Ryo Takata

Genome-wide association study identifies five new susceptibility loci for prostate cancer in the Japanese population

Prostate cancer is one of the most common malignancies in males throughout the world, and its incidence is increasing in Asian countries. We carried out a genome-wide association study and replication study using 4,584 Japanese men with prostate cancer and 8,801 control subjects. From the thirty-one associated SNPs reported in previous genome-wide association studies in European populations, we confirmed the association of nine SNPs at P < 1.0 × 10−7 and ten SNPs at P < 0.05 in the Japanese population. The remaining 12 SNPs showed no association (P > 0.05). In addition, we report here five new loci for prostate cancer susceptibility, at 5p15 (λ-corrected probability PGC = 3.9 × 10−18), GPRC6A/RFX6 (PGC = 1.6 × 10−12), 13q22 (PGC = 2.8 × 10−9), C2orf43 (PGC = 7.5 × 10−8) and FOXP4 (PGC = 7.6 × 10−8). These findings advance our understanding of the genetic basis of prostate carcinogenesis and also highlight the genetic heterogeneity of prostate cancer susceptibility among different ethnic populations.

Predicting Response to Methotrexate, Vinblastine, Doxorubicin, and Cisplatin Neoadjuvant Chemotherapy for Bladder Cancers through Genome-Wide Gene Expression Profiling

Purpose: Neoadjuvant chemotherapy for invasive bladder cancer, involving a regimen of methotrexate, vinblastine, doxorubicin, and cisplatin (M-VAC), can improve the resectability of larger neoplasms for some patients and offer a better prognosis. However, some suffer severe adverse drug reactions without any effect, and no method yet exists for predicting the response of an individual patient to chemotherapy. Our purpose in this study is to establish a method for predicting response to the M-VAC therapy. Experimental Design: We analyzed gene expression profiles of biopsy materials from 27 invasive bladder cancers using a cDNA microarray consisting of 27,648 genes, after populations of cancer cells had been purified by laser microbeam microdissection. Results: We identified dozens of genes that were expressed differently between nine “responder” and nine “nonresponder” tumors; from that list we selected the 14 “predictive” genes that showed the most significant differences and devised a numerical prediction scoring system that clearly separated the responder group from the nonresponder group. This system accurately predicted the drug responses of 8 of 9 test cases that were reserved from the original 27 cases. Because real-time reverse transcription–PCR data were highly concordant with the cDNA microarray data for those 14 genes, we developed a quantitative reverse transcription–PCR–based prediction system that could be feasible for routine clinical use. Conclusions: Our results suggest that the sensitivity of an invasive bladder cancer to the M-VAC neoadjuvant chemotherapy can be predicted by expression patterns in this set of genes, a step toward achievement of “personalized therapy” for treatment of this disease.

Molecular Features of the Transition from Prostatic Intraepithelial Neoplasia (PIN) to Prostate Cancer Genome-wide Gene-expression Profiles of Prostate Cancers and PINs

To characterize the molecular feature in prostate carcinogenesis and the putative transition from prostatic intraepithelial neoplasia (PIN) to invasive prostate cancer (PC), we analyzed gene-expression profiles of 20 PCs and 10 high-grade PINs with a cDNA microarray representing 23,040 genes. Considering the histological heterogeneity of PCs and the minimal nature of PIN lesions, we applied laser microbeam microdissection to purify populations of PC and PIN cells, and then compared their expression profiles with those of corresponding normal prostatic epithelium also purified by laser microbeam microdissection. A hierarchical clustering analysis separated the PC group from the PIN group, except for three tumors that were morphologically defined as one very-high-grade PIN and two low-grade PCs, suggesting that PINs and PCs share some molecular features and supporting the hypothesis of PIN-to-PC transition. On the basis of this hypothesis, we identified 21 up-regulated genes and 63 down-regulated genes commonly in PINs and PCs compared with normal epithelium, which were considered to be involved in the presumably early stage of prostatic carcinogenesis. They included AMACR, OR51E2, RODH, and SMS. Furthermore, we identified 41 up-regulated genes and 98 down-regulated genes in the transition from PINs to PCs; those altered genes, such as POV1, CDKN2C, EPHA4, APOD, FASN, ITGB2, LAMB2, PLAU, and TIMP1, included elements that are likely to be involved in cell adhesion or the motility of invasive PC cells. The down-regulation of EPHA4 by small interfering RNA in PC cells lead to attenuation of PC cell viability. These data provide clues to the molecular mechanisms underlying prostatic carcinogenesis, and suggest candidate genes the products of which might serve as molecular targets for the prevention and treatment of PC.

Association of a novel long non-coding RNA in 8q24 with prostate cancer susceptibility

Recent genome‐wide association studies reported strong and reproducible associations of multiple genetic variants in a large “gene‐desert” region of chromosome 8q24 with susceptibility to prostate cancer (PC). However, the causative or functional variants of these 8q24 loci and their biological mechanisms associated with PC susceptibility remain unclear and should be investigated. Here, focusing on its most centromeric region (so‐called Region 2: Chr8: 128.14‐128.28 Mb) among the multiple PC loci on 8q24, we performed fine mapping and re‐sequencing of this critical region and identified SNPs (single nucleotide polymorphisms) between rs1456315 and rs7463708 (chr8: 128,173,119‐128,173,237 bp) to be most significantly associated with PC susceptibility (P = 2.00 × 10−24, OR = 1.74, 95% confidence interval = 1.56–1.93). Importantly, we show that this region was transcribed as a ∼13 kb intron‐less long non‐coding RNA (ncRNA), termed PRNCR1 (prostate cancer non‐coding RNA 1), and PRNCR1 expression was upregulated in some of the PC cells as well as precursor lesion prostatic intraepithelial neoplasia. Knockdown of PRNCR1 by siRNA attenuated the viability of PC cells and the transactivation activity of androgen receptor, which indicates that PRNCR1 could be involved in prostate carcinogenesis possibly through androgen receptor activity. These findings could provide a new insight in understanding the pathogenesis of genetic factors for PC susceptibility and prostate carcinogenesis. (Cancer Sci 2011; 102: 245–252)

Molecular Features of Hormone-Refractory Prostate Cancer Cells by Genome-Wide Gene Expression Profiles

One of the most critical issues in prostate cancer clinic is emerging hormone-refractory prostate cancers (HRPCs) and their management. Prostate cancer is usually androgen dependent and responds well to androgen ablation therapy. However, at a certain stage, they eventually acquire androgen-independent and more aggressive phenotype and show poor response to any anticancer therapies. To characterize the molecular features of clinical HRPCs, we analyzed gene expression profiles of 25 clinical HRPCs and 10 hormone-sensitive prostate cancers (HSPCs) by genome-wide cDNA microarrays combining with laser microbeam microdissection. An unsupervised hierarchical clustering analysis clearly distinguished expression patterns of HRPC cells from those of HSPC cells. In addition, primary and metastatic HRPCs from three patients were closely clustered regardless of metastatic organs. A supervised analysis and permutation test identified 36 up-regulated genes and 70 down-regulated genes in HRPCs compared with HSPCs (average fold difference > 1.5; P < 0.0001). We observed overexpression of AR, ANLN, and SNRPE and down-regulation of NR4A1, CYP27A1, and HLA-A antigen in HRPC progression. AR overexpression is likely to play a central role of hormone-refractory phenotype, and other genes we identified were considered to be related to more aggressive phenotype of clinical HRPCs, and in fact, knockdown of these overexpressing genes by small interfering RNA resulted in drastic attenuation of prostate cancer cell viability. Our microarray analysis of HRPC cells should provide useful information to understand the molecular mechanism of HRPC progression and to identify molecular targets for development of HRPC treatment.

Common variants at 11q12, 10q26 and 3p11.2 are associated with prostate cancer susceptibility in Japanese

We have previously reported multiple loci associated with prostate cancer susceptibility in a Japanese population using a genome-wide association study (GWAS). To identify additional prostate cancer susceptibility loci, we genotyped nine SNPs that were nominally associated with prostate cancer (P < 1 × 10−4) in our previous GWAS in three independent studies of prostate cancer in Japanese men (2,557 individuals with prostate cancer (cases) and 3,003 controls). In a meta-analysis of our previous GWAS and the replication studies, which included a total of 7,141 prostate cancer cases and 11,804 controls from a single ancestry group, three new loci reached genome-wide significance on chromosomes 11q12 (rs1938781; P = 1.10 × 10−10; FAM111A-FAM111B), 10q26 (rs2252004; P = 1.98 × 10−8) and 3p11.2 (rs2055109; P = 3.94 × 10−8). We also found suggestive evidence of association at a previously reported prostate cancer susceptibility locus at 2p11 (rs2028898; P = 1.08 × 10−7). The identification of three new susceptibility loci should provide additional insight into the pathogenesis of prostate cancer and emphasizes the importance of conducting GWAS in diverse populations.

Oncogenic Role of MPHOSPH1, a Cancer-Testis Antigen Specific to Human Bladder Cancer

To disclose the molecular mechanism of bladder cancer, the second most common genitourinary tumor, we had previously done genome-wide expression profile analysis of 26 bladder cancers by means of cDNA microarray representing 27,648 genes. Among genes that were significantly up-regulated in the majority of bladder cancers, we here report identification of M-phase phosphoprotein 1 (MPHOSPH1) as a candidate molecule for drug development for bladder cancer. Northern blot analyses using mRNAs of normal human organs and cancer cell lines indicated this molecule to be a novel cancer-testis antigen. Introduction of MPHOSPH1 into NIH3T3 cells significantly enhanced cell growth at in vitro and in vivo conditions. We subsequently found an interaction between MPHOSPH1 and protein regulator of cytokinesis 1 (PRC1), which was also up-regulated in bladder cancer cells. Immunocytochemical analysis revealed colocalization of endogenous MPHOSPH1 and PRC1 proteins in bladder cancer cells. Interestingly, knockdown of either MPHOSPH1 or PRC1 expression with specific small interfering RNAs caused a significant increase of multinuclear cells and subsequent cell death of bladder cancer cells. Our results imply that the MPHOSPH1/PRC1 complex is likely to play a crucial role in bladder carcinogenesis and that inhibition of the MPHOSPH1/PRC1 expression or their interaction should be novel therapeutic targets for bladder cancers. [Cancer Res 2007;67(7):3276–85]

Cancer Peptide Vaccine Therapy Developed from Oncoantigens Identified through Genome-wide Expression Profile Analysis for Bladder Cancer

OBJECTIVE The field of cancer vaccine therapy is currently expected to become the fourth option in the treatment of cancer after surgery, chemotherapy and radiation therapy. We developed a novel cancer peptide vaccine therapy for bladder cancer through a genome-wide expression profile analysis. METHODS Among a number of oncoproteins that are transactivated in cancer cells, we focused on M phase phosphoprotein 1 and DEP domain containing 1, both of which are cancer-testis antigens playing critical roles in the growth of bladder cancer cells, as candidate molecules for the development of drugs for bladder cancer. In an attempt to identify the peptide epitope from these oncoantigens, we conducted a clinical trial using these peptides for patients with advanced bladder cancer. RESULTS We identified HLA-A24-restricted peptide epitopes corresponding to parts of M phase phosphoprotein 1 and DEP domain containing 1 proteins, which could induce peptide-specific cytotoxic T lymphocytes. Using these peptides, we found that M phase phosphoprotein 1- and DEP domain containing 1-derived peptide vaccines could be well tolerated without any serious adverse events, and effectively induced peptide-specific cytotoxic T lymphocytes in vivo. CONCLUSIONS The novel approach adopted in the treatment with peptide vaccines is considered to be a promising therapy for bladder cancer.

Cell-Permeable Peptide DEPDC1-ZNF224 Interferes with Transcriptional Repression and Oncogenicity in Bladder Cancer Cells

Bladder cancer is the second most common genitourinary cancer worldwide, yet its oncogenic origins remain poorly understood. The cancer-testis antigen DEPDC1 was shown recently to contribute to bladder cancer oncogenesis. In this study, we examined the biological functions of DEPDC1 and defined a potential therapeutic strategy to target this molecule. Coimmunoprecipitation and immunocytochemistry revealed that DEPDC1 interacted and colocalized with zinc finger transcription factor ZNF224, a known transcriptional repressor. Inhibiting this interaction with a cell-permeable peptide corresponding to the ZNF224-interacting domain in DEPDC1 induced apoptosis of bladder cancer cells in vitro and in vivo. By inhibiting DEPDC1-ZNF224 complex formation, this peptide triggered transcriptional activation of A20, a potent inhibitor of the NF-kappaB signaling pathway. Our findings indicate that the DEPDC1-ZNF224 complex is likely to play a critical role in bladder carcinogenesis.

Plasma low-molecular-weight proteome profiling identified neuropeptide-Y as a prostate cancer biomarker polypeptide.

In prostate cancer diagnosis, PSA test has greatly contributed to the early detection of prostate cancer; however, expanding overdiagnosis and unnecessary biopsies have emerged as serious issues. To explore plasma biomarkers complementing the specificity of PSA test, we developed a unique proteomic technology QUEST-MS (Quick Enrichment of Small Targets for Mass Spectrometry). The QUEST-MS method based on 96-well formatted sequential reversed-phase chromatography allowing efficient enrichment of <20 kDa proteins quickly and reproducibly. Plasma from 24 healthy controls, 19 benign prostate hypertrophy patients, and 73 prostate cancer patients were purified with QUEST-MS and analyzed by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed prostate cancer specific detection pattern, which included a neurotransmitter polypeptide neuropeptide-Y (NPY). We further validated the screening results by targeted multiple reaction monitoring technology using independent sample set (n = 110). The ROC curve analysis revealed that logistic regression-based combination of NPY, and PSA showed 81.5% sensitivity and 82.2% specificity for prostate cancer diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput profiling of plasma polypeptides and had potential to effectively uncover very low abundant tumor-derived small molecules, such as neurotransmitters, peptide hormones, or cytokines.