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Both siRNA and miRNA can serve as powerful gene-silencing reagents but their specific delivery to cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miRNA seed-targeting sequence to block the growth of prostate cancer in mouse models. Utilizing the thermodynamically ultra-stable three-way junction of the pRNA of phi29 DNA packaging motor, RNA nanoparticles were constructed by bottom-up self-assembly containing the anti-prostate-specific membrane antigen (PSMA) RNA aptamer as a targeting ligand and anti-miR17 or anti-miR21 as therapeutic modules. The 16 nm RNase-resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little or no accumulation in healthy organs 8 hours postinjection, and subsequently repressed tumor growth at low doses with high efficiency.

Background & Aims Vitamin D protects against colorectal cancer through unclear mechanisms. We investigated the effects of calcitriol (1α,25-dihydroxyvitamin D 3 ; the active form of vitamin D) on levels of different microRNAs (miRNAs) in colorectal cancer cells from humans and xenograft tumors in mice. Methods Expression of miRNAs in colorectal cancer cell lines was examined using the Ambion mirVana miRNA Bioarray. The effects of calcitriol on expression of miR-627 and cell proliferation were determined by real-time polymerase chain reaction and WST-1 assay, respectively; growth of colorectal xenograft tumors was examined in nude mice. Real-time polymerase chain reaction was used to analyze levels of miR-627 in human colon adenocarcinoma samples and nontumor colon mucosa tissues (controls). Results In HT-29 cells, miR-627 was the only miRNA significantly up-regulated by calcitriol. Jumonji domain containing 1A ( JMJD1A ), which encodes a histone demethylase, was found to be a target of miR-627. By down-regulating JMJD1A, miR-627 increased methylation of histone H3K9 and suppressed expression of proliferative factors, such as growth and differentiation factor 15. Calcitriol induced expression of miR-627, which down-regulated JMJD1A and suppressed growth of xenograft tumors from HCT-116 cells in nude mice. Overexpression of miR-627 prevented proliferation of colorectal cancer cell lines in culture and growth of xenograft tumors in mice. Conversely, blocking the activity of miR-627 inhibited the tumor suppressive effects of calcitriol in cultured colorectal cancer cells and in mice. Levels of miR-627 were decreased in human colon adenocarcinoma samples compared with controls. Conclusions miR-627 mediates tumor-suppressive epigenetic activities of vitamin D on colorectal cancer cells and xenograft tumors in mice. The messenger RNA that encodes the histone demethylase JMJD1A is a direct target of miR-627. Reagents designed to target JMJD1A or its messenger RNA, or increase the function of miR-627, might have the same antitumor activities of vitamin D without the hypercalcemic side effects.

The class I selective inhibitor of the histone deacetylases, mocetinostat, has promising antitumor activities in both preclinical studies and the clinical trials. To understand how mocetinostat induces apoptosis, we examined the effects of mocetinostat on miR-31, a proapoptotic microRNA that was previously found to be epigenetically silenced in prostate cancer. We found that miR-31 was significantly upregulated by mocetinostat in prostate cancer cells. Antiapoptotic protein E2F6, the target of miR-31, was decreased by mocetinostat treatment. When miR-31 was blocked with an inhibitor, the ability of mocetinostat to induce apoptosis was reduced. We further demonstrated that mocetinostat enhanced the activity of docetaxel in apoptosis induction. While siRNA knockdown of E2F6 sensitized cancer cells to mocetinostat-induced apoptosis, overexpression of E2F6 blocked mocetinostat-induced apoptosis. In an orthotopic xenograft model, we demonstrated that mocetinostat activated miR-31, decreased E2F6, induced apoptosis, and significantly reduced prostate cancer growth. Importantly, we found that mocetinostat also increased miR-31 expression, decreased E2F6, and induced apoptosis in the primary prostate cancer stem cells. Thus, activation of miR-31 and downregulation of E2F6 constitute an important mechanism in mocetinostat-induced apoptosis in prostate cancer.

Prostate cancer is the second leading cause of cancer-related mortality among men in the United States. However, our understanding of the cause of prostate cancer remains elusive. Recent studies have implicated that epigenetics, heritable changes in gene expression caused by mechanisms other than classical genetic alterations, is an important mechanism in tumorigenesis. MicroRNAs function as epigenetic regulators to alter gene expression, and very limited information is available regarding the roles of microRNAs in prostate cancer. Evasion of apoptosis is the hallmark of cancer, and it plays an important role in the growth and development of prostate cancer. We have recently demonstrated that miR-31 and miR-205 are downregulated in advanced prostate cancer. In addition, anti-apoptotic protein E2F6 was identified as the target of miR-31 and contributed resistance to chemotherapy-induced apoptosis. While we found that miR-205 is silenced by gene promoter methylation, the mechanism of the downregulation of miR-31 in prostate cancer remains poorly defined. In this study, we report that EZH2 (Enhancer of zeste 2), a histone methyltransferase and component of the polycomb repressive complex 2 (PRC2), is responsible for miR-31 silencing in prostate cancer. EZH2 plays an essential role in the epigenetic maintenance of the H3K27me3 repressive chromatin mark. Our results have shown that siRNA knockdown of EZH2 and DZNep (3-deazaneplanocin) treatment (an EZH2 inhibitor) in prostate cancer cells increased miR-31 expression. Furthermore, EZH2 is responsible for histone methylation at the promoter of miR-31 gene. In addition, siRNA knockdown of EZH2 and the treatment with DZNep sensitized prostate cancer cells to apoptosis. In contrast, EZH2 overexpression confers resistance to chemotherapy-induced apoptosis in prostate cancer cells. Thus, polycomb protein EZH2 suppresses the expression of miR-31 and consequently contributes to apoptosis resistance in prostate cancer. The results will improve our understanding of the mechanism of drug resistance in prostate cancer cells. Novel strategies targeting EZH2 and miR-31 can be developed to increase drug-induced apoptosis and improve cancer response to chemotherapy. Citation Format: Qunshu Zhang. Epigenetic regulation of miR-31 and miR-205 of apoptosis in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3535. doi:10.1158/1538-7445.AM2014-3535

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and suppresses gene expression by catalyzing histone H3 methylation on lysine 27. EZH2 is overexpressed in metastatic prostate cancer and has been shown to promote cell proliferation and metastasis. Here we show that EZH2 also suppresses prostate cancer apoptosis by coordinating the epigenetic silencing of two proapoptotic microRNAs (miRNA), miR-205 and miR-31. We previously reported that miR-205 promotes apoptosis by targeting antiapoptotic protein Bcl-w and miR-205 is silenced in prostate cancer through promoter methylation. In this study, we found that EZH2 suppresses miR-31 expression by trimethylation of lysine 27 on histone 3 on the miR-31 promoter. SiRNA knockdown of EZH2 increased miR-31 expression and decreased the antiapoptotic protein E2F6 (E2F transcription factor 6) (a target of miR-31), resulting in the sensitization of prostate cancer cells to docetaxel-induced apoptosis. Conversely, overexpression of EZH2 blocked docetaxel-induced apoptosis. We further demonstrated that miR-205 silencing is linked to miR-31 silencing through EZH2. Suppression of miR-205 with an miRNA inhibitor caused an increase of EZH2 protein, which in turn inhibited miR-31 expression. Conversely, overexpression of miR-205 decreased EZH2 protein and increased miR-31 expression. In paired human prostate cancer specimens and adjacent normal tissues, we observed that the decrease of miR-205 expression correlated with EZH2 overexpression and miR-31 silencing. Thus, EZH2 integrates the epigenetic silencing of miR-205 and miR-31 to confer resistance to chemotherapy-induced apoptosis.

Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2′-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer.