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Heterogeneous epigenetic regulation of TIMP3 in prostate cancer.

ABSTRACT: Tissue inhibitor of metalloproteinase-3 (TIMP3) is a tumor suppressor gene frequently downregulated in prostate cancer. The mechanisms involved in TIMP3 transcriptional repression are not fully understood, but evidence suggests that promoter hypermethylation may not be the predominant epigenetic alteration in prostate cancer. To clarify this issue, we examined the contribution of both CpG site promoter methylation and histone modifications on TIMP3 downregulation. Using publicly available data sets, we confirmed that TIMP3 mRNA expression is decreased in prostate tumors relative to normal glands. Immunohistochemical analysis also showed decreased TIMP3 levels in high-grade primary tumors, but promoter hypermethylation was only detected in 6 of 28 (21%) high-grade specimens. Similarly, in prostate cancer cells, TIMP3 hypermethylation was only observed in DU145 cells. Treatment of DU145 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored TIMP3 expression, and this was significantly amplified by co-treating the cells with the HDAC inhibitor trichostatin A (TSA). Alternatively, in cells that did not exhibit aberrant TIMP3 methylation (LNCaP and PC3), TIMP3 expression could be upregulated by the combination of histone methylation inhibitor 3-Deazaneplanocin A (DZNep) and TSA. This reversal of transcriptional repression was associated with decreased H3K27me3 and increased H3K9ac histone marks at the TIMP3 promoter, as demonstrated by chromatin immunoprecipitation. Collectively, these results indicate that histone modifications can contribute to TIMP3 repression in the absence of promoter hypermethylation, and suggest that the combination of histone modifying agents could restore TIMP3 expression in prostate tumors harboring aberrant histone modifications at the TIMP3 promoter.

SUBMITTER: Shinojima T

PROVIDER: S-EPMC3499329 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Heterogeneous epigenetic regulation of TIMP3 in prostate cancer.

Shinojima Toshiaki T Yu Qiang Q Huang Sharon K SK Li Michelle M Mizuno Ryuichi R Liu Edison T ET Hoon Dave S B DS Lessard Laurent L

Epigenetics 20120928 11

Tissue inhibitor of metalloproteinase-3 (TIMP3) is a tumor suppressor gene frequently downregulated in prostate cancer. The mechanisms involved in TIMP3 transcriptional repression are not fully understood, but evidence suggests that promoter hypermethylation may not be the predominant epigenetic alteration in prostate cancer. To clarify this issue, we examined the contribution of both CpG site promoter methylation and histone modifications on TIMP3 downregulation. Using publicly available data s ...[more]

PMID: 23023649

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Project description:BackgroundRecently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation.ResultsDifferentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1.ConclusionsUsing this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.

Project description:BackgroundProstate cancer (PCa) is a major type of cancer in man worldwide. Androgen deprivation therapy (ADT) and the next-generation androgen receptor (AR) pathway inhibitors have acquired great success in treating PCa. However, patients treated with ADT or AR targeted therapy are inevitably developing into castration-resistant prostate cancer (CRPC) or becoming drug resistance. The role of mRNA 5-methylcytosine (m5C) modification in cancers is largely unknown. This study aimed to explore the role of the m5C methyltransferase NSUN2 in Prostate cancer (PCa).MethodsThe expression of NSUN2 and its clinicopathological impact were evaluated in PCa cohorts. The effect of NSUN2 on the biological characteristics of PCa cells was investigated on the basis of gain-offunction and loss-of-function analyses. Subcutaneous models further uncovered the role of NSUN2 in tumor growth. Epi-transcriptome assays with RNA bisulfite sequencing (RNA-BisSeq) analysis and in vitro enzyme reaction assays were performed to validate the targeted effect of NSUN2 on AR. AR-binding sites in the NSUN2 promoter were investigated by ChIP and luciferase assays to uncover the interplay between NSUN2 and AR signaling. RIP-qPCR and EMSA methods were performed to confirm that YBX1 binds to AR m5 C sites.ResultsNSUN2 is highly expressed in PCa and predicts poor outcome. NSUN2 plays roles as a PCa oncogene both in vitro and in vivo. Depletion of NSUN2 results in decreased expression and activities of AR, including AR-V7. Mechanistically, NSUN2 posttranscriptionally stabilized AR by cluster m5 C modification in a m5CYBX1-dependent manner. Strikingly, treatment with enzalutamide, an effective AR inhibitor, reduces NSUN2 expression and decreases the m5C modification level in prostate cancer cells. Finally, we found that AR transcriptionally regulates NSUN2.ConclusionNSUN2 stabilizes AR mRNA through cluster 5-methylcytosine modification and activates a positive feedback loop to promote prostate cancer.

Project description:PurposeCell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer.Experimental designThe CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR.ResultsWe report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression.ConclusionsCADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers.

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Heterogeneous epigenetic regulation of TIMP3 in prostate cancer.

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Heterogeneous epigenetic regulation of TIMP3 in prostate cancer.

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