Abstract 4079: Role of HPVE6 oncoprotein in the maintenance of cervical cancer stem cells

Background & Objectives: Persistent infection of high-risk human papillomaviruses (HR-HPV) is a key etiological factor responsible for the development of cervical cancer. Deregulated expression of two viral oncoproteins E6 and E7 drive oncogenic transformation and tumour progression which may occur upon targeted infection of specific basal cervical cells with stem cell properties. However, how oncoproteins impact on putative CSCs, the stem cell signaling and their downstream functions, is not known. In this study, we have identified and characterized cervical cancer stem-like cells and unraveled the crosstalk between HPV oncoproteins and stem cell signaling that allowed maintenance of stemness in cancer cells. Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa, HeLa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness. Moreover, differential expression level and DNA-binding activity of CSL-specific Notch1 and its downstream targets in CaCxSLCs cells along with HPVE6 specific siRNA mediated gene silencing was evaluated by gel retardation assay, immunobloting qRT-PCR followed by in vivo tumor-initiating capacity in athymic nude mice. Results: CaCxSLCs cells isolated from HPV+ve cells showed high expression of CD49f, Lrig1, Nanog, Sox2, Oct4, ABCG2 and low Notch1 DNA binding activity as compared to non- CaCxSLCs cells. Enriched CaCxSLCs were also found to be more tumorigenic and showed high expression of HPVE6, HES1, CD133 transcripts with high percentage of SP cells. CaCxSLCs differed from their parental/non-CaCxSLCs as they overexpressed E6 oncogene. Knocking down of HPVE6 by RNA interference resulted in abolition of spheroid formation through downregulation of Hes1 and induction of re-differentiation while interference of Hes-1 expression resulted in reduced cervicosphere formation through down regulation of E6 and Nanog suggesting loss of self-renewing ability. Conclusions: Our findings suggest a potential regulatory role of HPVE6 in induction and maintenance of cervical cancer stem-like cells through Hes1 expression which could be utilized as a putative target for designing drugs that can promote differentiation of cancer stem cells making the therapy most effective. Note: This abstract was not presented at the meeting. Citation Format: Bhudev C. Das, Abhishek Tyagi, Kanchan Vishnoi, Sutapa Mahata, Gaurav Verma, Alok C. Bharti. Role of HPVE6 oncoprotein in the maintenance of cervical cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2015-4079