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UNC5D, suppressed by promoter hypermethylation, inhibits cell metastasis by activating death-associated protein kinase 1 in prostate cancer.

ABSTRACT: Prostate cancer (PCa) death primarily occurs due to metastasis of the cells, but little is known about the underlying molecular mechanisms. This study aimed to evaluate the expression of UNC5D, a newly identified tumor suppressor gene, analyze its epigenetic alterations, and elucidate its functional relevance to PCa metastasis. Meta-analysis of publicly available microarray datasets revealed that UNC5D expression was frequently downregulated in PCa tissues and inversely associated with PCa metastasis. These results were verified in clinical specimens by real-time PCR and immunohistochemistry assays. Through methylation analysis, the downregulated expression of UNC5D in PCa tissues and cell lines was found to be attributable to the hypermethylation of the promoter. A negative correlation was observed between methylation and UNC5D mRNA expression in PCa samples. The ectopic expression of UNC5D in PCa cells effectively reduced their ability to migrate and invade both in vitro and in vivo, and siRNA-mediated knockdown of UNC5D yielded consistent results. UNC5D can recruit and activate death-associated protein kinase 1, which remained to be essential for its metastatic suppressor function. In conclusion, these results suggested that UNC5D as a novel putative metastatic suppressor gene that is commonly down-regulated by hypermethylation in PCa.

SUBMITTER: Dong D

PROVIDER: S-EPMC6447834 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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UNC5D, suppressed by promoter hypermethylation, inhibits cell metastasis by activating death-associated protein kinase 1 in prostate cancer.

Dong Dong D Zhang Lufang L Bai Changsen C Ma Na N Ji Wei W Jia Li L Zhang Aimin A Zhang Pengyu P Ren Li L Zhou Yunli Y

Cancer science 20190220 4

Prostate cancer (PCa) death primarily occurs due to metastasis of the cells, but little is known about the underlying molecular mechanisms. This study aimed to evaluate the expression of UNC5D, a newly identified tumor suppressor gene, analyze its epigenetic alterations, and elucidate its functional relevance to PCa metastasis. Meta-analysis of publicly available microarray datasets revealed that UNC5D expression was frequently downregulated in PCa tissues and inversely associated with PCa metas ...[more]

PMID: 30632669

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Project description:BACKGROUND:Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells. METHODS:qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model. RESULTS:Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells. CONCLUSIONS:Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.

Project description:Promoter hypermethylation of tumor suppressor genes has been confirmed to serve a pivotal role in tumorigenesis. Protocadherin 8 (PCDH8), a novel tumor suppressor gene, has been reported to be inactivated by promoter hypermethylation a number of cancer types, including bladder cancer and renal cell carcinoma. The aim of the present study was to investigate the occurrence of PCDH8 hypermethylation in prostate cancer and its potential as a novel biomarker of prostate cancer. The transcriptional levels of PCDH8 were examined by quantitative polymerase chain reaction (PCR) in 82 prostate cancer tissues as well as 30 prostate hyperplasia tissues, and verified the protein level by western blot analysis of representative samples. PCDH8 expression levels were found to be reduced to 0.30±0.10 in 70.7% (58/82) of prostate cancer tissues. To identify the possible reason for mRNA downregulation, the methylation status of the PCDH8 promoter was assessed in prostate cancer tissues and prostate hyperplasia tissues by methylation-specific PCR (MSP). A total of 47 prostate cancer patients who exhibited reduced PCDH8 expression (57.3%; 47/82) also showed promoter hypermethylation (47/58). None of the samples (0/30) in the benign prostate hyperplasia group were positive on MSP. Furthermore, the associations between the methylation status of the PCDH8 promoter and various clinicopathological features of prostate cancer were analyzed, revealing that the methylation status of PCDH8 was closely associated with tumor size, tumor shape (papillary/non-papillary), tumor stage and tumor grade (all P<0.05), while there were no correlations with the age of the patients or the number of tumors (P>0.05). Additionally, patients with hypermethylation of the PCDH8 gene promoter had a relapse rate of 36.17% and a mortality rate of 29.79%, which were significantly higher than the hypermethylation-negative patients (P<0.05), indicating a poorer prognosis. Therefore, the methylation status of the PCDH8 gene in prostate cancer may be an important marker for use in the early diagnosis and prediction of prognosis in prostate cancer.

Project description:BackgroundDesmocollin 3 (DSC3), a member of the cadherin gene superfamily, is associated with pathogenesis of some cancers, but its role in prostate cancer (PCa) remains largely unknown.MethodsDSC3 gene expression level in available PCa microarray dataset was examined using the Oncomine database. DSC3 transcript expression in prostate cell line panel and an independent tissue cohort (n?=?52) was estimated by quantitative PCR (Q-PCR). Epigenetic status of DSC3 gene promoter in PCa was investigated by uploading three dataset (ENCODE Infinium 450K array data and two methylation sequencing) in UCSC genome browser. While pyrosequencing analysis measured promoter DNA methylation, Q-PCR estimates were obtained for DSC3 transcript re-expression after 5-Aza-deoxycytidine (5-Aza) treatment. Clinical relevance of DSC3 expression was studied by Kaplan-Meier survival analysis. Finally, functional studies monitoring cell proliferation, migration and invasion were performed in prostate cell lines after siRNA mediated DSC3 knockdown or following 5-Aza induced re-expression. EMT markers Vimentin and E-cadherin expression was measured by Western Blot.ResultsMicroarray data analyses revealed a significant decrease in DSC3 transcript expression in PCa, compared to benign samples. Q-PCR analysis of an independent cohort revealed DSC3 transcript down-regulation, both in PCa cell lines and tumor tissues but not in their benign counterpart. Examination of available NGS and Infinium data identified a role for epigenetic regulation DSC3 mRNA reduction in PCa. Pyrosequencing confirmed the increased DSC3 promoter methylation in cancer cell lines and restoration of transcript expression upon 5-Aza treatment further corroborated this epigenetic silencing mechanism. Importantly Kaplan-Meier analysis of an outcome cohort showed an association between loss of DSC3 expression and significantly increased risk of biochemical recurrence. Functional studies indicate a role for epithelial-mesenchymal transition in DSC3 regulated cell migration/invasion.ConclusionTaken together, our data suggests that DNA methylation contributes to down-regulation of DSC3 in prostate cancer, and loss of DSC3 predicts poor clinical outcome.

Dataset Information

UNC5D, suppressed by promoter hypermethylation, inhibits cell metastasis by activating death-associated protein kinase 1 in prostate cancer.

Publications

UNC5D, suppressed by promoter hypermethylation, inhibits cell metastasis by activating death-associated protein kinase 1 in prostate cancer.

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