Investigating the Expression of EZH2-Target Genes TM7SF2 and NTN4 in Triple Negative Breast Cancer

Breast cancer (BC) is the most diagnosed cancer worldwide and the leading cause of cancer-related deaths in women. Triple negative breast cancer (TNBC), a highly aggressive BC subtype currently lacks effective therapies. Due to its immunogenetic nature, immunotherapy has been recently approved for the treatment of TNBC; however only a small subset of patients within the current eligibility criteria respond, and half of the initial responders will develop resistance to immunotherapy and progress. This is partly due to the lack of reliable predictive biomarkers to identify TNBC patients who will benefit from immunotherapy. As methylation plays a vital role in anti-tumor immunity, Dr AL-Muftah’s lab hypothesized that genes downregulated by hypermethylation may serve as predictive biomarkers in TNBC. They identified a unique 30 -gene signature consisting of genes that are targets of the histone methyltransferase EZH2 including the genes TM7SF2 and NTN4. The aim of this project was to validate the link between EZH2 and the target genes TM7SF2 and NTN4 and investigate their expression in TNBC cell lines.

Bioinformatic analysis of BC cases in The Cancer Genome Atlas (TCGA) BC dataset demonstrated significantly high EZH2 expression and significantly low expression of TM7SF2 and NTN4 in TNBC compared to other BC subtypes. In vitro experiment demonstrated that both EZH2 inhibition using a potent inhibitor of EZH2 catalytic activity (GSK343) and EZH2 knockdown using shRNA can reduce the expression of EZH2 in TNBC cell lines and increase the expression of the target genes TM7SF2 and NTN4. This reverse correlation was validated at both the mRNA and protein expression levels. Furthermore, EZH2 knockdown (and the associated TM7SF2 and NTN4 upregulation) increased cells sensitivity to chemotherapy, reduced cells capability to form colonies and reduced cell migration in TNBC. These results underline the importance of further studies into the biological significance of TM7SF2 and NTN4.