HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes

Victoria Domínguez-Catzín, Alicia-María Reveles-Espinoza, Janet Sánchez-Ramos, Raúl Cruz-Cadena, Diana Lemus-Hernández, Efraín Garrido

Cervical cancer is the fourth cause of death worldwide by cancer in women and is a disease associated to persistent infection with human papillomavirus (HPV), particularly from two high-risk types HPV16 and 18. The virus initiates its replicative cycle infecting cells located in the basal layer of the epithelium, where a small population of epithelial stem cells is located performing important functions of renewal and maintenance of the tissue. Viral E2 gene is one of the first expressed after infection and plays relevant roles in the replicative cycle of the virus, modifying fundamental processes in the infected cells. Thus, the aim of the present study was to demonstrate the presence of hierarchic subpopulations in HaCaT cell line and evaluate the effect of HPV16-E2 expression, on their biological processes. HaCaT-HPV16-E2 cells were generated by transduction of HaCaT cell line with a lentiviral vector. The α6-integrin-CD71 expression profile was established by immunostaining and flow cytometric analysis. After sorting, cell subpopulations were analyzed in biological assays for self-renewal, clonogenicity and expression of stemness factors (RT-qPCR). We identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrin(dim)), transitory amplifying cells (α6-integrin(bri)/CD71(bri)) and progenitor cells (α6-integrin(bri)/CD71(dim)). The last subpopulation showed stem cell characteristics, such as self-renewal ability, clonogenicity and expression of the well-known stem cell factors SOX2, OCT4 and NANOG, suggesting they are stem-like cells. Interestingly, the expression of HPV16-E2 in HaCaT cells changed its α6-integrin-CD71 immunophenotype modifying the relative abundance of the cell subpopulations, reducing significantly the percentage of α6-integrin(bri)/CD71(dim) cells. Moreover, the expression of the stem cell markers was also modified, increasing the expression of SOX2 and NANOG, but decreasing notably the expression of OCT4. Our data demonstrated the presence of a small subpopulation with epithelial "progenitor cells" characteristics in the HaCaT cell line, and that HPV16-E2 expression on these cells induces early differentiation.