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.

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53^wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53^wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53^wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.

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.

Suppressive function of connexin(Cx)43 in carcinogenesis was recently contested by reports that showed a multifaceted function of Cx43 in cancer progression. These studies did not attempt to model the dynamics of intratumoral heterogeneity involved in the metastatic cascade. An unorthodox look at the phenotypic heterogeneity of prostate cancer cells in vitro enabled us to identify links between Cx43 functions and Snail-1-regulated functional speciation of invasive cells. Incomplete Snail-1-dependent phenotypic shifts accounted for the formation of phenotypically stable subclones of AT-2 cells. These subclones showed diverse predilection for invasive behavior. High Snail-1 and Cx43 levels accompanied high motility and nanomechanical elasticity of the fibroblastoid AT-2_Fi2 subclone, which determined its considerable invasiveness. Transforming growth factor-β and ectopic Snail-1 overexpression induced invasiveness and Cx43 expression in epithelioid AT-2 subclones and DU-145 cells. Functional links between Snail-1 function and Cx43 expression were confirmed by Cx43 downregulation and phenotypic shifts in AT-2_Fi2, DU-145 and MAT-LyLu cells upon Snail-1 silencing. Corresponding morphological changes and Snail-1 downregulation were seen upon Cx43 silencing in AT-2_Fi2 cells. This indicates that feedback loops between both proteins regulate cell invasive behavior. We demonstrate that Cx43 may differentially predispose prostate cancer cells for invasion in a coupling-dependent and coupling-independent manner. When extrapolated to in vivo conditions, these data show the complexity of Cx43 functions during the metastatic cascade of prostate cancer. They may explain how Cx43 confers a selective advantage during cooperative invasion of clonally evolving, invasive prostate cancer cell subpopulations.

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement—specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1–3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca²⁺ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca²⁺ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.