Bladder cancer is a global malignancy characterized by high recurrence rate and substantial tumor heterogeneity at genomic, transcriptional, and cellular levels (1). Frequent resistance to conventional and targeted therapies highlights the requirement for developing novel therapeutic options for bladder cancer patients.

As a key feature of the disease, bladder cancer cells harbor a highly enriched mutational load of genes associated with chromatin organization, especially those that oppose Polycomb-mediated repression (2-4). EZH2, the catalytic subunit of Polycomb repressive complex 2 (PRC2), functions as a transcriptional repressor through the deposition of H3K27me3 (5). The investigations have highlighted the role of EZH2 in bladder cancer by promoting invasion and metastasis and ultimately resulting in a worse prognosis (6-7). Consequently, EZH2 is portrayed as a potential therapeutic target for bladder cancer. However, the striking heterogeneity of bladder cancer indicates that targeting a single molecular pathway is unlikely to tackle the underlying mechanisms of molecular and cellular complexity. The retinoic acid derivatives, retinoids, have been recognized as tumor-suppressive and chemopreventive agents due to their central roles in apoptosis and differentiation (8–9). Furthermore, PRC2 is an important regulator of the retinoic acid signaling pathway (10). Thus, in this research, we aimed to utilize the actions of simultaneous activation of the retinoic acid pathway and EZH2 inhibition and hence potentiate the therapeutic response in bladder cancer. We applied a combinatorial strategy utilizing a specialized and potent EZH2 inhibitor, GSK-126, and a retinoid, fenretinide, in muscle-invasive bladder cancer (MIBC) cell lines.

We initiated the study by identifying the potential activity of drugs against MIBC. Next, we investigated the cooperation between retinoid activity and EZH2 inhibition. Drug combination analysis, as measured by the Bliss score, reflected the potent synergistic relationship between fenretinide and GSK-126 to decrease MIBC cell viability. Then, we conducted a series of molecular analyses to define the phenotypic changes related to the combination treatment. Co-treatment of drugs significantly induced apoptosis and reduced migratory capacity compared with single drugs. Furthermore, these two drugs cooperate to regulate the expression of a subset of genes critical for cell proliferation, apoptosis, and cell cycle processes. Collectively, these findings emphasized that EZH2 inhibition acts synergistically with retinoic acid pathway activation to stimulate the anti-oncogenic profile of MIBC.

To identify genome-wide effects on gene regulation induced by drugs, we performed RNA-seq. The gene ontology analysis revealed that the genes upregulated by combination treatment were mainly involved in apoptosis, unfolded protein response (UPR), and the endoplasmic reticulum (ER) stress response pathways. We hypothesized that the anti-oncogenic effects of drug combination might be generated through the activation of new transcriptional programs in the absence of EZH2-mediated repression. To test this theory, we proceeded with the characterization of the alterations in the H3K27me3 profile induced by drugs at the genome-wide level by performing ChIP-seq. Remarkably, ChIP-seq analysis showed that treatment with GSK-126 alone or in combination with fenretinide resulted in a decreased level of the repressive H3K27me3 chromatin mark at the promotor regions of genes involved in UPR and ER stress processes, emphasizing the role of GSK-126 in driving the UPR signature. Collectively, our findings demonstrated that combination treatment-enhanced genes, largely consisting of UPR-related genes, are directly targeted by EZH2-mediated H3K27me3 in MIBC, supported by robust changes in gene expression profiles.

We proceeded to identify potential transcription factors involved in the activation of anti-oncogenic transcriptional programs and the regulation of distinct classes of differentially expressed genes stimulated by drug combination. In this respect, the EnrichR database query unveiled the importance of the transcription factor CHOP (C/EBP homologous protein) for the genes upregulated by drug combination. The perturbations in ER homeostasis, including the accumulation of unfolded or misfolded proteins, cause the UPR (11). The prolonged stress causes apoptotic signals in this process. CHOP is one of the components of the ER stress-mediated apoptosis pathway (12). Thus, identification of CHOP as a key regulator of combinatorial treatment response was totally in line with our findings.

Determining CHOP as a key candidate transcription factor associated with the UPR and ER-stress responses inspired us to further strengthen the relevance of this factor in the anti-oncogenic programs induced by drug combination in MIBC cells. Knockdown analysis confirmed that CHOP depletion decreased the expression levels of the genes involved in the UPR, ER stress, and regulation of the apoptotic signaling pathways and increased the expression level of the anti-apoptotic gene BCL-2. All these findings point out that simultaneous activation of the retinoic acid pathway and EZH2 inhibition activates CHOP, ultimately stimulating ER stress-mediated anti-oncogenic transcriptional programs.

Collectively, in this study, we provide important insights revealing that EZH2 inhibition in MIBC tumors cooperates with retinoids, causing activation of a network of genes providing anti-oncogenic properties. Intriguingly, the network of co-regulatory factors recruited by RA activation and EZH2 inhibition cooperation has a principal role in response to ER stress. The crucial direction of our results is the positioning of CHOP as a unique downstream target of the EZH2/RA pathway interaction, which may reflect a molecular circuitry supporting apoptotic reprogramming. Our findings suggest that CHOP stimulates anti-oncogenic transcriptional reprogramming together with EZH2 inhibition, potentially reassembling accessible chromatin profiles in MIBC cells. The output of EZH2-mediated transcriptional rerouting of CHOP results in increased apoptosis and may thereby contribute to an improvement in susceptibility to treatment for bladder cancer.

Overall, our findings suggest a concerted mechanism between activation of the retinoic acid pathway and EZH2-mediated epigenetic signatures. Our data highlight the potential of RA/EZH2 targeting for reflection of the anti-oncogenic profiles. Consequently, our strategy appears to be a rational approach for improving the therapeutic efficacy in bladder cancer and contributes to targeted treatment paradigms.

References

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