Cystoscopy and the development of invasive bladder cancer in patients presenting with superficial urinary bladder cancer

The objective of this study was to determine the impact of different follow-up cystoscopy frequencies on time to development of invasive bladder cancer in a cohort of 3,658 eligible patients 65 and older with an initial diagnosis of superficial bladder cancer between 1994 and 1998. Bladder cancer patients in the Surveillance, Epidemiology, and End Results (SEER)-Medicare database were used as the study population. ^ It was hypothesized that superficial bladder cancer patients receiving less frequent cystoscopy follow-up would develop invasive bladder cancer sooner after initial diagnosis and treatment than patients seen more frequently for cystoscopy follow-up. Cox Proportional Hazard Regression revealed that patients seen for cystoscopy every 3 or more months were 83–89% less likely to develop invasive cancer than patients seen every 1 to 2 months. A comparison of the 2 groups (1 to 2 months vs. 3≥ months) revealed that the 1 to 2 month group may have had more aggressive disease, and they are seen more frequently as a result. ^ These findings suggest that there are two groups of superficial bladder cancer patients: those at high risk of developing invasive bladder cancer and those at low risk. Patients who developed invasive bladder cancer sooner after initial diagnosis and treatment were seen more frequently for cystoscopy follow-up. The recommendation is that cystoscopy should be based on disease status at 3 months. Standardized schedules give all patients the same number of cystoscopies regardless of their risk factors. This could lead to unnecessary cystoscopies in low risk patients, and fewer than optimal cystoscopies in high risk patients. ^

Genetic predictors of bladder cancer with an emphasis on methylation-related genes

Although tobacco exposure remains the prevailing risk factor for bladder cancer (BC), only a small percentage of exposed individuals develop cancer, suggesting that tobacco-related carcinogenesis is modulated by genetic susceptibility and possibly by DNA methylation-related events. Methylation patterns established by DNA methyltransferases (DNMTs) are influenced by dietary folate and genetic polymorphisms in the methylene-tetrahydrofolate reductase gene (MTHFR). Therefore, we hypothesized that DNA methylation-related genes, such as DNMT3B and MTHFR, might modulate BC risk. ^ In a study of 514 Caucasian BC cases and 498 healthy Caucasian controls examining the DNMT3B C46359T polymorphism, CC genotype was found to be a risk factor in women (Odds Ratio (OR) = 1.79), but not in men. This risk was further increased among women who were never smokers, consumed low dietary folate, and had adverse variants of MTHFR. In addition, higher DNMT3B expression among smokers was a risk factor (OR = 4.27) and correlated with genetic variants of the DNMT3B C46359T polymorphism, providing salient evidence for the risk associated with the CC variant. This suggests that the DNMT3B CC variant may confer a predisposition toward aberrant de novo methylation of CpG islands in critical tumor suppressor genes. ^ The convergence of alterations in DNMT3B, associated with promoter methylation, and reduced dietary folate consumption, accompanying global hypomethylation and genetic instability, may act synergistically to promote bladder carcinogenesis, especially in women. The results of this study unveiled new gender-specific paradigms of BC risk for women and demonstrated that this risk can be modified by folate consumption as well as polymorphisms in the folate pathway. ^

Bortezomib modulates TRAIL sensitivity in human bladder and prostate cancer

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the TNF superfamily of cytokines that can induce cell death through engagement of cognate death receptors. Unlike other death receptor ligands, it selectively kills tumor cells while sparing normal cells. Preclinical studies in non-human primates have generated much enthusiasm regarding its therapeutic potential. However, many human cancer cell lines exhibit significant resistance to TRAIL-induced apoptosis, and the molecular mechanisms underling this are controversial. Possible explanations are typically cell-type dependent, but include alterations of receptor expression, enhancement of pro-apoptotic intracellular signaling molecules, and reductions in anti-apoptotic proteins. We show here that the proteasome inhibitor bortezomib (Velcade, PS-341) produces synergistic apoptosis in both bladder and prostate cancer cell lines within 4-6 hours when co-treated with recombinant human TRAIL which is associated with accumulation of p21 and cdk1/2 inhibition. Our data suggest that bortezomib's mechanism of action involves a p21-dependent enhancement of caspase maturation. Furthermore, we found enhanced tumor cell death in in vivo models using athymic nude mice. This is associated with increases in caspase-8 and caspase-3 cleavage as well as significant reductions in microvessel density (MVD) and proliferation. Although TRAIL alone had less of an effect, its biological significance as a single agent requires further investigations. Toxicity studies reveal that the combination of bortezomib and rhTRAIL has fatal consequences that can be circumvented by altering treatment schedules. Based on our findings, we conclude that this strategy has significant therapeutic potential as an anti-cancer agent. ^

Mechanism of IFN-induced apoptosis and sensitization by the proteasome inhibitor bortezomib in bladder cancer cells

Bladder cancer is the fifth most common cancer with more than 50,000 cases diagnosed each year. Interferon-α (IFNα) is mostly used in combination with BCG for the treatment of transitional cell carcinoma (TCC). To examine the effects of IFNα on bladder cancer cells, I analyzed a panel of 20 bladder cancer cell lines in terms of their sensitivity to IFNα-induced apoptosis and the underlying mechanisms. I identified three categories: cells that die after 48hr, after 72h, and cells resistant even after 72hr of IFNα treatment. Examination of the IFN-signal transduction pathway revealed that the defect was not due to abrogation of IFN signaling. Further analysis demonstrated dependency of IFN-induced apoptosis on caspase-8, implicating the role of death receptors in IFN-induced cell death. Of the six most-IFN-sensitive cell lines, the majority upregulated Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at the mRNA and protein level and IFN-induced cell death was mediated through TRAIL, while a minority of the most IFN-sensitive cells undergo apoptosis through a TNFα-dependent mechanism. IFNα resistance was due to either absence of TRAIL upregulation at the mRNA or protein level, resistance to exogenous rhTRAIL itself or lack of sensitization to IFN-induced cell death. Downregulation of XIAP, or XIAP inactivation through its regulator NFκB has been reported to sensitize tumor cells to death receptor-induced cell death. Baseline and IFN-inducible XIAP levels were examined in the most and least IFN-sensitive cells, knocking down XIAP and the p65 subunit of NFκB enhanced IFN-induced cell death, implicating XIAP downregulation as a mechanism through which bladder cancer cells are sensitized to IFN-induced apoptosis. To determine whether or not the proteasome inhibitor Bortezomib (BZ) sensitizes bladder cancer cells to IFN-induced cell death, the combined effects of IFN+BZ and the underlying molecular mechanisms were examined both in vitro and in vivo using two bladder xenograft models. In both models, tumor growth inhibition was the result of either increased cell death of tumor cells exerted by the two agents and/or inhibition of angiogenesis. In vitro, MAP downregulation in response to the combined treatment of IFN+BZ accounts for one of the mechanisms mediating IFN+BZ cell death in bladder cancer cells. ^

Genetic variations in the PI3K-AKT-mTOR pathway and bladder cancer susceptibility and clinical outcomes

Bladder cancer is the fourth most common cancer in men in the United States. There is compelling evidence supporting that genetic variations contribute to the risk and outcomes of bladder cancer. The PI3K-AKT-mTOR pathway is a major cellular pathway involved in proliferation, invasion, inflammation, tumorigenesis, and drug response. Somatic aberrations of PI3K-AKT-mTOR pathway are frequent events in several cancers including bladder cancer; however, no studies have investigated the role of germline genetic variations in this pathway in bladder cancer. In this project, we used a large case control study to evaluate the associations of a comprehensive catalogue of SNPs in this pathway with bladder cancer risk and outcomes. Three SNPs in RAPTOR were significantly associated with susceptibility: rs11653499 (OR: 1.79, 95%CI: 1.24–2.60), rs7211818 (OR: 2.13, 95%CI: 1.35–3.36), and rs7212142 (OR: 1.57, 95%CI: 1.19–2.07). Two haplotypes constructed from these 3 SNPs were also associated with bladder cancer risk. In combined analysis, a significant trend was observed for increased risk with an increase in the number of unfavorable genotypes (P for trend<0.001). Classification and regression tree analysis identified potential gene-environment interactions between RPS6KA5 rs11653499 and smoking. In superficial bladder cancer, we found that PTEN rs1234219 and rs11202600, TSC1 rs7040593, RAPTOR rs901065, and PIK3R1 rs251404 were significantly associated with recurrence in patients receiving BCG. In muscle invasive and metastatic bladder cancer, AKT2 rs3730050, PIK3R1 rs10515074, and RAPTOR rs9906827 were associated with survival. Survival tree analysis revealed potential gene-gene interactions: patients carrying the unfavorable genotypes of PTEN rs1234219 and TSC1 rs704059 exhibited a 5.24-fold (95% CI: 2.44–11.24) increased risk of recurrence. In combined analysis, with the increasing number of unfavorable genotypes, there was a significant trend of higher risk of recurrence and death (P for trend<0.001) in Cox proportional hazard regression analysis, and shorter event (recurrence and death) free survival in Kaplan-Meier estimates (P log rank<0.001). This study strongly suggests that genetic variations in PI3K-AKT-mTOR pathway play an important role in bladder cancer development. The identified SNPs, if validated in further studies, may become valuable biomarkers in assessing an individual's cancer risk, predicting prognosis and treatment response, and facilitating physicians to make individualized treatment decisions. ^

Understanding treatment-related symptoms, symptom management techniques, and symptom management self-efficacy in a sample of non-muscle invasive bladder cancer patients

The current literature available on bladder cancer symptom management from the perspective of the patients themselves is limited. There is also limited psychosocial research specific to bladder cancer patients and no previous studies have developed and validated measures for bladder cancer patients’ symptom management self-efficacy. The purpose of this study was to investigate non-muscle invasive bladder cancer patients’ health related quality of life through two main study objectives: (1) to describe the treatment related symptoms, reported effectiveness of symptom-management techniques, and the advice a sample of non-muscle invasive bladder cancer patients would convey to physicians and future patients; and (2) to evaluate Lepore’s symptom management self-efficacy measure on a sample of non-muscle invasive bladder cancer patients. Methods. A total of twelve (n=12) non-muscle invasive bladder cancer patients participated in an in-depth interview and a sample of 46 (n=4) non-muscle invasive bladder cancer patients participated in the symptom-management self-efficacy survey. Results. A total of five symptom categories emerged for the participants’ 59 reported symptoms. Four symptom management categories emerged out of the 71 reported techniques. A total of 62% of the participants’ treatment related symptom-management techniques were reported as effective in managing their treatment-related symptoms. Five advice categories emerged out of the in-depth interviews: service delivery; medical advice; physician-patient communication; encouragement; and no advice. An exploratory factor analysis indicated a single-factor structure for the total population and a multiple factor structure for three subgroups: all males, married males, and all married participants. Conclusion. These findings can inform physicians and patients of effective symptom-management techniques thus improving patients’ health-related quality of life. The advice these patients’ impart can improve service-delivery and patient education.^

Genetic variants within the Wnt/ B-catenin signaling pathway as indicators of bladder cancer clinical outcomes

The genetic factors that influence bladder cancer clinical outcomes are largely unknown. In this clinical outcomes study, I assessed genetic variations in the Wnt/β-catenin stem-cell pathway genes for association with recurrence and progression. A total of 230 SNPS in 40 genes from the Wnt/β-catenin pathway were genotyped in 419 histologically confirmed non-muscle invasive bladder cancer cases. Several significant associations were observed in the clinical outcomes analysis. Under the dominant model WNT8B: rs4919464 (HR: 1.55, 95% CI: 1.17-2.06, P=2.2x10-3) and WNT8B: rs3793771 (HR: 1.54, 95% CI: 1.09-1.62, P=4.6x10-3 ) were statistically significantly associated with an increase risk of recurrence while two other variants, APC2: rs11668593 (HR: 2.50, 95% CI: 1.43-4.35, P=1.2x10-3) and LRP5 : rs312778 (HR: 1.81, 95% CI: 1.23-2.65, P=2.7x10-3), were significantly associated with recurrence risk under the recessive model of inheritance. Four SNPs in the recessive model were associated with an increased risk of progression (AXIN2: rs1544427, LRP5: rs312778, AXIN1: rs370681, AXIN1: rs2301522). LRP5: rs312778 had the most significant increased risk of progression with a 2.68 (95% CI: 1.52-4.72, P=6.4x10-4)-fold increased risk. Stratification analysis based on treatment regimen (transurethral resection (TUR) and Bacillus Calmette-Guérin (BCG)) was also performed. Individuals with at least one variant in AXIN2: rs2007085 were found to have a 2.09 (95% CI: 1.24-3.52, P=5.4x10-3) -fold increased risk of recurrence in those that received TUR only, and no statistically significant effect was seen in those that received BCG. Individuals who received TUR with at least one variant in LEF1: rs10516550 were found to have a 2.26 (95% CI: 1.22-4.18, P=9.7x10-3)-fold increase risk of recurrence and no statistically significant effect was found in individuals who received BCG. Also, the recessive model of LRP6: rs2302684 in TUR only treatment was shown to have a 1.95 (95%CI: 1.18-3.21, P=8.8x10 -3)-fold increased risk of recurrence, and a suggested protective effect associated with a (HR: 0.83, 95% CI: 0.51-1.37, P=0.468) decreased risk of recurrence. Together, these findings implicate the Wnt/β-catenin stem-cell pathway as playing a role in bladder cancer clinical outcomes and have important implications for personalization of future treatment regimens. ^

THE p63 ISOFORM ∆Np63α INHIBITS EPITHELIAL – MESENCHYMAL TRANSITION BY PROMOTING THE EXPRESSION OF MIR-205 IN HUMAN BLADDER CANCER CELLS

p63, a p53 family member, is a transcription factor that has complex roles in cancer. This study focuses on the role of the ∆Np63α isoform in bladder cancer (BC). Epithelial – mesenchymal transition (EMT) is a physiological process that plays an important part in metastasis and drug resistance. At the molecular level, EMT is characterized by the loss of the epithelial marker E-cadherin, and the acquisition of the transcriptional repressors of E-cadherin (ZEB1, ZEB2, TWIST, SNAI1 and SNAI2). Recent publications highlight the role of microRNAs belonging to the miR-200 family and miR-205 in preventing EMT through suppression of ZEB1 and ZEB2. p53, the homologue of p63, is implicated in regulating EMT by modulating the expression of miR-200c; however, the mechanisms underlying miR-205 control remain unclear. Here we show that ∆Np63α regulates the transcription of miR-205 and controls EMT in human BC cells. We observed a strong correlation between the expression of ∆Np63α, miR-205 and E-cadherin in a panel of BC cell lines (n=28) and also in bladder primary tumors from a cohort of patients (n=98). A remarkably inverse correlation is observed between ∆Np63α and ZEB1/2 in cell lines. Stable knockdown (KD) ∆Np63α in UC6, an “epithelial” BC cell line, decreased the expression of miR-205 and induced ZEB1/2 expression, the effects that were reversed by expression of exogenous miR-205. Moreover, overexpressing ∆Np63α in UC3, a “messenchymal” BC cell line, brought about opposite results, an increase in miR-205 expression and a reduction in ZEB1/2 expression. Modulation of ∆Np63α expression resulted in a parallel change in the expression of miR-205 and miR-205 “host” gene (miR-205HG). Nuclear run-on and chromatin immunoprecipitation experiments demonstrated that ∆Np63α regulates the transcription of miR-205 through controlling the recruitment of RNA Polymerase II to the promoter of miR-205HG. Interestingly, high miR-205 expression correlated with poor clinical outcome in BC patients, consistent with our recent publication highlighting the enrichment of ∆Np63 in a lethal subset of muscle invasive BC. In summary, our data present the important roles of ∆Np63α in preventing EMT mediated by miR-205. Our study also identifies miR-205 as a potential molecular marker to predict clinical outcome in BC patients.