Suppressor effect of catechol-O-methyltransferase gene in prostate cancer.

Catechol-estrogens can cause genetic mutations and to counteract their oncogenicity, the catechol-O-methyltransferase (COMT) gene is capable of neutralizing these reactive compounds. In this study, we determined the functional effects and regulation of COMT in prostate cancer. Both the Cancer Genome Atlas (TCGA) and immunohistochemical analysis of clinical specimens demonstrated a reduction of COMT expression in prostate cancer. Also, western analyses of prostate cancer cell lines show COMT levels to be minimal in DuPro and DU145 and thus, these cells were used for further analyses. Re-expression of COMT led to suppressed migration ability (wound healing assay) and enhanced apoptosis (flow cytometric analyses), and when challenged with 4-hydroxyestradiol, a marked reduction of cell proliferation (MTT assay) was observed. Xenograft growth in athymic mice also resulted in inhibition due to COMT. As a mechanism, western analyses show cleaved CASP3 and BID were increased whereas XIAP and cIAP2 were reduced due to COMT. As COMT expression is low in prostate cancer, its regulation was determined. Databases identified several miRNAs capable of binding COMT and of these, miR-195 was observed to be increased in prostate cancer according to TCGA. Real-time PCR validated upregulation of miR-195 in clinical prostate cancer specimens as well as DuPro and DU145 and interestingly, luciferase reporter showed miR-195 capable of binding COMT and overexpressing miR-195 could reduce COMT in cells. These results demonstrate COMT to play a protective role by activating the apoptosis pathway and for miR-195 to regulate its expression. COMT may thus be a potential biomarker and gene of interest for therapeutic development for prostate cancer.

Role of miR-182/PDCD4 axis in aggressive behavior of prostate cancer in the African Americans.

BACKGROUND: Prostate cancer is one of the most commonly diagnosed cancers among men. African Americans (AA) are at an increased risk of developing prostate cancer compared to European Americans (EA). miRNAs play a critical role in these tumors, leading to tumor progression. In this study, we investigated the role of miR-182 in racial disparity in prostate cancer. RESULTS: We found significantly increased levels of miR-182 in prostate cancer tissues compared to BPH. Also, miR-182 shows increased expression in AA prostate cancer cell line and tissue samples compared to EA. We performed biochemical recurrence (BCR) - free survival time in AA and EA patients and found that high miR-182 expression had significantly shorter BCR-free survival than patients with low miR-182 expression (P = 0.031). To elucidate the role of miR-182, we knocked down miR-182 in EA (DU-145 and LNCaP) and AA (MDA-PCa-2b) cell lines and found an increase in apoptosis, arrest of the cell cycle, and inhibition of colony formation in the AA cell line to a greater extent than EA cell lines. CONCLUSIONS: Our results showed that PDCD4 is a direct miR-182 target and its inhibition is associated with aggressiveness and high Gleason grade in prostate cancer among AA. These findings show that miR-182 is highly expressed in AA patients and miR-182 may be a target for effective therapy in AA patients.

A lncRNA TCL6-miR-155 Interaction Regulates the Src-Akt-EMT Network to Mediate Kidney Cancer Progression and Metastasis.

Metastasis is the leading cause of mortality from kidney cancer, and understanding the underlying mechanism of this event will provide better strategies for its management. Here we investigated the biological, functional, and clinical significance of lncTCL6 and its interacting miR-155 in clear cell renal cell carcinoma (ccRCC). We employed a comprehensive approach to investigate the lncTCL6-miR-155-Src/Akt-mediated epithelial-to-mesenchymal transition (EMT) pathway as a novel regulatory mechanism in ccRCC progression. Expression analyses revealed that lncTCL6 is downregulated in ccRCC compared with normal tissues. Overexpression of lncTCL6 in ccRCC cell lines impaired their oncogenic functions, such as cell proliferation and migration/invasion, and induced cell-cycle arrest and apoptosis; conversely, depletion of lncTCL6 rescued these phenotypic effects. Furthermore, lncTCL6 directly interacted with miR-155. Unlike lncTCL6, miR-155 was overexpressed in ccRCC. Stable knockdown of miR-155 phenocopied the effects of lncTCL6 overexpression. Conversely, reconstitution of miR-155 and suppression of lncTCL6 in noncancerous renal cell HK2 induced tumorigenic characteristics. Patients with higher expression of lncTCL6 and lower expression of miR-155 had better survival probability. When overexpressed, lncTCL6 recruited STAU1 and mediated decay of Src mRNA, followed by a marked downregulation of an integrated network of Src target genes involved in migration, invasion, and EMT. However, the interaction between miR-155 and lncTCL6 attenuated the regulatory role of lncTCL6 on Src-mediated EMT. In conclusion, this study is the first report documenting the lncTCL6-miR155-Src/Akt/EMT network as a novel regulatory mechanism in aggressive ccRCC and a promising therapeutic target to inhibit renal cancer. SIGNIFICANCE: This study's investigation of noncoding RNA interactions in renal cell carcinoma identify miRNA-155-lncRNA TCL6-mediated regulation of the Src-Akt-EMT network as a novel mechanism of disease progression and metastasis.

Role of the PI3K/Akt pathway in cadmium induced malignant transformation of normal prostate epithelial cells.

This study investigated the role of the PI3K/Akt pathway in cadmium (Cd) induced malignant transformation of normal prostate epithelial (PWR1E and RWPE1) cells. Both PWR1E and RWPE1 cells were exposed to 10 µM Cd for one year and designated as Cd-PWR1E and Cd-RWPE1. Cd-RWPE1 cells robustly formed tumors in athymic nude mice. Functionally, Cd-exposure induced tumorigenic attributes indicated by increased wound healing, migration and invasion capabilities in both cell lines. RT2-array analysis revealed many oncogenes including P110α, Akt, mTOR, NFKB1 and RAF were induced whereas tumor suppressor (TS) genes were attenuated in Cd-RWPE1. This was validated by individual quantitative-real-time-PCR at transcriptional and by immunoblot at translational levels. These results were consistent in Cd-PWR1E vs parental PWR1E cells. Gene Set Enrichment Analysis revealed that five prostate cancer (PCa) related pathways were enriched in Cd-exposed cells compared to their normal controls. These pathways include the KEGG- Pathways in cancer, Prostate Cancer Pathway, ERBB, Apoptosis and MAPK pathways. We selected up- and down-regulated genes randomly from the PI3K/Akt pathway array and profiled these in the TCGA/GDC prostate-adenocarcinoma (PRAD) patient cohort. An upregulation of oncogenes and downregulation of TS genes was observed in PCa compared to their normal controls. Taken together, our study reveals that the PI3K/Akt signaling is one of the main molecular pathways involved in Cd-driven transformation of normal prostate epithelial cells to malignant form. Understanding the molecular mechanisms involved in the Cd-driven malignant transformation of normal prostate cells will provide a significant insight to develop better therapeutic strategies for Cd-induced prostate cancer.

LncRNA CDKN2B-AS1/miR-141/cyclin D network regulates tumor progression and metastasis of renal cell carcinoma.

The molecular heterogeneity of renal cell carcinoma (RCC) complicates the therapeutic interventions for advanced metastatic disease and thus its management remains a significant challenge. This study investigates the role of the lncRNA CDKN2B-AS1 and miR-141-3p interactions in the progression and metastasis of kidney cancer. Human renal cancer cell lines (ACHN and Caki1), normal RPTEC cells, tissue cohorts, and a series of in vitro assays and in vivo mouse model were used for this study. An overexpression of CDKN2B-AS1 was observed in RCC compared to normal samples in TCGA and our in-house SFVAMC tissue cohorts. Reciprocally, we observed reduced expression of miR-141 in RCC compared to normal in the same cohorts. CDKN2B-AS1 shares regulatory miR-141 binding sites with CCND1 and CCND2 genes. Direct interactions of CDKN2B-AS1/miR-141/Cyclin D1-D2 were confirmed by RNA immunoprecipitation and luciferase reporter assays indicating that CDKN2B-AS1/miR-141/Cyclin D1-D2 acts as a ceRNA network in RCC. Functionally, attenuation of CDKN2B-AS1 and/or overexpression of miR-141 inhibited proliferation, clonogenicity, migration/invasion, induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model. Further, overexpression of CDKN2B-AS1 is positively correlated with poor overall survival of RCC patients. Expression of miR-141 also robustly discriminated malignant from non-malignant tissues and its inhibition in normal RPTEC cells induced pro-cancerous characteristics. CDKN2B-AS1 attenuation or miR-141 overexpression decreased CCND1/CCND2 expression, resulting in reduced RAC1/pPXN that are involved in migration, invasion and epithelial-mesenchymal transition. This study, for the first time, deciphered the role of CDKN2B-AS1/miR-141/Cyclin D axis in RCC and highlights this network as a promising therapeutic target for the regulation of EMT driven metastasis in RCC.

Activation of the Erk/MAPK signaling pathway is a driver for cadmium induced prostate cancer.

PURPOSE: Cadmium (Cd) is reported to be associated with carcinogenesis. The molecular mechanisms associated with Cd-induced prostate cancer (PCa) remain elusive. MATERIALS AND METHODS: RWPE1, PWR1E and DU 145 cells were used. RT2 Profiler Array, real-time-quantitative-PCR, immunofluorescence, cell cycle, apoptosis, proliferation and colony formation assays along with Gene Set Enrichment Analysis (GSEA) were performed. RESULT: Chronic Cd exposure of non-malignant RWPE1 and PWR1E cells promoted cell survival, proliferation and colony formation with inhibition of apoptosis. Even a two-week Cd exposure of PCa cell line (DU 145) significantly increased the proliferation and decreased apoptosis. RT2 profiler array of 84 genes involved in the Erk/MAPK pathway revealed induction of gene expression in Cd-RWPE1 cells compared to RWPE1. This was confirmed by individual TaqMan gene expression analysis in both Cd-RWPE1 and Cd-PWR1E cell lines. GSEA showed an enrichment of the Erk/MAPK pathway along with other pathways such as KEGG-ERBB, KEGG-Cell Cycle, KEGG-VEGF, KEGG-Pathways in cancer and KEGG-prostate cancer pathway. We randomly selected upregulated genes from Erk/MAPK pathway and performed profile analysis in a PCa data set from the TCGA/GDC data base. We observed upregulation of these genes in PCa compared to normal samples. An increase in phosphorylation of the Erk1/2 and Mek1/2 was observed in Cd-RWPE1 and Cd-PWR1E cells compared to parental cells, confirming that Cd-exposure induces activation of the Erk/MAPK pathway. CONCLUSION: This study demonstrates that Erk/MAPK signaling is a major pathway involved in Cd-induced malignant transformation of normal prostate cells. Understanding these dominant oncogenic pathways may help develop optimal therapeutic strategies for PCa.

Genistein Represses HOTAIR/Chromatin Remodeling Pathways to Suppress Kidney Cancer.

BACKGROUND/AIMS: Genistein, a soy isoflavone, has been shown to have anti-cancer effects in various cancers including renal cancer. Long non-coding RNA, HOX transcript antisense RNA (HOTAIR), is involved in cancer progression and metastasis, such as renal cancer. Our aim was to investigate the effects of genistein on HOTAIR chromatin remodeling functions. METHODS: We used MTS assays and Transwell migration assays to study the effects of genistein on cell proliferation and migration respectively in human renal cell carcinoma (RCC) cell lines. We used Western blots to analyze SNAIL and ZO-1 expression. We performed chromatin immunoprecipitation (ChIP) assays to study recruitment of the polycomb repressive complex 2 (PRC2) to the ZO-1 promoter. We performed RNA immunoprecipitation (RIP) assays to study interaction between HOTAIR and PRC2, SMARCB1 or ARID1A. We also performed transfection experiments to overexpress EED, HOTAIR and knockdown SMARCB1. RESULTS: Genistein reduced cell proliferation and migration of human renal cell carcinoma cell lines. ChIP assays indicated that genistein reduces recruitment of the PRC2 to the ZO-1 promoter and increased its expression. RIP assays showed that genistein inhibits HOTAIR interaction with PRC2, leading to tumor suppression. Immunoprecipitation also revealed that genistein reduced EED levels in PRC2, suggesting that decreased EED levels suppress HOTAIR interaction with PRC2. EED overexpression in the presence of genistein restored PRC2 interaction with HOTAIR and reduced ZO-1 transcription, suggesting genistein activates ZO-1 by inhibiting HOTAIR/PRC2 functions. RIP assays also showed that HOTAIR interacts with SMARCB1 and ARID1A, subunits of the human SWI/SNF chromatin remodeling complex and genistein reduces this interaction. Combination of HOTAIR overexpression and SMARCB1 knockdown in the presence of genistein revealed that genistein inhibits SNAIL transcription via the HOTAIR/SMARCB1 pathway. CONCLUSION: Genistein suppresses EED levels in PRC2 and inhibits HOTAIR/PRC2 interaction. Genistein suppresses HOTAIR/PRC2 recruitment to the ZO-1 promoter and enhances ZO-1 transcription. Genistein also inhibits SNAIL transcription via reducing HOTAIR/SMARCB1 interaction. We demonstrate that the reduction of HOTAIR interaction with chromatin remodeling factors by genistein represses HOTAIR/chromatin remodeling pathways to suppress RCC malignancy.

Upregulation of miR-130b Contributes to Risk of Poor Prognosis and Racial Disparity in African-American Prostate Cancer.

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.

Hyaluronan-CD44 interaction promotes HPV 16 E6 oncogene-mediated oropharyngeal cell carcinoma survival and chemoresistance.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy that often involves the oral cavity, pharynx, larynx, or paranasal sinuses. There is a compelling evidence of the human papilloma virus including HPV16 E6 oncogene drives cell transformation and oncogenic processes of HPV positive (HVP+) HNSCC [in particular, Oropharyngeal Squamous Cell Carcinoma (OPSCC)]. In this study, we determined that human OPSCC-derived, HPV16 E6+ cells (UMSCC-104 and UMSCC-47 cell lines) express CD44 and a regulatory transcription factor, c-Jun. Importantly, interaction between matrix hyaluronan (HA) and CD44 (an HA receptor) promotes c-Jun phosphorylation followed by phospho-c-Jun nuclear translocation and co-localization with HPV16 E6 in the nucleus of both UMSCC-104 and UMSCC-47 cells. Further analyses revealed that HPV16 E6 expression is regulated by an upstream promoter containing AP1/c-Jun binding site(s), and chromatin immunoprecipitation (ChIP) assays demonstrated that stimulation of HPV16 E6 expression by HA-CD44 interaction is phospho-c-Jun dependent in these HPV16+ UMSCC-104 and UMSCC-47 cells. This process results in an upregulation of survival proteins, inhibitors of the apoptosis family of proteins (IAPs) and chemoresistance in these HPV16+ cells. Treatment of UMSCC-104 or UMSCC-47 cells with c-Jun-specific or HPV16 E6-specific small interfering RNAs effectively blocks HA/CD44-mediated c-Jun signaling and abrogates HPV16 E6 expression as well as causes downregulation of survival proteins (cIAP-1 and cIAP-2) expression and enhancement of chemosensitivity. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in HPV16 E6 upregulation leading to survival protein (cIAP-1/cIAP-2) production and chemoresistance in HPV16+ UMSCC-104 and UMSCC-47 cells. Most importantly, using a mouse xenograft model, we have observed that Cisplatin chemotherapy combined with the suppression of CD44, c-Jun and HPV16 E6 (by treating both UMSCC-104 cells and UMSCC-47 cells with CD44shRNA or c-Jun shRNA or HPV16 E6 shRNA) appears to be more effective in tumor size reduction than chemotherapy alone. Thus, these newly-discovered HA/CD44-c-Jun/HPV16E6 signaling pathways may provide new drug targets for overcoming cisplatin chemoresistance in HPV16E6-positive OPSCC cells.

Elevated miR-182-5p Associates with Renal Cancer Cell Mitotic Arrest through Diminished MALAT-1 Expression.

The molecular heterogeneity of clear cell renal carcinoma (ccRCC) makes prediction of disease progression and therapeutic response difficult. Thus, this report investigates the functional significance, mechanisms of action, and clinical utility of miR-182-5p and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1/NEAT2), a long noncoding RNA (lncRNA), in the regulation of kidney cancer using human kidney cancer tissues as well as in vitro and in vivo model systems. Profiling of miR-182-5p and MALAT-1 in human renal cancer cells and clinical specimens was done by quantitative real-time PCR (qPCR). The biological significance was determined by series of in vitro and in vivo experiments. The interaction between miR-182-5p and MALAT-1 was investigated using luciferase reporter assays. In addition, the effects of miR-182-5p overexpression and MALAT-1 downregulation on cell-cycle progression were assessed in ccRCC cells. The data indicate that miR-182-5p is downregulated in ccRCC; the mechanism being CpG hypermethylation as observed from 5-Aza CdR treatment that decreased promoter methylation and expression of key methylation regulatory genes like DNMT1, DNMT3a, and DNMT3b Overexpression of miR-182-5p-inhibited cell proliferation, colony formation, apoptosis, and led to G2-M-phase cell-cycle arrest by directly targeting MALAT-1 Downregulation of MALAT-1 led to upregulation of p53, downregulation of CDC20, AURKA, drivers of the cell-cycle mitotic phase. Transient knockdown of MALAT-1 mimicked the effects of miR-182-5p overexpression. Finally, overexpression of miR-182-5p decreased tumor growth in mice, compared with controls; thus, demonstrating its antitumor effect in vivo Implications: This is the first study that offers new insight into role of miR-182-5p/MALAT-1 interaction on inhibition of ccRCC progression. Mol Cancer Res; 16(11); 1750-60. ©2018 AACR.

MicroRNA-203 Inhibits Long Noncoding RNA HOTAIR and Regulates Tumorigenesis through Epithelial-to-mesenchymal Transition Pathway in Renal Cell Carcinoma.

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.

Activation of Matrix Hyaluronan-Mediated CD44 Signaling, Epigenetic Regulation and Chemoresistance in Head and Neck Cancer Stem Cells.

Head and neck squamous cell carcinoma (HNSCC) is a solid tumor composed by a genotypically and phenotypically heterogeneous population of neoplastic cells types. High recurrence rate and regional metastases lead to major morbidity and mortality. Recently, many studies have focused on cellular and molecular mechanisms of tumor progression that can help to predict prognosis and to choose the best therapeutic approach for HNSCC patients. Hyaluronan (HA), an important glycosaminoglycan component of the extracellular matrix (ECM), and its major cell surface receptor, CD44, have been suggested to be important cellular mediators influencing tumor progression and treatment resistance in head and neck cancer. HNSCC contains a small subpopulation of cells that exhibit a hallmark of CD44-expressing cancer stem cell (CSC) properties with self-renewal, multipotency, and a unique potential for tumor initiation. HA has been shown to stimulate a variety of CSC functions including self-renewal, clone formation and differentiation. This review article will present current evidence for the existence of a unique small population of CD44v3highALDHhigh-expressing CSCs in HNSCC. A special focus will be placed on the role of HA/CD44-induced oncogenic signaling and histone methyltransferase, DOT1L activities in regulating histone modifications (via epigenetic changes) and miRNA activation. Many of these events are essential for the CSC properties such as Nanog/Oct4/Sox2 expression, spheroid/clone formation, self-renewal, tumor cell migration/invasion, survival and chemotherapeutic drug resistance in HA-activated head and neck cancer. These newly-discovered HA/CD44-mediated oncogenic signaling pathways delineate unique tumor dynamics with implications for defining the drivers of HNSCC progression processes. Most importantly, the important knowledge obtained from HA/CD44-regulated CSC signaling and functional activation could provide new information regarding the design of novel drug targets to overcome current therapeutic drug resistance which will have significant treatment implications for head and neck cancer patients.

A novel microRNA regulator of prostate cancer epithelial-mesenchymal transition.

The most frequent alteration in the prostate oncogenome is loss of chromosome (chr) 8p21 that has been associated with loss of NKX3.1 homeobox gene. Chr8p21 deletions increase significantly with tumor grade and are associated with poor prognosis in prostate cancer (PCa), suggesting critical involvement of this region in tumor progression. Recent studies suggest that apart from NKX3.1, this region harbors alternative tumor suppressors that are yet undefined. We proposed a novel, paradigm shifting hypothesis that this locus is associated with a miRNA gene cluster-miR-3622a/b- that plays a crucial suppressive role in PCa. Here we demonstrate the crucial role of miR-3622a in prostate cancer epithelial-to-mesenchymal transition (EMT). MicroRNA expression profiling in microdissected human PCa clinical tissues showed that miR-3622a expression is widely downregulated and is significantly correlated with poor survival outcome and tumor progression. To understand the functional significance of miR-3622a, knockdown and overexpression was performed using non-transformed prostate epithelial and PCa cell lines, respectively, followed by functional assays. Our data demonstrate that endogenous miR-3622a expression is vital to maintain the epithelial state of normal and untransformed prostate cells. miR-3622a expression inhibits EMT, progression and metastasis of PCa in vitro and in vivo. Further, we found that miR-3622a directly targets EMT effectors ZEB1 and SNAI2. In view of these data, we propose that frequent loss of miR-3622a at chr8p21 region leads to induction of EMT states that in turn, promotes PCa progression and metastasis. This study has potentially significant implications in the field of prostate cancer as it identifies an important miRNA component of a frequently lost chromosomal region with critical roles in prostate carcinogenesis which is a highly significant step towards understanding the mechanistic involvement of this locus. Also, our study indicates that miR-3622a is a novel PCa biomarker and potential drug target for developing therapeutic regimens against advanced PCa.

Versican Promotes Tumor Progression, Metastasis and Predicts Poor Prognosis in Renal Carcinoma.

The proteoglycan versican (VCAN) promotes tumor progression and enhances metastasis in several cancers; however, its role in clear cell renal cell carcinoma (ccRCC) remains unknown. Recent evidence suggests that VCAN is an important target of chromosomal 5q gain, one of the most prevalent genetic abnormalities in ccRCC. Thus, we investigated whether VCAN expression is associated with the pathogenesis of ccRCC. VCAN expression was analyzed using three RCC and normal kidney cell lines as well as a clinical cohort of 84 matched ccRCC and normal renal tissues. Functional analyses on growth and progression properties were performed using VCAN-depleted ccRCC cells. Microarray expression profiling was employed to investigate the target genes and biologic pathways involved in VCAN-mediated ccRCC carcinogenesis. ccRCC had elevated VCAN expression in comparison with normal kidney in both cell lines and clinical specimens. The elevated expression of VCAN was significantly correlated with metastasis (P < 0.001) and worse 5-year overall survival after radical nephrectomy (P = 0.014). In vitro, VCAN knockdown significantly decreased cell proliferation and increased apoptosis in Caki-2 and 786-O cells, and this was associated with alteration of several TNF signaling-related genes such as TNFα, BID, and BAK Furthermore, VCAN depletion markedly decreased cell migration and invasion which correlated with reduction of MMP7 and CXCR4. These results demonstrate that VCAN promotes ccRCC tumorigenesis and metastasis and thus is an attractive target for novel diagnostic, prognostic, and therapeutic strategies.Implications: This study highlights the oncogenic role of VCAN in renal cell carcinogenesis and suggests that this gene has therapeutic and/or biomarker potential for renal cell cancer. Mol Cancer Res; 15(7); 884-95. ©2017 AACR.

The role of miR-24 as a race related genetic factor in prostate cancer.

The incidence of prostate cancer (PCa) among African-Americans (AfA) is significantly higher than Caucasian-Americans (CaA) but the genetic basis for this disparity is not known. To address this problem, we analyzed miRNA expression in AfA (n = 81) and CaA (n = 51) PCa patients. Here, we found that miR-24 is differentially expressed in AfA and CaA PCa patients and attempt to clarify its role in AfA patients. Also, the public sequencing data of the miR-24 promoter confirmed that it was highly methylated and down-regulated in PCa patients. Utilizing a VAMCSF and NDRI patient cohorts, we discovered that miR-24 expression was linked to a racial difference between AfA/CaA PCa patients. Interestingly, miR-24 was restored after treatment of PCa cells with 5Aza-CdR in an AfA cell line (MDA-PCa-2b), while restoration of miR-24 was not observed in CaA cells, DU-145. Ectopic expression of miR-24 showed decreased growth and induced apoptosis, though the effect was less in the CaA cell line compared to the AfA cell line. Finally, we found unique changes in biological pathways and processes associated with miR-24 transfected AfA cells by quantitative PCR-based gene expression array. Evaluation of the altered pathways showed that AR, IGF1, IGFBP5 and ETV1 were markedly decreased in the AfA derived cell line compared with CaA cells, and there was a reciprocal regulatory relationship of miR-24/target expression in prostate cancer patients. These results demonstrate that miR-24 may be a central regulator of key events that contribute to race-related tumorigenesis and has potential to be a therapeutic agent for PCa treatment.

Differential expression of miR-34b and androgen receptor pathway regulate prostate cancer aggressiveness between African-Americans and Caucasians.

African-Americans are diagnosed with more aggressive prostate cancers and have worse survival than Caucasians, however a comprehensive understanding of this health disparity remains unclear. To clarify the mechanisms leading to this disparity, we analyzed the potential involvement of miR-34b expression in African-Americans and Caucasians. miR-34b functions as a tumor suppressor and has a multi-functional role, through regulation of cell proliferation, cell cycle and apoptosis. We found that miR-34b expression is lower in human prostate cancer tissues from African-Americans compared to Caucasians. DNA hypermethylation of the miR-34b-3p promoter region showed significantly higher methylation in prostate cancer compared to normal samples. We then sequenced the promoter region of miR-34b-3p and found a chromosomal deletion in miR-34b in African-American prostate cancer cell line (MDA-PCA-2b) and not in Caucasian cell line (DU-145). We found that AR and ETV1 genes are differentially expressed in MDA-PCa-2b and DU-145 cells after overexpression of miR-34b. Direct interaction of miR-34b with the 3' untranslated region of AR and ETV1 was validated by luciferase reporter assay. We found that miR-34b downregulation in African-Americans is inversely correlated with high AR levels that lead to increased cell proliferation. Overexpression of miR-34b in cell lines showed higher inhibition of cell proliferation, apoptosis and G1 arrest in the African-American cells (MDA-PCa-2b) compared to Caucasian cell line (DU-145). Taken together, our results show that differential expression of miR-34b and AR are associated with prostate cancer aggressiveness in African-Americans.

Oncogenic microRNA-4534 regulates PTEN pathway in prostate cancer.

Prostate carcinogenesis involves alterations in several signaling pathways, the most prominent being the PI3K/AKT pathway. This pathway is constitutively active and drives prostate cancer (PCa) progression to advanced metastatic disease. PTEN, a critical tumor and metastasis suppressor gene negatively regulates cell survival, proliferation, migration and angiogenesis via the PI3K/Akt pathway. PTEN is mutated, downregulated/dysfunctional in many cancers and its dysregulation correlates with poor prognosis in PCa. Here, we demonstrate that microRNA-4534 (miR-4534) is overexpressed in PCa and show that miR-4534 is hypermethylated in normal tissues and cell lines compared to PCa tissues/cells. miR-4534 exerts its oncogenic effects partly by downregulating the tumor suppressor PTEN gene. Knockdown of miR-4534 impaired cell proliferation, migration/invasion and induced G0/G1 cell cycle arrest and apoptosis in PCa. Suppression of miR-4534 and its effects on tumor growth was confirmed in a xenograft mouse model. We performed parallel experiments in non-cancer RWPE1 cells by overexpessing miR-4534 followed by functional assays. Overexpression of miR-4534 induced pro-cancerous characteristics in this non-cancer cell line. Statistical analyses revealed that miR-4534 has potential to independently distinguish malignant from normal tissues and positively correlated with poor overall and PSA recurrence free survival. Taken together, our results show that depletion of miR-4534 in PCa induces a tumor suppressor phenotype partly through induction of PTEN. These results have important implications for identifying and defining the role of new PTEN regulators such as microRNAs in prostate tumorigenesis. Understanding aberrantly overexpressed miR-4534 and its downregulation of PTEN will provide mechanistic insight and therapeutic targets for PCa therapy.

Functional role and tobacco smoking effects on methylation of CYP1A1 gene in prostate cancer.

Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2'-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment.

Up-regulation of Histone Methyltransferase, DOT1L, by Matrix Hyaluronan Promotes MicroRNA-10 Expression Leading to Tumor Cell Invasion and Chemoresistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma.

Human head and neck squamous cell carcinoma is a solid tumor malignancy associated with major morbidity and mortality. In this study, we determined that human head and neck squamous cell carcinoma-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by a high level of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. Importantly, matrix hyaluronan (HA) induces the up-regulation of stem cell markers that display the hallmark CSC properties. Histone methyltransferase, DOT1L, is also up-regulated by HA in CSCs (isolated from HSC-3 cells). Further analyses indicate that the stimulation of microRNA-10b (miR-10b) expression is DOT1L-specific and HA/CD44-dependent in CSCs. This process subsequently results in the overexpression of RhoGTPases and survival proteins leading to tumor cell invasion and cisplatin resistance. Treatment of CSCs with DOT1L-specific small interfering RNAs (siRNAs) effectively blocks HA/CD44-mediated expression of DOT1L, miR-10b production, and RhoGTPase/survival protein up-regulation as well as reduces tumor cell invasion and enhances chemosensitivity. CSCs were also transfected with a specific anti-miR-10b inhibitor to silence miR-10b expression and block its target functions. Our results demonstrate that the anti-miR-10 inhibitor not only decreases RhoGTPase/survival protein expression and tumor cell invasion, but also increases chemosensitivity in HA-treated CSCs. Taken together, these findings strongly support the contention that histone methyltransferase, DOT1L-associated epigenetic changes induced by HA play pivotal roles in miR-10 production leading to up-regulation of RhoGTPase and survival proteins. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, tumor cell invasion, and chemotherapy resistance in HA/CD44-activated head and neck cancer.

Selective Activation of Cancer Stem Cells by Size-Specific Hyaluronan in Head and Neck Cancer.

We determined that human head and neck cancer cells (HSC-3 cell line) contain a subpopulation displaying cancer stem cell (CSC) properties and are very tumorigenic. Specifically, we investigated whether different sizes of hyaluronan (HA) (e.g., 5 kDa, 20 kDa, 200 kDa, or 700 kDa-HA-sizes) play a role in regulating these CSCs. First, we observed that 200 kDa-HA (but not other sizes of HA) preferentially induces certain stem cell marker expression resulting in self-renewal and clonal formation of these cells. Further analyses indicate that 200 kDa-HA selectively stimulates the expression of a panel of microRNAs (most noticeably miR-10b) in these CSCs. Survival protein (cIAP-1) expression was also stimulated by 200 kDa-HA in these CSCs leading to cisplatin resistance. Furthermore, our results indicate that the anti-miR-10 inhibitor not only decreases survival protein expression, but also increases chemosensitivity of the 200 kDa-HA-treated CSCs. These findings strongly support the contention that 200 kDa-HA plays a pivotal role in miR-10 production leading to survival protein upregulation and chemoresistance in CSCs. Together, our findings suggest that selective activation of oncogenic signaling by certain sizes of HA (e.g., 200 kDa-HA) may be instrumental in the formation of CSC functions leading to tumor cell survival and chemoresistance in head and neck cancer progression.