Stem Cell Properties of Normal Human Keratinocytes Determine Transformation Responses to Human Papillomavirus 16 DNA

Abstract

Human papillomavirus (HPV) infection of the genital tract is common; however, only about 10 to 15% of infections persist, and approximately 10 to 15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. However, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA. Normal human keratinocyte isolates (NHKc) derived from different donors were cultured in three-dimensional anchorage-free suspension to assess their spheroid-forming ability. NHKc spheroids were then plated back into plastic monolayer culture and transfected with full-length HPV16 DNA, which we have previously shown to integrate into the host cell genome upon transfection. Spheroid-derived NHKc (SD-NHKc) and fluorescence-activated cell sorting-purified populations of basal stem-like keratinocytes, expressing low levels of epidermal growth factor receptor and high levels of integrin alpha 6 (EGFR/ITGα6), responded to transfection with HPV16 DNA with more vigorous proliferation, greater immortalization efficiency, and faster progression to differentiation resistance than autologous mass-cultured cells. Conversely, cells committed to terminal differentiation (EGFR/ITGα6) grew slowly after transfection with HPV16 and failed to generate immortalized or DR clones. HPV16 DNA induced stem cell properties in mass-cultured NHKc. We conclude that HPV16 preferentially immortalizes basal keratinocytes with stem cell properties and that these cells readily achieve a differentiation-resistant phenotype upon immortalization by HPV16. This paper explores the relationship between the stem cell properties of normal human epidermal cells in culture and these cells' susceptibility to transformation by HPV16 DNA, the HPV type present in about 50% of cervical cancers. We report variable susceptibilities to HPV16-mediated transformation among different keratinocyte isolates derived from neonatal foreskin. Our findings provide strong experimental evidence that HPV16 preferentially transforms basal keratinocytes with stem cell properties. Insights gained from these studies increase our understanding of the host cell-specific factors influencing individual susceptibility to HPV-driven transformation and the contributing factors leading to preneoplastic and neoplastic progression of HPV-positive lesions.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition

“…High-risk HPVs have been proposed to infect stem cells (65, 66). A recent study showed that HPV16 preferentially transforms basal keratinocytes with stem cell properties (67) and that the HPV E7 oncoprotein, which inhibits Rb similarly to MCV LT, activates pluripotency genes, including Oct4, Sox2, and Nanog (68). Thus, the targeting of cellular reprogramming factors, such as Sox2 and Oct4, may be a common mechanism for stem cell transformation by tumor viruses.…”

Putative Role of Circulating Human Papillomavirus DNA in the Development of Primary Squamous Cell Carcinoma of the Middle Rectum: A Case Report

“…It is also possible that the virus by itself induces an immunomodulation as previously demonstrated for other viruses (37, 38). Notably, we identified the reservoir of HPV in non-neoplastic epithelial cells within the crypts of colonic normal mucosa adjacent to the neoplastic mucosa and we demonstrated that these cells were stem-cells, which are known to be susceptible to transformation by HPV 16 (39, 40). It can be argued that following inflammatory response to a number of stimuli (also including dysbiosis) (41), non-cancer stem cells may be activated into cancer stem cells, sustaining a feed-forward circuit of self-renewal, proliferation, differentiation and neoplastic transformation in a condition of immune evasion, as previously demonstrated (40, 42–44).…”

Self-assembling 3D spheroid cultures of human neonatal keratinocytes have enhanced regenerative properties

“…NHKc were isolated from neonatal foreskin as previously described ( Akerman et al, 2001 ) and cultured according to Woappi et al ( Woappi et al, 2018 ). Briefly, KSFM media (Thermo Fisher Scientific Waltham, MA) was supplemented with 1% PenStrep/10 mg/ml gentamycine, 50 μg/mL bovine pituitary extract, 20 ng/ml EGF, 10 ng/ml basic fibroblast growth factor, 0.4% bovine serum albumin (BSA), and 4 μg/ml insulin.…”