Cervical Cancer Stem Cells Selectively Overexpress HPV Oncoprotein E6 that Controls Stemness and Self-Renewal through Upregulation of HES1

Abstract

Purpose:Perturbation of keratinocyte differentiation by E6/E7 oncoproteins of high-risk human papillomaviruses that drive oncogenic transformation of cells in squamocolumnar junction of the uterine cervix may confer 'stem cell like' characteristics. However, the crosstalk between E6/E7 and stem cell signaling during cervical carcinogenesis is not well understood. We have therefore examined the role of viral oncoproteins in stem cell signalling and maintenance of stemness in cervical cancer. Experimental Design:Isolation and enrichment of cervical cancer stem-like cells (CaCxSLCs) was done from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media for assessing sphere formation and expression of self renewal and stemness markers by FACS, confocal microscopy and qRT-PCR. Differential expression level and DNA-binding activity of Notch1 and its downstream targets in CaCxSLCs as well as silencing of HPVE6/Hes1 by siRNA was evaluated by gel retardation assay, FACS, immunoblotting and qRT-PCR followed by in-silico and in-vivo xenograft analysis. Results:CaCxSLCs showed spheroid forming ability and expressed self-renewal and stemness markers Oct4, Sox2, Nanog, Lrig1 and CD133 and selectively overexpressed E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enriched CaCxSLCs were highly tumorigenic and did recapitulated primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulates AP-1-STAT3 signaling and induced re-differentiation. Conclusions:Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1.