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Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

ABSTRACT: Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI.To identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation.Analyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.The mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.

SUBMITTER: Kunnev D

PROVIDER: S-EPMC2749059 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

Kunnev Dimiter D Ivanov Igor I Ionov Yurij Y

BMC cancer 20090908

<h4>Background</h4>Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant ...[more]

PMID: 19737411

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Project description:Partitioning-defective protein 6 (Par6) family proteins have been demonstrated to be closely associated with the occurrence and development of cancers. It is well accepted that dysregulation of epithelial-mesenchymal transition (EMT) greatly contributes to carcinogenesis and metastases of ovarian cancer. So far, the roles of Par6 in EMT of ovarian cancer are not clear. Functional experiments were carried out to study the roles of PARD6A in EMT of ovarian cancer in vitro and in vivo, and EMT pathways potentially affected by PARD6A expression were screened. We found that PARD6A was significantly highly expressed in tissues of ovarian cancer patients in III-IV stages, poorly differentiated or with lymphatic metastases versus I-II stages, moderately or well differentiated, or without lymphatic metastases, respectively. PARD6A knockdown suppressed EMT of SKOV3 and A2780 cells in vitro and ovarian cancer metastasis in vivo, while overexpression of PARD6A promoted EMT in HO8910 and OVCAR8 cells. It was indicated that PARD6A affected EMT of ovarian cancer cells through SNAIL1 signaling pathway and subsequently modulated the expression of VIMENTIN and E-cadherin, which was further confirmed by knockdown and overexpression of SNAIL1 experiments. PARD6A was also demonstrated to regulate expression of SNAIL1 by modulating integrin β1 and ILK proteins, specifically it was shown that the transcription of SNAIL1 was regulated by ILK in this study. In addition, expression of ILK in ovarian cancer tissues was demonstrated to be correlated with tumor stages and lymphatic metastases clinically. In this study, we identified a novel role of PARD6A as an inducer of cell migration and invasion, which is likely to play an important role in metastasis of ovarian cancer. The molecular pathways of EMT mediated by PARD6A-Integrin β1-ILK-SNAIL1 and finally implemented by E-cadherin and VIMENTIN may provide a novel strategy for drug development for ovarian cancer therapy in the near future.

Project description:BackgroundCastration resistant prostate cancer (CRPC) develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR) despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh) signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI) prostate cancer cells.ResultsTreatment of a variety of androgen-deprived or AI prostate cancer cells with the Hh inhibitor, cyclopamine, resulted in dose-dependent modulation of the expression of genes that are regulated by androgen. The effect of cyclopamine on endogenous androgen-regulated gene expression in androgen deprived and AI prostate cancer cells was consistent with the suppressive effects of cyclopamine on the expression of a reporter gene (luciferase) from two different androgen-dependent promoters. Similarly, reduction of smoothened (Smo) expression with siRNA co-suppressed expression of androgen-inducible KLK2 and KLK3 in androgen deprived cells without affecting the expression of androgen receptor (AR) mRNA or protein. Cyclopamine also prevented the outgrowth of AI cells from androgen growth-dependent parental LNCaP cells and suppressed the growth of an overt AI-LNCaP variant whereas supplemental androgen (R1881) restored growth to the AI cells in the presence of cyclopamine. Conversely, overexpression of Gli1 or Gli2 in LNCaP cells enhanced AR-specific gene expression in the absence of androgen. Overexpressed Gli1/Gli2 also enabled parental LNCaP cells to grow in androgen depleted medium. AR protein co-immunoprecipitates with Gli2 protein from transfected 293T cell lysates.ConclusionsCollectively, our results indicate that Hh/Gli signaling supports androgen signaling and AI growth in prostate cancer cells in a low androgen environment. The finding that Gli2 co-immunoprecipitates with AR protein suggests that an interaction between these proteins might be the basis for Hedgehog/Gli support of androgen signaling under this condition.

Dataset Information

Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

Publications

Par-3 partitioning defective 3 homolog (C. elegans) and androgen-induced prostate proliferative shutoff associated protein genes are mutationally inactivated in prostate cancer cells.

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