO3A may have clinical significance in the development of novel strategies for treating aggressive prostate cancer. Clin Cancer Res; 21(21); 4970–84. ©2015 AACR.

Alternative splicing promotes tumour aggressiveness and drug resistance in African American prostate cancer

Clinical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 (idelalisib), in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa.

Circulating Tumor Cells in Biochemical Recurrence of Prostate Cancer

OBJECTIVE Circulating tumor cells (CTCs) have known prognostic implications in metastatic castration-resistant prostate cancer, but little is known regarding its utility in biochemical recurrence (BR) of prostate cancer. The primary objectives were to determine whether CTCs are measurable in patients with BR and whether it can reliably predict prostate-specific antigen (PSA) increase and PSA doubling times (PSADTs). METHODS BR was identified in patients after prostatectomy or radiation or both, with a PSA increase of ≥ 0.2 for prior prostatectomy or > 2 mg/dL increase for post-nadir in prior radiotherapy. CTCs were enumerated at baseline at the time of study entry using the CellSearch (Janssen Diagnostics, Raritan, NJ) test. RESULTS The median age for all 36 patients accrued was 69.5 years (range, 51-91) with a median PSA of 1.65 ng/mL (range, 0.2-65.8). Gleason scores ranged from 5 to 9 (median, 7). The majority had prostatectomy (n = 25), external beam radiotherapy (n = 9), CyberKnife (Accuray, Sunnyvale, CA) (n = 1), and combined radiohormonal therapy (n = 1). PSADT ranged from 0.35 to 55 months, with a median of 7.43 months. The incidence of positive CTCs was 8.3% (3 patients), of whom 2 had biopsy-proven bony lesions on presenting with equivocal scans and PSADTs of 2.27 and 3.08 months, respectively. The third CTC-positive patient had a PSADT of 4.99 months. CONCLUSIONS Obtaining CTCs in unselected patients presenting with BR has a relatively low yield. However, obtaining a positive CTC raises the suspicion of the presence of metastatic disease and may have utility for longitudinal follow-ups of patients with BR.

Circulating tumor cells (CTCs) in biochemical recurrence (BR) of prostate cancer: Final results.

179 Background: CTCs have known prognostic implications in metastatic prostate cancer. We presented initial data on the role of CTCs in BR of prostate cancer (Aragon-Ching et. al., ASCO GU 2011). The primary aim of this study was to determine whether there is a correlation between the number of CTCs with prostate specific antigen (PSA) levels and PSA doubling time (PSADT) in men with BR. METHODS BR was defined as patients who have undergone primary treatment with prostatectomy or radiation or both, with a rise to ≥ 0.2 from a prior undetectable level for prior prostatectomy or > 2 mg/dl rise for post-nadir radiotherapy. 36 patients (pts) were enrolled from May 2010 to May 2012. CTCs were evaluated in 7.5 mL of peripheral blood using the CTC CellSearch test. RESULTS The median age for 36 pts was 69.5 (range: 51 - 91) with a median PSA of 1.65 ng/mL (range 0.2 - 65.8) and testosterone levels of 315 ng/dL (range: 31 - 727). Gleason scores ranged from 5 to 9 (median=7). Prostatectomy was the primary treatment in 25 pts, radiotherapy in 9 pts, Cyberknife in 1 pt and combined radiohormones in 1 pt. PSADT varied between 0.35 to 55 months, with a median of 7.43 mos. The incidence of positive CTCs was 8.3% (3 out of 36 pts) of whom 2 had biopsy-proven bony lesions upon presenting with equivocal scans. The 3 pts who had measurable CTCs had PSADT of 2.27, 4.99 and 3.08 months, respectively. CONCLUSIONS Obtaining CTCs in unselected patients presenting with biochemical recurrence have low yield. However, obtaining a positive CTC raises the suspicion of presence of metastatic disease and may have utility for longitudinal follow-ups of patients with BR. Supported by IRG-08-091-01 from ACS to GWU Cancer Institute.

Role of circulating tumor cells (CTCs) in biochemical recurrence (BR) of prostate cancer.

60 Background: CTCs have an established role in the prognosis of metastatic prostate cancer. Little data exists regarding the role of CTCs in BR of prostate cancer. The aim of this study was to determine whether there is a correlation between the number of CTCs in men with BR with varying prostate specific antigen (PSA) and PSA doubling time (PSADT) categories. Secondary endpoints looking at correlation of the CTCs with clinical or laboratory factors (Gleason scores, testosterone, hemoglobin, alkaline phosphatase, BMI, imaging results) will also be assessed. METHODS BR was defined as patients (pts) who have undergone primary treatment with prostatectomy or radiation or both, with rise to >/= 0.2 from a prior undetectable level for prior prostatectomy or > 2 mg/dl rise from post-nadir radiotherapy. The study was powered to detect a Pearson correlation of .46 with a sample size of 36. Eleven of planned accrual goal of 36 pts were enrolled from May to September 2010. PSADT was obtained and correlated with the CTC values, categorized as PSADT of < 3 months, 3-14.9 months and > 15 months. CTCs were evaluated in 7.5 mL of peripheral blood using the CTC CellSearch test. RESULTS The median age for 11 patients was 75 y/o (range: 57-91) with a median PSA of 1.6 ng/mL (range 0.2-6.5) and testosterone levels of 309 ng/dL (range: 31-471). Gleason scores were 8 (n=1), 7 (n=5), 6 (n=2), 5 (n=3). Prostatectomy was the primary treatment in 6 pts, radiotherapy in 5 pts and Cyberknife in 1 pt. Median hemoglobin was 12.43 g/dL, BMI was 26.79 and alkaline phosphatase was 69 IU/L. PSADT varied between 3 to 55 months. All pts accrued had 0 CTC levels. The latter result translates into a 95% confidence interval upper bound of approximately .27 for the proportion of patients in this population who have non-zero CTC levels. CONCLUSIONS Prostate cancer pts with BR have negative blood CTCs and does not appear to correlate with PSA or PSADT. However, the limited number of patients precludes sufficient interpretation at this time and further accrual is ongoing. The absence of CTC levels in this patient population, if supported through further data collection, could emerge as an important unanticipated finding from this study. Supported by IRG-08-091-01 from ACS to GWU Cancer Institute. No significant financial relationships to disclose.

Abstract LB-265: Genomic analysis reveals alternative mRNA splicing, differential mRNA expression and gene networks associated with prostate cancer disparities in African American and Caucasian American populations

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Prostate cancer (PCa) is a disease conferred by gene mutations, numerous alternations in gene expression and aberrant changes in genome composition/architecture. An area of research that continues to garner attention is PCa health disparities, wherein the African American (AA) population exhibits higher incidence and mortality rates compared to Caucasian Americans (CA). To identify the genetic predispositions and oncogenic networks associated with the observed PCa disparities, we applied a systems biology approach by integrating exon, microRNA and SNP information from PCa specimens of AA and CA patients. RNA and DNA purified from PCa and patient-matched normal prostate needle biopsies from AAs and CAs were processed and hybridized onto Affymetrix human Exon 1.0 ST, SNP 6.0 arrays or Agilent human microRNA arrays. A 4-way statistical design (10% FDR, >1.5 fold-change) was employed to identify differentially expressed mRNAs in the following comparisons: AA normal vs. CA normal, AA cancer vs. CA cancer, AA cancer vs. AA normal, and CA cancer vs. CA normal. Pathway analyses of the differentially expressed genes have revealed several critical network-level rewiring of gene interactions accounting for PCa disparities between AA and CA. For example, the mis-regulated testosterone metabolism network in normal AA prostate tissues may represent a genetic predisposition factor in the AA population. Whereas, the inflammatory response (NF-κB network), activated oncogenic signaling pathways ( ERK , JNK and p38 ), and up-regulated cancer promoting genes ( RHOA and STAT1 ) may be associated with the higher recurrence and death rates in AA patients. Moreover, our exon and SNP profiling results have identified hundreds of genes exhibiting differential splicing patterns and/or copy number variations (CNVs) in AA and CA patients. Notably, at least 18 genes residing within the 5 oncogenic signaling pathways have been identified as exhibiting either alternative mRNA splicing, differential microRNA regulation or CNV between AA and CA PCa specimens. Using exon-specific siRNA knockdowns for selectively suppressing isoforms of 4 oncogenes ( PIK3CD, FGFR3 , MET and RASGRP2 ), we have further revealed that the alternative spliced variants have differential functions on regulating cell proliferation and invasive migration. Taken together, our data suggest that mRNA splicing, CNV, deregulated microRNA expression and gene-network rewiring may be critical in PCa health disparities between the AA and CA populations. These findings should advance our knowledge on the molecular mechanisms underlying PCa disparities, and may facilitate the development of novel strategies for PCa detection, diagnosis, prognosis, and therapy in AA population. This work was supported by NCI grant 5U01-CA-116937 and ACS-IRG-08–091–01. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-265. doi:10.1158/1538-7445.AM2011-LB-265

Abstract B37: Differential mRNA expression, microRNA regulation and gene networks are associated with the prostate cancer disparities in African American and Caucasian American populations

Prostate cancer (PCa) is a disease conferred by multiple gene mutations, numerous alternations in gene expression and aberrant changes in genome composition/architecture. An area of research that continues to garner attention is PCa health disparities, wherein the African American (AA) population exhibits higher incidence and mortality rates compared to Caucasian Americans (CA). Although accumulating evidences have suggested that the widespread microRNA deregulation may play crucial role in cancer development, the relationship between population-specific microRNAs and PCa disparities remains largely unknown. To identify the genetic predispositions and oncogenic networks associated with the observed PCa disparities, we applied a systems biology approach by combining mRNA expression profiling, microRNA profiling and microRNA target searches to characterize the genetic portraits of PCa in AA and CA populations. Affymetrix human exon 1.0ST arrays and Agilent human miRNA V2 arrays were used to analyze the global mRNA and microRNA expression profiles in AA and CA prostate tissue samples. From the mRNA profiling results, a 4-way statistical analysis (t-test with 10% FDR; AA normal vs. CA normal, AA cancer vs. CA cancer, AA cancer vs. AA normal, and CA cancer vs. CA normal) has identified 275 to 987 genes with differential expression in the 4-way comparisons. Furthermore, pathway analysis revealed that the mis-regulated testosterone metabolic pathway, activated inflammatory response and up-regulated oncogenic pathways (ERK, JNK and p38) are associated with the biological differences when comparing AA normal to CA normal, AA cancer to CA cancer and AA cancer to AA normal. In addition, a microRNA/mRNA-regulated network, generated by integrating microRAN-targeted mRNAs and mRNA profiling data, suggested that the population-specific microRNAs may contribute to differential cell-cycle control, protein synthesis and cell apoptosis in AA and CA prostate cancers. Our study has demonstrated that differential gene-network regulation, driven by the population-specific mRNA and microRNA expression, may explain in part the biological differences between AA and CA prostate cancers. This work was supported by NCI grant 5U01-CA-116937 and ACS-IRG-08-091-01. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B37.

Abstract 2227: Genomic study on prostate cancer disparities reveals the differential transcriptomes, microRNA profiles, alternative splicing patterns and copy number variations between Caucasian and African American populations

Prostate cancer (PCa) is a disease conferred by gene mutations, numerous alternations in gene expression and aberrant changes in genome composition/architecture. An area of research that continues to garner attention is PCa health disparities, wherein the African American (AA) population exhibits higher incidence and mortality rates compared to Caucasian Americans (CA). To identify the genetic predispositions and oncogenetic networks associated with the observed PCa disparities, we applied integrated genomic technologies to investigate RNA and microRNA expressions, alternative splicing events, and DNA copy number variations (CNVs) in AA and CA populations. RNA and DNA purified from PCa (Gleason score > 6) and paired adjacent normal prostate needle biopsies from AAs and CAs were processed and hybridized onto Affymetrix human Exon 1.0 ST and SNP 6.0 arrays. A 4-way statistical design (t-test with 10% false discovery rate) was employed to identify differentially expressed mRNAs in the following comparisons: AA normal vs. CA normal, AA cancer vs. CA cancer, AA cancer vs. AA normal, and CA cancer vs. CA normal. Pathway analyses of the differentially expressed genes (275 to 987 genes in the 4-way comparison) revealed network-level rewiring of gene interactions (e.g. gene networks or circuits) accounting for the PCa disparities between AA and CA. For example, the mis-regulated testosterone metabolism network in normal AA compared with normal CA tissues may represent a predisposition factor in the AA population; whereas the activated inflammatory response (NF-κB network), up-regulated oncogenic signaling pathways (ERK, JNK and p38) and more aggressive cancer invasion and metastasis (highly expressed RHOA and STAT1) in AA carcinomas may associated with the higher recurrence and death rates in AA patients. In addition, our expression and CNV atlas have identified hundreds of genes exhibiting differential splicing patterns or copy number aberrations (deletions or amplifications) between the AAs and CAs, representing candidate genes mediating PCa disparities. Notably, at least 13 genes residing within the 5 oncogenic signaling pathways have been identified as exhibiting either differential splicing (in FGFR3, PDGFRA, MET, EPHA3, NF1, RASGRP2, GSK3, TSC2, ATM, RAF, and RB1) or CNVs (at EPHA, PTEN and APC) between AA and CA PCa specimens. Taken together, our data suggest that gene-network rewiring, mRNA splicing and CNV may play important roles in the PCa health disparities between AA and CA populations. Further identification of these critical genetic elements related to PCa disparities may facilitate the development of biomarkers for screening, early detection, and prediction of clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2227.

Abstract B55: Integrative genomic analysis of prostate cancer disparities between African American and Caucasian American populations

Prostate cancer (PCa) is a disease conferred by gene mutations, numerous alternations in gene expression and aberrant changes in genome composition/architecture. An area of research that continues to garner attention is PCa health disparities, wherein the African American (AA) population exhibits higher incidence and mortality rates compared to Caucasian Americans (CA). To identify the genetic predispositions and oncogenic networks associated with the observed PCa disparities, we applied a systems biology approach by integrating exon, microRNA and SNP information from PCa specimens of AA and CA patients. RNA and DNA purified from PCa (with Gleason score ≥6) and patient-matched normal prostate needle biopsies from AAs and CAs were processed and hybridized onto Affymetrix human Exon 1.0 ST, SNP 6.0 arrays or Agilent human microRNA arrays. A 4-way statistical design (10% FDR, >1.5 fold-change) was employed to identify differentially expressed mRNAs in the following comparisons: AA normal vs. CA normal, AA cancer vs. CA cancer, AA cancer vs. AA normal, and CA cancer vs. CA normal. Pathway analyses of the differentially expressed genes have revealed several critical network-level rewiring of gene interactions accounting for PCa disparities between AA and CA. For example, the mis-regulated testosterone metabolism network in normal AA prostate tissues may represent a genetic predisposition factor in the AA population. Whereas, the inflammatory response (NF-?B network), activated oncogenic signaling pathways (ERK, JNK and p38), and up-regulated cancer promoting genes (RHOA and STAT1) may be associated with the higher recurrence and death rates in AA patients. Moreover, our exon and SNP profiling results have identified hundreds of genes exhibiting differential splicing patterns and/or copy number variations (CNVs) in AA and CA patients. Notably, at least 13 genes residing within the 5 oncogenic signaling pathways have been identified as exhibiting either differential splicing (in FGFR3, PDGFRA, MET, EPHA1, NF1, RASGRP2, GSK3, TSC2, ATM, RAF and RB1) or CNVs (at EPHA, PTEN and APQ between AA and CA PCa specimens. In addition, micro RNA profiling further revealed that 14 micro RNAs (e.g. miR-125b-2∗, miR-34a, miR-100, miR-99a, miR-200a) were differentially expressed in AA and CA cancer samples, potentially related to cell proliferation, anti-apoptosis, cell cycle regulation in AA cancers. Taken together, our data suggest that mRNA splicing, CNVs, deregulated microRNA expression and gene-network rewiring may be critical in PCa health disparities between the AA and CA populations. These findings should advance our knowledge on the molecular mechanisms underlying PCa disparities, and may facilitate the development of novel strategies for PCa detection, diagnosis, prognosis, and therapy in AA population. This work was supported by NCI grant 5U01-CA-116937. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B55.