Endometrial cancer is the most common gynecologic malignancy in the U.S. Fortunately, many are diagnosed at an early stage and the overall prognosis for women with endometrial cancer is excellent. Salvage treatments such as hormonal agents or cytotoxic chemotherapy, which provide short-term remission, represent the typical treatment strategy for endometrial cancer.
However, patients with recurrent or advanced-stage disease have much poorer clinical outcomes (Gynecol Oncol Res Pract 2017; https://doi.org/10.1186/s40661-017-0056-7). Consequently, there is an urgent need for novel therapeutic options.
To identify new targets, researchers have explored the relevance of epigenetic modifiers in cancer therapeutics. One such potential candidate is enhancer of zeste homolog 2 (EZH2). An important constituent of polycomb repressive complex 2 histone methyltransferase complex, EZH2 mediates epigenetic silencing of tumor suppressor genes. It is commonly overexpressed in several solid tumors and has been shown to be a prognostic biomarker.
Given that its expression is increased in multiple cancers and is critical for pathways that control cellular proliferation, angiogenesis, and survival, EZH2 is an attractive target for drug development in several malignancies, including breast, bladder, prostate, and gastric cancers (J Hematol Oncol 2020; https://doi.org/10.1186/s13045-020-00937-8). This has led to the development of oral EZH2 inhibitors that have demonstrated activity in early clinical trials with a favorable safety profile (Leuk Lymphoma 2018; doi: 10.1080/10428194.2018.1430795).
However, EZH2's clinical relevance in endometrial cancer remains unclear. Consequently, a recent study published in Cancer Biology & Therapy, explores this issue (2020;21(2):147-156).
Speaking to Oncology Times, Anil Sood, MD, Professor and Vice Chair for Translational Research in the Departments of Gynecologic Oncology and Cancer Biology at The University of Texas MD Anderson Cancer Center and senior author of the study, explained the rationale of the study.
"This was an initial study to look at the potential clinical and biological significance of EZH2 expression and the therapeutic efficacy of its silencing in endometrial cancer," he noted. In addition, the team assessed the mechanism mediating the effect of EZH2 knockdown on tumor growth.
In the present analysis, the biological roles of EZH2 were examined in vitro, in vivo, and in tissue samples. EZH2 expression was evaluated in a total of 150 samples of endometrial cancer tissue using a tissue microarray and correlated with clinical outcomes. Due to lack of sample availability, non-endometrioid histologic types such as serous carcinoma, clear cell carcinoma, and carcinosarcoma were omitted. Patients age ranged from 30 years to 75 years, with an average age of 52.4 years. Of the 150 endometrioid carcinomas, 90 were 2018 International Federation of Gynecology and Obstetrics (FIGO) grade 1, 34 were grade 2, and 26 were grade 3.
To identify the molecular mechanism underlying the roles of EZH2 in endometrial cancer, hierarchical clustering analysis was performed. To determine biological characteristics, gene network analysis was performed with Ingenuity Pathways Analysis software. Moreover, expression levels of EZH2 and related genes affected by EZH2 silencing were examined in various endometrial cancer cell lines. Using a small-interfering RNA (siRNA) to knockdown EZH2 expression, in vitro experiments included western blots, cell viability, and congenic, migration and apoptosis assays. In vivo therapeutic effects of EZH2 knockdown were evaluated using orthotopic mouse models.
The study findings revealed that EZH2 expression was associated with FIGO grade, recurrence, and death due to disease. Survival analysis revealed that EZH2 overexpression was significantly correlated with disease-free and overall survival of patients with endometrial cancer.
The in vitro studies revealed EZH2 silencing using siRNA incorporated into chitosan nanoparticles (siRNA/CN) inhibited cell survival, tumor growth, and angiogenesis, while increasing apoptosis. Furthermore, silencing EZH2 expression increased the cytotoxicity of taxanes and cisplatin in Hec-1A and Ishikawa endometrial cancer cells. EZH2 knockdown also led to a significant anti-tumor effect compared with that observed in controls (66.6% reduction in Hec-1A cells and 63.2% reduction in Ishikawa cells, p <.05 for both). Importantly, EZH2 silencing in combination with taxanes elicited a more enhanced anti-tumor effect versus those produced by monotherapies (77.0% for Hec-1A cells and 57.7% for Ishikawa cells, p <.05 for both).
Strikingly, the in vivo effects of EZH2 knockdown were greater than the in vitro effects. This finding was not surprising to the researchers, as EZH2 silencing not only produces indirect effects (mediated by reducing angiogenesis), but also exerts direct effects on cancer cells such as decreased proliferation and increased apoptosis.
Genomic analyses revealed that 32 genes showing differential expression due to EZH2 silencing were highly associated with cell cycle progression, developmental disorders, cellular growth, and proliferation. Among the identified differentially expressed genes, the team focused on peroxiredoxin 6 (PRDX6) as a novel candidate gene linked to EZH2 expression.
Interestingly, the analysis also revealed that increased tumor cell death after EZH2 silencing may be mediated, at least in part, via PRDX6 suppression. PRDX6, a member of the thiol-dependent antioxidant peroxidase protein family, is involved in carcinogenesis and chemoresistance of various solid tumors. One of the observed effects of PRDX6 downregulation is the subsequent stimulation and activation of the extrinsic caspase pathway in tumor cells. Specifically, levels of caspase-3, -8, and -10, but not those of caspase-9, were found to increase after EZH2 silencing.
Sood noted that there is still much to explore. "Additional work would be needed in many aspects: 1) we used siRNA-based approaches-there are now EZH2 inhibitors available in clinical testing-and those could be evaluated; 2) understanding the role of EZH2 in microenvironment is important; 3) elucidate the detailed molecular mechanism underlying the interaction between EZH2 and PRDX6 in endometrial cancer; and 4) getting a deeper understanding of potential toxicities, etc. "
Taken together, the findings reveal that increased EZH2 expression significantly correlated with poor prognosis of endometrial cancer. "Collectively, these results support that EZH2 silencing is a potentially valuable strategy for the therapeutic management of endometrial cancer," Sood concluded.
Dibash Kumar Das is a contributing writer.