A Functional Study of an Orphan Nuclear Receptor TLX in Prostate Cancer.

摘要

Background and aims of the study. Cellular senescence represents an irreversible form of permanent cell-cycle arrest and it acts a key process of tumor suppression, while targeting to pathways involved in this process can provide potential and promising therapeutic strategies to cancer treatments. TLX belongs to the NR2E1 orphan nuclear receptor subfamily. The overall aim of this study was to elucidate the functional role of TLX in prostate cancer cell growth.;Materials and methods In order to elucidate the functional roles of TLX in prostate cancer growth and the involved mechanisms, the following experiments were conducted: 1) To investigate and determine the expression pattern of TLX in clinical prostatic tissues by immunohistochemistry, and to survey the expression profile of TLX in a panel of prostatic immortalized epithelial and prostate cancer cell lines by quantitative real-time PCR analysis; 2) To generate stable TLX-knockdown prostate cancer cells by lentiviral transduction and TLX-stable expressing cells by retroviral transduction in both hormone-sensitive (LNCaP) and -insensitive (DU145 and PC-3) prostate cancer lines with different expression status of p53; and to conduct growth phenotype characterization studies (including cell growth, cell cycle, cellular senescence, cell migration and invasion, resistance to chemotherapy drugs, hypoxic cell growth assays, and tumorigenesis) on these TLX-transfectants in vitro and in vivo; 3) To characterize cellular senescence phenotype of TLX-infectants by senescence-associated beta-galactosidase (SA-beta-Gal) staining method with or without senescence inducers; 4) To investigate the expression status of markers involved in cellular senescence in TLX-infectants by immunoblotting; 5) To demonstrate the transcriptional regulation targets of TLX by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay; 6) To confirm the cellular function of TLX in prostatic and non-prostatic cells expressing different TLX deletion mutants (DeltaZF1 and DeltaLBD-AF2).;Results Results obtained in this study are summarized as follows: 1) TLX displayed an increased expression pattern in many prostate cancer cell lines and also high-grade (Gleason score ≥ 7) prostate cancer tissues; 2) Depletion of TLX mRNA by RNA interference dramatically suppressed in vitro and in vivo tumor cell growth and triggered cellular senescence (SA-beta-Gal histochemical marker) in prostate cancer cells; 3) On the contrary, TLX overexpression significantly enhanced multiple advanced malignant growth capacities (including enhanced anchorage-dependent and -independent cell growth, cell migration and invasion, hypoxia adaptation, resistance to chemotherapy drug Doxorubicin as well as in vivo tumorigenicity) in prostate cancer cells; 4) TLX overexpression significantly suppressed cellular senescence and protected cells against doxorubicin-induced or oncogenic H-RAS (H-RASG12V)-induced senescence; 5) TLX could directly bind to p21WAF1/CIP1 gene (hereafter p21) promoter and repress the transcriptional activity of p21 promoter, while ectopic restoration of p21 expression in TLX-overexpressed cells could rescue cellular senescence with enhanced SA-beta-Gal staining; 6) protein deacetylase SIRT1 gene was also activated by TLX through its direct transcriptional regulation, while knockdown of SIRT1 in TLX-overexpressed cells could rescue cellular senescence; 7) TLX-induced suppression of cellular senescence and also its direct gene regulation would require an intact DBD and LBD domain, as truncated deletion of DBD or LBD domain could both abolish the cellular function and transcriptional activity of TLX in prostatic and non-prostatic cells.;Conclusions The results obtained in this study suggested that TLX could play a positive growth regulatory or tumor-promoting role in prostate cancer development by its suppression of cellular senescence and this senescence suppression was mediated via its direct transcriptional regulation of both p21 (repression) and SIRT1 (transactivation) genes. Moreover, this study also showed for the first time that TLX, which was overexpressed in prostate cancer tissues, might function to suppress premature senescence in prostate cancer progression and also targeting to TLX could be a potential therapeutic approach for prostate cancer treatment. (Abstract shortened by UMI.).