Yohei Horikawa

Single nucleotide polymorphisms of microRNA-machinery genes modify the risk of renal cell carcinoma

Purpose: MicroRNAs (miRNA) are a class of small noncoding RNA molecules that have been implicated in a wide variety of basic cellular functions through posttranscriptional regulations on their target genes. Compelling evidence has shown that miRNAs are involved in cancer initiation and progression. We hypothesized that genetic variations of the miRNA machinery genes could be associated with the risk of renal cell carcinoma. Experimental Design: We genotyped 40 single nucleotide polymorphisms (SNP) from 11 miRNA processing genes (DROSHA, DGCR8, XPO5, RAN, DICER1, TARBP2, AGO1, AGO2, GEMIN3, GEMIN4, HIWI) and 15 miRNA genes in 279 Caucasian patients with renal cell carcinoma and 278 matched controls. Results: We found that two SNPs in the GEMIN4 gene were significantly associated with altered renal cell carcinoma risks. The variant-containing genotypes of Asn929Asp and Cys1033Arg exhibited significantly reduced risks, with odds ratios (OR) of 0.67 [95% confidence interval (95% CI), 0.47-0.96] and 0.68 (95% CI, 0.47-0.98), respectively. Haplotype analysis showed that a common haplotype of GEMIN4 was associated with a significant reduction in the risk of renal cell carcinoma (OR, 0.66; 95% CI, 0.45-0.97). We also conducted a combined unfavorable genotype analysis including five promising SNPs showing at least a borderline significant risk association. Compared with the low-risk reference group with one unfavorable genotype, the median-risk and high-risk groups exhibited a 1.55-fold (95% CI, 0.96-2.50) and a 2.49-fold (95% CI, 1.58-3.91) increased risk of renal cell carcinoma, respectively (P for trend < 0.001). Conclusions: Our results suggested that genetic polymorphisms of the miRNA-machinery genes may affect renal cell carcinoma susceptibility individually and jointly.

Genetic variations in microRNA-related genes are associated with survival and recurrence in patients with renal cell carcinoma.

We took a polygenic approach to evaluate the effects of 41 potentially functional single-nucleotide polymorphisms (SNPs) in microRNAs (miRNAs)-related genes on survival and recurrence among renal cell carcinoma (RCC) patients. During a median follow-up of 21.8 months, among 316 RCC patients, 64 died and 56 developed recurrence. In single-SNP analysis, we identified seven SNPs significantly associated with RCC survival and five SNPs with recurrence. The most significant associations were SNPs in GEMIN4 with the variant alleles of both rs7813 and rs910925 associated with 1.74-fold [95% confidence interval (CI) = 1.15-2.62] increased risk of death, whereas the variant allele of rs3744741 conferred a decreased risk of death [hazard ratio (HR) = 0.39; 95% CI = 0.19-0.77]. Several SNPs belonging to the pre-miRNA and were identified to be significantly associated with RCC recurrence. Haplotypes of DICER and DROSHA were also associated with altered patient survival and recurrence. More importantly, we observed cumulative effects of multiple SNPs on RCC survival. Compared with subjects carrying zero to two unfavorable genotypes, those carrying three to five and six and more unfavorable genotypes had an increased risk of death with a HR of 2.49 (95% CI = 1.24-5.00) and 6.66 (95% CI = 2.49-17.86), respectively, with significant dose-response trend (P for trend<0.001). As the first study of miRNA-related genetic polymorphisms on RCC clinical outcome, our results strongly suggested that miRNA-related SNPs may impact the recurrence and survival in RCC patients. Future investigation in larger populations and functional characterizations are necessary to validate these results.

A novel strategy for evasion of NK cell immunity by tumours expressing core2 O‐glycans

The O‐glycan branching enzyme, core2 β‐1,6‐N‐acetylglucosaminyltransferase (C2GnT), forms O‐glycans containing an N‐acetylglucosamine branch connected to N‐acetylgalactosamine (core2 O‐glycans) on cell‐surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT‐expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT‐expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK‐activating receptor, NKG2D, by its tumour‐associated ligand, Major histocompatibility complex class I‐related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT‐expressing bladder tumour cells, poly‐N‐acetyllactosamine was present on core2 O‐glycans on MICA, and galectin‐3 bound the NKG2D‐binding site of MICA through this poly‐N‐acetyllactosamine. Galectin‐3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT‐expressing bladder tumour cells, resulting in tumour metastasis.

Clinical and genetic risk factors for posttransplant diabetes mellitus in adult renal transplant recipients treated with tacrolimus.

Background. The present study investigated the incidence of posttransplant diabetes mellitus (PTDM) and calculated the risk of developing PTDM under a tacrolimus-based immunosuppression based on clinical characteristics, tacrolimus pharmacokinetics, and genetic polymorphisms related to tacrolimus pharmacokinetics or diabetes mellitus. Methods. Seventy nondiabetic adult kidney recipients were studied. Patients with continuous high plasma glucose levels, over 6.5 mg/dl of hemoglobin A1c, or requiring insulin and/or oral antidiabetic agents for more than 3 months after transplantation 6 months postoperatively were diagnosed as having PTDM. Twelve genomic polymorphisms were assessed. Results. Six months after transplantation, 10 recipients (14.3%) developed PTDM. Positive risk factors were age (P=0.019) and body mass index (P=0.038). There were no significant differences in acute rejection rate, total steroid doses, tacrolimus pharmacokinetics or its related to genetic polymorphisms between the two groups. The frequency of PTDM was significantly higher in patients with the vitamin D receptor (VDR) TaqI t allele than in those with the TT genotype (P=0.013). On multivariate analysis, age over 50 years (odds ratio 9.28, P=0.003) and the presence of the VDR TaqI t allele (odds ratio 7.05, P=0.048) were correlated with the development of PTDM. Conclusion. The incidence of PTDM was 14.3% in our cohort. Age over 50 years was a risk factor. The presence of the VDR TaqI t allele may also be a risk factor for PTDM, suggesting that genotyping of diabetes-related polymorphisms is a possible method of predicting a patients risk for developing PTDM and would be a valuable asset in selecting appropriate immunosuppressive regimens for individuals.

Short-term clinicopathological outcome of neoadjuvant chemohormonal therapy comprising complete androgen blockade, followed by treatment with docetaxel and estramustine phosphate before radical prostatectomy in Japanese patients with high-risk localized prostate cancer

BackgroundTo assess the outcome of neoadjuvant chemohormonal therapy comprising complete androgen blockade followed by treatment with docetaxel and estramustine phosphate before radical prostatectomy in Japanese patients with a high risk of localized prostate cancer (PCa).MethodsComplete androgen blockade followed by 6 cycles of docetaxel (30 mg/m2) with estramustine phosphate (560 mg) were given to 18 PCa patients before radical prostatectomy. Subsequently, the clinical and pathological outcomes were analyzed.ResultsNo patients had severe adverse events during chemohormonal therapy, and hence they were treated with radical prostatectomy. Two patients (11.1%) achieved pathological complete response. Surgical margins were negative in all patients. At a median follow-up of 18 months, 14 patients (77.8%) were disease-free without PSA recurrence. All 4 patients with PSA recurrence had pathologic T3b or T4 disease and 3 of these 4 patients had pathologic N1 disease.ConclusionWe found that neoadjuvant chemohormonal therapy with complete androgen blockade followed by treatment with docetaxel and estramustine phosphate before radical prostatectomy was safe, feasible, and associated with favorable pathological outcomes in patients with a high risk of localized PCa.

Prognostic significance of HIF-1α polymorphisms in transitional cell carcinoma of the bladder

Recently, two single nucleotide polymorphisms in the hypoxia‐inducible factor‐1α (HIF‐1α) gene, P582S and A588T, were shown to cause significantly higher transcriptional activity than the wild type. We investigated the association between the HIF‐1α polymorphisms and the incidence and progression of transitional cell carcinoma of the bladder, and the relationship between the polymorphisms and the tissue vascular endothelial growth factor (VEGF) level or microvessel density (MVD). A total of 219 patients with bladder cancer and 464 healthy native Japanese control subjects were enrolled. Tissue VEGF and HIF‐1α expression levels and the mean MVD were evaluated in 73 radical cystectomy specimens by immunohistochemistry. The HIF‐1α genotype did not significantly influence the incidence or disease status of bladder cancer. Among patients who underwent radical cystectomy, those with a variant allele had significantly worse disease‐free survival (p = 0.001) and disease‐specific survival (p = 0.006) than those without a variant allele. Multivariate analysis using a Cox proportional hazard model revealed that the presence of a variant allele was an independent predictor of disease‐free survival (HR = 3.10, 95%CI = 1.38–6.99, p = 0.006). Although not statistically significant, the moderate/high expression levels of VEGF in tumor tissues were more frequently observed in patients with a HIF‐1α variant allele (11/13, 84.6%) than in those without (33/60, 55%, p = 0.063). The HIF‐1α polymorphisms may have a significant influence on the poor prognosis of the patients undergoing radical cystectomy for bladder cancer, while they seem to have no relation to the bladder cancer occurrence.

Overexpression of Fn14 promotes androgen-independent prostate cancer progression through MMP-9 and correlates with poor treatment outcome

Fibroblast growth factor-inducible 14 (Fn14), a transmembrane receptor binding to the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is known to modulate many cellular activities including cancer progression. Here, we demonstrated the significant role of Fn14 in invasion, migration and proliferation of androgen-independent prostate cancer (AIPC) cells. Fn14 and its ligand TWEAK were highly expressed in two AIPC cell lines, DU 145 and PC-3, whereas expression was weak in androgen-sensitive LNCaP cells. Fn14 knockdown using small-interfering RNAs attenuated migration, invasion and proliferation and enhanced apoptosis in the AIPC cell lines. Both forced overexpression of Fn14 by stable Fn14 complementary DNA transfection to PC-3 cells (PC-3/Fn14) and ligand activation by recombinant TWEAK in PC-3 cells enhanced invasion. Fn14 was shown to modulate expression of matrix metalloproteinase (MMP)-9, and MMP-9 mediated the invasive potential influenced by Fn14 in PC-3 cells. In vivo, subcutaneous xenografts of PC-3/Fn14 grew significantly faster than xenograft of PC-3/Mock, and the invasive capacity in PC-3/Fn14 was found to be higher than that of PC-3/Mock as evaluated in an invasion model of the diaphragm. Furthermore, the messenger RNA expressions of MMP-9 in PC-3/Fn14 xenografts were significantly higher than those in PC-3/Mock xenografts. Clinically, high expression of Fn14 was significantly associated with higher prostate-specific antigen recurrence rate in patients who underwent radical prostatectomy. In conclusion, the overexpression of Fn14 may contribute to multiple malignant cellular phenotypes associated with prostate cancer (PCa) progression, in part via MMP-9. TWEAK-Fn14 signaling may be a novel therapeutic target of PCa.

N-Acetylglucosaminyltransferase V and β1-6 Branching N-Linked Oligosaccharides Are Associated with Good Prognosis of Patients with Bladder Cancer

Purpose:N-acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes β1-6 branching of N-acetylglucosamine on asparagine (N)-linked oligosaccharides (N-glycan) of cell proteins. We examined the relationship between GnT-V expression and clinicopathologic features of the patients with bladder cancer. Experimental Design: We immunohistochemically examined GnT-V expression in paraffin-embedded bladder cancer specimen using anti-GnT-V monoclonal antibody. We compared GnT-V expression with cause-specific survival of the patients with bladder cancer treated by radical cystectomy. Kaplan-Meier survival curves were generated to show the cause-specific survival. Univariate and multivariate analyses were carried out to compare GnT-V expression with other clinical and pathologic variables. We also evaluated mRNA expression of GnT-V and N-linked oligosaccharide structure in bladder cancer specimens. Results: Immunohistochemistry revealed that GnT-V expression inversely correlated with tumor grade and stage. The incidence of positive GnT-V expression in bladder cancer was significantly higher in low-grade/superficial cancer than in high-grade/invasive cancer. The patients whose tumor was positive for GnT-V survived significantly longer than those whose tumor was negative for GnT-V. Univariate and multivariate analyses revealed that GnT-V expression was an independent predictor of prognosis of the patient. The expression of GnT-V mRNA determined by reverse transcription-PCR was consistent with the results with immunohistochemistry for tumor samples. Carbohydrate structural analysis revealed that superficial bladder cancer is rich in branched N-linked oligosaccharides, for which biosynthesis GnT-V is responsible. Conclusions: GnT-V and its resultant β1-6 branching N-linked oligosaccharides are closely related to low malignant potential and good prognosis of the patients with bladder cancer.

A high‐fat diet enhances proliferation of prostate cancer cells and activates MCP‐1/CCR2 signaling

Dietary patterns including high‐fat diet (HFD) and high‐carbohydrate diet (HCD) play an important role in prostate cancer progression. However, which of these diets have the greatest effect on tumor progression and its underlying mechanisms remains unclear.

Polymorphisms of fibroblast growth factor receptor 4 have association with the development of prostate cancer and benign prostatic hyperplasia and the progression of prostate cancer in a Japanese population

Fibroblast growth factor receptor 4 (FGFR4) is a member of a family of transmembrane receptors with ligand‐induced tyrosine kinase activity. The Glycine (Gly) to Arginine (Arg) polymorphism at codon 388 (Gly388Arg), which encodes an amino acid in the transmembrane part of the FGFR4 gene, was reported to be associated with an increased risk in some carcinomas. We investigated the association between the Gly388Arg polymorphism or the G or A polymorphism at intron 11 (rs2011077) of FGFR4, which was located 1,213 base pairs apart from the Gly388Arg polymorphism, and the risk of prostate cancer or benign prostate hyperplasia (BPH), and the prostate cancer disease status in Japanese men. Genotypes of Gly388Arg and rs2011077 polymorphisms of FGFR4 were determined in 492 patients with prostate cancer, 165 patients with BPH and 179 male controls. Regarding the Gly388Arg polymorphism, individuals with the ArgArg genotype had a 2.207‐ and 1.958‐fold increased risk of prostate cancer and BPH, and a 1.804‐fold increased risk of metastatic prostate cancer compared with those with the GlyGly genotype. Regarding the rs2011077 polymorphism, individuals with the GG genotype had a 6.260‐ and 3.033‐fold increased risk of prostate cancer and BPH, and a 5.550‐fold increased risk of metastatic prostate cancer compared with those with the AA genotype. Our results indicate that the FGFR4 Arg allele of the Gly388Arg polymorphism and the G allele of the rs2011077 polymorphism have a significant impact on the development of prostate cancer and BPH, and the progression of prostate cancer in a Japanese population.