P. Elliott1, G. Kaushik1, B. Roy1, S. Umar1, S. Anant1, J. M. Mammen1 1University Of Kansas,Surgery,Kansas City, KS, USA
Introduction: Epigenetic mechanisms and chromatin modifications emerged as key players in cancer development. Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase, is the main component of the polycomb-repressive complex 2 (PRC2) involved in epigenetic modification. Increased activity of EZH2 has been reported to be associated with different cancer types including melanoma (mel). Evidence for functional role of EZH2 in melanomagenesis, in particular to stem signaling, is poorly understood. We focused on the interplay between epigenetic modifier Enhancer of Zeste Homolog 2 (EZH2) and stem cells regulation via notch stem cell signaling pathway.
Methods: We used various mel lines and normal melanocytes (melcytes). The hexoseaminidase assay, colony formation assay (CFA), migration assay and spheroid assay (SA) were used to determine cell growth, proliferation and stemness. Protein expression studies and mRNA levels in cells were done by using standard Immunoblotting technique and qPCR. A lentiviral expression system was used to knockdown as well as to ectopic overexpress the gene of interest in various mel lines and melcytes respectively.
Results:From TCGA database, we observed higher expression levels (> 60%) EZH2 mRNA in cutaneous as compared to uveal mel. EZH2 protein is highly expressed in most mel cell types as compared to normal melcytes with very low or undetected EZH2 levels (Figure 1) (n=3, p < 0.01). This higher expression level of EZH2 was further confirmed by IHC staining of tissue slides. We studied the effects of EZH2 knockdown by using shRNA virus on growth/proliferation (approximately 40%), CFA, SA and migration of SKMEL-28 mel cells. We observed reduction in cell growth, spheroid size and cell migration in shRNA expressed cells. EHZ2 knockdown showed reduction in colony size in shRNA expressed mel. Notch receptor proteins play an important role in mel stemness and signaling. We studied the correlation of EZH2 with Notch signaling pathway. We further studied mRNA levels of various Notch receptors in UACC275 cells with Ezh2 knockdown using siRNAs. Knockdown significantly reduced the levels of Notch-1 mRNA . Other Notch mRNAs (Notch-2, Notch-3 and Notch-4) were not significantly affected. Higher expression pattern of Notch-1 protein in mel lines was similar to Ezh2 expression patterns. EZH2 was overexpressed ectopically in normal melcytes to study physiological effect on proliferation. Over expression of EZH2 in melcytes increased the proliferation of melcytes. EZH2 overexpression also affected the protein levels of Notch-1 in melcytes.
Conclusion:Ezh2 and notch signaling showed cross talk in mel cells. More studies required to support association of EZH2 with notch signaling in mel stem cells.
