The carcinogenic properties of naturally occurring human papillomavirus 16 E6 oncogene variants

Abstract

Human papillomavirus is the causative agent of the 2nd highest occurring cancer in women, cervical cancer, which is the result of expression of the E6 and E7 oncogenes. Here we challenged the dogma that the oncogenic protein E6 requires co-expression of E7 for malignant transformation of human keratinocytes using common HPV16 E6 variants that are highly associated with cervical cancer: the Asian-American E6 variant (AAE6) and L83V variant of HPV16. The E6 variants containing the L83V amino acid mutation are more frequently detected in cervical cancer than the corresponding prototype HPV 16 as evidenced by independent epidemiological data. We set out to analyze in a cell culture study how these variants in the absence of E7 perform during vital steps of carcinogenesis and assessed their ability to immortalize and transform primary human foreskin keratinocytes (PHFKs). Their migration ability, a hallmark for invasiveness and/or metastasis, was also investigated. Immortalization capability was based on population doublings, number of passages, surpassing Mortality Stages 1 and 2, telomerase reverse transcriptase expression and the ability to overcome G1 arrest via p53 degradation. Transformation and migration efficiency were analyzed by a combination of functional cell-based assays. For the first time, we observed that all E6 proteins alone were sufficient to immortalize PHFKs, with the AAE6 variant being the most proficient. The AAE6 variant protein alone also pushed PHFKs through transformation and significantly increased their migration ability over that of the PHFKs expressing L83V and E6 prototype proteins. Our findings are in line with epidemiological data that the AAE6 variant confers an increased risk over the prototype for cervical cancer as evidenced by a superior immortalization, transformation and metastasis potential.