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 therefore examined the role of viral oncoproteins in stem cell signaling 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, 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 recapitulate primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulated AP-1-STAT3 signaling, and induced redifferentiation.Conclusions: Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1.

Cervical cancer stem cells manifest radioresistance: Association with upregulated AP-1 activity

“…Therefore to use these sorted NSP cells as reference, alternate surface and condition were standardised using adherent plates coated with LMP-agarose in complete media (CM) and were used as reference (see Supplementary Fig. S1) as described earlier 6 . Confocal microscopy analysis of cervicospheres derived from CaCxSLCs and non-CaCxSLCs cultures revealed CD133 and ABCG2 expression in peripheral cells of the cervicosphere and these punctate markers were found to co-localize (Fig.…”

“…In contrast to these studies that employed single phenotypic/functional marker for isolation of CSC, we employed sequential gating and intermittent culturing of CaCxSLCs and enriched CaCxSLCs 6 . The process resulted in gradual enrichment of CSCs and was helpful in collecting higher number of CSCs for subsequent examination of their AP-1 response to UV exposure.…”

“…Emerging data suggest presence of a hierarchically-organized small population of cancer stem cells (CSCs) that are inherently resistant to radiation and other anti-cancer drugs 3 and variable degree of their presence/activity may determine the extent of the chemo-radioresistance 4 . The CSCs derived from primary cervical tumors 5, 6 and cervical cancer cell lines 6–8 demonstrated an increased radioresistance 7, 9 . However, the mechanism(s) responsible for manifestation of radioresistance by these CSCs are poorly understood.…”

Targeting therapy-resistant cancer stem cells by hyperthermia

“…This may be caused by production of the viral oncoproteins E6 and E7 that interfere with tumour suppressor proteins p53 and retinoblastoma protein (Rb), abolishing cell cycle regulation and apoptosis [146][147][148]. Overexpression of E6 was found to promote stemness and self-renewal in HPV þ cervical CSCs [149] and the ALDH1 þ CSC population in HPV þ tumours was considerably higher, than in HPV À tumours [150]. Furthermore, various transcription factors that are overexpressed in non-CSCs and CSCs, such as Oct4, Nanog and Notch -essential for self-renewal and for re-establishing pluripotency -are altered by HPV infections [21].…”

Manipulation of the innate immune response by human papillomaviruses

“…Furthermore, inhibition of STAT-3 activities by siRNAs or pharmacological inhibitors lead to an accumulation of p53 as well as pRb and reduced HPV gene expression (Shukla et al, 2013). Silencing of HPV E6 by E6-specific siRNAs results in abrogation of STAT-3 signaling (Tyagi et al, 2016), whereas HPV E7 controls CDC91L1 (Guo et al, 2004) which in turn increases STAT-3 phosphorylation.…”