Sex and gender disparities in bladder cancer have long been a subject of interest to the cancer research community, wherein men have a 4 times higher incidence rate than women, and female patients often present with higher-grade disease and experience worse outcomes. Despite the known differences in disease incidence and clinical outcomes between male and female bladder cancer patients, clinical management remains the same. In this review, we critically analyze studies that report on the biological differences between men and women and evaluate how these differences contribute to sex and gender disparities in bladder cancer. Distinct characteristics of the male and female immune systems, differences in circulating hormone levels and hormone receptor expression, and different genetic and epigenetic alterations are major biological factors that all likely contribute to disparate incidence rates and outcomes for male and female bladder cancer patients. Future preclinical and clinical studies in this area should employ experimental approaches that account for and consider sex and gender disparities in bladder cancer, thereby facilitating the development of precision medicine for the effective treatment of bladder cancer in all patients.

Lysine-specific demethylase 6A (KDM6A) and members of the Switch/Sucrose Non-Fermentable (SWI/SNF) family are known to counteract the activity of Enhancer of Zeste Homolog 2 (EZH2), which is often overexpressed and is associated with poor prognosis in muscle-invasive bladder cancer. Here we provide evidence that alterations in chromatin modifying enzymes, including KDM6A and members of the SWI/SNF complex, are frequent in muscle-invasive bladder cancer. We exploit the loss of function mutations in KDM6A and SWI/SNF complex to make bladder cancer cells susceptible to EZH2-based epigenetic therapy that activates an immune response to drive tumor cell differentiation and death. We reveal a novel mechanism of action of EZH2 inhibition, alone and in combination with cisplatin, which induces immune signaling with the largest changes observed in interferon gamma (IFN-γ). This upregulation is a result of activated natural killer (NK) signaling as demonstrated by the increase in NK cell-associated genes MIP-1α, ICAM1, ICAM2, and CD86 in xenografts treated with EZH2 inhibitors. Conversely, EZH2 inhibition results in decreased expression of pluripotency markers, ALDH2 and CK5, and increased cell death. Our results reveal a novel sensitivity of muscle-invasive bladder cancer cells with KMD6A and SWI/SNF mutations to EZH2 inhibition alone and in combination with cisplatin. This sensitivity is mediated through increased NK cell-related signaling resulting in tumor cell differentiation and cell death.

Aberrant DNA methylation observed in cancer can provide survival benefits to cells by silencing genes essential for anti-tumor activity. DNA-demethylating agents such as Decitabine (DAC)/Azacitidine (AZA) activate otherwise silenced tumor suppressor genes, alter immune response and epigenetically reprogram tumor cells. In this study, we show that non-cytotoxic nanomolar DAC concentrations modify the bladder cancer transcriptome to activate NOTCH1 at the mRNA and protein level, increase double-stranded RNA sensors and CK5-dependent differentiation. Importantly, DAC treatment increases ICN1 expression (the active intracellular domain of NOTCH1) significantly inhibiting cell proliferation and causing changes in cell size inducing morphological alterations reminiscent of senescence. These changes were not associated with β-galactosidase activity or increased p16 levels, but instead were associated with substantial IL-6 release. Increased IL-6 release was observed in both DAC-treated and ICN1 overexpressing cells as compared to control cells. Exogenous IL-6 expression was associated with a similar enlarged cell morphology that was rescued by the addition of a monoclonal antibody against IL-6. Treatment with DAC, overexpression with ICN1 or addition of exogenous IL-6 showed CK5 reduction, a surrogate marker of differentiation. Overall this study suggests that in MIBC cells, DNA hypomethylation increases NOTCH1 expression and IL-6 release to induce CK5-related differentiation.

African Americans (AAs) have lower circulating 25-hydroxyvitamin D3 [25(OH)D3] concentrations and higher prostate cancer (CaP) aggressiveness than other racial/ethnic groups. The purpose of the current study was to examine the relationship between plasma 25(OH)D3, African ancestry and CaP aggressiveness among AAs and European Americans (EAs). Plasma 25(OH)D3 was measured using LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) in 537 AA and 663 EA newly-diagnosed CaP patients from the North Carolina-Louisiana Prostate Cancer Project (PCaP) classified as having either ‘high’ or ‘low’ aggressive disease based on clinical stage, Gleason grade and prostate specific antigen at diagnosis. Mean plasma 25(OH)D3 concentrations were compared by proportion of African ancestry. Logistic regression was used to calculate multivariable adjusted odds ratios (OR) and 95% confidence intervals (95%CI) for high aggressive CaP by tertile of plasma 25(OH)D3. AAs with highest percent African ancestry (>95%) had the lowest mean plasma 25(OH)D3 concentrations. Overall, plasma 25(OH)D3 was associated positively with aggressiveness among AA men, an association that was modified by calcium intake (OR_T3vs.T1: 2.23, 95%CI: 1.26–3.95 among men with low calcium intake, and OR_T3vs.T1: 0.19, 95%CI: 0.05–0.70 among men with high calcium intake). Among EAs, the point estimates of the ORs were <1.0 for the upper tertiles with CIs that included the null. Among AAs, plasma 25(OH)D3 was associated positively with CaP aggressiveness among men with low calcium intake and inversely among men with high calcium intake. The clinical significance of circulating concentrations of 25(OH)D3 and interactions with calcium intake in the AA population warrants further study.

Background: African‐American (AA) men tend to present with more aggressive prostate cancer (Gleason score >7) than European‐American (EA) men. Vitamin D and its metabolites are implicated in prostate cancer biology with vitamin D deficiency, indicated by its metabolite levels in serum or plasma, usually observed in AA men.Objective: To determine if 1, 25‐dihydroxy vitamin D3 [1,25(OH)₂D] plasma levels in AA and EA prostate cancer patients alter the risk of having aggressive prostate cancer.Design: Research subjects from the North Carolina‐Louisiana Prostate Cancer Project (AA n = 435 and EA n = 532) were included. Plasma metabolites 1,25(OH)₂D and 25‐hydroxyvitamin D3 [25(OH)D] were measured using liquid chromatography with tandem mass spectrophotometry. Research subjects were classified into low (Gleason sum < 7, stage T1‐T2, and Prostate‐specific antigen (PSA) < 9 ng/mL) or high (Gleason sum > 8 or Gleason sum = 7 with 4 + 3, or PSA > 20 ng/mL, or Gleason sum = 7 and stage T3‐T4) aggressive disease.Results: Research subjects in the second and third tertiles of plasma levels of 1, 25(OH)₂D had lower odds of high aggressive prostate cancer (AA [OR_T2vsT1: 0.66, 95%CI: 0.39‐1.12; OR_T3vsT1: 0.83, 95%CI: 0.49‐1.41] and EA [OR_T2vsT1: 0.68, 95%CI: 0.41‐1.11; OR_T3vsT1: 0.67, 95%CI: 0.40‐1.11]) compared with the first tertile, though confidence intervals included the null. Greater 1,25(OH)₂D/25(OH)D molar ratios were associated with lower odds of high aggressive prostate cancer more evidently in AA (OR_Q4vsQ1: 0.45, CI: 0.24‐0.82) than in EA (OR_Q4vsQ1: 0.64, CI: 0.35‐1.17) research subjects.Conclusions: The 1,25(OH)₂D/25(OH)D molar ratio was associated with decreased risk of high aggressive prostate cancer in AA men, and possibly in EA men. Further studies analyzing vitamin D polymorphisms, vitamin D binding protein levels, and prostatic levels of these metabolites may be useful. These studies may provide a better understanding of the vitamin D pathway and its biological role underlying health disparities in prostate cancer.