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Knockdown of HOTAIR inhibits EMT

Figure S1 depicts the location of CpG islands in promoter region of miR-182-5p

Prostate cancer incidence and mortality rates are higher in African-American (AA) than in European-American (EA) men. The main objective of this study was to elucidate the role of miR-130b as a contributor to prostate cancer health disparity in AA patients. We also determined whether miR-130b is a prognostic biomarker and a new therapeutic candidate for AA prostate cancer. A comprehensive approach of using cell lines, tissue samples, and the TCGA database was employed. We performed a series of functional assays such as cell proliferation, migration, invasion, RT2-PCR array, qRT-PCR, cell cycle, luciferase reporter, immunoblot, and IHC. Various statistical approaches such as Kaplan–Meier, uni-, and multivariate analyses were utilized to determine the clinical significance of miR-130b. Our results showed that elevated levels of miR-130b correlated with race disparity and PSA levels/failure and acted as an independent prognostic biomarker for AA patients. Two tumor suppressor genes, CDKN1B and FHIT, were validated as direct functional targets of miR-130b. We also found race-specific cell-cycle pathway activation in AA patients with prostate cancer. Functionally, miR-130b inhibition reduced cell proliferation, colony formation, migration/invasion, and induced cell-cycle arrest. Inhibition of miR-130b modulated critical prostate cancer–related biological pathways in AA compared with EA prostate cancer patients. In conclusion, attenuation of miR-130b expression has tumor suppressor effects in AA prostate cancer. miR-130b is a significant contributor to prostate cancer racial disparity as its overexpression is a risk factor for poor prognosis in AA patients with prostate cancer. Thus, regulation of miR-130b may provide a novel therapeutic approach for the management of prostate cancer in AA patients.

Supplementary Figure 4. miR-130b cluster miRNA and MET expression level in TCGA PRAD samples.

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PDF - 277K, Prostate cancer cell lines (LNCaP, PC3, Du145) treated with docetaxel at varying concentrations and time periods (6 hrs, 24 hrs) followed by miR-3607 expression analysis by real-time PCR.

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Table S1 is for primer sequences

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Prostate cancer is one of the most prevalent cancers in males and ranks as the second most common cause of cancer-related deaths. 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, is a promising anticancer agent for various types of cancers. Although 2-ME has been shown to activate c-Jun-NH2-kinase (JNK) and mitochondrial-dependent apoptotic signaling pathways, the underlying mechanisms, including downstream effectors, remain unclear. Here, we report that the human Bcl-2 homology 3 (BH3)-only protein harakiri (Hrk) is a critical effector of 2-ME–induced JNK/mitochondria–dependent apoptosis in prostate cancer cells. Hrk mRNA and protein are preferentially upregulated by 2-ME, and Hrk induction is dependent on the JNK activation of c-Jun. Hrk knockdown prevents 2-ME–mediated apoptosis by attenuating the decrease in mitochondrial membrane potential, subsequent cytochrome c (cyt c) release, and caspase activation. Involvement of the proapoptotic protein Bak in this process suggested the possible interaction between Hrk and Bak. Thus, Hrk activation by 2-ME or its overexpression displaced Bak from the complex with antiapoptotic protein Bcl-xL, whereas deletion of the Hrk BH3 domain abolished its interaction with Bcl-xL, reducing the proapoptotic function of Hrk. Finally, Hrk is also involved in the 2-ME–mediated reduction of X-linked inhibitor of apoptosis through Bak activation in prostate cancer cells. Together, our findings suggest that induction of the BH3-only protein Hrk is a critical step in 2-ME activation of the JNK-induced apoptotic pathway, targeting mitochondria by liberating proapoptotic protein Bak. Mol Cancer Ther; 12(6); 1049–59. ©2013 AACR.

Patient characteristics

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PDF file - 321K, Figure S1. Induction of BH3-only protein family members by 2-ME in prostate cancer cells. Figure S2. Hrk knockdown prevents 2-ME-mediated cell death in time-dependent manner. Figure S3. Mitochondrial localization of Hrk. Figure S4. No change in total cell number after 2-ME treatment. Figure S5. Bax is not involved in 2-ME-mediated apoptotic cell death. Figure S6. Endogenous Bak interacts with Bcl-xL but not with Bcl-2 in prostate cancer cells. Figure S7. Hrk requires the BH3 domain for pro-apoptotic function.

Supplemental Figure Legends

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PDF - 176K, Relative miR-3607 expression levels in PC3/Du145/LNCaP cells in mock/miR-CON/ miR-3607 transfected cells as assessed by quantitative RT-PCR analysis.

Bladder cancer is often associated with recurrence and progression to invasive metastatic disease that have palliative therapeutic options. The use of traditional chemotherapeutic agents for bladder cancer management often suffers from toxicity and resistance concerns. This emphasizes the need for development of safer, natural, nontoxic compounds as chemotherapeutic/chemopreventive agents. Curcumin (diferuloylmethane) is a natural compound that has been known to possess anticancer properties in various cancers, including bladder cancer. However, the biological targets of curcumin are not well defined. Recently, it has been proposed that curcumin may mediate epigenetic modulation of expression of microRNAs (miRNA). In this article, we define for the first time, that curcumin directly induces a tumor-suppressive miRNA, miR-203, in bladder cancer. miR-203 is frequently downregulated in bladder cancer due to DNA hypermethylation of its promoter. We studied the functional significance of miR-203 in bladder cancer cell lines and found that miR-203 has tumor suppressive properties. Also, we define Akt2 and Src as novel miR-203 targets in bladder cancer. Curcumin induces hypomethylation of the miR-203 promoter and subsequent upregulation of miR-203 expression. This leads to downregulation of miR-203 target genes Akt2 and Src that culminates in decreased proliferation and increased apoptosis of bladder cancer cells. This is the first report that shows a direct effect of curcumin on inducing epigenetic changes at a miRNA promoter with direct biological consequences. Our study suggests that curcumin may offer a therapeutic advantage in the clinical management of refractory bladder cancer over other standard treatment modalities. Cancer Prev Res; 4(10); 1698–709. ©2011 AACR.

This study aims to investigate the role of miR-203–HOTAIR interaction in the suppression of renal cell carcinoma (RCC). We employed series of in vitro assays such as proliferation, invasion, migration, and colony formation along with in vivo tumor xenograft model. Profiling of miR-203 and HOTAIR expression revealed that miR-203 was significantly underexpressed, whereas HOTAIR was overexpressed in RCC cell lines and clinical specimens compared with normal cell line and tissue. Both miR-203 and HOTAIR expression significantly distinguished malignant from normal tissues and significantly correlated with clinicopathologic characteristics of patients. Overexpression of miR-203 significantly inhibited proliferation, migration, and invasion with an induction of apoptosis and cell-cycle arrest. However, HOTAIR suppression resulted in the similar functional effects in the same RCC cell lines. In silico, RNA-22 algorithm showed a binding site for miR-203 in HOTAIR. We observed a direct interaction between miR-203 and HOTAIR by RNA-immunoprecipitation (RIP) and luciferase reporter assays. We show that miR-203–HOTAIR interaction resulted in the inhibition of epithelial-to-mesenchymal transition (EMT) and metastatic genes as indicated by induction of key metastasis-suppressing proteins E-cadherin, claudin (epithelial markers), and PTEN along with induction of tumor suppressor genes p21 and p27. A significant decrease in vimentin (mesenchymal marker), KLF4, and Nanog (stemness markers) was also observed. This is the first report demonstrating miR-203–mediated regulation of HOTAIR induces tumor suppressor effects in RCC by regulating EMT and metastatic pathway genes. Thus, the study suggests that therapeutic regulation of HOTAIR by miR-203 overexpression may provide an opportunity to regulate RCC growth and metastasis. Mol Cancer Ther; 17(5); 1061–9. ©2018 AACR.

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PDF - 91K, Supplemental materials and methods and figure legends to Fig. S1, S2, S3 and Table S1.

PDF - 76K, Relative miR-3607 expression levels in normal immortalized prostate epithelial cell lines and prostate cancer cell lines as assessed by quantitative RT-PCR analysis.

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PDF file - 425K, MS-PCR analysis of miR-203 promoter methylation in T24 cell line following treatment with epigenetic drugs.

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PDF file - 503K, Restoration of miR-203 expression suppresses tumorigenicity in vitro in J82 cell line.

PDF - 68K, Clinicopathological data for matched LCM-microdissected tissues used for real-time PCR analysis of miR-3607 expression in Figure 1.

PDF file - 620K, miR-203 promoter is hypermethylated in bladder cancer cell lines.

PDF file - 130K, Curcumin augments miR-203 expression in bladder cancer cell lines J82 and TCCSUP.

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Primer Sequences

Merged file with following figues: Fig. S1 Characterization of serum exosomal preparations Fig. S2 Biological pathways potentially influenced by dysregulated miRNAsin aggressive PCa as compared to BPH Fig. S3 Exosomal miR-1246 expression patterns differ from circulating miR-1246 expression in sera of prostate cancer patients Fig. S4 ex-miR-1246 expression as a measure of PCa aggressiveness

Because of high heterogeneity, molecular characterization of prostate cancer based on biopsy sampling is often challenging. Hence, a minimally invasive method to determine the molecular imprints of a patient's tumor for risk stratification would be advantageous. In this study, we employ a novel, digital amplification-free quantification method using the nCounter technology (NanoString Technologies) to profile exosomal serum miRNAs (ex-miRNA) from aggressive prostate cancer cases, benign prostatic hyperplasia, and disease-free controls. We identified several dysregulated miRNAs, one of which was the tumor suppressor miR-1246. miR-1246 was downregulated in prostate cancer clinical tissues and cell lines and was selectively released into exosomes. Overexpression of miR-1246 in a prostate cancer cell line significantly inhibited xenograft tumor growth in vivo and increased apoptosis and decreased proliferation, invasiveness, and migration in vitro. miR-1246 inhibited N-cadherin and vimentin activities, thereby inhibiting epithelial–mesenchymal transition. Ex-miR-1246 expression correlated with increasing pathologic grade, positive metastasis, and poor prognosis. Our analyses suggest ex-miR-1246 as a promising prostate cancer biomarker with diagnostic potential that can predict disease aggressiveness.Significance: Dysregulation of exosomal miRNAs in aggressive prostate cancer leads to alteration of key signaling pathways associated with metastatic prostate cancer. Cancer Res; 78(7); 1833–44. ©2018 AACR.

Supplementary Figures S1-S2; Supplementary Data.

PDF file - 76K, Supplemental Table 1. Clinicopathologic characteristics of prostate cancer patients. Supplemental Table 2. Complimentary sequences in 3'UTRs of genes for miR-34b and sequences of primers and probes.

List of microRNAs significantly dysregulated in A. BPH vs aggressive PCa B. normal vs aggressive PCa

Purpose: Advanced metastatic prostate cancer (PCa) is a fatal disease, with only palliative therapeutic options. Though almost 80% of cases of metastatic PCa present bone metastasis, our current understanding of the molecular mechanisms that govern this metastatic dissemination remains fragmentary. The main objective of the present study was to identify microRNA (miRNA) genes that regulate metastatic PCa.Experimental Design: miRNA expression profiling was done in human prostate cell lines to identify dysregulated miRNA components of advanced PCa. miR-203 expression was assessed in prostate carcinoma cell lines and clinical specimens by real-time PCR and in situ hybridization. To assess the biological significance of miR-203, miR-203 was reexpressed in bone metastatic PCa cell lines followed by in vitro and in vivo functional assays.Results: miR-203 expression is specifically attenuated in bone metastatic PCa suggesting a fundamental antimetastatic role for this miRNA. Reintroduction of miR-203 in bone metastatic PCa cell lines suppresses metastasis via inhibition of several critical steps of the metastatic cascade including epithelial-mesenchymal transition, invasion, and motility. Ectopic miR-203 significantly attenuated the development of metastasis in a bone metastatic model of PCa. Importantly, miR-203 regulates a cohort of pro-metastatic genes including ZEB2, Bmi, survivin, and bone-specific effectors including Runx2, a master regulator of bone metastasis.Conclusions: miR-203 is an “antimetastatic” miRNA in PCa that acts at multiple steps of the PCa metastatic cascade via repression of a cohort of prometastatic targets. miR-203 may be an attractive target for therapeutic intervention in advanced PCa. Clin Cancer Res; 17(16); 5287–98. ©2011 AACR.

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Supplementary figures S1-S6

Includes supplemental methods, figure and table legends

PDF file - 325K, Supplemental Figure 1: Downregulation of miR-34b expression, its potential diagnostic and prognostic significance and methylation status in PCa. A) Box plot representation of mir-34b expression in a cohort of unmatched tissue samples. B) Box plot representation of ISH results of tissue samples. (Ave- Average; T/N- Tumor/Normal). C) Chi-square test showing correlation of clinicopathological characteristics with miR-34b expression. D) CpG islands within the 1.0 kb region upstream of the miR-34b gene. Arrow indicates the start of the miR-34b gene. Locations for primers and probes are also shown on the miR-34b gene. F- Forward, R-Reverse, M/U Methylation and Unmethylation probes, CF1, CF2, CR1, CR2- ChiP Forward1-2, ChIP Reverse 1-2. E) Correlation of miR-34b expression with average percent methylation indicating cases with low miR-34b expression have higher methylation and vice versa.

Antibody used with their catalog numbers

Clinicopathological features

PDF file - 141K, miR-23b directly targets Akt in prostate cancer