Taku Kato

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.

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.

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.

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.

Influence of lifestyle choices on risks of CYP1B1 polymorphisms for prostate cancer

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5′ untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G‐C‐T‐G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non‐smokers and non‐drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G‐C‐T‐G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

MP14-06 RISKS OF CYTOCHROME P450 1B1 POLYMORPHISMS AND LIFESTYLE CHOICES ON PROSTATE CANCER

METHODS: We identified 35,968 adult patients aged 18-89 years who underwent open or minimally invasive RP from 2010-2015 in the National Surgical Quality Improvement Program (NSQIP) database. Age was modeled as a categorical variable. Thirty-day complications and perioperative outcomes were assessed using a standardized protocol as part of the NSQIP. The associations of age with 30-day complications and perioperative outcomes were evaluated using logistic regression, adjusted for patient features. RESULTS: Age at surgery was distributed as follows: <60 years in 12,172 (33.8%) patients, 60-69 years in 18,076 (50.3%) patients, 70-79 years in 5,480 (15.2%) patients, and 80-89 years in 240 (0.7%) patients. Median operative time was 191 (IQR 151, 191) minutes. There were statistically significant differences in several baseline characteristics across age strata, with higher American Society of Anesthesiology (ASA) class and greater prevalence of diabetes, chronic obstructive pulmonary disease, hypertension, and renal failure among older patients. Overall, 30-day complications occurred in 1,798 (5%) patients. In multivariable analyses adjusted for patient features and surgical approach, ages 70-79 and 80-89 years were statistically significantly associated with increased risks of 30-day complications (OR 1.24, p1⁄40.01; OR 2.83, p<0.01, respectively), perioperative blood transfusion (OR 1.25, p1⁄40.01; OR 3.89, p<0.01, respectively) and 30-day mortality (OR 2.24, p1⁄40.05; OR 10.02, p<0.01, respectively). Only the 80-89 years age group was associated with an increased risk of readmissions (OR 1.75, p1⁄40.03). CONCLUSIONS: In this national, surgical cohort, older age was independently associated with increased risks of 30-day complications, perioperative blood transfusion, hospital readmissions, and 30-day mortality. However, there were no statistically significant differences among men younger than 70 years for all perioperative endpoints. These results have implications for patient counseling and decision making.

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.

Abstract 5749: VCAN promotes clear cell renal cell carcinoma tumor progression and metastasis, and predicts poor prognosis

Introduction and Objectives: Although versican (VCAN) is known to promote tumor progression and enhance metastasis of several types of cancers, its role in clear cell renal cell carcinoma (ccRCC) remains unknown. Interestingly, a recent report showed that VCAN is an important target of chromosomal 5q gain, one of the most prevalent genetic abnormalities in ccRCC cases. In the present study, we investigated whether VCAN expression is associated with the pathogenesis of ccRCC. Methods: VCAN expression was analyzed in 3 RCC and normal kidney cell lines, as well as 84 matched ccRCC and normal renal tissues. We also performed various functional analyses of growth and progression properties using VCAN-depleted ccRCC cells. Microarray analysis was then employed to investigate the target genes of the pathway involved in ccRCC tumorigenesis and development. Results: There are 4 isoforms of VCAN containing the N-terminal globular (G1 domain) and C-terminal globular (G3 domain) domains, each of which was found to be over-expressed in the ccRCC samples as compared to the controls. Higher VCAN expression was significantly correlated with metastasis (p Conclusion: Our results demonstrate that VCAN promotes ccRCC tumorigenesis and metastasis, showing it to be an attractive novel target for diagnostic, prognostic, and therapeutic strategies for affected patients. Note: This abstract was not presented at the meeting. Citation Format: Yozo Mitsui, Taku Kato, Shigekatsu Maekawa, Yutaka Hashimoto, Marisa Shiina, Mitsuho Imai Sumida, Ryan Kenji Wong, Soichiro Yamamura, Varahram Shahryari, Shahana Majid, Sharanjot Saini, Guoren Deng, Rajvir Dahiya, Koichi Nakajima, Yuichiro Tanaka. VCAN promotes clear cell renal cell carcinoma tumor progression and metastasis, and predicts poor prognosis [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 5749. doi:10.1158/1538-7445.AM2017-5749

Abstract 3449: Genistein inhibits renal cancer progression through long non-coding RNA HOTAIR suppression

Genistein, a soy isoflavone, has been shown to have anticancer effects on various cancers in vitro and in vivo including renal cancer. Long non-coding RNAs (lncRNAs) are differentially expressed in various tissues and have important functions in cellular processes such as cell proliferation, motility and apoptosis in various malignancies. HOX transcript antisense RNA (HOTAIR) is a lncRNA localized in the Homeobox C gene cluster on chromosome 12. HOTAIR interacts with the polycomb repressive complex 2 (PRC2), which enhances H3K27 trimethylation and represses the expression of tumor suppressors. In various cancers, HOTAIR is highly expressed and involved in their progression and metastasis. In this study, we investigated the molecular mechanisms of genistein action through a novel pathway that represses HOTAIR. We found that HOTAIR expression is higher in renal cancer cell lines compared to normal controls. Genistein treatment was found to significantly decrease HOTAIR expression in renal cancer cells (786-O and ACHN cells). Genistein treatment also reduced expression of epithelial-to-mesenchyme transition (EMT)-related proteins (ZEB1, Vimentin and Snail), causing reduced cell migration, invasion, and increased apoptosis. We performed RNA immunoprecipitation assays, and found that genistein inhibits HOTAIR binding to PRC2. One of the other EMT markers, a tight junction protein ZO-1, is upregulated by genistein. We are currently investigating if genistein represses PRC2 recruitment to the ZO-1 promoter by inhibiting binding of HOTAIR to PRC2. Our results indicate that genistein is a potent therapeutic agent for renal cancer. Citation Format: Mitsuho Imai-Sumida, Shahana Majid, Pritha Dasgupta, Priyanka Kulkarni, Sharanjot Saini, Divya Bhagirath, Taku Kato, Shigekatsu Maekawa, Yutaka Hashimoto, Marisa Shiina, Guoren Deng, Varahram Shahryari, Yuichiro Tanaka, Rajvir Dahiya, Soichiro Yamamura. Genistein inhibits renal cancer progression through long non-coding RNA HOTAIR suppression [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 3449. doi:10.1158/1538-7445.AM2017-3449

Abstract 2288: Effects of tobacco smoking and alcohol consumption on risks of CYP1B1 polymorphisms for prostate cancer

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