Human Prostate Cancer Gene Research Articles

Epigenetic regulation of pluripotency inducer genes NANOG and SOX2 in human prostate cancer.

The cells of multicellular organisms are genetically homogeneous but heterogenous in structure and function by virtue of differential gene expression. During embryonic development, differential gene expression by modification of chromatin (DNA and histone complex) regulates the developmental proceedings before and after the germ layers are formed. Post-replicative DNA modification, where the fifth carbon atom of the cytosine gets methylated (hereafter, DNA methylation), does not incorporate mutations within the DNA. In the past few years, a boom has been observed in the field of research related to various epigenetic regulation models, which includes DNA methylation, post-translational modification of histone tails, control of chromatin structure by non-coding RNAs, and remodeling of nucleosome. Epigenetic effects like DNA methylation or histone modification play a cardinal role in development but also be able to arise stochastically, as observed during aging, in tumor development and cancer progression. Over the past few decades, researchers allured toward the involvement of pluripotency inducer genes in cancer progression and apparent for prostate cancer (PCa); also, PCa is the most diagnosed tumor worldwide and comes to the second position in causing mortality in men. The anomalous articulation of pluripotency-inducing transcription factor; SRY-related HMG box-containing transcription factor-2 (SOX2), Octamer-binding transcription factor 4 (OCT4) or POU domain, class 5, transcription factor 1 (POU5F1), and NANOG have been reported in different cancers which includes breast cancer, tongue cancer, and lung cancer, etc. Although there is a variety in gene expression signatures demonstrated by cancer cells, the epigenetic mode of regulation at the pluripotency-associated genes in PCa has been recently explored. This chapter focuses on the epigenetic control of NANOG and SOX2 genes in human PCa and the precise role thereof executed by the two transcription factors.

PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation.

RationaleFructose-1,6-bisphosphatase (FBP1) is a tumor suppressor and a key enzyme negatively regulating Warburg effect in cancer. However, regulation of FBP1 protein expression and its exact role in prostate cancer (PCa) is largely unclear. Phosphatase and tensin homolog (PTEN) is one of the most frequently deleted tumor suppressor genes in human PCa. However, the role of PTEN loss in aberrant Warburg effect in cancer remains poorly understood.MethodsExpression of PTEN and FBP1 was analyzed in several PCa cell lines and prostate tumor tissues in mice. Western blot (WB) and RT-PCR approaches were used to examine how PTEN regulates FBP1 expression. Co-immunoprecipitation (co-IP) and in vivo ubiquitination assays were used to define the regulatory mechanisms. A PCa xenograft model was employed to determine the impact of PTEN regulation of FBP1 on PCa growth in vivo.ResultWe demonstrated that in a manner dependent of PI3K/AKT signal pathway PTEN regulated FBP1 expression in various PCa cell lines and tumors in mice. We confirmed that this regulation took place at the protein level and was mediated by SKP2 E3 ubiquitin ligase. Mechanistically, we showed that serine 271 phosphorylation of FBP1 by cyclin-dependent kinases (CDKs) was essential for SKP2-mediated degradation of FBP1 protein induced by PTEN loss. Most importantly, we further showed that loss of PTEN expression enhanced Warburg effect and PCa growth in mice in a manner dependent, at least partially on FBP1 protein degradation.ConclusionsOur results reveal a novel tumor-suppressive feature of PTEN in restraining FBP1 degradation and the Warburg effect. These results also suggest that prohibiting FBP1 protein degradation could be a viable therapeutic strategy for PTEN-deficient PCa.

PTEN deletion drives aberrations of DNA methylome and transcriptome in different stages of prostate cancer.

Phosphatase and tensin homolog located on chromosome 10 (PTEN) is a tumor suppressor gene and one of the most frequently mutated/deleted genes in human prostate cancer (PCa). However, how PTEN deletion would impact the epigenome and transcriptome alterations remain unknown. This hypothesis was tested in a prostate-specific PTEN-/- (KO) mouse prostatic adenocarcinoma model through DNA methyl-Seq and RNA-Seq analyses. Examination of cancer genomic datasets revealed that PTEN is expressed at lower levels in PTEN-deleted tumor samples than in normal solid tissue samples. Methylome and transcriptome profiling identified several inflammatory responses and immune response signaling pathways, including NF-kB signaling, IL-6 signaling, LPS/IL-1-mediated inhibition of RXR Function, PI3K in B lymphocytes, iCOS-iCOSL in T helper cells, and the role of NFAT in regulating the immune response, were affected by PTEN deletion. Importantly, a small subset of genes that showed DNA hypermethylation or hypomethylation was correlated with decreased or increased gene expression including CXCL1. quantitative polymerase chain reaction analyses of representative genes validated the RNA-Seq results. Histopathological examinations showed that the severity of prostatic intraepithelial neoplasia and inflammation development gradually increased as PTEN null mice aged. Collectively, these findings suggest that loss of PTEN drives global changes in DNA CpG methylation and transcriptomic gene expression and highly associated with several inflammatory and immune molecular pathways during PCa development. These biomarkers could be valuable molecular targets for cancer drug discovery and development against PCa.

CXCL1-LCN2 paracrine axis promotes progression of prostate cancer via the Src activation and epithelial-mesenchymal transition

BackgroundMechanisms driving the progression of castration-resistant prostate cancer are believed to relate substantially to the tumor microenvironment. However, the cross-talks between tumor epithelial cell, stromal cells, and immune cells are yet to be fully elucidated. The present study aims to determine the role of chemokine and neutrophil derived cytokine paracrine axis in mediating the interaction between tumor cells, stromal myofibroblasts, and neutrophils in the tumor microenvironment of prostate cancer.MethodsTo identify myofibroblasts and neutrophil derived specific proteins affecting progression of prostate cancer, bioinformatics analyses were firstly performed in independent human prostate cancer gene expression data sets from the GEO data bank. Expression of stromal myofibroblasts secretory chemokine CXCL1 and neutrophil derived cytokine LCN2 was evaluated in prostate tissues via immunohistochemistry assay. We further investigated the effect of CXCL1 and LCN2 on prostate cancer using in vivo and in vitro models, and explored the underlying signal transduction pathways.ResultsA CXCL1-LCN2 paracrine network was confirmed in prostate cancer tissue samples, which was correlated with the biochemical recurrence of prostate cancer. Of note, CXCL1-LCN2 axis activates Src signaling, triggers the epithelial-mesenchymal transition (EMT), consequently promotes the migration of prostate cancer cells, leading to enhanced tumor metastasis.ConclusionsOur findings may provide enhanced insight into the interactions of carcinoma-stromal cells and immune cells linked to prostate cancer progression, wherein CXCL1-LCN2 axis is a key contributor to prostate cancer cells migration. These data indicate tumor microenvironment and Src signaling pathway may be potential therapeutic targets of prostate cancer treatment.

Effect of PTEN loss on metabolic reprogramming in prostate cancer cells.

The tumor suppressor gene PTEN is one of the most often deleted genes in human prostate cancer. Loss of PTEN is an important event in prostate carcinogenesis. Metabolic reprogramming induced by PTEN loss fuels malignant growth and proliferation of prostate cancer cells. Targeted metabolomics analysis was used to investigate the effects of PTEN loss on intracellular metabolic pathways in prostate cancer cells. DU-145 cells were transfected with PTEN siRNAs (siRNA-1 and siRNA-2) for 48 h, and endogenous PTEN expression was monitored by western blotting. Changes in intracellular metabolites were determined by liquid chromatography-tandem mass chromatography (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS). Most intracellular metabolites involved in glycolysis and glutaminolysis were increased in PTEN knockdown prostate cancer cells. In addition, most intracellular metabolites involved in fatty acid de novo synthesis, fatty acid beta oxidation and branched chain amino acid catabolism were also increased in PTEN knockdown prostate cancer cells. These results revealed that PTEN loss induced the metabolic reprogramming of prostate cancer cells and promoted the malignant proliferation of prostate cancer cells. The present metabolomics analysis indicates that tumor suppressor gene PTEN mutation or deletion can induce metabolic reprogramming in prostate cancer cells and tumorigenesis by altering the metabolic flux of glycolysis, glutaminolysis, fatty acid metabolism and branched chain amino acid catabolism pathways. Metabolic reprogramming is one of the contributors to PTEN-loss driven prostate cancer.

Integrated Analysis of Genetic Abnormalities of the Histone Lysine Methyltransferases in Prostate Cancer.

BackgroundThe histone methyltransferase (HMT) family includes histone lysine methyltransferases (HKMTs) and histone/protein arginine methyltransferases (PRMTs). The role of HMT gene variants in prostate cancer remains unknown. Therefore, this study aimed to evaluate HMT gene variants in the pathogenesis and prognosis of human prostate cancer, using in vitro cell studies and bioinformatics analysis.Material/MethodsIntegrative bioinformatics analysis of the expression of 51 HMT genes in human prostate cancer was based on datasets from the Cancer Genome Atlas (TCGA). Correlation and regression analysis were used to identify critical HMTs in prostate cancer. Kaplan-Meier and the area under the receiver operating characteristics curve (AUROC) were performed to evaluate the function of the HMTs on prognosis. Gene expression and function of 22Rv1 human prostate carcinoma cells were studied.ResultsThe HMT genes identified to have a role in the pathogenesis of prostate cancer included the EZH2, SETD5, PRDM12, NSD1, SETD6, SMYD1, and the WHSC1L1 gene. The EZH2, SETD5, and SMYD1 genes were selected as a prognostic panel, with the SUV420H2 HMT gene. SETD2, NSD1, and ASH1L were identified as critical genes in the development of castration-resistant prostate cancer (CRPC), similar to mixed-lineage leukemia (MLL) complex family members. Knockdown of the SETD5 gene in 22Rv1 prostate carcinoma cells in vitro inhibited cancer cell growth and migration.ConclusionsHMT gene variants may have a role in the pathogenesis of prostate cancer. Future studies may determine the role of HMT genes as prognostic biomarkers in patients with prostate cancer.

MP64-04 SLCO2B1 HIGH EXPRESSED TUMOR IN HIGH GLEASON SCORE PROSTATE CANCER PATIENTS RECUR EARLIER AFTER RADICAL PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2018MP64-04 SLCO2B1 HIGH EXPRESSED TUMOR IN HIGH GLEASON SCORE PROSTATE CANCER PATIENTS RECUR EARLIER AFTER RADICAL PROSTATECTOMY Tomoaki Terakawa, Eriko Katsuta, Khurshid Guru, Kazuaki Takabe, and Masato Fujisawa Tomoaki TerakawaTomoaki Terakawa More articles by this author , Eriko KatsutaEriko Katsuta More articles by this author , Khurshid GuruKhurshid Guru More articles by this author , Kazuaki TakabeKazuaki Takabe More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2049AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Solute carrier organic anion (SLCO) genes encode organic anion transport proteins, which is a family of transport proteins that influx number of substrates into the cells including androgens. Among them, high expression of SLCO genes, including SLCO1B3 and SLCO2B1, has been shown to be associated with the resistance to androgen deprivation therapy in prostate cancer. However, the significance of the expression of SLCO genes in the recurrence after radical prostatectomy has not been elucidated. Here, we hypothesized that the high expression of these SLCO genes in human prostate cancer is associated with the recurrence after radical prostatectomy. METHODS Clinical and RNA-seq data were all obtained from the Cancer Genome Atlas (TCGA). Patients were classified as either high or low expression of SLCO1B3 or SLCO2B1 by the mean value. Survival analysis and gene set enrichment analysis (GSEA) was conducted between high and low expression group in whole cohort, as well as by Gleason score (GS=6, =7 or =8). RESULTS Patients were classified with the expression level of SLOC1B3 or SLCO2B1 by mean value of each gene. In overall survival, either of these genes was not related to survival. However, high expression group of SLCO2B1 showed significantly worse disease-free survival after radical prostatectomy (p=0.026), whereas the expression level of SLCO1B3 was not related to disease-free survival. The patients with higher Gleason score had significantly higher levels of SLCO2B1 expression (GS<6 vs GS=7; p=0.045, GS=7 vs GS>8; p=0.002). Significant difference in disease-free survival between high and low expression groups were only observed in the patients with GS=8 (p=0.005), and not in the patients with GS=7 (GS<6; p=0.640, GS=7; p=0.923). GSEA demonstrated that in the high expression group of SLCO2B1 enriched epithelial mesenchymal transition signaling related genes. CONCLUSIONS High expression of SLCO2B1 in the prostate cancer patients associated with the aggressive cancer characteristics and recurrence after radical prostatectomy, however no relation was found with the expression level of SLCO1B3. Furthermore, the high recurrence rate of the patients with high expression of SLCO2B1 may be able to be explained by its metastatic potential with up-regulated EMT pathways. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e851 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Tomoaki Terakawa More articles by this author Eriko Katsuta More articles by this author Khurshid Guru More articles by this author Kazuaki Takabe More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PRUNE2 and PCA3 expression in paired non-malignant and tumor specimens from radical prostatectomy patients with gleason score 7 prostate cancer.

e16582 Background: PCA3 is an FDA-approved biomarker for prostate cancer. We have recently reported that PRUNE2 is a tumor suppressor gene in human prostate cancer that it is inactivated through a PCA3-mediated regulatory axis. Based upon this previous work, here we investigated if PRUNE2 and PCA3expression levels would be different in non-malignant versus tumor paired samples obtained from Gleason 7 Score prostate cancer patients undergoing radical prostatectomy. Methods: We examined Gleason Score 7 prostate cancer specimens, obtained from a cohort of patients (n=24) who have undergone radical prostatectomy at UNM Hospital. Paired non-malignant and tumor tissues were recovered from these specimens after tissue microdissection and processed for RNA extraction. PRUNE2 and PCA3transcripts were measured by qRT-PCR using three and two different primer sets, respectively, and normalized using appropriate internal controls. Each experiment was carried out in triplicates. The Wilcoxon signed rank test was used to test whether the differences between the paired tumor and non-malignant tissue samples were significant. Results: Gene expression analyses showed PRUNE2 levels to be consistently lower in tumor samples compared to non-malignant areas of the same gland (P&lt;0.0001). In contrast, the opposite trend was seen for PCA3, which has a marked upregulation in tumor areas (P&lt;0.0001). Conclusions: Both PRUNE2 and PCA3 expression levels were significantly different between matched non-malignant and malignant tissue pairs in Gleason Score 7 prostate cancer patients post radical prostatectomy. This retrospective dataset provides additional support for a mechanistic role for the molecular interplay between PRUNE2 and PCA3 in human prostate cancer, which may lead to the development of novel diagnostic and therapeutic approaches in this disease. Prospective studies targeting our original finding are either ongoing or planned. [Table: see text]

Overexpression of KAI1/CD82 suppresses invitro cell growth, migration, invasion and xenograft growth in oral cancer.

KAI1/CD82 is a metastatic suppressor gene in human prostate cancer and several other types of cancer in humans. The present study aimed to examine the role of the overexpression of KAI1 in the progression of oral cancer. Human KAI1/CD82 cDNA was transfected into OSCC-15 and 293T cell lines, and its effects on OSCC-15 cell proliferation, invasion and apoptosis were assessed by performing a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, Matrigel invasion and Annexin V-FITC staining, respectively. In addition, a xenograft model was used to assess the effect of KAI1/CD82 on the in vivo growth of tumors. The overexpression of KAI1/CD82 inhibited the proliferation and invasion of OSCC-15 cells. It also enhanced the apoptotic rate of the OSCC-15 cells. Furthermore, the overexpression of KAI1/CD82 inhibited tumor growth in the xenograft model. The results demonstrated that the overexpression of KAI1/CD82 significantly inhibited the proliferation and invasion of human oral cancer cells, and inhibited tumor growth in the xenograft model. Therefore, KAI1/CD82 may be considered as a potential therapeutic target in oral cancer.

Abstract 2674: High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring

Abstract Introduction: The mechanisms underlying the association between high dietary fat intake and prostate cancer (PCa) are unknown. Using a MYC-driven PCa mouse model, we sought to identify metabolic and epigenomic alterations driven by high fat diet (HFD) that facilitate PCa progression. Additionally, we investigated whether these alterations were relevant to PCa progression and lethality in humans. Material and Methods: Wild-type (WT) and transgenic Hi-MYC (MYC) mice were assigned either a HFD or control diet and were sacrificed at 12, 24, and 36 weeks of age for histologic and phenotypic characterization. Metabolic and epigenomic analyses were carried on the ventral prostates of 12-week old mice. Human PCa gene expression profiling data were obtained from 319 men with PCa and well-annotated post-diagnostic saturated fat intake (SFI) data from the Physicians’ Health Study and Health Professionals Follow-up Study prospective cohorts. Results: HFD does not affect the incidence of MYC-induced murine prostate intraepithelial neoplasia (mPIN) at 12 weeks, but increases mPIN proliferative index (Ki-67) at 24 weeks and tumor burden at 36 weeks. MYC overexpression, as expected, induces a significant metabolic reprogramming and HFD further enhances this rewiring to provide additional anabolic metabolites to sustain the increased proliferation of MYC prostate while having little effect on the WT prostate. Moreover, MYC altered key metabolites of the methionine cycle in a direction suggestive of a global hypomethylation, again amplified by HFD. Targeted quantitative histone mass spectrometry revealed a robust MYC-driven signature, including a global demethylation of H3K27 and H4K20 marks, the latter enhanced by HFD. Moreover, ChIP-seq revealed an intricate crosstalk between MYC and the H4K20me1 demethylase PHF8, resulting in enhanced genomic instability in the context of HFD. Finally, RNA-seq and ATAC-seq analyses showed that HFD rewires MYC-driven PCa through the alteration of genes implicated in chromatin function and remodeling. In humans, SFI was associated with enrichment in genes associated with increased MYC transcriptional activity in the prostate. Furthermore, this MYC transcriptional signature was associated with PCa lethality overall (OR = 3.21; 95% CI = 1.47, 7.35 comparing extreme score tertiles), and the association was stronger among men with high post-diagnostic SFI (OR = 1.32; 95%CI = 1.11, 1.66) than those with low SFI (OR = 1.05; 95%CI = 0.98, 1.12). Conclusions: HFD supports a coordinated metabolomic and epigenomic rewiring to increase epigenomic plasticity and MYC transcriptional activity prior to the appearance of phenotypic alterations in the prostate. Importantly, HFD requires MYC-mediated transformation to trigger its deleterious effects. In humans, SFI also enhances MYC transcriptional activity, which is associated with increased PCa lethality. Citation Format: David P. Labbe’, Giorgia Zadra, Meng Yang, Charles Y. Lin, Jaime M. Reyes, Stefano Cacciatore, Ericka M. Ebot, Maura B. Cotter, Amanda L. Creech, Jacob D. Jaffe, Philip W. Kantoff, James E. Bradner, Lorelei A. Mucci, Jorge E. Chavarro, Massimo Loda, Myles Brown. High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2674.

Predicting Drug Response in Human Prostate Cancer from Preclinical Analysis of In Vivo Mouse Models.

Although genetically engineered mouse (GEM) models are often used to evaluate cancer therapies, extrapolation of such preclinical data to human cancer can be challenging. Here, we introduce an approach that uses drug perturbation data from GEM models to predict drug efficacy in human cancer. Network-based analysis of expression profiles from invivo treatment of GEM models identified drugs and drug combinations that inhibit the activity of FOXM1 and CENPF, which are master regulators of prostate cancer malignancy. Validation of mouse and human prostate cancer models confirmed the specificity and synergy of a predicted drug combination to abrogate FOXM1/CENPF activity and inhibit tumorigenicity. Network-based analysis of treatment signatures from GEM models identified treatment-responsive genes in human prostate cancer that are potential biomarkers of patient response. More generally, this approach allows systematic identification of drugs that inhibit tumor dependencies, thereby improving the utility of GEM models for prioritizing drugs for clinical evaluation.

Abstract 2261: DLGAP5, MAT1A, SKA3, and ZMYM5 are novel susceptibility genes for aggressive prostate cancer

Abstract Prostate cancer is a common disease with approximately 233,000 men estimated to be diagnosed in the United States alone in 2014. Yet, it is usually an indolent disease with only 13% of patients succumbing to prostate cancer, and the molecular determinants of aggressive prostate cancer remain unclear. Previously, we reported that germline variation influences disease aggressiveness in the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model. These mice develop neuroendocrine prostate tumors similar to a subset of human prostate tumors associated with poor outcomes. Here, we used a multifaceted approach to identify candidate genes for susceptibility to aggressive prostate tumorigenesis and metastasis using the TRAMP mouse model. Candidate prostate cancer metastasis susceptibility genes were identified through quantitative trait locus (QTL) mapping in 201 (TRAMP x PWK/PhJ) F2 males. Two aggressive disease QTLs were identified; one for lymph node metastasis burden on chromosome 12 (LOD = 5.86) and one for distant metastasis-free survival on chromosome 14 (LOD = 4.41). Correlation analysis using microarray data derived from 27 (TRAMP x PWK/PhJ) F2 prostate tumors identified 35 metastasis-associated transcripts within the two loci. The role of these genes in susceptibility to aggressive human prostate cancer was analyzed in two different datasets. First, logistic regression and survival analyses in human prostate cancer gene expression datasets demonstrated that the expression levels of 5 of the 35 candidate genes was associated with both an increased risk of aggressive disease and a poorer disease-free survival. Second, four of these genes - DLGAP5, MAT1A, SKA3, and ZMYM5 - harbored SNPs associated with aggressive tumorigenesis in the PLCO/CGEMS GWAS cohort of 1,172 prostate cancer patients. This approach, novel to the prostate cancer field, demonstrates how mouse models can be used to identify aggressive disease susceptibility genes, and gives new insight into the molecular mechanisms of aggressive disease. Citation Format: Minnkyong Lee, Kendra A. Williams, Ying Hu, Jonathan Andreas, Shashankkumar J. Patel, Suiyuan Zhang, Nigel PS Crawford. DLGAP5, MAT1A, SKA3, and ZMYM5 are novel susceptibility genes for aggressive prostate cancer. [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 2261. doi:10.1158/1538-7445.AM2015-2261

PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3

Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.

Methylation of Integrin α4 and E-Cadherin Genes in Human Prostate Cancer.

Prostate cancer is the second most common malignancy in men worldwide. Abnormal epigenetic alterations such as DNA methylation and histone modification play an important role in tumor initiation, progression and regulation of cancer-related genes such as integrin α4 and E-cadherin. Expression of these genes was determined by semi-quantitative reverse transcriptase-PCR in prostate cancer cell lines, DU145 and PC3, before and after treatment with 5-aza-2-deoxycytidine and trichostatin A. Laser capture microdissection microscopy was used to obtain exclusively affected epithelial cells from prostate gland biopsies of 30 patients with prostate cancer and 40 with benign prostate hyperplasia. DNA bisulfite modifications followed by methylation-specific PCR were used to evaluate the promoter methylation status of E-cadherin and α4 integrin genes in extracted DNA from patients and aforementioned cell lines. The integrin α4 promoter in DU145 was fully methylated, whereas in PC3 cells, partial methylation was detected. E-cadherin was expressed in both cell lines; trichostatin A and 5-aza-2-deoxycytidine treatment had no effect on E-cadherin expression, however the combined treatment of both drugs or 5-aza-2-deoxycytidine alone increased integrin α4 expression. Integrin α4 and E-cadherin were hypermethylated in 66.6 % and 6.6 % of prostate cancer cases, respectively; no hypermethylation was observed in patients with benign prostate hyperplasia. These results together suggest that aberrant DNA methylation is one of the mechanisms involved in integrin α4 expression and may play an important role in human prostate carcinogenesis. In addition, the higher rate of integrin α4 gene methylation in prostate cancer patients elects it as a potential molecular tumor marker.

Epigenetic DNA methylation of antioxidative stress regulator NRF2 in human prostate cancer.

Epigenetic control of NRF2, a master regulator of many critical antioxidative stress defense genes in human prostate cancer (CaP), is unknown. Our previous animal study found decreased Nrf2 expression through promoter CpG methylation/histone modifications during prostate cancer progression in TRAMP mice. In this study, we evaluated CpG methylation of human NRF2 promoter in 27 clinical prostate cancer samples and in LNCaP cells using MAQMA analysis and bisulfite genomic DNA sequencing. Prostate cancer tissue microarray (TMA) containing normal and prostate cancer tissues was studied by immunohistochemistry. Luciferase reporter assay using specific human NRF2 DNA promoter segments and chromatin immunoprecipitation (ChIP) assay against histone modifying proteins were performed in LNCaP cells. Three specific CpG sites in the NRF2 promoter were found to be hypermethylated in clinical prostate cancer samples (BPH<ADT-RCaP<AS-CaP). NRF2 staining in human prostate cancer TMA showed a decreasing trend for both intensity and percentage of positive cells from normal tissues to advanced-stage prostate cancer (Gleason score from 3-9). Reporter assays in the LNCaP cells containing these three CpG sites showed methylation-inhibited transcriptional activity of the NRF2 promoter. LNCaP cells treated with 5-aza/TSA restored the expression of NRF2 and NRF2 downstream target genes, decreased expression levels of DNMT and HDAC proteins, and ChIP assays showed increased RNA Pol II and H3Ac with a concomitant decrease in H3K9me3, MBD2, and MeCP2 at CpG sites of human NRF2 promoter. Taken together, these findings suggest that epigenetic modification may contribute to the regulation of transcription activity of NRF2, which could be used as prevention and treatment target of human prostate cancer.

Abstract B06: Abi1 levels regulate prostate tumor progression in mice downstream from Pten inactivation

Abstract With over 200,000 new cases and nearly 30,000 deaths every year, prostate cancer remains one of the most critical healthcare issues in the United States. Prostate cancer is progressive, eventually leading to an aggressive and metastatic disease. Although indolent disease can be left untreated (watchful waiting or symptomatically treated), predicting which tumors will progress to the metastatic and lethal stage of the disease is problematic. More comprehensive knowledge of disease-progression mechanisms in prostate cancer treatment decisions would offer considerable saving to society in treatment costs and effort, and would likely improve patient quality of life by reducing the number of patients treated with radical therapies. Mouse models recapitulate well many stages of human pathology associated with prostate tumor progression. Tumor invasiveness involves dysregulation of actin cytoskeleton. Abi1 is the key component of actin polymerization regulatory WAVE complex. WAVE complex plays important role in a variety of cellular processes, including protrusion formation during cell migration and the formation of adherens junctions, indicating the critical importance of Abi1 for cell-to-cell adhesion and cell motility. ABI1 is implicated in variety of human cancers including initiation of prostate cancer (Oncogenesis. 2012; 1:e26) but its role in tumor progression is unknown. In a search for molecular mechanism of tumor progression here we modeled the effect of ABI1 inactivation in absence of PTEN gene, which is the most commonly mutated gene in human prostate cancer. Prostate specific inactivation of Pten and Abi1 genes in mice led to establishment of progression-specific prostate tumorigenesis models in mice. Preliminary data indicate that Abi1 controls tumor invasiveness resulting from Pten loss in a dose dependent manner. By modifying Abi1 and Pten gene dose using the double Abi1/Pten KO mouse, three models of prostate cancer progression were produced: Model 1, a model of invasive carcinoma (complete Pten and Abi1 loss, mice with the genotype: [Abi1(fl/fl); Pten(fl/fl); PbCre+]); Model 2, a model of changes consistent with high grade PIN with partial progression to invasive carcinoma (complete Pten loss, single copy of Abi1 present, mice with the genotype: [Abi1(wt/fl); Pten(fl/fl); PbCre+]); and, Model 3, a model of noninvasive prostate cancer with development of changes similar to low grade PIN (complete Abi1 loss, single copy of Pten present, mice with the genotype: [Abi1(fl/fl); Pten(wt/fl); PbCre+]). Histopathological changes observed in Abi1/Pten double KO are being compared to Pten KO alone to evaluate the influence of Abi1 on dynamics of tumor progression due to Pten loss. Analysis of Abi1/Pten double KO mice indicates that activation of PI-3 kinase activity is coincident with abnormal regulation of actin cytoskeleton. Examination of molecular and biochemical changes indicate that Pten inactivation alone resulted in up-regulation of Abi1 levels. Hence, Abi1 inactivation-mediated abrogation of oncogenic effects due to Pten loss in the double Abi1/Pten KO mouse is consistent with Abi1's tumor suppressor function. We propose that Abi1 levels control tumor progression by controlling level of actin dysregulation associated with tumor invasion. As Abi1/WAVE complex and Pten dysregulation are observed in human pathology it is hoped that the analysis of Abi1/Pten KO mouse model will lead to better understanding of tumor progression in human prostate cancer and to translation of the findings from bench to bedside. Citation Format: Anna Chorzalska, Xiaoling Xiong, Andrea Lunardi, Julie R. White, Janet A. Sawicki, Gennady Bratslavsky, Pier Paolo Pandolfi, Leszek Kotula. Abi1 levels regulate prostate tumor progression in mice downstream from Pten inactivation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B06.

Loss of somatostatin receptor subtype 2 in prostate cancer is linked to an aggressive cancer phenotype, high tumor cell proliferation and predicts early metastatic and biochemical relapse.

Somatostatin receptor subtype 2 (SSTR2) is the most frequently expressed SSTR subtype in normal human tissues. SSTR2 expression is differentially regulated in various tumor types and therapeutic somatostatin analogs binding to SSTR2 are in clinical use. In prostate cancers highly contradictory results in terms of SSTR2 expression and its consequences have been published over the past years. The aim of this study was to clarify prevalence and clinical significance of SSTR2 expression in prostate cancer. Therefore, quantitative immunohistochemistry (IHC) using a tissue microarray containing samples from 3,261 prostate cancer patients with extensive clinical and molecular cancer characteristics and oncological follow-up data was performed. IHC data was compared to publicly available Gene Expression Omnibus datasets of human prostate cancer gene expression arrays. While membranous SSTR2 staining was always seen in normal prostate epithelium, SSTR2 staining was absent in more than half (56.1%) of 2,195 interpretable prostate cancer samples. About 13% of all analyzed prostate cancers showed moderate to strong cytoplasmic and membranous SSTR2 staining. Staining intensities were inversely correlated with high Gleason grade, advanced pT category, high tumor cell proliferation (p<0.0001 each), high pre-operative PSA levels, (p = 0.0011) and positive surgical margins (p = 0.006). In silico analysis confirmed lower SSTR2 gene expression in prostate cancers vs. normal adjacent tissue (p = 0.0424), prostate cancer metastases vs. primary cancers (p = 0.0011) and recurrent vs. non-recurrent prostate cancers (p = 0.0438). PSA-free survival gradually declined with SSTR2 staining intensity (p<0.0001). SSTR2-negative cancers were more likely to develop metastases over time (p<0.05). In conclusion, most prostate cancers are indeed SSTR2-negative and loss of SSTR2 strongly predicts an unfavorable tumor phenotype and poor prognosis. Therefore, SSTR2 expression seems an important factor in the pathogenesis of prostate cancer and re-introduction of the receptor in SSTR2-negative prostate cancers may feature a promising target for novel gene therapy approaches.

Deletion of Atbf1/Zfhx3 In Mouse Prostate Causes Neoplastic Lesions, Likely by Attenuation of Membrane and Secretory Proteins and Multiple Signaling Pathways

The ATBF1/ZFHX3 gene at 16q22 is the second most frequently mutated gene in human prostate cancer and has reduced expression or mislocalization in several types of human tumors. Nonetheless, the hypothesis that ATBF1 has a tumor suppressor function in prostate cancer has not been tested. In this study, we examined the role of ATBF1 in prostatic carcinogenesis by specifically deleting Atbf1 in mouse prostatic epithelial cells. We also examined the effect of Atbf1 deletion on gene expression and signaling pathways in mouse prostates. Histopathologic analyses showed that Atbf1 deficiency caused hyperplasia and mouse prostatic intraepithelial neoplasia (mPIN) primarily in the dorsal prostate but also in other lobes. Hemizygous deletion of Atbf1 also increased the development of hyperplasia and mPIN, indicating a haploinsufficiency of Atbf1. The mPIN lesions expressed luminal cell markers and harbored molecular changes similar to those in human PIN and prostate cancer, including weaker expression of basal cell marker cytokeratin 5 (Ck5), cell adhesion protein E-cadherin, and the smooth muscle layer marker Sma; elevated expression of the oncoproteins phospho-Erk1/2, phospho-Akt and Muc1; and aberrant protein glycosylation. Gene expression profiling revealed a large number of genes that were dysregulated by Atbf1 deletion, particularly those that encode for secretory and cell membrane proteins. The four signaling networks that were most affected by Atbf1 deletion included those centered on Erk1/2 and IGF1, Akt and FSH, NF-κB and progesterone and β-estradiol. These findings provide in vivo evidence that ATBF1 is a tumor suppressor in the prostate, suggest that loss of Atbf1 contributes to tumorigenesis by dysregulating membrane and secretory proteins and multiple signaling pathways, and provide a new animal model for prostate cancer.

Abstract B157: Understanding the role of transcription factor ETV1 in metabolic reprogramming of prostate cells as a route to novel therapeutics.

Abstract ETV1 is the second most frequently rearranged ETS transcription factor gene in human prostate cancer. It is widely believed that ectopically expressed ETV1 (due to gene fusions) plays a similar role in prostate tumorigenesis to that of ERG. However, further analysis of clinical data revealed that high levels of ETV1 expression are often found in more advanced prostate cancers, whereas ERG overexpression is mainly associated with localized prostate cancer and elevated androgen receptor signaling. High expression of ETV1, rather than ERG, in the prostate has been associated with disease recurrence in some patients. Interestingly, many metabolic pathways often associated with cancer cells are enriched in the prostates of mice engineered to carry a Tmprss2-ETV1 knockin allele and ETV1-expressing human prostate cancer samples. To determine the dominant metabolic programs that accompany ETV1 expression, we have performed metabolic profiling, and integrated the data with human prostate cells and mouse transcriptome data. Our analyses have identified ETV1 as a regulator of lipid metabolism, glycolysis and glutamine metabolism. Of note, ETV1 expression reprograms the steroid biosynthesis pathway, leading to an increase in testosterone production at least in part through 17-β hydroxysteorid dehydrogenases activation. Indeed, one of the main causes of prostate cancer recurrence is local androgen production due to metabolic changes in prostate cancer cells. This novel finding may explain, at least in part, how ETV1 expression promotes castration-resistant disease, opening a new path to the development of novel therapies directed to progressive and lethal prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B157. Citation Format: Esther Baena, Shahan Mamoor, Zhe Li, Stuart H. Orkin. Understanding the role of transcription factor ETV1 in metabolic reprogramming of prostate cells as a route to novel therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B157.

Deletion of the Olfactomedin 4 Gene Is Associated with Progression of Human Prostate Cancer

The olfactomedin 4 (OLFM4) gene is located on chromosome 13q14.3, which frequently is deleted in human prostate cancer. However, direct genetic evidence of OLFM4 gene alteration in human prostate cancer has not yet been obtained. In this study, we investigated the genetics, protein expression, and functions of the OLFM4 gene in human prostate cancer. We found overall 25% deletions within the OLFM4 gene in cancerous epithelial cells compared with adjacent normal epithelial cells that were microdissected from 31 prostate cancer specimens using laser-capture microdissection and genomic DNA sequencing. We found 28% to 45% hemizygous and 15% to 57% homozygous deletions of the OLFM4 gene via fluorescence in situ hybridization analysis from 44 different prostate cancer patient samples. Moreover, homozygous deletion of the OLFM4 gene significantly correlated with advanced prostate cancer. By using immunohistochemical analysis of 162 prostate cancer tissue array samples representing a range of Gleason scores, we found that OLFM4 protein expression correlated inversely with advanced prostate cancer, consistent with the genetic results. We also showed that a truncated mutant of OLFM4 that lacks the olfactomedin domain eliminated suppression of PC-3 prostate cancer cell growth. Together, our findings indicate that OLFM4 is a novel candidate tumor-suppressor gene for chromosome 13q and may shed new light on strategies that could be used for the diagnosis, prognosis, and treatment of prostate cancer patients.