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

Abstract

BackgroundCervical 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.MethodsHaCaT-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).ResultsWe identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrindim), transitory amplifying cells (α6-integrinbri/CD71bri) and progenitor cells (α6-integrinbri/CD71dim). 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-integrinbri/CD71dim 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0736-2) contains supplementary material, which is available to authorized users.

Octamer binding transcription factor-4 expression is associated with cervical cancer malignancy and histological differentiation: a systematic review and meta-analysis

“…CC is a common gynecological tumor that threatens women’s health. CSCs are characterized by self-renewal, multi-directional differentiation, radiotherapy and chemotherapy resistance, high tumorigenicity and so on, and may affect the invasion, metastasis, and recurrence of CC [30–32].…”

CD71+ Population Enriched by HPV-E6 Protein Promotes Cancer Aggressiveness and Radioresistance in Cervical Cancer Cells

“…Nonetheless, some reports have indicated that CD71 À or CD71 dim populations also display stem cell characteristics in some cancers. Elevated expression of "stemness" genes was detected in a side population (a6-integrin bri /CD71 dim ) isolated from spontaneously immortalized keratinocytes in the HaCaT cell line that showed epithelial progenitor cell characteristics (25). Furthermore, an enriched CD71 À subpopulation was found in the gastric adenosquamous carcinoma cell line MKN-1 after 5-fluorouracil treatment, and the CD71 À subpopulation exhibited elevated migratory and invasive ability, enhanced selfrenewal ability, and tumorigenicity (26).…”

Cervical cancer stem cells and other leading factors associated with cervical cancer development (Review)

“…In Mexico, CC is the second most common cause of cancer-associated mortality in women (2018), primarily due to poor clinical diagnosis during the early stages of disease (7,10). Previous reports between 2015 and 2017 revealed that HPV-16 and HPV-18 are responsible for 65–75% of precancerous cervical lesions and are present in 99% of patients with CC worldwide (9,11–13). However, it has also been established that HPV infections do not necessarily trigger CC (9,12,13).…”

“…Although a number of genetic and molecular events require further clarification in cervical carcinogenesis, it is currently accepted that HPV viruses depend on epithelial cell differentiation to establish their progeny and spread their viral genes (11–13). HPV has a more significant opportunity to infect basal cells with stem cell-like properties and integrate its viral DNA into the genome of these cells, driving the oncogenic transformation into CCSCs (13,23).…”

“…E2 is considered the central transcriptional regulator of the papilloma viruses as it interacts with E1 when DNA replication begins, and because this protein scatters the viral episomal genomes during the division of the infected cells. E2 mediates the interactions between the viral genome and chromatin adapter proteins at the point of mitotic division and indirectly regulates transcription of E6 and E7 producing several effects on the cell cycle that affect proliferation, differentiation, apoptosis and senescence (11,34–36).…”