title: Cervical Cancer Stem Cells Selectively Overexpress HPV Oncoprotein E6 that Controls Stemness and Self-Renewal through Upregulation of HES1.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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. Clin Cancer Res; 22(16); 4170-84. ©2016 AACR.
authors.affiliation: Stem Cell and Cancer Research Laboratory, Amity Institute of Molecular Medicine and Stem Cell Research, Amity University Campus, Noida, Uttar Pradesh, India. Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, Uttar Pradesh, India. Molecular Oncology Laboratory, B.R. Ambedkar Centre for Biomedical Research (ACBR), University of Delhi, Delhi, India.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Tyagi
authors.affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, Uttar Pradesh, India.
authors.forename: Kanchan
authors.initials: K
authors.lastname: Vishnoi
authors.affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, Uttar Pradesh, India.
authors.forename: Sutapa
authors.initials: S
authors.lastname: Mahata
authors.affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, Uttar Pradesh, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Verma
authors.affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, Uttar Pradesh, India.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Srivastava
authors.affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, Uttar Pradesh, India.
authors.forename: Shashank
authors.initials: S
authors.lastname: Masaldan
authors.affiliation: Institute of Nuclear Medicine and Allied Sciences, DRDO, Delhi, India.
authors.forename: Bal Gangadhar
authors.initials: BG
authors.lastname: Roy
authors.affiliation: Division of Molecular Oncology, Institute of Cytology & Preventive Oncology (ICMR), Noida, Uttar Pradesh, India. Molecular Oncology Laboratory, Department of Zoology, University of Delhi, Delhi, India. bcdas@amity.edu alokchandrab@yahoo.com.
authors.forename: Alok C
authors.initials: AC
authors.lastname: Bharti
authors.affiliation: Stem Cell and Cancer Research Laboratory, Amity Institute of Molecular Medicine and Stem Cell Research, Amity University Campus, Noida, Uttar Pradesh, India. Molecular Oncology Laboratory, B.R. Ambedkar Centre for Biomedical Research (ACBR), University of Delhi, Delhi, India. bcdas@amity.edu alokchandrab@yahoo.com.
authors.forename: Bhudev C
authors.initials: BC
authors.lastname: Das
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D045744:Cell Line, Tumor; D000066673:Cell Self Renewal / Q000235:genetics*; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002472:Cell Transformation, Viral; D004195:Disease Models, Animal; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016762:Genes, fos; D016758:Genes, jun; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D008954:Models, Biological; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D060066:Protein Interaction Maps; D034622:RNA Interference; D051881:Receptor, Notch1 / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000072056:Transcription Factor HES-1 / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000209:etiology* / Q000378:metabolism / Q000473:pathology
chemical_list: D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D009856:Oncogene Proteins, Viral; D051881:Receptor, Notch1; D050796:STAT3 Transcription Factor; D000072056:Transcription Factor HES-1; C078493:HES1 protein, human