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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.

Table S1 is for primer sequences

Supplemental Figure Legends

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.

Antibody used with their catalog numbers

Primer Sequences

Supplementary figures S1-S6

Clinicopathological features

The proteasome machinery regulates the levels of most cellular proteins. However, the factors determining rate of proteasomal degradation remain poorly understood. A rate-limiting step of degradation is the mechanical unfolding of substrates by proteasome ATPases before they enter the core peptidase chamber. Posttranslational modification with the protein FAT10 serves as a proteasomal degradation signal. FAT10 degradation is distinct from the Ubiquitin-proteasome degradation pathway since FAT10 is degraded with the substrate, whereas Ubiquitin is recycled. Hence, both FAT10 and FAT10lyated substrates unfold at the proteasome, and their unfolding kinetics are critical to the degradation rate. Here, we have characterized the thermodynamics and kinetics of unfolding FAT10 and FAT10 conjugated substrate proteins. Our results highlight that fundamental electrostatic interactions of the Ubiquitin fold are absent in FAT10, resulting in a conformationally flexible and malleable native state with low free energy of unfolding. FAT10 substantially modulates substrate stability and induces partially unfolded forms in the substrate to accelerate its unfolding and degradation. The substrate’s energy landscape and the FAT10 binding site determine the change in free energy of the substrate. These results highlight a strong dependence of proteasomal degradation rate on the plasticity of proteasomal signal FAT10.

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.

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.

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.

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.

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.

AimsTo survey the effect of COVID-19 on mental health of both medical professionals and the general population, as well as attitudes surrounding the disclosure of mental illness.MethodAn online survey comprised of two questionnaires, one for medical professionals and one for the general population, were conducted via social media. Both questionnaires asked respondents of the effect of COVID-19 on their mental health, and the former asked respondents about the effect of COVID-19 on their patient group's mental health. The questionnaires went on to ask respondents about their attitudes to mental health disclosure in various scenarios, to varying groups of people. The general population group was also asked how they would react if someone else disclosed their mental illness to them.ResultThe questionnaire for the medical professionals gained 62 respondents and the one for the general population had 122 respondents, with responses from multiple nations. Overall, COVID-19 has affected everyone's mental health to a degree, and all groups had reservations about disclosing their mental health issues to others. The medical professionals were especially reluctant to disclose mental illness to their patients, but were more comfortable when it came to disclosing mental illness to colleagues. The general population, however, was much more reluctant to disclose mental health issues to their colleagues. The general population were, on the whole, willing to listen to and help anyone who came to them with mental health concerns. Both groups surveyed showed reluctance toward disclosure to the wider community.ConclusionCOVID-19 appears to significantly affect not only physical health, but mental health as well. There is at least some degree of stigma surrounding the disclosure of mental health issues. While most would be happy to help anyone who came to them with their mental health problems, there seems to be an attitude shift when people must contend with mental health issues of their own.

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.

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.

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.

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.

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.

Novel approaches to nanoparticle synthesis using herbal products and their potential application in treatments are now in the limelight of recent cancer research. Diallyl disulfide (DADS), a bioactive component of garlic has been used for centuries as an effective remedy for different ailments including cancer. In the present study, DADS is used to synthesize less toxic, eco-friendly gold nanoparticles having anti-proliferative effects against cancer cells. DADS induced gold nanoparticles (D-GNPs) display a characteristic surface plasmon band near 551 nm which is 22 nm red shifted compared to conventionally prepared gold nanoparticles (GNPs) using tri-sodium citrate. Moreover, the hydrodynamic diameter of D-GNP ranges from 70 to 77 nm and its zeta potential is −24.6 mV. A nearly spherical ultra-structure of D-GNPs was visualized under atomic force microscopy and transmission electron microscopy. FT-IR analysis confirms the association of the sulfur group of DADS with these nanoparticles. D-GNPs show dose dependent cytotoxicity in human leukemic cell-lines U937 and K562. Cellular uptake of D-GNPs in U937 leading to nuclear fragmentation and DNA ladder formation are other insightful findings. This report therefore details the synthesis of stable gold nanoparticles using DADS and reveals D-GNPs to be a highly effective anti-proliferative agent showing apoptosis in human leukemia cell-lines.

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.

// Marisa Shiina 1, * , Yutaka Hashimoto 1, * , Taku Kato 1 , Soichiro Yamamura 1 , Yuichiro Tanaka 1 , Shahana Majid 1 , Sharanjot Saini 1 , Shahryari Varahram 1 , Priyanka Kulkarni 1 , Pritha Dasgupta 1 , Yozo Mitsui 1 , Mitsuho Sumida 1 , Laura Tabatabai 1 , Guoren Deng 1 , Deepak Kumar 2 , Rajvir Dahiya 1 1 Department of Urology, Veterans Affairs Medical Center, San Francisco and University of California San Francisco, San Francisco, California, USA 2 Division of Science and Mathematic, Cancer Research Laboratory, University of the District of Columbia, Washington, DC, USA * These authors have contributed equally to this work Correspondence to: Rajvir Dahiya, email: rdahiya@ucsf.edu Keywords: miR-34b, prostate cancer, african-americans, caucasians, androgen receptor Received: October 04, 2016 Accepted: November 23, 2016 Published: December 25, 2016 ABSTRACT 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.

Cadmium (Cd) has been implicated in cancer development and classified as a type I carcinogen by the International Agency for Cancer Research. The etiology of prostate cancer development is associated with multitude of causative risk factors including exposure to cadmium. However, the molecular mechanisms associated with Cd-induced prostate cancer remain elusive. This study provides evidence that PI3K/Akt signaling is a major molecular pathway involved in Cd induced malignant transformation of normal prostate epithelial cells. Functionally, Cd exposure induced aggressive behavior as indicated by increased proliferation, migration and invasion in Cd-RWPE1 cells compared to parental RWPE1. PI3K/Akt pathway is constitutively activated in prostate cancer driving the most aggressive forms of cancer and metastasis. Consistent with these findings, the RT2 PCR array analysis of 84 genes involved in the PI3K/Akt pathway revealed induction of gene expression in catalytic units (P110α, Akt, mTOR, NFKB1, RAF etc.) with a concomitant reduction in expression of regulatory units (PIK3R1, PIK3R2, PTEN etc.) of the PI3K/Akt pathway in Cd-RWPE1 cells compared to parental RWPE1. This was confirmed by individual quantitative real-time PCR analysis using TaqMan gene expression assay probes. Effect of Cd on the translation of the PI3K/Akt pathway genes was examined by immunoblot assays. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes in Cd-RWPE1 showed 5 overlapping pathways that were enriched in Cd-treated cells (Cd-RWPE1) and negatively correlated with parental RWPE1. The overlapping pathways include KEGG Apoptosis pathway (ES=0.56, NES=1), KEGG ERBB pathway (ES=0.25, NES=1), KEGG MAPK pathway (ES=0.48, NES=1), KEGG Pathways in cancer (ES=0.33, NES=1) and KEGG Prostate Cancer pathway (ES=0.35, NES=1). Interestingly, all these pathways are implicated in prostate cancer progression and metastasis. We randomly selected up- and down-regulated genes from the PI3K/Akt pathway in PCR array and performed profile analysis in a prostate adenocarcinoma data set (n=534) from the TCGA/GDC data base. We observed upregulation of the oncogenes along with downregulation of tumor suppressors in prostate cancer compared to normal prostate samples. Taken together, these data reveal that Cd exposure induced aggressive malignant characteristics in normal prostate epithelial cells via modulation of the PI3K/Akt signaling pathway. Understanding the molecular mechanisms involved in the malignant transformation of normal prostate epithelial cells may help develop optimal therapeutic strategies for advanced prostate cancer. Citation Format: Priyanka Kulkarni, Pritha Dasgupta, Nadeem S. Bhat, Yutaka Hashimoto, Sharanjot Saini, Altaf A. Dar, Varahram Shahryari, Marisa Shiina, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya, Shahana Majid. Involvement of PI3K/Akt pathway in cadmium triggered aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5040.

Objective: Clear cell renal cell carcinoma (ccRCC), a common histological subtype of renal cell carcinoma (RCC) are categorized by their aggressive nature and comprise 90% of metastatic RCCs. Despite recent advances, management of the disease in the advanced metastatic phase is a significant challenge. Expression of miR-141-3p (miR-141) is low and function as tumor suppressor in various cancers. However, its association with long non-coding RNAs (lncRNAs) in RCC is not well understood. This study shows that miR-141 interacts with lncRNAs, and plays vital role in the regulation of stemness and epithelial-to-mesenchymal transition (EMT) in RCC. Experimental Design: Human renal cancer cell lines (ACHN and Caki-1), normal renal epithelial cells, RPTEC and tumor tissues were used for this study. We analyzed the expression of miR-141 in tissue samples and cell lines, and studied the function of miR-141 in kidney cancer progression using in vitro and in vivo models. Statistical analysis was performed to determine the clinical significance of miR-141 in kidney cancer patients. Results: Reduced expression of miR-141 was observed in ccRCC clinical specimens and cell lines. To elucidate the epigenetic role of miR-141 silencing in RCC, methylation status of CpG islands in its putative promoter region was analyzed. Result showed significant increase in miR-141 expression after 5-Aza-CdR treatment indicating that promoter hypermethylation is responsible for its inactivation. Ectopic expression of miR-141 reduced cell proliferation, clonogenicity, migration, invasion and induced apoptosis and cell cycle arrest compared to controls. An increase in cleaved PARP, cleaved caspase-3 and epithelial marker (CLDN1) was observed with a concomitant decrease in stemness (KLF4, Nanog) and EMT markers (FN1, VIM). We further investigated the biological significance of miR-141 in RCC. Loss of miR-141 function in RPTEC cells induced pro-cancerous characteristics. In addition, we examined lncRNAs (CDKN2B-As1, PCAT1 and PVT1) that bind to miR-141 and are overexpressed in RCC clinical samples compared to controls. Reduced expression of these lncRNAs was observed in RCC cells with overexpression of miR-141, supporting the notion that miR-141 interacts with these lncRNAs in RCC. Finally, in vivo experiment in nude mice revealed that intra-tumoral administration of miR-141 in the established tumors significantly suppressed tumor growth compared to controls. Furthermore, statistical analysis of patient samples showed that miR-141 may serve as a RCC diagnostic biomarker. Conclusion: Our results demonstrate that miR-141 overexpression inhibits RCC progression and inhibits epithelial-to-mesenchymal transition. These studies also show that miR-141 may be a useful RCC biomarker for early detection and monitoring RCC progression. Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Shahana Majid, Varahram Shahryari, Nadeem S. Bhat, Yutaka Hashimoto, Marisa Shiina, Guoren Deng, Sharanjot Saini, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. Elevated miR-141-3p inhibits renal cell carcinoma aggressiveness by targeting epithelial-to-mesenchymal transition pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3549.

Purpose: Prostate cancer (PC) is the second most diagnosed cancer in men. African-American (AfA) men have higher incidence and twice the PC mortality rates compared to Caucasian-American (CaA) men. In this study, we investigated the biochemical role of miR-10a in prostate cancer racial disparity. Methods: We used PCa cell lines from CaA (DU145, LNCaP, PC3), AfA (MDA-PCa-2b, E006AA-hT) and a normal epithelial cell line (PWR1E) to examine miR-10a expression levels. Total RNA was extracted from total 138 human prostate cancer clinical samples (AfA: n=75, CaA: n=63) and cell lines. Gene expression levels were determined by quantitative real-time PCR (qPCR). A miRNA target database (miRWalk that integrates mainly three databases, miRDB, TargetScan and miRTarBase) was used for miR-10a target prediction. Cell proliferation and apoptosis were monitored after miR-10a knockdown by using anti-miR-10a. Results: miR-10a was significantly up-regulated in AfA prostate cancer cell line, E006AA-hT compared to CaA cells. Utilizing SFVAMC and NDRI patient cohorts, we confirmed that miR-10a expression was linked to a racial difference between AfA/CaA PCa patients. Knockdown of miR-10a showed decreased growth and apoptosis, though the effect was less in CaA compared to AfA cells. We found NCOR2 which is frequently deleted in castration- resistant prostate cancer patient specimens and tumor-suppressor PTEN as miR-10a targets that overlapped in multiple miRNA target prediction databases. Conclusion: These results demonstrate that miR-10a may be a central regulator of crucial events that contribute to racial differences in prostate cancer. Regulation of miR-10a expression may be a new therapeutic strategy for AfA prostate cancer patients. Citation Format: Yutaka Hashimoto, Marisa Shiina, Yuichiro Tanaka, Pritha Dasgupta, Priyanka Kulkarni, Taku Kato, Ryan K. Wong, Varahram Shahryari, Shigekatsu Maekawa, Soichiro Yamamura, Divya Bhagiratha, Sharanjot Saini, Guoren Deng, Laura Tabatabai, Shahana Majid, Rajvir Dahiya, Rajvir Dahiya. Up-regulation of miR-10a affect on prostate cancer racial disparity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 761.

Background: African-Americans have higher risk for developing prostate cancer and when diagnosed, the cancer is more aggressive and African-Americans are more than twice as likely to die of the disease compared to Caucasians. To clarify the mechanisms involved in this disparity, we analyzed the role of miR-155 in African-American and Caucasian prostate cancer tissues and cell lines. Materials and Methods: Human tissue samples from African-Americans and Caucasians clinical FFPE (Formaldehyde Fixed Paraffin Embedded) were used to analyzed miR-155 expression by Real Time PCR. We selected prostate cancer cell lines from Caucasians (DU-145, LNCaP, PC3), African-American (E006AA-hT) and normal epithelial cell line (PWR-1E) which express different levels of miR-155, to mimic the tissue samples and help identify the mechanisms related to racial disparity. We knocked down miR-155 in cells lines and performed cell viability assays using a CellTiter-Glo luminescent assay. For migration analysis, miR-155 knockdown cells were seeded in chambers and we measured the capacity of cell motility towards a chemo-attractant. To identify miR-155 mediated downstream regulators, three target prediction algorithms (miRDB, Targetscan and miRecords) were used. Results: We found miR-155 expression to be higher in African-American prostate cancer tissues compared to Caucasian patients with localized disease. Also, expression of miR-155 in African-American cell line, E006AA-hT, was significantly higher compared to Caucasian cell lines, DU-145, LNCaP and PC3. To determine the role of miR-155 in the differences between African-Americans and Caucasians, miR-155 was knocked down in E006AA-hT and DU-145 cells. miR-155 knockdown in E006AA-hT cells caused a decrease in cell viability compared with negative control, while there was no difference in DU-145 cells. We observed inhibition of migration E006AA-hT miR-155 knockdown cells but not on DU-145 miR-155 knockdown cells, suggesting that miR-155 knockdown have the more potent effect on African-American cell line. We analyzed three target prediction algorithms and found that four genes overlapped, SPI1, JARID2, ETS1 and CEBPB that may play a pivotal role in the treatment of aggressive African-American prostate cancers. Conclusion: We conclude that high levels of miR-155 is associated with prostate cancer aggressiveness in African-Americans. Citation Format: Marisa Shiina, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Varahram Shahryari, Guoren Deng, Divya Bhagirath, Laura Tabatabai, Sharanjot Saini, Shahana Majid, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 780.

Background: Kidney cancer is among the 10 most common cancers in both men and women, with clear cell carcinoma (ccRCC) being the most common histologic subtype. It has become increasingly apparent that non-coding RNAs are involved in the regulation of a wide variety of diseases. However, the functional role of non-coding RNAs, such as long noncoding RNA (lncRNAs) and microRNAs (miRNA) in the initiation and progression of kidney tumorigenesis is unclear. Here we show an oncogenic role for miR-155-5p in ccRCC and its novel regulation of RCC through LncRNA TCL6. Methods: Profiling of TCL6 and miR-155-5p was performed in microdissected renal cancer tissues, matched adjacent normal regions and in human renal cancer cell lines by quantitative real-time PCR. To assess the functional significance of miR-155-5p in RCC, we generated stable miR-155-5p knockdown RCC cells (786-O, Caki-1) and performed functional assays. We also examined the therapeutic potential of miR-155-5p in vivo using a renal cancer xenograft mouse model injected with these stable cells. We performed qPCR and Western analysis to confirm the interaction between miR-155-5p and its targets (TCL6, PIK3R1, FOXO3a, RhoA). Results: Expression analyses in a cohort of renal cancer clinical specimens showed that LncRNA TCL6 is frequently downregulated and miR-155-5p expression is upregulated in ccRCC. We observed that knockdown of miR-155-5p inhibited cell proliferation, colony formation, migration, invasion and led to G2/M arrest, supporting its oncogenic role in vitro. In ccRCC cells (786-O and Caki-1), inhibition of miR-155-5p significantly upregulated expression, whereas in normal kidney epithelial cell line HK2 over-expression of miR-155-5p decreased levels of its target genes. Over-expression of miR-155-5p in HK2 cells induced cell proliferation, colony formation and enhanced its tumorogenic properties. Furthermore, we found that miR-155-5p may play an important role in the genesis of ccRCC through the PI3K/AKT pathway. In vivo studies demonstrated that the tumor volumes for the mice injected with stable miR-155-5p knockdown cells were significantly less as compared to the mice injected with control parental cells. Conclusions: Collectively, these data suggest that miR-155-5p plays an oncogenic role in ccRCC. These findings offer new insight into role of lncRNA TCL6/miR-155 axis as a key regulator of the PI3K/AKT pathway in the progression of ccRCC. Thus, inhibition of miR-155 may be an effective therapeutic strategy for treating RCC. Citation Format: Priyanka Kulkarni, Pritha Dasgupta, Shahana Majid, Marisa Shiina, Yutaka Hashimoto, Varahram Shahryari, Sharanjot Saini, Soichiro Yamamura, Laura Tabatabai, Guoren Deng, Yuichiro Tanaka, Rajvir Dahiya. LncRNA TCL6 / microRNA155 axis regulates the PI3K/ AKT pathway in clear cell renal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1827.

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.

Background: Renal cell carcinoma (RCC) is among the ten leading cancer types in United States. The 5-year relative survival rate of regional or localized RCC is 65-92%, while that of metastatic RCC is only 12%. Tumor recurrence and metastasis represent two major obstacles in the successful treatment of cancer. Emerging lines of evidence suggest that cancer aggressiveness is associated with epithelial to mesenchymal transition (EMT). Therefore, it is of critical importance to regulate EMT and to develop effective therapeutic strategies for the treatment of recurrent and metastatic cancer. Critical regulators of EMT include transcription repressors and microRNAs that target key proteins involved in EMT. MicroRNAs are implicated in regulating cancer progression and metastasis. Here we show that miR-720 is positively associated with RCC by negatively regulating key metastasis suppressing genes. Methods: We performed a series of in vitro and in vivo experiments including qRT-PCR, FACS cell cycle and apoptosis, chemotactic transwell migration and invasion, immunoblotting and luciferase reporter assays along with intratumoral xenograft mouse model. Results: Elevated levels of miR-720 were observed in a panel of RCC cell lines and clinical tissues compared to non-malignant cell line and normal samples. Loss of miR-720 function inhibited proliferation, migration and invasion and induced apoptosis in RCC cell lines in vitro and repressed tumor growth in xenograft mouse model. Conversely, gain of miR-720 function in non-malignant HK-2 cells induced pro-cancerous characteristics. Silencing of miR-720 caused a marked induction in the levels of endogenous αE-catenin and E-cadherin protein levels in anti720 transfected cells compared to control. Whereas, miR-720 overexpression in RCC cell lines reduced activity of a luciferase reporter gene fused to the wild-type αE-catenin or E-cadherin 3' UTR compared to non-specific 3' UTR control indicating that αE-catenin-E-cadherin complex is a direct and functional target of miR-720 in RCC. We also observed attenuation of β-Catenin, CD44 and Akt expression in RCC cells transfected with miR-720 inhibitor compared to control. Further, miR-720 exhibited clinical significance in RCC. Expression of miR-720 significantly distinguished malignant from normal samples. Elevated miR-720 levels positively correlated with higher Fuhrman grade, pathological stage and poor overall survival of RCC patients. Conclusion: These findings uncover a new regulatory network in RCC involving metastasis-promoting miR-720 that directly targets expression of key metastasis-suppressing proteins E-cadherin and αE-catenin complex. These results suggest that therapeutic regulation of miR-720 may provide an opportunity to regulate EMT and metastasis in RCC. Citation Format: Nadeem S. Bhat, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Soichiro Yamamura, Yuichiro Tanaka, Taku Kato, Yutaka Hashimoto, Marisa Shiina, Priyanka Kulkarni, Pritha Dasgupta, Z Laura Tabatabai, Guoren Deng, Rajvir Dahiya, Shahana Majid. Role of a prometastatic miRNA as a negative regulator of the key metastasis suppressor genes in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1111.

Purpose: Prostate cancer (PCa) is a common cancer in men. African-American (AfA) men have higher incidence and significantly higher prostate cancer mortality rates than Caucasian-American (CaA) men. In this study, we investigated the biochemical role of miR-130b in this disparity. Methods: We used meta-analyses and data from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). PCa cell lines from CaA (DU145, LNCaP, PC3), AfA (MDA-PCa-2b, E006AA-hT) and a normal epithelial cell line (PWR1E) were used for examining miR-130b expression levels and/or establishing stable miR-130b knock-down clones. Total RNA was extracted from clinical samples and cell lines. miR-130b and mRNA levels of its target genes were determined by quantitative real-time PCR (qPCR). qPCR based gene expression arrays were used to identify miR-130b target genes. Cell proliferation, apoptosis and cell cycle were monitored after stable miR-130b knock down. Western blotting was performed to detect miR-130b target protein expression levels. Results: TCGA data showed up-regulation of miR-130b in AfA PCa tissue samples compared with CaA samples. miR-130b was significantly up-regulated in AfA prostate cancer tissues and cell lines compared to CaA cells and tissues. Utilizing SFVAMC and NDRI patient cohorts, we confirmed that miR-130b expression was linked to a racial difference between AfA/CaA PCa patients. Knock down of miR-130b showed decreased growth and cell cycle arrest, though the effect was less in CaA compared to AfA cells. We found unique changes in biological pathways associated with miR-130b knock down in AfA cells by qPCR array. Evaluation of the altered pathways showed that Fragile Histidine Triad (FHIT) was markedly changed in the AfA compared with CaA cells. Conclusion: These results demonstrate that miR-130b may be a central regulator of key events that contribute racial differences in prostate cancer. FHIT may also be a new AfA specific tumor-suppressive pathway that may be of therapeutic importance in AfA prostate cancer patients. Citation Format: Yutaka Hashimoto, Masrisa Shiina, Taku Kato, Soichiro Yamamura, Yuichiro Tanaka, Shahana Majid, Sharanjot Saini, Varahram Shahryari, Priyanka Kulkarni, Pritha Dasgupta, Divya Bhagiratha, Nadeem Bhat, Guoren Deng, Laura Tabatabai, Deepak Kumar, Rajvir Dahiya. Up-regulation of miR-130b is involved in prostate cancer racial disparity through FHIT pathway inactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 489.

Background: Renal cell carcinoma (RCC) is a leading cause of death, accounting for nearly 14,000 (~2.4%) deaths in the United States in 2017, with clear cell carcinoma (ccRCC) being the most common histologic subtype. New and precise disease progression biomarkers are needed for early detection and follow-up. Characterization of these new biomarkers offers a new approach for the identification of novel therapeutic targets and drugs for RCC treatment. MicroRNAs (miRNAs) act as either onco-miRs or tumor suppressors in cancer. Here we show a tumor-suppressor role for miR-182-5p in ccRCC and novel regulation of cell proliferation through MALAT-1. Methods: Profiling of miR-182-5p and MALAT-1 was performed in microdissected renal cancer tissues, matched adjacent normal regions and in human renal cancer cell lines by quantitative real-time PCR. To assess the functional significance of miR-182-5p in RCC, we overexpressed miR-182-5p/control miRNA (miR-CON) in RCC cell lines (ACHN, Caki-1) followed by functional assays. We also examined the therapeutic potential of synthetic miR-182-5p mimics in vivo using a renal cancer xenograft mouse model. We performed luciferase reporter assay and Ago2-RIP assay to investigate the interaction between miR-182-5p and MALAT-1. In addition, we assessed the effects of miR-182-5p overexpression and MALAT-1 downregulation on cell cycle progression in ccRCC cell lines. Results: Expression analyses in a cohort of renal cancer clinical specimens showed that miR-182-5p expression is frequently downregulated in ccRCC. We observed that overexpression of miR-182-5p inhibited cell proliferation, colony formation, apoptosis and led to G2/M arrest, supporting an antiproliferative role for this microRNA. Overexpression of miR-182-5p led to decreased expression of CDC20 and AURKA, drivers of the cell cycle mitotic phase. Also, overexpression of miR-182-5p directly lowered the expression of MALAT-1 and knockdown of MALAT-1 mimicked the effects of miR-182-5p overexpression. Downregulation of MALAT-1 led to upregulation of p53 that ultimately downregulated CDC20, AURKA. In vivo studies demonstrated that administration of miR-182-5p caused regression of established renal tumor xenografts. Conclusions: Collectively, these data suggest that miR-182-5p plays a tumor-suppressive role in ccRCC. These findings offer new insight into role of miR-182-5p in the inhibition of ccRCC tumor growth through MALAT-1 downregulation. This study may provide rationale for development of new strategies targeting MALAT-1 through miR-182-5p for treatment of ccRCC. Citation Format: Priyanka Kulkarni, Pritha Dasgupta, Shahana Majid, Varahram Shahryari, Marisa Shiina, Yutaka Hashimoto, Nadeem Bhat, Guoren Deng, Sharanjot Saini, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. miR-182-5p suppresses progression of renal cancer through cell cycle arrest by targeting lncRNA MALAT-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2478.

Background: MicroRNAs are small noncoding RNAs that regulate the expression of >60% of all human genes, either inhibiting target mRNA translation or inducing its degradation. MicroRNAs act as tumor suppressors or oncogenes in various cancers. The main objective of this study was to investigate the role of microRNA-588 (miR-588) in prostate cancer (PC). Methods: The methods employed in this study include quantitative-real time PCR; western blot; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viability, migration and invasiveness of prostate cancer cells. Luciferase reporter assays and in-vivo study in nude mice was also performed. Results: The expression of miR-588 was significantly suppressed or silenced in PC tissue samples and cell lines when compared with normal tissues and a non-malignant cell line. Similar results were observed by analyzing the publicly available TCGA data sets for prostate adenocarcinoma. Functionally ectopic expression of miR-588 induced G0/G1 cell cycle arrest and apoptosis and suppressed cell proliferation. miR-588 exerted these functional effects by directly targeting the oncogenic Cyclin A2 that is involved in cancer cell cycle and proliferation. In silico algorithm showed a complimentary binding sequence in the 3'UTR of Cyclin A2 for miR-588. Over-expression of miR-588 significantly suppressed the luciferase activity of reporter plasmid containing the wild type 3'UTR sequences of cyclin A2 complementary to miR-588, which was abolished by mutations in these 3'UTR regions. miR-588 overexpression also significantly reduced the expression of cyclin A2 at both mRNA and protein levels. A significant decrease in the expression of various cell cycle pathway genes such as CycE1, MCM2, MCM4, CDC7 and CDT1 was also observed. These genes are involved in promoting cell cycle and proliferation and are overexpressed in prostate cancer. In vivo intracardiac implantation of PC3-MLuc-C6 prostate cancer cells constitutively expressing miR-588 showed a significant inhibition of the metastatic dissemination of these cells compared to the control miR expressing cells. Conclusion: This is the first study demonstrating that miR-588 is significantly silenced and acts as a tumor suppressor in prostate cancer. Reconstitution of silenced miR-588 may contribute to novel therapeutic approaches for regulating prostate cancer. Citation Format: Nadeem Bhat, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Priyanka Kulkarni, Pritha Dasgupta, Soichiro Yamamura, Yuichiro Tanaka, Taku Kato, Yutaka Hashimoto, Marisa Shiina, Z. Laura Tabatabai, Guoren Deng, Rajvir Dahiya, Shahana Majid. Negative regulation of oncogenes by a novel tumor suppressor microRNA in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4292.

Background: African-Americans (AfA) have higher risk for developing prostate cancer and when diagnosed, the cancer is more aggressive with worse survival compared to Caucasians (CA). To clarify the mechanisms involved in this disparity, we analyzed the role of miR-182 in AfA and CA prostate cancer tissues and cells. Materials and Methods: We analyzed miR-182 expression in AfA and CA prostate cancer tissue samples by Real Time PCR. We selected two cell lines, DU-145 (CA) and MDA-PCa-2b (AfA), which express different levels of miR-182, to mimic the tissue samples and help identify the mechanisms related to racial disparity. We generated lentivirus stable miR-182 knockdown cells lines and performed cell viability assays using a CellTiter-Glo luminescent assay. For cell cycle analysis, cells were stained with PI/RNase staining buffer and analyzed for DNA content by BD FACSVerse. Cancer pathway-focused gene expression profiling was done using a human RT2 Profiler Cancer Pathway Finder and Oncogenes and Tumor Suppressor Genes PCR Array. Results: We found miR-182 expression to be higher in AfA prostate cancer tissues compared to CA patients with localized disease. Also, expression of miR-182 in AfA cell line, MDA-PCa-2b, was significantly higher compared to CA cell line, DU-145. To determine the role of miR-182 in the differences between AfA and CA, miR-182 was knocked down in MDA-PCa-2b and DU- 145 cells. miR-182 knockdown in MDA-PCa-2b cells effectively inhibited cell migration, invasion and colony formation compared to control cells, while there was no difference in DU-145 cells. Knockdown of miR-182 caused a decrease in MDA-PCa-2b cell viability compared with negative control. In addition, the cell cycle profile showed an increase in the G1 phase of MDA-PCa-2b cells (control 7.3% compared to miR-182 knockdown cells 18.6%), whereas no change was observed in DU-145 cells. Similar to these results, wound healing was significantly slower in the miR-182 knockdown MDA-PCa-2b cells compared to controls. PCR array analysis was performed to determine the molecular effects of miR-182 knockdown in MDA-PCa-2b and DU-145 cells. We found from our PCR array data some potential targets genes regulated by miR-182. Conclusion: We conclude that high levels of miR-182 is associated with prostate cancer aggressiveness in African-Americans. Citation Format: Marisa Shiina, Yutaka Hashimoto, Guoren Deng, Varahram Shahryari, Majid Shahana, Soichiro Yamamura, Priyanka Kulkarni, Pritha Dasgupta, Divya Bhagirath, Sharanjot Sharan, Yuichiro Tanaka, Rajvir Dahiya. High expression of miR-182 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 471.

Background: Tumor metastasis and recurrence are major obstacles in renal cell carcinoma (RCC) treatment. RCC aggressiveness is highly associated with stemness and epithelial-to-mesenchymal transition (EMT) which involves increased cellular migration, invasion, inhibition of apoptosis and senescence. HOX transcript antisense RNA (HOTAIR), a lncRNA, is reported to be over-expressed, whereas miR-203 has low expression and is a tumor suppressor in various cancers. However, their association and role in RCC is not well understood. The aim of the present study was to examine the interaction of HOTAIR and miR-203 in the regulation of stemness and EMT in RCC. Experimental Procedure: ACHN, Caki-1 (human RCC cell lines), normal renal epithelial cells HK-2 and clinical specimens were used for this study. Profiling of HOTAIR and miR203 expression were done by quantitative real-time PCR and luciferase assay was performed to confirm their interaction. Attenuation of HOTAIR (25nM siRNA) and overexpression of miR-203 (10nM mimic) for 72 hours were used for functional studies. The biological role of miR-203 and its interaction with HOTAIR was also investigated using nude mouse models. Results: HOTAIR was observed to be overexpressed in RCC cell-lines and clinical specimens whereas, miR-203 was significantly under-expressed when compared to normal cells and tissues. Overexpression of miR-203 altered the cell cycle, inhibited epithelial-to-mesenchymal transition (EMT) and decreased cell proliferation along with induction of epithelial marker proteins and decrease in mesenchymal and stemness marker proteins. Conversely, knockdown of miR-203 in non-malignant HK-2 cells induced pro-cancerous characteristics. Direct binding of miR-203 to HOTAIR was shown by RNA immunoprecipitation and luciferase assay. Attenuation of HOTAIR expression reduced cell migration, invasion, and induced apoptosis, mimicking the effects of miR-203 overexpression. Administration of miR-203 mimic to established tumors in nude mice significantly suppressed tumor growth compared to controls. Conclusion: Our results show that HOTAIR-miR-203 interaction inhibits EMT and stemness to regulate RCC progression. Statistical analysis of the clinicopathological data from kidney cancer patients suggests that HOTAIR and miR-203 may be useful in RCC diagnostics and therapeutics. Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Shahana Majid, Varahram Shahryari, Yutaka Hashimoto, Nadeem S. Bhat, Marisa Shiina, Guoren Deng, Sharanjot Saini, Soichiro Yamamura Yamamura, Yuichiro Tanaka, Rajvir Dahiya. Epithelial-to-mesenchymal transition and stemness: Key targets of HOTAIR-miR-203 interaction in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2457.

Women in management, today are performing important tasks but very few have advanced to the top echelons of corporate power. Work place policies have adjusted somewhat towards accommodating women managers, but they have not produced a situation that most observors would label as equal for women and men. Yet avenues for positive changes do exist, Women have to recognise and develop their inherent qualities and innate skills to satisfy the future knowledge world. Self empowerment appears to be the answer for women managers.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and polymorphic variants have been shown to increase activity levels. Lifestyle choices such as tobacco smoking and alcohol consumption are known to enhance the carcinogenesis process and in this study, how these factors may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population consisting of 405 healthy men and 400 prostate cancer patients were obtained. Of these, 507 were current or former smokers and 407 were alcohol drinkers. Eight polymorphic sites of the promoter region of CYP1B1 (rs2551188 G to A, rs2567206 G to A, rs2567207 A to G, rs162556 A to G, rs10175368 C to T, rs163090 T to A, rs162330 T to G, and rs162331 A to G) were analyzed in samples using Taqman genotyping assays and real-time PCR. Lifestyle factors and its influence on CYP1B1 polymorphisms toward cancer risks were also evaluated. Overall, both alcohol (P=0.006) and smoker (P=0.069) status were associated with prostate cancer. CYP1B1 variants were also risks for cancer at rs2551188 (P=0.043), rs2567206 (P=0.008), and rs10175368 (P=0.001). Evaluation of linkage disequilibrium show rs2551188, rs2567206, rs2567207, and rs10175368 to be linked and interestingly, the G-G-A-C haplotype (wildtype at respective sites) was significantly reduced in cancer (P=0.0282). When classified by lifestyle factors, no associations for CYP1B1 variants were found for cancer among non-smokers with rs10175368 (P=0.051) being a risk among non-drinkers. On the other hand, variants at both rs2567206 and rs10175368 showed increased cancer risk among smokers (P=0.032 and 0.002, respectively) as well as drinkers (P=0.044 and 0.019, respectively). No genotyping differences were observed when analyzing cancers by pathological grades. These results demonstrate smoker and alcoholic drinker status to modify the risks of CYP1B1 polymorphisms for prostate cancer and this is of importance in understanding their role in the pathogenesis of this disease. Citation Format: Taku Kato, Yutaka Hashimoto, Shigekatsu Maekawa, Marisa Shiina, Mitsuho Imai-Sumida, Pritha Dasgupta, Priyanka Kulkarni, Soichiro Yamamura, Shahana Majid, Sharanjot Saini, Varahram Sharryari, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. Effects of tobacco smoking and alcohol consumption on risks of CYP1B1 polymorphisms for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2288. doi:10.1158/1538-7445.AM2017-2288

Loss of chromosome (chr) 8p21 is a frequent genomic alteration in prostate cancer (PCa). Genomic deletions of this region increase significantly with tumor grade and are associated with tumor progression and poor prognosis. A common region of loss of heterozygosity (LOH) has been mapped to the chr8p21 locus that primarily harbors prostate-specific NKX3.1 homeobox gene. We recently demonstrated that this frequently deleted locus is associated with a cluster of microRNA genes- miR-3622a/b- that are lost in prostate cancer and play an important mechanistic role in PCa progression and metastasis. MicroRNA expression profiling in microdissected human PCa clinical tissues showed that miR-3622a/b expression is widely downregulated and is correlated with poor survival outcome in prostate cancer. Our analyses suggested that miR-3622a has potential as a prognostic and diagnostic marker for prostate cancer. Extending these findings, we explored the prognostic potential of serum miR-3622a in prostate cancer patients. Since exosomes provide an important source of non-invasive, circulating biomarkers and represent an enriched source of microRNAs for biomarker profiling, we profiled the microRNA content of exosomes derived from sera of prostate cancer patients. In a pilot study, we profiled exosomal miRNAs from a training cohort of individuals with benign prostate hyperplasia (BPH), an indolent form of PCa or aggressive metastatic PCa. Exosomes were isolated from sera of prostate cancer patients and integrity of exosomal preparations was confirmed by nanoparticle tracking analysis and Western blot analyses for exosomal markers. Expression analyses of exosomal miR-3622a expression showed that this miRNA is enriched in exosomes. Higher levels of miR-3622a were significantly associated with tumor stage and lymph node metastasis in a cohort of prostate cancer clinical specimens. Further, ROC (Receiver Operating Characteristic) analyses showed that exosomal miR-3622a expression can be a single significant parameter to discriminate between BPH and prostate cancer. Overall, our data suggests that exosomal miR-3622a is a promising prognostic biomarker for prostate cancer. patients. Citation Format: Thao Yang, Divya Bhagirath, Kirandeep Sekhon, Nathan Bucay, Shahana Majid, Varahram Shahryari, Marisa Shiina, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Mitsuho Imai-Sumida, Soichiro Yamamura, Z Laura Tabatabai, Kirsten Greene, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng, Sharanjot Saini. Exosomal miR-3622a as prognostic marker in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2017-4435

Prostate tumors are highly heterogeneous and molecular characterization based on biopsy sampling is often challenging. Hence, it is desirable to have an easily accessible, minimally invasive way to determine the molecular imprints of a patient’s tumor. Exosomes in blood are an important source of non-invasive, circulating biomarkers and provide an enriched source of microRNAs. With an objective of identifying microRNA based markers, we developed a comprehensive procedure for exosomal microRNA profiling analyses from sera of prostate cancer patients employing Nanostring nCounter technology. In a pilot study, we profiled exosomal miRNAs from a training cohort of normal individuals, patients with benign prostate hyperplasia (BPH), indolent form of PCa and aggressive metastatic prostate cancer. Exosomes were extracted from serum and the integrity of exosomal preparations was confirmed by nanoparticle tracking analysis and Western blot analyses for exosomal markers CD63, CD9 and TSG101. Levels of two miRNAs were significantly upregulated and several candidate miRNAs were markedly downregulated in metastatic PCa serum samples compared with controls. The candidate microRNAs were further validated in a larger cohort by real time PCR based microRNA assays. Of significance, we validated miR-1246 as an exosomal marker for prostate cancer. miR-1246 levels were specifically altered in prostate cancer patients and not significantly altered in BPH. Our analyses suggest that miR-1246 is a promising diagnostic biomarker for prostate cancer. Citation Format: Divya Bhagirath, Thao Yang, Kirandeep Sekhon, Nathan Bucay, Shahana Majid, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Marisa Shiina, Varahram Shahryari, Mitsuho Imai-Sumida, Soichiro Yamamura, Z Laura Tabatabai, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng, Sharanjot Saini. An exosomal biomarker for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2017-5448