Background: A cross-talk between different receptor tyrosine kinases (RTKs) plays an important role in the pathogenesis of human cancers.Methods: Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA) silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients.Results: A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α) with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-αin vitrowas through aras- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (p< 0.05), and overexpression of c-Met/Axl/PDGFR-α or c-Met alone showed the most significant correlation with poor survival (p< 0.01).Conclusions: In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy.

Increased urinary tract malignancy has been reported in end-stage renal disease (ESRD). However, little is known about chronic kidney disease (CKD). This study is designed to explore the association between CKD and upper tract urothelial carcinoma (UTUC). Using Taiwan's Longitudinal Health Insurance Database, we studied CKD patients between January 2000 and December 2011. The non-CKD controls were selected at a ratio of 4:1 and frequency matched by gender, age group, and index date. We used the chi-square test and t test to analyze the sociodemographic information and comorbidities. Cox regression analysis was used to calculate the hazard ratio (HR) and 95% confidence interval (CI). The selected cases included 45,321 CKD cases and 181,284 controls. A significantly higher incidence of UTUC was noted in the CKD group (0.22% vs 0.07%, P < 0.001). In univariate analysis, CKD, female gender, age, hypertension, hematuria, repeated urinary tract infection, bladder cancer, and ESRD were all associated with UTUC. In multivariate analysis, only CKD, female gender, age, hematuria, bladder cancer, and ESRD were significantly associated. The HR for CKD was 1.63 (95% CI: 1.26–2.13). Females had a higher HR of 1.38 (95% CI: 1.11–1.71). After excluding those patients who progressed to dialysis or kidney transplantation, the risk for CKD was still high, with an HR of 1.72 (95% CI: 1.33–2.33). CKD is a significant factor associated with UTUC. We should pay attention to the possibility of UTUC for CKD patients before they progress to ESRD.

Background: Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Previous studies have identified several tumor-related genes that are hypermethylated in bladder cancer; however the DNA methylation profile of bladder cancer in Taiwan is not fully understood. Methods: In this study, we compared the DNA methylation profile of multiple tumor suppressor genes (APC, DAPK, E-cadherin, hMLH1, IRF8, p14, p15, RASSF1A, SFRP1 and SOCS-1) in bladder cancer patients from different Chinese sub-populations including Taiwan (104 cases), Hong Kong (82 cases) and China (24 cases) by MSP. Two normal human urothelium were also included as control. To investigate the diagnostic potential of using DNA methylation in non-invasive detection of bladder cancer, degree of methylation of DAPK, IRF8, p14, RASSF1A and SFRP1 was also accessed by quantitative MSP in urine samples from thirty bladder cancer patients and nineteen non-cancer controls. Results: There were distinct DNA methylation epigenotypes among the different sub-populations. Further, samples from Taiwan and China demonstrated a bimodal distribution suggesting that CpG island methylator phentotype (CIMP) is presented in bladder cancer. Moreover, the number of methylated genes in samples from Taiwan and Hong Kong were significantly correlated with histological grade (P < 0.01) and pathological stage (P < 0.01). Regarding the samples from Taiwan, methylation of SFRP1, IRF8, APC and RASSF1A were significantly associated with increased tumor grade, stage. Methylation of RASSF1A was associated with tumor recurrence. Patients with methylation of APC or RASSF1A were also significantly associated with shorter recurrence-free survival. For methylation detection in voided urine samples of cancer patients, the sensitivity and specificity of using any of the methylated genes (IRF8, p14 or sFRP1) by qMSP was 86.7% and 94.7%. Conclusions: Our results indicate that there are distinct methylation epigenotypes among different Chinese sub-populations. These profiles demonstrate gradual increases with cancer progression. Finally, detection of gene methylation in voided urine with these distinct DNA methylation markers is more sensitive than urine cytology.

Our previous study demonstrated that the glutathione S-transferase Mu 5 (GSTM5) gene is highly CpG-methylated in bladder cancer cells and that demethylation by 5-aza-dC activates GSTM5 gene expression. The aim of the present study was to investigate the role of GSTM5 in bladder cancer. The levels of GSTM5 gene expression and DNA methylation were analyzed in patients with bladder cancer, and functional studies of GSTM5 were conducted using GSTM5 overexpression in cultured bladder cancer cells. Clinical analysis revealed that the GSTM5 mRNA expression was lower in bladder cancer tissues than in normal tissues and that the level of GSTM5 DNA methylation was higher in bladder cancer tissues than in normal urine pellets. Overexpression of GSTM5 decreased cell proliferation, migration and colony formation capacity. Glutathione (GSH) assay results indicated that cellular GSH concentration was decreased by GSTM5 expression and that GSH supplementation reversed the decrease in proliferation and migration of cells overexpressing GSTM5. By contrast, a GSH synthesis inhibitor significantly decreased 5637 cell GSH levels, survival and migration. Furthermore, GSTM5 overexpression inhibited the adhesion of cells to the extracellular matrix protein fibronectin. To elucidate the effect of GSTM5 on anticancer drugs used to treat bladder cancer, cellular viability was compared between cells with or without GSTM5 overexpression. GSTM5-overexpressed cells showed no significant change in the cytotoxicity of cisplatin or mitomycin C in 5637, RT4 and BFTC 905 cells. Though a degree of resistance to doxorubicin was noted in 5637 cells overexpressing GSTM5, no such resistance was observed in RT4 and BFTC 905 cells. In summary, GSTM5 plays a tumor suppressor role in bladder cancer cells without significantly affecting chemoresistance to cisplatin and mitomycin C, and the cellular GSH levels highlight a key mechanism underlying the cancer inhibition effect of GSTM5. These findings suggest that low gene expression and high DNA methylation levels of GSTM5 may act as tumor markers for bladder cancer.

Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter–driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter’s tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP’s gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter–driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk–carrying JCPyV VLPs. In mice injected with pSPB-tk–carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.