Nobuyuki Kikuno

Genistein mediated histone acetylation and demethylation activates tumor suppressor genes in prostate cancer cells.

The above article, published online on 22 April 2008 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor in Chief, Prof. Peter Lichter, and John Wiley & Sons Ltd. The retraction has been agreed due to errors identified in Figs, 3, 4, and 5. The concerns about these figures cannot be resolved because the original data can no longer be retrieved.

Epigenetic inactivation of wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt/β-catenin signaling pathway

Purpose: Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis. Experimental Design: To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/β-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa. Results: Wif-1 mRNA expression was significantly enhanced after 5-aza-2′-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of β-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/β-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth. Conclusion: This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/β-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.

Combination Analysis of Hypermethylated Wnt-Antagonist Family Genes as a Novel Epigenetic Biomarker Panel for Bladder Cancer Detection

Purpose: Aberrant promoter hypermethylation of Wnt-antagonist genes contributes to the pathogenesis of several cancers. We hypothesized that combined methylation analysis of Wnt-antagonist genes could improve their use as a panel of biomarkers for diagnosing and staging of bladder cancers. Experimental Design: Samples (54 total) of bladder tumor and corresponding normal bladder mucosa were analyzed for the methylation and expression levels of six Wnt-antagonist genes (sFRP-1, sFRP-2, sFRP-4, and sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of bladder tumor detection, the methylation score (M score), a new method for multigene methylation analysis, was developed. The M score of each sample was calculated as the sum of the corresponding log hazard ratio coefficients derived from multivariate logistic regression analysis of the methylation status for each Wnt-antagonist gene. Receiver operator characteristic (ROC) curve analysis was used to determine the optimal sensitivity/specificity of the M score. Urine DNA from 24 matched patients with bladder tumor and 20 cancer-free volunteers was also used to investigate the methylation status of Wnt-antagonist genes. Results: The methylation levels of Wnt-antagonists were significantly higher and mRNA levels were significantly lower in bladder tumor than in bladder mucosa. Each methylation level was inversely correlated with the corresponding mRNA level. In multivariate regression analysis, the methylation levels of sFRP-2 and Dkk-3 were significant independent predictors of bladder tumor (P < 0.05 and P < 0.01, respectively), whereas with sFRP-1, sFRP-5, and Wif-1 there was a trend towards significance as independent predictors. The M score of Wnt-antagonist genes was significantly higher in bladder tumor than in bladder mucosa (P < 0.05). Overall, the M score had a sensitivity of 77.2% and a specificity of 66.7% as a diagnostic biomarker (areas under the curve, 0.763). The M score could distinguish superficial from invasive bladder tumors with a sensitivity of 72.2% and a specificity of 61.1% as a staging biomarker (areas under the curve, 0.671). In patients with bladder tumor, 80.6% of the methylation-specific PCR results had identical methylation in samples of tumor- and urine-derived DNA. Most urine DNA in normal controls showed no aberrant methylation of the Wnt-antagonist genes. Conclusions: Hypermethylation of Wnt-antagonist genes plays an important role in the pathogenesis of bladder tumor and can be detected using cellular DNA extracted from urine samples. This is the first report demonstrating that M score analysis of Wnt-antagonist genes could serve as an excellent epigenetic biomarker panel for bladder tumors.

Bcl-2 Expression as a Predictive Marker of Hormone-Refractory Prostate Cancer Treated with Taxane-Based Chemotherapy

Purpose: Bcl-2 inhibits apoptosis, and its overexpression is associated with hormone refractory prostate cancer (HRPC). Bak and Bax are in the Bcl-2 family and counteract the antiapoptotic function of Bcl-2. Taxane-induced (paclitaxel and its analogue docetaxel) phosphorylation of Bcl-2 abolishes the potential antiapoptotic effect of Bcl-2. We hypothesized that (a) survival benefit in HRPC patients treated with taxanes is determined by the presence of Bcl-2 protein and (b) altered expression of Bak and Bax protein caused by genetic mutation is associated with biological aggressiveness of prostate cancer. Experimental Design: Forty localized prostate cancer and 30 HRPC cases were used in this study. Surgical specimens of localized prostate cancer and biopsy specimens of HRPC were used for immunostaining of Bcl-2, Bak, and Bax as well as DNA extraction. Mutations in the Bak and Bax genes were screened by single-strand conformational polymorphism, and confirmed by direct DNA sequencing. Results: Bcl-2–positive HRPC showed longer cause-specific survival in comparison with the counterparts. Multivariate analysis revealed that the level of Bcl-2 expression before treatment with taxane-based chemotherapy was an independent predictor for cause-specific survival (P < 0.01) and baseline prostate-specific antigen level was an independent predictor for progression-free survival (P < 0.01). Bax gene mutation was found in only one HRPC specimen. Conclusions: Bcl-2 expression in addition to prostate-specific antigen measurement before treatment could identify HRPC patients who may benefit from taxane-based chemotherapy. Mutation of the Bak and Bax genes is a rare event in prostate cancer.

Wnt antagonist family genes as biomarkers for diagnosis, staging, and prognosis of renal cell carcinoma using tumor and serum DNA

Purpose: We hypothesized that combined methylation analysis of Wnt antagonist genes could serve as a panel of biomarkers for diagnosis, staging, and prognosis in renal cell carcinoma (RCC). Experimental Design: Samples (n = 62) of RCC and corresponding normal renal tissue (NRT) were analyzed using methylation-specific PCR for methylation of six Wnt antagonist genes (sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of RCC detection, the methylation score (M score) for multigene methylation analysis was developed. Receiver operator characteristic curve analysis was used to determine the optimal sensitivity/specificity of the M score. In addition, the M score was compared with the clinicopathologic outcome. Thirty-three serum DNA samples were also used to investigate the methylation status of Wnt antagonist genes. Results: The methylation levels of all Wnt antagonists were significantly higher in RCC than in NRT. In multivariate regression analysis, the methylation level of sFRP-1 was a significant independent predictor of RCC, whereas for sFRP-2 and sFRP-4 there was a trend toward significance as independent predictors. The M score of Wnt antagonist genes was significantly higher in RCC than in NRT. Overall, the M score had a sensitivity of 79.0% and a specificity of 75.8% (area under the curve, 0.808) as a diagnostic biomarker. In addition, the M score could significantly distinguish grade, pT category, M category, and overall survival of RCC patients. The M score was independent of age and gender in predicting overall survival by the Cox proportional hazards model. In RCC patients, 72.7% of the methylation-specific PCR results had identical methylation in samples of tumor and serum DNA. No serum DNA in normal controls showed aberrant methylation of the Wnt antagonist genes. In addition, the methylation status of Wnt antagonist genes in serum DNA was significantly correlated with tumor grade and stage. Conclusions: This is the first report showing that M score analysis of Wnt antagonist genes can serve as an excellent epigenetic biomarker panel for detection, staging, and prognosis of RCC using serum DNA.

MDM2 SNP309 Polymorphism as Risk Factor for Susceptibility and Poor Prognosis in Renal Cell Carcinoma

Purpose: MDM2 is a major negative regulator of p53, and a single nucleotide polymorphism in the MDM2 promoter region SNP309 (rs2279744) has been shown to increase the affinity of the transcriptional activator Sp1, resulting in elevated MDM2 transcription and expression in some cancers. There is currently no information about the role of MDM2 polymorphism in renal cell carcinoma (RCC). We investigated polymorphisms in p53-related genes, including MDM2, and their interactions in renal cancer. Experimental Design: We genotyped three single nucleotide polymorphisms of three genes (p53 Arg72Pro, p21 Ser31Arg, and MDM2 SNP309) in 200 patients with renal cancer and 200 age- and gender-matched healthy subjects. Genotyping was confirmed by direct DNA sequencing. Samples that showed significant polymorphic variants were analyzed for MDM2 expression by immunohistochemistry. Association of polymorphic variants on survival of RCC patients was analyzed by Kaplan-Meier curves. Results: A significant increase in the GG genotype of the MDM2 SNP309 was observed in RCC patients compared with healthy controls (odds ratio, 1.80; 95% confidence interval, 1.14-2.84). To investigate the effect of the MDM2 SNP309 polymorphism on MDM2 expression, immunohistochemistry was done in genotyped RCC tissues. Positive staining for MDM2 was detected in 2 of 15 (13%) TT genotype, 4 of 15 (26%) TG genotype, and 5 of 10 (50%) GG genotype carriers. The frequency of MDM2 expression in GG genotype carriers was significantly higher than that in TT genotype carriers. Polymorphisms of p53 Arg72Pro and p21 Ser31Arg did not show significant association with RCC. In univariate and multivariate analysis, MDM2 SNP309 GG genotype was independently associated with poor prognosis. Kaplan-Meier curve analysis showed that survival of patients with GG carriers was significantly worse than that of carriers with TG + TT genotypes. Conclusions: This is the first report to show a significant association between functional polymorphisms in MDM2 and increased risk of developing renal cancer. In addition, the MDM2 polymorphism was shown to be an independent adverse prognostic factor for RCC. Patients with MDM2 309GG genotype showed worse prognosis and low survival.

CXCL12 G801A Polymorphism Is a Risk Factor for Sporadic Prostate Cancer Susceptibility

Purpose: The chemokine CXCL12 and its receptor CXCR4 have been found to be associated with cancer metastasis. A single nucleotide polymorphism of CXCL12 G801A has been described and is regarded as a target for cis-acting factor that has the ability to up-regulate CXCL12 expression. Currently, there are no reports investigating the role of CXCL12 G801A polymorphism in prostate cancer (PC). Experimental Design: We genotyped CXCL12 G801A and p53Arg72Pro in 167 PC patients and 167 age-matched healthy subjects. Genotyping was done with PCR-RFLP and confirmed by direct DNA sequencing. To investigate the effect of the CXCL12 G801A polymorphism on CXCL12 and CXCR4 expression, immunohistochemistry was done in genotyped PC tissues. Results: A significant increase in the GA + AA genotype of the CXCL12 G801A polymorphism was observed in PC patients compared with healthy controls. The frequency of CXCL12 AA genotype was significantly higher in a group of patients with lymph node metastasis (23%) compared with those without metastasis (7%). The frequency of CXCL12 expression in AA + GA genotype carriers was significantly higher than that in GG genotype carriers. Among the carriers with CXCL12 GA + AA genotypes, CXCR4 expression was also significantly higher compared with those with the GG genotype. Moreover, among the groups with both CXCL12- and CXCR4-positive staining, the frequency of the CXCL12 GA + AA genotype was high. Although we did not find a significant relationship between the frequency of the Arg/Pro + Pro/Pro genotype of p53 Arg72Pro and susceptibility in PC, there was a combined effect of CXCL12 GA + AA genotype and the p53 72Arg/Pro + Pro/Pro genotype on the frequency of PC. These results indicate that the p53 codon 72 polymorphism may interact with CXCL12 G801A. Conclusions: This is the first report showing that CXCL12 G801A polymorphism may be a risk factor for PC. Moreover, this study suggests that this polymorphism can be an important marker for detecting microinvasion and PC metastasis.

Wnt antagonist DKK1 acts as a tumor suppressor gene that induces apoptosis and inhibits proliferation in human renal cell carcinoma

The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5‐Aza‐2′‐deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5‐Aza‐2′‐deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T‐cell factor/lymphoid enhancer factor activity and nuclear beta‐catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c‐Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta‐catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.

COMBINATION CHEMOTHERAPY WITH PACLITAXEL, ESTRAMUSTINE AND CARBOPLATIN FOR HORMONE REFRACTORY PROSTATE CANCER

PURPOSE The activity of estramustine phosphate is synergistic with paclitaxel against hormone refractory prostate cancer. Moreover, the single agent activity of carboplatin has demonstrated a 17% response rate in measurable disease. Therefore, we conducted a prospective trial to establish more effective chemotherapy consisting of paclitaxel, estramustine phosphate and carboplatin for hormone refractory prostate cancer. MATERIALS AND METHODS The study included 32 patients with hormone refractory prostate cancer. Prior chemotherapy was accepted. Patients were treated with 100 mg./m.2 paclitaxel intravenously weekly, 10 mg./kg. estramustine phosphate orally daily and carboplatin intravenously to an area under the curve of 6 on day 1 of every 4-week cycle. Treatment was continued until disease progression or excessive toxicity. RESULTS Of the 32 patients 30 were assessable for response. A median of 7 consecutive cycles was administered per patient. Ten patients had received prior cytotoxic chemotherapy. Levels of prostate specific antigen decreased by greater than 50% in 100% of patients and by greater than 90% in 56.7%. Partial response was obtained in 61.1% of measurable lesions. Consumption of medication for cancer induced pain was reduced in 89.5% of patients. Tumor volume reduction and/or antitumor therapeutic effects were exhibited in 81.0% of patients with positive biopsy. At a median followup of 48 weeks median time to progression was 48 weeks and median overall survival was 95 weeks. Two patients suffered myocardial infarction and hepatic insufficiency, respectively, and discontinued treatment during the first cycle. Major toxicities were grade 3 or 4 anemia in 59.4% of patients, leukopenia in 37.5%, thrombocytopenia in 28.1% and neuropathy in 12.5%. However, all toxicity was temporary and reversible with dose reduction or temporary cessation of chemotherapeutic agents. CONCLUSIONS Paclitaxel, estramustine phosphate and carboplatin chemotherapy was extremely effective for hormone refractory prostate cancer. Although hematological and neurotoxicity were modest, this therapy may be more manageable with lower doses.

Wnt Antagonist Gene DKK2 Is Epigenetically Silenced and Inhibits Renal Cancer Progression through Apoptotic and Cell Cycle Pathways

Purpose: Wnt/β-catenin signaling is involved in renal cancer. DKK2, a Wnt antagonist, is silenced in some cancers, although its function has not been investigated. We hypothesized that DKK2 may be epigenetically silenced and inhibits progression of renal cell carcinoma (RCC). Experimental Design: RCC cell lines and a normal kidney cell line were used for methylation and chromatin immunoprecipitation assays. To assess various functions of DKK2, we established stable DKK2-transfected cells and examined them with regard to cell viability, colony formation, apoptosis, cell cycle, and invasive capability. A total of 52 patients with confirmed conventional RCC were enrolled in this study. Results: RCC cell lines had decreased levels of DKK2, which were significantly increased after treatment with 5-Aza-2′-deoxycytidine alone or 5-Aza-2′-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the levels of acetyl H3, acetyl H4, and dimethylated H3K4 were decreased, whereas the level of dimethylated H3K9 was increased in RCC cell lines compared with HK2 cells. Increased methylation in RCC tissues was associated with higher grades, pathologic stages, and pathologic tumor in RCC. Functional analysis showed that the numbers of viable A498 cells were significantly decreased in DKK2-transfected cells compared with mock cells. The number of apoptotic cells and S/G2-M phase cells was significantly increased and decreased after DKK2 transfection, respectively. Corresponding to these results, Bcl2 and cyclin D1 expression were also decreased in DKK2-overexpressing cells. Conclusion: DKK2 is epigenetically silenced by methylation in higher grades and stages of RCC. These results suggest that DKK2 inhibits renal cancer progression through apoptotic and cell cycle pathways. (Clin Cancer Res 2009;15(18):5678–87)