ABSTRACT
Castration resistant prostate cancer (CRPC) remains an incurable disease stage with ineffective treatments options. Here, the androgen receptor (AR) coactivators CBP/p300, which are histone acetyltransferases, were identified as critical mediators of DNA damage repair (DDR) to potentially enhance therapeutic targeting of CRPC. Key findings demonstrate that CBP/p300 expression increases with disease progression and selects for poor prognosis in metastatic disease. CBP/p300 bromodomain inhibition enhances response to standard of care therapeutics. Functional studies, CBP/p300 cistrome mapping, and transcriptome in CRPC revealed that CBP/p300 regulates DDR. Further mechanistic investigation showed that CBP/p300 attenuation via therapeutic targeting and genomic knockdown decreases homologous recombination (HR) factors in vitro, in vivo, and in human prostate cancer (PCa) tumors ex vivo. Similarly, CBP/p300 expression in human prostate tissue correlates with HR factors. Lastly, targeting CBP/p300 impacts HR-mediate repair and patient outcome. Collectively, these studies identify CBP/p300 as drivers of PCa tumorigenesis and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed and DDR therapies.
ABSTRACT
Growing evidence indicates the involvement of a genetic component in prostate cancer (CaP) susceptibility and clinical severity. Studies have reported the role of germline mutations and single nucleotide polymorphisms (SNPs) of TP53 as possible risk factors for cancer development. In this single institutional retrospective study, we identified common SNPs in the TP53 gene in AA and CA men and performed association analyses for functional TP53 SNPs with the clinico-pathological features of CaP. The SNP genotyping analysis of the final cohort of 308 men (212 AA; 95 CA) identified 74 SNPs in the TP53 region, with a minor allele frequency (MAF) of at least 1%. Two SNPs were non-synonymous in the exonic region of TP53: rs1800371 (Pro47Ser) and rs1042522 (Arg72Pro). The Pro47Ser variant had an MAF of 0.01 in AA but was not detected in CA. Arg72Pro was the most common SNP, with an MAF of 0.50 (0.41 in AA; 0.68 in CA). Arg72Pro was associated with a shorter time to biochemical recurrence (BCR) (p = 0.046; HR = 1.52). The study demonstrated ancestral differences in the allele frequencies of the TP53 Arg72Pro and Pro47Ser SNPs, providing a valuable framework for evaluating CaP disparities among AA and CA men.
ABSTRACT
In prostate cancer, emerging data highlight the role of DNA damage repair genes (DDRGs) in aggressive forms of the disease. However, DDRG mutations in African American men are not yet fully defined. Here, we profile germline mutations in all known DDRGs (N = 276) using whole genome sequences from blood DNA of a matched cohort of patients with primary prostate cancer comprising of 300 African American and 300 European Ancestry prostate cancer patients, to determine whether the mutation status can enhance patient stratification for specific targeted therapies. Here, we show that only 13 of the 46 DDRGs identified with pathogenic/likely pathogenic mutations are present in both African American and European ancestry patients. Importantly, RAD family genes (RAD51, RAD54L, RAD54B), which are potentially targetable, as well as PMS2 and BRCA1, are among the most frequently mutated DDRGs in African American, but not in European Ancestry patients.
Subject(s)
Black or African American , Prostatic Neoplasms , Black or African American/genetics , DNA Damage/genetics , Germ-Line Mutation , Humans , Male , Mutation , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathologyABSTRACT
PURPOSE: Prostate cancer is predominantly indolent at diagnosis with a small fraction (15% to 25%) representing aggressive subtype (Gleason score 7-10), which is prone to metastatic progression. It is critical to explore noninvasive assays for the early detection of this aggressive subtype, when it still can be treated effectively. Additionally, there is an emerging need to develop markers that perform equally well across races, as racial differences in the prevalence and mortality of prostate cancer has become evident. MATERIALS AND METHODS: First catch, nondigital rectal examination urine specimens were collected from patients undergoing diagnostic biopsy. Total RNA was extracted from urinary exosomes and a quantitative expression assay protocol using droplet digital polymerase chain reaction was developed for detection of candidate genes in exosomal mRNAs from urine. Clinical performance for the gene expression assay was evaluated to predict high grade cancer (Gleason score 7-10) from low grade cancer (Gleason score 6) and cancer negative cases at biopsy. Assay performance was examined in combination with standard of care to determine improvement in model prediction. RESULTS: In a racially diverse patient cohort a 2-gene panel (PCA3, PCGEM1), in combination with standard of care variables, significantly improved the prediction of high grade cancer at diagnosis compared to standard of care variables alone (AUC 0.88 vs 0.80, respectively, p=0.016). Decision curve analysis showed that there is a benefit of adopting the gene panel for detection of high grade cancer compared to standard of care alone. CONCLUSIONS: This study highlights the potential for developing broadly applicable prostate cancer diagnostic biomarker panels for aggressive prostate cancer using our novel gene expression assay platform.
Subject(s)
Exosomes/genetics , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Biopsy , Cohort Studies , Diagnosis, Differential , Humans , Male , Middle Aged , Prostatic Neoplasms/urineABSTRACT
INTRODUCTION: Oncogenic activation of ERG resulting from TMPRSS2-ERG gene fusion is a key molecular genetic alteration in prostate cancer (CaP). The frequency of ERG fusion is variable by race; however, there are limited data available on germline polymorphisms associating with ERG fusion status. The goal of this study is to identify the inherited risk variants associating with ERG status of CaP. MATERIALS AND METHODS: SNP genotyping was performed on the Illumina platform using Infinium Oncoarray SNP chip on blood derived genomic DNA samples from 400 patients treated by radical prostatectomy at a single military institution. ERG status was determined in whole mounted prostate specimens by immuno-histochemistry (IHC) for ERG protein expression. Data analysis approaches included association analyses based on EMMAX and imputation by IMPUTE2. Imputed SNPs were validated by ddPCR. RESULTS: SNP genotyping analysis using imputation identified rs34349373 (p 4.68 × 10 -8 ) and rs2055272 (p 5.62 × 10-8) in TBC1D22B to be significantly associated with ERG fusion status in index tumor and non-index tumor foci. Imputed SNP rs2055272 was further experimentally validated by ddPCR with 98.04% (100/102) concordance. Initial discovery analysis based on SNPs on Oncoarray SNP chip, showed significant (p 10-5) association for SNPs (rs6698333, rs1889877, rs3798999, rs10215144, rs3818136, rs9380660 and rs1792695) with ERG fusion status. The study also replicated two previously known ERG fusion associated SNPs (rs11704416 in chromsome 22; rs16901979 in chromosome 8). CONCLUSIONS: This study identified SNPs associated with ERG status of CaP. IMPACT: The findings may contribute towards defining the underlying genetics of ERG positive and ERG negative CaP patients.
ABSTRACT
Dysfunctions of androgen/TGF-ß signaling play important roles in prostate tumorigenesis. Prostate Transmembrane Protein Androgen Induced 1 (PMEPA1) inhibits androgen and TGF-ß signaling via a negative feedback loop. The loss of PMEPA1 confers resistance to androgen signaling inhibitors and promotes bone metastasis. Conflicting reports on the expression and biological functions of PMEPA1 in prostate and other cancers propelled us to investigate isoform specific functions in prostate cancer (PCa). One hundred and twenty laser capture micro-dissection matched normal prostate and prostate tumor tissues were analyzed for correlations between quantitative expression of PMEPA1 isoforms and clinical outcomes with Q-RT-PCR, and further validated with a The Cancer Genome Atlas (TCGA) RNA-Seq dataset of 499 PCa. Cell proliferation was assessed with cell counting, plating efficiency and soft agar assay in androgen responsive LNCaP and TGF-ß responsive PC3 cells. TGF-ß signaling was measured by SMAD dual-luciferase reporter assay. Higher PMEPA1-a mRNA levels indicated biochemical recurrence (p = 0.0183) and lower PMEPA1-b expression associated with metastasis (p = 0.0173). Further, lower PMEPA1-b and a higher ratio of PMEPA1-a vs. -b were correlated to higher Gleason scores and lower progression free survival rate (p < 0.01). TGF-ß-responsive PMEPA1-a promoted PCa cell growth, and androgen-responsive PMEPA1-b inhibited cancer cell proliferation. PMEPA1 isoforms -a and -b were shown to be promising candidate biomarkers indicating PCa aggressiveness including earlier biochemical relapse and lower disease specific life expectancy via interrupting androgen/TGF-ß signaling.
ABSTRACT
BACKGROUND: As a major cause of morbidity and mortality among men, prostate cancer is a heterogenous disease, with a vast heterogeneity in the biology of the disease and in clinical outcome. While it often runs an indolent course, local progression or metastasis may eventually develop, even among patients considered "low risk" at diagnosis. Therefore, biomarkers that can discriminate aggressive from indolent disease at an early stage would greatly benefit patients. We hypothesized that tissue specimens from early stage prostate cancers may harbor predictive signatures for disease progression. METHODS: We used a cohort of radical prostatectomy patients with longitudinal follow-up, who had tumors with low grade and stage that revealed no signs of future disease progression at surgery. During the follow-up period, some patients either remained indolent (non-BCR) or progressed to biochemical recurrence (BCR). Total RNA was extracted from tumor, and adjacent normal epithelium of formalin-fixed-paraffin-embedded (FFPE) specimens. Differential gene expression in tumors, and in tumor versus normal tissues between BCR and non-BCR patients were analyzed by NanoString using a customized CodeSet of 151 probes. RESULTS: After controlling for false discovery rates, we identified a panel of eight genes (ERG, GGT1, HDAC1, KLK2, MYO6, PLA2G7, BICD1 and CACNAID) that distinguished BCR from non-BCR patients. We found a clear association of ERG expression with non-BCR, which was further corroborated by quantitative RT-PCR and immunohistochemistry assays. CONCLUSIONS: Our results identified ERG as the strongest predictor for BCR and showed that potential prognostic prostate cancer biomarkers can be identified from FFPE tumor specimens.
ABSTRACT
BACKGROUND: Germline mutations in BRCA2 have been linked to a higher risk of prostate cancer (PCa), and high frequency of BRCA1 and BRCA2 (BRCA1/2) gene alterations was recently reported in metastatic castration-resistant PCa specimens. Mutations in BRCA2 vary in racial and ethnic groups including African-American (AA) and Caucasian-American (CA) populations. METHODS: BRCA1 and BRCA2 genes were sequenced (Ion AmpliSeq targeted sequencing) in archived blood DNA specimens in 1240 PCa patients, including 30% AA patients, in three different cohorts: localized early stage (T2) PCa (N = 935); advanced PCa (50% T3-4) (N = 189); and metastatic PCa (N = 116). The sequences were analyzed for known and novel mutations in BRCA1/2. Statistical analyses were performed to determine associations of the mutations with clinico-pathological parameters. RESULTS: BRCA2 mutations with known pathogenic annotation were significantly more prevalent in men with advanced and metastatic PCa (3.1%) compared to patients with an organ-confined disease (0.7%). AA patients carried more frequently BRCA1/2 variants of unknown significance (VUS) when compared to Caucasian Americans (4.6 vs. 1.6%, respectively). Significantly, pathogenic BRCA2 mutations in men with localized early stage PCa increased the risk of distant metastasis. CONCLUSIONS: Germline variants of unknown significance in BRCA1/2 are more frequent in AA than CA PCa patients; however, the prevalence of pathogenic mutations were similar across the races. Patients carrying BRCA2 pathogenic mutations are more likely to progress to metastasis.
Subject(s)
BRCA2 Protein/genetics , Neoplasm Recurrence, Local/genetics , Prostatectomy , Prostatic Neoplasms/genetics , Adult , Black or African American/genetics , BRCA1 Protein/genetics , Case-Control Studies , DNA Mutational Analysis , Disease Progression , Follow-Up Studies , Germ-Line Mutation , Humans , Male , Middle Aged , Neoplasm Grading , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prostate/pathology , Prostate/surgery , Prostatic Neoplasms/pathology , Prostatic Neoplasms/surgery , Time Factors , White People/geneticsABSTRACT
Oncogenic activation of the ETS-related gene (ERG) by recurrent gene fusions (predominantly TMPRSS2-ERG) is one of the most validated and prevalent genomic alterations present in early stages of prostate cancer. In this study, we screened small-molecule libraries for inhibition of ERG protein in TMPRSS2-ERG harboring VCaP prostate cancer cells using an In-Cell Western Assay with the highly specific ERG-MAb (9FY). Among a subset of promising candidates, 1-[2-Thiazolylazo]-2-naphthol (NSC139021, hereafter ERGi-USU) was identified and further characterized. ERGi-USU selectively inhibited growth of ERG-positive cancer cell lines with minimal effect on normal prostate or endothelial cells or ERG-negative tumor cell lines. Combination of ERGi-USU with enzalutamide showed additive effects in inhibiting growth of VCaP cells. A screen of kinases revealed that ERGi-USU directly bound the ribosomal biogenesis regulator atypical kinase RIOK2 and induced ribosomal stress signature. In vivo, ERGi-USU treatment inhibited growth of ERG-positive VCaP tumor xenografts with no apparent toxicity. Structure-activity-based derivatives of ERGi-USU recapitulated the ERG-selective activity of the parental compound. Taken together, ERGi-USU acts as a highly selective inhibitor for the growth of ERG-positive cancer cells and has potential for further development of ERG-targeted therapy of prostate cancer and other malignancies.Significance: A highly selective small-molecule inhibitor of ERG, a critical driver of early stages of prostate cancer, will be imperative for prostate cancer therapy. Cancer Res; 78(13); 3659-71. ©2018 AACR.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Azo Compounds/pharmacology , Oncogene Proteins, Fusion/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Azo Compounds/therapeutic use , Benzamides , Cell Line, Tumor , Humans , Male , Mice , Mice, Nude , Nitriles , Oncogene Proteins, Fusion/genetics , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Phenylthiohydantoin/therapeutic use , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/metabolism , Small Molecule Libraries , Transcriptional Regulator ERG/antagonists & inhibitors , Transcriptional Regulator ERG/genetics , Xenograft Model Antitumor AssaysABSTRACT
The oncogenic activation of the ETS-related gene (ERG) due to gene fusions is present in over half of prostate cancers in Western countries. Because of its high incidence and oncogenic role, ERG and components of ERG network have emerged as potential drug targets for prostate cancer. Utilizing gene expression datasets, from matched normal and prostate tumor epithelial cells, an association of NOTCH transcription factors with ERG expression status was identified, confirming that NOTCH factors are direct transcriptional targets of ERG. Inhibition of ERG in TMPRSS2-ERG-positive VCaP cells led to decreased levels of NOTCH1 and 2 proteins and downstream transcriptional targets and partially recapitulated the phenotypes associated with ERG inhibition. Regulation of NOTCH1 and 2 genes by ERG were also noted with ectopic ERG expression in LNCaP (ERG-negative prostate cancer) and RWPE-1 (benign prostate-derived immortalized) cells. Furthermore, inhibition of NOTCH by the small-molecule γ-secretase inhibitor 1, GSI-1, conferred an increased sensitivity to androgen receptor (AR) inhibitors (bicalutamide and enzalutamide) or the androgen biosynthesis inhibitor (abiraterone) in VCaP cells. Combined treatment with bicalutamide and GSI-1 showed strongest inhibition of AR, ERG, NOTCH1, NOTCH2, and PSA protein levels along with decreased cell growth, cell survival, and enhanced apoptosis. Intriguingly, this effect was not observed in ERG-negative prostate cancer cells or immortalized benign/normal prostate epithelial cells. These data underscore the synergy of AR and NOTCH inhibitors in reducing the growth of ERG-positive prostate cancer cells.Implications: Combinational targeting of NOTCH and AR signaling has therapeutic potential in advanced ERG-driven prostate cancers. Mol Cancer Res; 15(10); 1308-17. ©2017 AACR.
Subject(s)
Androgen Antagonists/pharmacology , Oligopeptides/pharmacology , Prostatic Neoplasms/genetics , Receptors, Notch/genetics , Androstenes/pharmacology , Anilides/pharmacology , Benzamides , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Synergism , Humans , Male , Nitriles/pharmacology , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Receptors, Notch/metabolism , Tosyl Compounds/pharmacology , Transcriptional Regulator ERG/genetics , Transcriptional Regulator ERG/metabolismABSTRACT
Overdiagnosis and overtreatment of prostate cancer (CaP) is attributable to widespread reliance on PSA screening in the US. This has prompted us and others to search for improved biomarkers for CaP, to facilitate early detection and disease stratification. In this regard, autoantibodies (AAbs) against tumor antigens could serve as potential candidates for diagnosis and prognosis of CaP. Towards this, our goals were: i) To investigate whether AAbs against ERG oncoprotein (overexpressed in 25-50% of Caucasian American and African American CaP) are present in the sera of CaP patients; ii) To evaluate an AAb panel to enhance CaP detection. The results using an enzyme-linked immunosorbent assay (ELISA) showed that anti-ERG AAbs are present in a significantly higher proportion in the sera of CaP patients compared to healthy controls (p = 0.0001). Furthermore, a panel of AAbs against ERG, AMACR and human endogenous retrovirus-K Gag successfully differentiated CaP patient sera from healthy controls (AUC = 0.791). These results demonstrate for the first time that anti-ERG AAbs are present in the sera of CaP patients. In addition, the data also suggest that AAbs against ERG together with AMACR and HERV-K Gag may be a useful panel of biomarkers for diagnosis and prognosis of CaP.
ABSTRACT
BACKGROUND: Over one million men undergo prostate biopsies annually in the United States, a majority of whom due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of both the PSA test and the biopsy procedure have led to the development for more precise CaP detection assays in urine (e.g., PCA3, TMPRSS2-ERG) or blood (e.g., PHI, 4K). Here, we describe the development and evaluation of the Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine. METHODS: The cellular content of the post-DRE urine was captured on a translucent filter membrane, which is placed on Cytoclear slides for direct evaluation by microscopy and immuno-cytochemistry (ICC). Cells captured on the membrane were assayed for PSA and Prostein expression to identify prostate epithelial cells, and for ERG and AMACR to identify prostate tumor cells. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy. RESULTS: The assay was optimized for single cell capture sensitivity and downstream evaluations by spiking a known number of cells from established CaP cell lines, LNCaP and VCaP, into pre-cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR (CaP specific), and Prostein or PSA (prostate epithelium specific) rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy. CONCLUSIONS: This pilot study assessing a minimally invasive CaP detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this novel assay platform. Prostate 75: 969-975, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.
Subject(s)
Biomarkers, Tumor/urine , Prostatic Neoplasms/urine , Cell Line, Tumor , Cohort Studies , Humans , Immunohistochemistry/methods , Kallikreins/urine , Male , Membrane Proteins/urine , Pilot Projects , Prostate-Specific Antigen/urine , Racemases and Epimerases/urine , Reproducibility of Results , Sensitivity and Specificity , Trans-Activators/urine , Transcriptional Regulator ERGABSTRACT
Evaluation of cancer genomes in global context is of great interest in light of changing ethnic distribution of the world population. We focused our study on men of African ancestry because of their disproportionately higher rate of prostate cancer (CaP) incidence and mortality. We present a systematic whole genome analyses, revealing alterations that differentiate African American (AA) and Caucasian American (CA) CaP genomes. We discovered a recurrent deletion on chromosome 3q13.31 centering on the LSAMP locus that was prevalent in tumors from AA men (cumulative analyses of 435 patients: whole genome sequence, 14; FISH evaluations, 101; and SNP array, 320 patients). Notably, carriers of this deletion experienced more rapid disease progression. In contrast, PTEN and ERG common driver alterations in CaP were significantly lower in AA prostate tumors compared to prostate tumors from CA. Moreover, the frequency of inter-chromosomal rearrangements was significantly higher in AA than CA tumors. These findings reveal differentially distributed somatic mutations in CaP across ancestral groups, which have implications for precision medicine strategies.
Subject(s)
Black or African American/genetics , Cell Adhesion Molecules, Neuronal/genetics , Genetic Association Studies , Genetic Variation , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Aged , Biomarkers, Tumor , Cluster Analysis , Disease Progression , GPI-Linked Proteins/genetics , Gene Deletion , Gene Rearrangement , Genetic Loci , Genomics , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Mutation , Neoplasm Grading , Neoplasm Staging , Oncogene Proteins, Fusion/genetics , PTEN Phosphohydrolase , Polymorphism, Single Nucleotide , Prostatic Neoplasms/metabolism , Reproducibility of ResultsABSTRACT
The prostate transmembrane protein androgen induced 1 (PMEPA1) gene is highly expressed in prostate epithelial cells and is a direct transcriptional target for the androgen receptor (AR). AR protein levels are controlled by the AR-PMEPA1 negative feedback loop through NEDD4-E3 ligase. Reduced expression of PMEPA1 observed in prostate tumors, suggests that loss of PMEPA1 may play critical roles in prostate tumorigenesis. This study focuses on epigenetic mechanisms of reduced PMEPA1 expression in the cancer of the prostate (CaP). Benign (n = 77) and matched malignant (n = 77) prostate epithelial cells were laser capture micro-dissected from optimum cutting temperature embedded frozen prostate sections from 42 Caucasian American (CA) and 35 African American (AA) cases. Purified DNA specimens were analyzed for CpG methylation of the PMEPA1 gene. PMEPA1 mRNA expression levels were evaluated by qRT-PCR. Analysis of PMEPA1 methylation and mRNA expression in the same tumor cell populations indicated a significant inverse correlation between mRNA expression and methylation in CaP (P = 0.0115). We noted higher frequency of CpG methylation within the evaluated first intronic region of the PMEPA1 gene in prostate tumors of CA men as compared with AA. In CaP cell lines, PMEPA1 expression was induced and AR protein levels were diminished in response to treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (decitabine). Cell culture-based studies demonstrated that decitabine restores PMEPA1 expression in AR-positive CaP cell lines. This report reveals the potential role of PMEPA1 gene methylation in the regulation of AR stability. Thus, downregulation of PMEPA1 may result in increased AR protein levels and function in CaP cells, contributing to prostate tumorigenesis.
Subject(s)
DNA Methylation , Membrane Proteins/genetics , Prostatic Neoplasms/pathology , Receptors, Androgen/metabolism , Black or African American , Aged , Antimetabolites, Antineoplastic/pharmacology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Base Sequence , Cell Line, Tumor , CpG Islands , Decitabine , Gene Expression , Humans , Introns , Male , Membrane Proteins/metabolism , Middle Aged , Molecular Sequence Data , Prostatic Neoplasms/metabolism , RNA, Messenger/metabolism , White PeopleABSTRACT
PURPOSE: A region on chromosome 8q24 was recently identified as a novel prostate cancer risk locus. Inherited variation in this region is associated with prostate cancer risk in the general population (21-58%), and specific alleles show a strong association in African-American men. This study was designed to evaluate associations between 8q24 risk alleles and clinical variables, such as pathologic stage, age at diagnosis, and recurrence, in a case series of African-American men. EXPERIMENTAL DESIGN: Peripheral blood DNA samples from 114 African-American men with prostate cancer, including 106 who had undergone radical prostatectomy, were genotyped for six single-nucleotide polymorphisms on three 8q24 regions. The presence of these single-nucleotide polymorphisms was compared with clinicopathologic and follow-up data after radical prostatectomy. RESULTS: The mean age of diagnosis and follow-up time were 57.4 (+/-8.9) years and 49.1 (+/-31.6) months, respectively. Patients carrying the Broad11934905 A risk allele, which is specific for African ancestry, were more likely to have a higher pathologic stage (pT(3-4)) than individuals with the wild type (odds ratio, 4.48; 95% confidence interval, 1.42-14.14; P = 0.011). A trend toward increased frequency of and shorter time to biochemical recurrence was noted in patients with this risk allele on Kaplan-Meier unadjusted survival analysis (P = 0.076). CONCLUSIONS: The Broad11934905 polymorphism at 8q24, which is only found in people of African ancestry, is associated with an increase in non-organ-confined prostate cancer at prostatectomy. In addition, for those with this risk allele, there is a trend toward early biochemical recurrence that requires validation in larger studies.
Subject(s)
Black or African American/genetics , Chromosomes, Human, Pair 8/genetics , Genetic Predisposition to Disease/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Alleles , Genome-Wide Association Study , Genotype , Humans , Male , Middle Aged , Neoplasm Staging , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Prostatectomy , Prostatic Neoplasms/surgery , Risk FactorsABSTRACT
PURPOSE: The expression of the ETS-related gene (ERG) is low or undetectable in benign prostate epithelial cells. High prevalence of ERG overexpression in prostate cancer cells due to TMPRSS2-ERG fusions suggest for causal roles of ERG protein in the neoplastic process. TMPRSS2-ERG fusion junctions have been extensively studied in prostate cancer. However, virtually nothing is known about the nature of full-length transcripts and encoded proteins. This study focuses on qualitative and quantitative features of full-length TMPRSS2-ERG transcripts in prostate cancer. EXPERIMENTAL DESIGN: Full-length TMPRSS2-ERG transcripts were cloned and sequenced from a cDNA library generated from pooled RNA of six TMPRSS2-ERG fusion-positive prostate tumors. The encoded ERG proteins were analyzed in HEK293 cells. Copy numbers of TMPRSS2-ERG splice variants were determined by quantitative reverse transcription-PCR in laser capture microdissected prostate cancer cells. RESULTS: Two types of TMPRSS2-ERG cDNAs were identified: type I, which encodes full-length prototypical ERG protein (ERG1, ERG2, ERG3), and type II, encoding truncated ERG proteins lacking the ETS domain (ERG8 and a new variant, TEPC). In microdissected prostate tumor cells from 122 patients, relative abundance of these variants was in the following order: ERG8 > TEPC > ERG 3 > ERG1/2 with combined overexpression rate of 62.3% in prostate cancer. Increased ratio of type I over type II splice forms showed a trend of correlation with less favorable pathology and outcome. CONCLUSIONS: Qualitative and quantitative features of specific ERG splice variants defined here promise to enhance the utility of ERG as a biomarker and therapeutic target in prostate cancer.
Subject(s)
Alternative Splicing , Gene Expression Regulation, Neoplastic , Oncogene Proteins, Fusion/genetics , Prostatic Neoplasms/metabolism , Serine Endopeptidases/genetics , Trans-Activators/genetics , Biomarkers, Tumor , Cell Line, Tumor , Cloning, Molecular , DNA, Complementary/metabolism , Female , Gene Library , Humans , Male , Oncogene Proteins, Fusion/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Serine Endopeptidases/biosynthesis , Trans-Activators/biosynthesis , Transcriptional Regulator ERGABSTRACT
PURPOSE: Alterations of the androgen receptor (AR)-mediated signaling through numerous mechanisms are increasingly recognized in prostate cancer (CaP) progression. We hypothesized that the assessment of well-defined AR transcriptional targets (e.g., PSA/HK3 mRNA) in CaP tissues will provide in vivo readout of AR dysfunctions. Moreover, quantitative expression features of PSA/HK3 mRNA in prostate tumor cells may serve as a prognostic indicator of disease progression. EXPERIMENTAL DESIGN: Paired benign and malignant epithelial cells (242 specimens) were obtained from laser capture microdissection of frozen OCT-embedded tissue sections prepared from radical prostatectomy specimens of 121 patients. Quantitative expression of PSA/HK3 mRNA in the matched malignant and benign cells was analyzed by real-time reverse transcription-PCR. RESULTS: CaP cells express significantly lower PSA/HK3 mRNA levels than matched benign cells (P = 0.0133). Moreover, low PSA/HK3 mRNA expression in malignant cells was associated with increased risk of biochemical recurrence (P = 0.0217), as well as with time to recurrence (P = 0.0371), in patients with intermediate preoperative serum prostate-specific antigen levels (2-10 ng/mL). The expression of androgen-dependent genes in clinical samples correlates with each other in patients with higher expression of PSA/HK3 mRNA but not in patients with lower expression of PSA/HK3 mRNA reflecting AR pathway dysfunction. CONCLUSIONS: Our study has unraveled a novel prognostic utility of quantitative measurements of PSA/HK3 mRNA reflecting AR transcriptional activity in CaP cells, which is independent of serum prostate-specific antigen. It also has potential in stratifying subsets of patients exhibiting progressive disease associated with dampened AR transcriptional functions who may be targeted by tailored therapeutic strategies.
Subject(s)
Prostate-Specific Antigen/genetics , Prostatic Neoplasms/genetics , RNA, Messenger/genetics , Receptors, Androgen/genetics , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Male , Prognosis , Prostatic Neoplasms/pathology , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
TMPRSS2-ERG gene fusion leading to the androgenic induction of the ERG proto-oncogene expression is a highly prevalent oncogenic alteration in prostate tumor cells. Prostate cancer is a multi-focal disease, and the origins as well as biological contribution of multiple cancer foci remain unclear with respect to prostate cancer onset or progression. To assess the role of TMPRSS2-ERG alteration in prostate cancer onset and/or progression, we have evaluated the status of fusion transcripts in benign glands, prostatic intraepithelial neoplasia (PIN) and multiple cancer foci of each prostate. Quantitative expression of TMPRSS2-ERG fusion type A and C transcripts was analyzed in benign, tumor and PIN areas, selected from whole-mount radical prostatectomy slides. TMPRSS2-ERG expression was correlated with clinicopathological features. Overall, 30 of 45 (67%) patients exhibited TMPRSS2-ERG fusion transcripts in at least one tumor focus. Of 80 tumor foci analyzed, 39 had TMPRSS2-ERG fusion (type A only: 30, type C only: 2, both types A and C: 7), with predominant detection of the TMPRSS2-ERG fusion type A (27/30, 90%) in the index tumors. Of 14 PIN lesions, 2 were positive for type A fusion. Frequent presence of the TMPRSS2-ERG in index tumors suggests critical roles of ERG alterations in the onset and progression of a large subset of prostate cancer. However, heterogeneity of the TMPRSS2-ERG detection in the context of multiple cancer foci and its frequency in PIN also support the role of other genomic alterations in the origins of prostate cancer.
Subject(s)
Adenocarcinoma/genetics , Oncogene Proteins, Fusion/genetics , Precancerous Conditions/genetics , Prostatic Intraepithelial Neoplasia/genetics , Prostatic Neoplasms/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Disease-Free Survival , Gene Expression Regulation, Neoplastic , Humans , Male , Microdissection , Middle Aged , Neoplasm Recurrence, Local , Oncogene Proteins, Fusion/metabolism , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Prostate/metabolism , Prostate/pathology , Prostate/surgery , Prostate-Specific Antigen/blood , Prostatectomy , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Mas , RNA, Neoplasm/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVES: Alterations of androgen receptor (AR) functions caused by overexpression, amplification, or mutation have been described in a significant subset of advanced prostate cancer (CaP). Because AR mutations or amplification are rare in early stage CaP, we hypothesized that altered AR expression in prostate tumor cells may provide a prognostic indicator of disease progression. METHODS: RNA from laser capture microdissected (LCM) tumor and benign epithelial cells from radical prostatectomy specimens of 115 hormone-naive patients were studied. Expression of AR and GAPDH genes were measured by duplex quantitative real-time polymerase chain reaction (RT-PCR) in 230 specimens. A ratio of the expression of AR gene, normalized to GAPDH gene expression in the same specimens, was compared in tumor and benign epithelial cells (tumor-to-benign ratio) and correlated with clinicopathologic features. RESULTS: Paired t test analysis revealed a 62% lower AR expression in tumor tissue compared with benign tissue (P = 0.0005). However, multivariate Cox proportional hazards regression analysis of time to PSA recurrence revealed that higher tumor cell associated AR expression (continuous, log-transformed), significantly increases odds of prostate-specific antigen (PSA) recurrence (P = 0.0139) when controlling for age at surgery, race, time from diagnosis to surgery, risk stratification, pathologic T stage, Gleason sum, and margin status. CONCLUSIONS: Quantitative determination of AR gene expression levels in prostate epithelial cells may be useful for predicting PSA recurrence. This study supports the accumulating data suggesting that gain of AR function may contribute to CaP progression.
Subject(s)
Prostate/metabolism , Prostatectomy , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Adult , Disease Progression , Gene Expression , Humans , Male , Prognosis , Prostate-Specific Antigen/blood , Prostatic Neoplasms/surgery , RNA, Messenger/analysis , RNA, Messenger/metabolism , Receptors, Androgen/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Cancer cells gain selection advantages by the coordinated silencing of protective and by the activation of cell proliferation/cell survival genes. Evaluations of epithelial cell transcriptome of benign and malignant prostate glands by laser capture microdissection (LCM) identified Lactotransferrin (LTF) as the most significantly downregulated gene in prostate cancer (CaP) cells (p < 10(-6)). Frequent downregulation, significant association of LTF with PSA recurrence-free survival in CaP patients and the established anti-tumorigenic effects of LTF in experimental cancer models have provided impetus to evaluate LTF expression features and mechanisms in CaP specimens. METHODS: LTF mRNA expression analysis was performed in LCM derived benign and malignant prostate epithelial cells by using Affymetrix GeneChip and QRT-PCR. LTF protein expression was assessed in tissue specimens by immunohistochemistry and in serum samples from CaP patients compared to healthy male control by using ELISA. Mechanism of LTF downregulation was analyzed in 5-azadeoxycytidine treated LNCaP and LAPC4 cells using MALDI-TOF MS. Proliferation and cell cycle analysis of CaP cells by FACS flow cytrometry was assessed in LNCaP cell cultures. RESULTS: Quantitative analysis of LTF mRNA expression in tumor cells revealed marked downregulation of LTF with significant associations to decreased PSA recurrence-free survival of CaP patients (n = 100, p < or = 0.0322). Moreover, low levels of LTF protein expression was observed in tumor tissues as well as in sera from CaP patients (p < or = 0.0001). LTF promoter downstream CpG island methylation was found in LNCaP and LAPC4 cells. Furthermore, replenishing of LTF by supplementing growth media with LTF protein resulted in reduced cell growth. Cell cycle analysis revealed robust increases in apoptosis in response to LTF treatment. CONCLUSION: This study highlights the potential for LTF in chemoprevention and to become a biologically relevant prognostic marker of CaP, suggesting that silencing of the LTF gene may be causally linked to CaP progression.