Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor and the second leading cause of cancer deaths in American men, the mechanisms explaining the development and progression of CaP remain largely unknown. Recent studies have shown that some aberrantly expressed microRNAs (miRNAs) are involved in tumorigenesis. Although aberrant expression of certain miRNAs has been discovered in CaP, their function in this disease has not yet been defined. In this study, we found differential expression of miR-125b in androgen-dependent and independent CaP cells, as well as in benign and malignant prostate tissues. Furthermore, androgen signaling was able to up-regulate the expression of miR-125b. In addition, transfection of synthetic miR-125b stimulated androgen-independent growth of CaP cells and down-regulated the expression of Bak1. Our results suggest that miR-125b acts as an oncogene, contributing to the pathogenesis of CaP.microRNA ͉ miR-125b ͉ ISH ͉ LNCaP

BACKGROUND Increasing evidence demonstrates that aberrantly-regulated microRNAs (miRNAs) contribute to the initiation and progression of human cancer. We previously have demonstrated that miR-125b stimulated the growth of prostate cancer (CaP) cells. In this study, we further determined the influence of miR-125b on the pathogenesis of prostate cancer. METHODS To evaluate the effect of miR-125b on xenograft tumor growth, male athymic mice were subcutaneously injected with PC-346C-miR-125b cells that stably overexpressed miR-125b. Potential direct target transcripts of miR-125b were identified using a bioinformatics approach and three miR-125b targeted molecules were confirmed by means of biochemical analyses. RESULTS Enforced expression of miR-125b promoted tumor growth in both intact and castrated male nude mice. In an effort to define the molecular mechanism(s) mediating its tumor growth properties, we found that miR-125b directly targets eight transcripts, including three key pro-apoptotic genes: p53, Puma and Bak1. Increasing the abundance of miR-125b resulted in a dramatic decrease in the levels of these three proteins in prostate cancer cells. A direct repressive effect on each of these was supported by the ability of miR-125b to significantly reduce the activity of luciferase reporters containing their 3′-untranslated regions of each gene encompassing the miR-125b-binding sites. Additionally, we found that repression of miR-125b activity was able to sensitize prostate cancer cells to different therapeutic interventions. CONCLUSION Data obtained in this study demonstrate that miR-125b promotes growth of prostatic xenograft tumors by down-regulating three key pro-apoptotic genes. This suggests that miR-125b is oncogenic and makes it an attractive therapeutic target in prostate cancer.

PurposeProstate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.Experimental DesignLevels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.ResultsWe identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.ConclusionsThese results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.

Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor in American men, the mechanisms underlying the development and progression of CaP remain largely unknown. Recent studies have shown that downregulation of miR-124 occurs in several types of human cancer, suggesting a tumor suppressive function of miR-124. Until now, however, it has been unclear whether miR-124 is associated with CaP. In the present study, we completed a series of experiments to understand the functional role of miR-124 in CaP. We detected the expression level of miR-124 in clinical CaP tissues, evaluated the influence of miR-124 on the growth of CaP cells, and investigated the mechanism underlying the dysregulation of miR-124. We found that i) miR-124 directly targets the androgen receptor (AR) and subsequently induces a upregulation of p53; ii) miR-124 is significantly down-regulated in malignant prostatic cells compared to that in benign cells and DNA methylation causes the reduced expression of miR-124; and iii) miR-124 can inhibit the growth of CaP cells in vitro and in vivo. Data from this study revealed that loss of miR-124 expression is a common event in CaP, which may contribute to pathogenesis of CaP. Our studies also suggest that miR-124 is a potential tumor suppressive gene in CaP, and restoration of miR-124 expression may represent a novel strategy for CaP therapy.

BACKGROUND Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer. METHODS The effects of docetaxel on human DU145, PC3, LNCaP, and C4-2 prostate cancer cells were examined in cell culture, and p53 expression were analyzed by Western blot analysis. The potential role of p53 in docetaxel sensitivity in prostate cancer cells was tested by either p53 silencing using shRNA or p53 overexpression by introducing wild-type p53. RESULTS We found that DU145 (mutant p53) and PC3 (p53 null) cells were less sensitive than LNCaP and C4-2 cells expressing functional p53 in response to docetaxel. Docetaxel treatment induces considerably higher apoptosis in LNCaP and C4-2 cells than in DU145 and PC3 cells in a dose dependent manner. Docetaxel increases the levels of ser15 phosphorylation of p53 in a dose dependent manner in both LNCaP and C4-2 cells, while has no effect on the levels of ser15 phosphorylation of p53 in DU145 cells. These results suggest that p53 phosphorylation is associated with docetaxel sensitivity in prostate cancer cells. To further confirm whether p53 activation can induce cell sensitivity to docetaxel treatment, we used p53 shRNA to knock down p53 expression in C4-2 cells and determined the cells response to docetaxel treatment. Knockdown of p53 significantly down regulated p53 phosphorylation and blocked docetaxel induced apoptotic cell death compared to the vector control. To further confirm this observation, we established a stable knock out p53 in C4-2 cells. Down regulation of p53 in the stable p53 knock out C4-2 cells significantly inhibited docetaxel induced apoptotic cell death. We also used wild-type (WT) p53 to over express p53 in DU145 cells, and found that expression of WT-p53 in DU145 cells increased their sensitivity to docetaxel. CONCLUSIONS These results demonstrate that docetaxel induces p53 phosphorylation and that p53 status is a crucial determinant of docetaxel sensitivity in prostate cancer cells.

Exosomes are membrane-enclosed nanovesicles that shuttle active cargoes, such as mRNAs and microRNAs (miRNAs), between different cells. Mesenchymal stem cells (MSCs) are able to migrate to the tumor sites and exert complex functions over tumor progress. We investigated the effect of human bone marrow-derived MSC (BMSC)-derived exosomal miR-143 on prostate cancer. During the co-culture experiments, we disrupted exosome secretion by the inhibitor GW4869 and overexpressed exosomal miR-143 using miR-143 plasmid. miR-143 was involved in the progression of prostate cancer via trefoil factor 3 (TFF3). Moreover, miR-143 was downregulated while TFF3 was upregulated in prostate cancer cells and tissues, and miR-143 was found to specifically inhibit TFF3 expression. Human MSC-derived exosomes enriched miR-143 and transferred miR-143 to prostate cancer cells. Furthermore, elevated miR-143 or exosome-miR-143 or silencing TFF3 inhibited the expression of TFF3, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2, and MMP-9 and PC3 cell proliferation, migration, invasion, and tumor growth, whereas it promoted apoptosis. In conclusion, hMSC-derived exosomal miR-143 directly and negatively targets TFF3 to suppress prostate cancer.

We have shown a novel role of miR-106b, in the setting of radiation treatment, in regulating the p21-activated cell cycle arrest. Our finding that miR-106b is able to override radiation-induced cell cycle arrest and cell growth inhibition points to a potential therapeutic target in certain prostate cancer cells whose radiation resistance is likely due to consistently elevated level of miR-106b.