PDLIM2 suppression efficiently reduces tumor growth and invasiveness of human castration-resistant prostate cancer-like cells.

Although PDLIM2 gene may have a context-dependent role in various human malignancies and can be a potential therapeutic target, only a limited number of in vitro studies addressed the molecular functions of PDLIM2 in prostate cancer. Here, we aimed to explore the role of PDLIM2 and the effect of the PDLIM2 gene suppression on oncogenic phenotypes of human castration-resistant prostate cancer (CRPC)-like cells. We used human CRPC-like cell lines (PC3, DU145, and C4-2B) for our experiments. Transcription levels of PDLIM2 and relevant genes were measured by real time-PCR and protein expression was analyzed by western blot. Cell viability, proliferation, clonogenic growth, and tumor sphere formation were examined after a specific inhibition of PDLIM2 using RNA interference. Flow cytometry was used to examine apoptotic cell death and cell cycle disturbances. Wound healing and transwell migration assays were performed to investigate the invasion capabilities of CRPC-like cells. Additionally, key oncogenic signaling pathways were examined using western blot. Lastly, we evaluated the in vivo efficacy of PDLIM2 suppression on tumor growth of human CRPC xenografts in mice. We observed a significant enhancement of PDLIM2 expression in human CRPC-like cell lines, while a specific inhibition of PDLIM2 reduced cell viability and proliferation due to apoptotic cell death. Conversely, PDLIM2 overexpression significantly reduced cell proliferation compared to the negative control in androgen-sensitive LNCaP cells. Moreover, PDLIM2 suppression led to a decrease of clonogenic growth and tumor sphere formation in three-dimensional cultures with the G2/M cell cycle arrest in human CRPC-like cells. PDLIM2 inhibition also attenuated cellular migration and invasion capabilities of human CRPC-like cells, and reduced the expression of mesenchymal marker. Among several oncogenic signaling pathways, only the MAPK/ERK signaling cascade was decreased by PDLIM2 inhibition and reciprocally, ERK inhibition down-regulated PDLIM2 expression. Importantly, PDLIM2 inhibition remarkably compromised tumor growth in a human CRPC xenograft model. In summary, the suppression of PDLIM2 significantly reduced such oncogenic phenotypes as proliferation, clonogenicity, invasiveness, and tumor cell growth in human CRPC-like cells both in vitro and in vivo, indicating that PDLIM2 may be considered a novel therapeutic target gene for treating human CRPC.

Kang M ，Lee KH ，Lee HS ，Park YH ，Jeong CW ，Ku JH ，Kim HH ，Kwak C ... -  《-》

S100A3 suppression inhibits in vitro and in vivo tumor growth and invasion of human castration-resistant prostate cancer cells.

To investigate the role of S100A3 and the effect of S100A3 inhibition on human castration-resistant prostate cancer (CRPC) cells by using in vitro and in vivo functional assays. Using human CRPC cells (PC3 and DU145), S100A3 expression levels were assessed by reverse transcription-polymerase chain reaction and Western blot analysis. After S100A3-specific small interfering ribonucleic acid (RNA) treatment, cell viability was determined by Cell Counting Kit-8 assay, and apoptotic cell fractions were evaluated by flow cytometry. The invasive properties of these cells and the expression pattern of matrix metalloproteinases (MMPs) were assessed using transwell migration assays, reverse transcription-polymerase chain reaction, and gelatin zymography. Finally, the in vivo efficacy of S100A3 inhibition on human CRPC cells was investigated using human tumor xenograft models in nude mice. Human CRPC cells showed overexpression of S100A3, and its suppression reduced cell viability owing to apoptotic cell death. Additionally, S100A3 inhibition decreased the invasiveness of human CRPC cells. Moreover, MMP-2 and MMP-9 were downregulated in PC3, whereas only MMP-9 was downregulated in D145 after S100A3 inhibition. In human CRPC xenograft models, we noted a marked reduction in tumor growth in mice injected with S100A3 short hairpin RNA-transfected PC3 and DU145 cells. Human CRPC cells showed upregulation of S100A3 expression, and S100A3 inhibition reduced tumor cell viability. S100A3 inhibition reduced invasion capability with downregulation of MMP expression. More importantly, S100A3 inhibition resulted in tumor growth suppression in human CRPC xenograft models, suggesting S100A3 could serve as a novel therapeutic target for the treatment of human CRPC.

Kang M ，Lee HS ，Lee YJ ，Choi WS ，Park YH ，Jeong CW ，Ku JH ，Kim HH ，Kwak C ... -  《-》

Suppression of LIM and SH3 Domain Protein 1 (LASP1) Negatively Regulated by Androgen Receptor Delays Castration Resistant Prostate Cancer Progression.

LIM and SH3 domain protein 1 (LASP1) has been implicated in several human malignancies and has been shown to predict PSA recurrence in prostate cancer. However, the anti-tumor effect of LASP1 knockdown and the association between LASP1 and the androgen receptor (AR) remains unclear. The aim of this study is to clarify the significance of LASP1 as a target for prostate cancer, and to test the effect of silencing LASP1 in vivo using antisense oligonucleotides (ASO). A tissue microarray (TMA) was performed to characterize the differences in LASP1 expression in prostate cancer treated after hormone deprivation therapy. Flow cytometry was used to analyze cell cycle. We designed LASP1 ASO for knockdown of LASP1 in vivo studies. The expression of LASP1 in TMA was increased after androgen ablation and persisted in castration resistant prostate cancer (CRPC). Also in TMA, compared with LNCaP cell, LASP1 expression is elevated in CRPC cell lines (C4-2 and VehA cells). Interestingly, suppression of AR elevated LASP1 expression conversely, AR activation decreased LASP1 expression. Silencing of LASP1 reduced cell growth through G1 arrest which was accompanied by a decrease of cyclin D1. Forced overexpression of LASP1 promoted cell cycle and induced cell growth which was accompanied by an increase of cyclin D1. Systemic administration of LASP1 ASO with athymic mice significantly inhibited tumor growth in CRPC xenografts. These results indicate that LASP1 is negatively regulated by AR at the transcriptional level and promotes tumor growth through induction of cell cycle, ultimately suggesting that LASP1 may be a potential target in prostate cancer treatment. Prostate 77:309-320, 2017. © 2016 Wiley Periodicals, Inc.

Dejima T ，Imada K ，Takeuchi A ，Shiota M ，Leong J ，Tombe T ，Tam K ，Fazli L ，Naito S ，Gleave ME ，Ong CJ ... -  《-》

NPRL2 enhances autophagy and the resistance to Everolimus in castration-resistant prostate cancer.

Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and involved in the mTOR signaling and drug resistance in several cancers. However, the role of NPRL2 in regulating the resistance to Everolimus (EVS), an inhibitor of the mTOR, in castration-resistant prostate cancer (CRPC) is still unclear. Therefore, in present study, we evaluated the role of NPRL2 and its potential resistance to EVS in CRPC. NPRL2 expression levels in prostate tissues, including benign prostate hyperplasia (BPH) tissues, primary prostate cancer (PCa) tissues, CRPC tissues, and several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) were be evaluated by immunohistochemistry, RT-PCR, and Western blot. Furthermore, we employed the loss or gain function of NPRL2 to determine the role of NPRL2 in regulating the proliferation, sensitivity to EVS, the mTOR signaling, autophagy in CRPC. Lastly, relationship between NPRL2 expression level and the efficacy of EVS were evaluated in mice tumor xenograft models. NPRL2 expression level is upregulated in PCa, particularly in the CRPC. NPRL2 over-expression promoted the proliferation, resistance to EVS, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to EVS in PC3 and LNPER cells. Moreover, NPRL2-silencing increased the activity of mTOR signaling, and the autophagy attenuation induced by NPRL2-silencing in EVS-treated CRPC cells was associated with the increase of apoptosis. In addition, the growth prevention of NPRL2-silencing LNPER tumors in mice induced by EVS-treatment was associated with the autophagy attenuation and apoptosis increase. NPRL2 may act as a pro-growth factor in PCa. The high levels of NPRL2 expression in CRPC promote resistance to EVS by enhancing autophagy. NPRL2 may be a new therapeutic target for intervention of CRPC and a biomarker for predicting resistance to EVS in CRPC.

Chen Z ，Jiang Q ，Zhu P ，Chen Y ，Xie X ，Du Z ，Jiang L ，Tang W ... -  《-》

KLF9, a transcription factor induced in flutamide-caused cell apoptosis, inhibits AKT activation and suppresses tumor growth of prostate cancer cells.

Kruppel-like factors (KLFs) are involved in various biological processes; emerging studies have indicated that KLF9 plays a critical role in regulating tumorigenesis. The role of KLF9 in prostate cancer (PCa), however, has not yet been investigated. The expression of KLF members, AKT- and apoptosis-related proteins were analyzed by Western blot or qRT-PCR. Tet-On inducible KLF9 expression was established for the evaluation of the effects of KLF9 on cell proliferation, apoptosis, and xenograft tumor growth in nude mice. Cell cycle and apoptosis were determined by flow cytometry. KLF9 was induced in a time-dependent manner in flutamide-caused apoptosis, and knockdown of KLF9 significantly decreased flutamide-induced growth inhibition and apoptosis in LNCaP cells. The levels of KLF9 were relatively lower in PCa cell lines, particularly in androgen-independent cell lines compared with those in nontumorous prostate epithelial cell lines. Overexpression of KLF9 dramatically suppressed cell proliferation and caused cell cycle arrest in the G2/M phase and cell apoptosis in the androgen-independent cell lines, PC3 and DU145. Intriguingly, KLF9 expression severely suppressed the activation of AKT and its downstream targets. AKT reactivation partially rescued the KLF9-mediated inhibitory effects on the proliferation of PCa cells. More importantly, we found that KLF9 overexpression efficiently inhibited the xenograft tumor growth of PCa cells. These data collectively showing that KLF9 substantially inhibits AKT activation and abrogates tumor growth of PCa cells, suggest the potential of either genetic or pharmacological activation of KLF9 in the therapeutic treatment of castration-resistant PCa.

Shen P ，Sun J ，Xu G ，Zhang L ，Yang Z ，Xia S ，Wang Y ，Liu Y ，Shi G ... -  《-》