Estudio preclínico de potenciales estrategias terapéuticas basadas en la epigenética para el cancer de vejiga

Abstract

Bladder cancer (BC) represents a major clinical problem due to its incidence, prevalence, recurrence and progression in recurrences. The recurrences account for a high mortality rates and also contributes to the development of major comorbidities and increasing costs to health systems. Although many of the oncogenic events leading to BC are known, their significance is still under debate. Previous data from our group pointed towards an involvement of EZH2- dependent signaling in recurrence and progression of BC, whereas the frequent alterations of PIK3CA gene (amplification and/or mutations) were predictive of better clinical outcome. In the present work we demonstrated a functional interaction between EZH2 and PIK3CA-dependent signaling pathways by transcriptional and post-transcriptional mechanisms. Using clinical samples and bladder cancer cell lines, we found that PIK3CA alterations opposed to EZH2 signaling through the induction of two microRNAs, miR-101 and miR-138, which posttranscriptionally downregulate EZH2 expression. In addition, PIK3CA gene alterations facilitate the activation of AKT, which phosphorylates EZH2 on Ser21, avoiding the catalytic activity of EZH2 to trimethylated the histone 3 in lysine 27 (H3K27me3). In agreement with these findings, the increased expression of miR-101 or miR-138, and the expression of Ser21-phosphorylated EZH2 are good prognostic factors regarding recurrence and progression of BC. Based on the recent discoveries regarding the role of chromatin remodeling in BC pathogenesis (including EZH2-dependent processes), we also aimed to analyze their use as target for BC therapy. Since we observed that the augmented expression of G9a, which dimethylates the histone 3 in lysine 9 (H3K9me2) and displays cooperative roles with EZH2 and DNMTs, is associated with a poor clinical outcome, we have evaluated the potential therapeutic effect of a novel dual inhibitor of G9a/DNMT (CM-272). We found that this inhibitor induced cell cycle arrest, apoptosis, and immunogenic cell death in BC cells bearing wt PIK3CA gene through a process partially dependent on EZH2 expression or activity. In addition, this agent showed synergy in vivo and in vitro with Cisplatin (CDDP), the standard of care for advanced BC patients. The treatment with CM-272 in combination with CDDP improved survival in an immunocompetent mouse model of metastatic aggressive BC. This model has been generated by the specific ablation of Rb1, Pten and TrP53 tumor suppressor genes in the basal urothelial cells of the bladder of Rbl1-null background mice. Histological and molecular features of the tumors developed in the mouse model, resembled basal-squamous molecular subtype of human BC. The combined treatment of the transgenic mice with CM-272 and CDDP resulted in a significant tumor and metastasis regression, without major effects in major oncogenic pathways such as ERK, AKT or STAT3, which are permanently activated in the model. Based on transcriptional and in silico analyses, we uncovered a novel process in which the inhibitor induced an endogenous retroviral response leading to interferon γ signaling and the induction of activating NK cell ligands in tumor cells. This produces the immune infiltration of tumors and metastasis, and the immune cell-mediated debulking of tumors and metastasis, thus representing a novel and potential new avenue in advanced BC management. Remarkably, we also observed that the absence of clinical response in a series of advanced urothelial cancer patients treated with a PD-1 inhibitor as a first line of treatment, is associated with increased levels of G9a, EZH2 and their respective methylation marks H3K9me2 and H3K27me3. These observations indicate that G9a and/or EZH2 expression could be potential candidate biomarkersof immunotherapy response, and their inhibition may increase the efficacy of immune checkpoint inhibition- based therapies. In summary, our results pointed to key molecular processes clearly involved in BC progression that should be considered in the management of patients, and in the design of novel targeted therapeutic approaches. Our findings support new and promising opportunities for epigenetic therapy in patients with BC.