HPV16 E6 and E7 are involved in inhibition of autophagy and maintenance of cancer stem cell properties in HPV16‐positive oral squamous cell carcinoma cells

Abstract

AbstractAimThe involvement of human papillomavirus (HPV) E6/E7 in autophagy has not been fully elucidated in oral squamous cell carcinoma (OSCC) cells. The objective of this study was to clarify the association between HPV E6/E7 and autophagy and cancer stem cell properties such as self‐renewal ability and chemotherapy resistance in HPV16‐positive OSCC cells.MethodsUM‐SCC‐104 HPV16‐positive OSCC cells were cultured on laminin 332‐coated silicone gel. The impact of HPV16 E6 or E7 siRNA transfection on cell proliferation, spheroid formation, 5‐fluorouracil (5‐FU)‐induced cell death, and autophagy was investigated. After treatment with rapamycin, DAPGreen was used to detect autophagosome formation.ResultsHPV16 E6 or E7 siRNA transfection resulted in knockdown of both E6 and E7 in UM‐SCC‐104 cells. HPV16 E6/E7 knockdown inhibited cell proliferation, mRNA expression of stem cell markers (i.e., CD44, ALDH1, BMI1, OCT4, and NANOG), and spheroid formation. Furthermore, HPV16 E6/E7 knockdown enhanced 5‐FU‐induced cell death. Additionally, mRNA expression of autophagy‐related genes such as ATG5 and BECN1 was significantly increased after HPV16 E6/E7 knockdown. Furthermore, HPV16 E6/E7 knockdown augmented rapamycin‐induced autophagy. Next, we found that miR‐30a‐5p is indicated to interact with the 3′‐untranslated region of ATG5 and BECN1 using TargetScan. However, significant induction of autophagy was not found after miR‐30a‐5p inhibitor transfection. Importantly, 5‐FU‐induced cell death was significantly attenuated by chloroquine autophagy inhibitor in HPV16 E6/E7 knockdown cells.ConclusionHPV16 E6/E7 is involved in autophagy regulation as well as in the maintenance of cancer stem cell features in OSCC cells.