Investigating the effects of EZH2 inhibitors on targeting epigenetic changes in head and neck squamous cell carcinoma

Introduction: Head and neck squamous cell carcinoma (HNSCC) affect both men and women worldwide with approximately 880,000 new cases annually. The main risk factors involved in the pathogenesis of HNSCC include smoking tobacco, alcohol consumption and infection with human papillomavirus (HPV). There are over 200 different HPV types, with HPV16 accounting for approximately 90% of HPV positivity in HNSCC. HPV oncogenes E6 and E7 are the main causative agents for cancer progression in HPV positive HNSCC, due to their interactions with tumour suppressor proteins P53 and Rb and the HPV oncogene’s ability to inactivate them. Similarly, in HPV negative HNSCC mutations in the p53 and Rb tumour suppressors are often seen. Since epigenetic modifications were identified from a large number of epithelial cancer cases, EZH2 inhibitors has been shown to be effectively targeting those cancers, and HPV relevance has been also taken into consideration. However, the results were not consistent and whether or not HPV infection is related to the treatment outcome and the mechanisms of the treatment were not clearly understood. Therefore, in this study, two EZH2 inhibitors, GSK343 and GSK126, have been investigated on their therapeutic effects on HNSCC cell lines with different HPV status. The mechanisms of their action were also evaluated accordingly.

Methods: Two HNSCC cell lines, one HPV negative, HN5, and one HPV positive, SCC47, were investigated in this study. MTT assay was used to calculate the IC50 values of two EZH2 inhibitors, GSK343 and GSK126, and chemotherapeutic agent, cisplatin, which was used as the treatment dosage for further experiments. Following 48-hour incubation, apoptosis assay via flow cytometry was carried out; immunocytochemistry staining, and Western blotting were used to analyse various protein expressions of epigenetic, EMT, HPV16 E6/E7 and tumour suppressor markers in HNSCC cell lines. Furthermore, the analysis of gene expression of these markers was conducted by qPCR analysis.

Results: Both EZH2 inhibitors reduced protein expressions of epigenetic markers EZH2 and
H3K27me3 and EZH2 gene expression also significantly decreased following 48-hour incubation. Cisplatin had similar effects for EZH2 although an opposite effect was observed from H3K27me3 protein expression. A reversal of EMT was observed at the protein level, following treatment with EZH2 inhibitors in HNSCC cells following 48-hour treatment. Additionally, HPV oncoproteins E6 and E7 had decreased expressions following treatment with EZH2 inhibitors in SCC47 cell line and only HPV positive SCC47 had an increase in tumour suppressor, p53 and Rb gene expression following incubation with GSK343 and GSK126, no significant changes were observed for HPV negative HN5 cell line.

Conclusion: EZH2 inhibitors, GSK343 and GSK126, demonstrated their potential as desirable chemotherapeutic agents for the treatment of HNSCC in vitro. They displayed reduced cytotoxicity in normal cell line in comparison of HNSCC cell lines, which overcomes the drawbacks of current chemotherapeutic agent, cisplatin. They showed significant reductions in epigenetic protein and gene expressions, a reversal of EMT, and a reduction in HPV16 oncogenes. Additionally, EZH2 inhibitors demonstrated the ability to increase tumour suppressor gene expression in HPV positive HNSCC cell line SCC47.