A214: Salinomycin targets EZH2 driven epigenetic repression of death receptors in colon cancer stem cells
1CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
Poly comb group proteins (PcG) mediated dynamic epigenetic alternations in histone methylation landscapes are emerging as driving player for cancer stem cell (CSC) associated phenotypes including drug resistance. Enhancer of Zeste homologue 2 (EZH2), the catalytic component of Polycomb repressive complex 2 (PRC2) has been reported to be over expressed in CSCs and linked with poor outcome in disparate tumors including colorectal cancer. Recently, Salinomycin has found renewed interest as an agent for prevention of cancer recurrence through selectively targeting cancer stem cells. Although emerging body of reports indicates the ability of salinomycin to upregulate death receptors, its exact mechanism of action is largely unknown.
CSC rich colon cancer cells were treated with salinomycin and other epigenetic drugs. Human apoptotic proteome profiler was used to assess changes in various apoptotic proteins after salinomycin treatment. Effect of salinomycin on CCSC associated markers was analyzed using flow cytometry. Methylation status of DR4/DR5 promoter region and global histone methylation were analyzed by methylation-specific PCR. H3K27me3 and EZH2 levels were determined using western blotting as well as confocal microscopy. ChIP was performed to investigate specific histone modifications (H3K27me3) and EZH2 occupancy at the DR4/DR5 promoters.
Expression of DR4, DR5 repression in CSCs was found to be epigenetically repressed in colon CSCs due to EZH2 mediated H3K27me3, independent of promoter DNA methylation. Treatment of colon cancer cells with salinomycin or EZH2 inhibitor DZNep increases the DR4/DR5 expression with a concomitant decrease in the EZH2 level. p53 level were also found to increase which may caused restoration of their TRAIL (ligand for DR4/DR5) sensitivity. Transcriptional activation of DR4/DR5 was found to be associated with decreased EZH2 localization in the nucleus and hence reduced EZH2 recruitment and histone methylation near promoters.
TRAIL resistance in colon CSCs may be attributed due to EZH2 mediated epigenetic suppression of DR4/DR5 expression caused by histone trimethylation in promoter region; and salinomycin is able to restore their TRAIL sensitivity by targeting EZH2 and subsequent histone trimethylation.