E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

Highlights

• E6D25E HPV16 specifically modulates protein profile of human keratinocytes.

• E6D25E HPV16 modulates protein profile which involves in TLR signalling and transformation of squamocolumnar junction cells.

• E6D25E oncoprotein may correlate to impair of immune response against viral infection and cells transformation.

Abstract

HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant.

Introduction

A persistent infection with high-risk human papillomaviruses (HR-HPVs) is required for development of cervical carcinoma [1]. Among those HR-HPVs, HPV16 is the most common in invasive cervical cancers worldwide [2]. Viral genes, E6 and E7 of HR-HPVs play vital roles in carcinogenesis. The E7 oncoprotein interacts with and inactivates the tumor suppressor pRb family proteins, and thus interrupts the interaction of pRb with E2Fs and promotes cell cycle progression. The E6 oncoprotein promotes degradation of tumor suppressor p53 by forming ternary complexes with the E3 ubiquitin-ligase E6-associated protein (E6AP). These complexes lead to the ubiquitination of p53 and degradation via a proteasome pathway [3].

E6 protein can interact with various cellular proteins involved in apoptosis, cell proliferation, differentiation and transformation. It can block apoptosis by inactivating p53, p300/CBP, Bak, FADD and pro-caspase 8 [4]. E6 is also known to activate telomerase by interacting with several cellular proteins including NFX1-91, a repressor of the hTERT promoter [5], MYC and the telomerase complex itself [6], [7]. Moreover, E6 can suppress the immune system by binding to IRF-3 [8] and TYK2 [9].

Numerous studies reported that HPV16 variants differ in risks of viral persistence, progression to cervical cancers and immunogenicity [10], [11]. HPV16 variants have been classified into four phylogenetic lineages according to different geographic regions such as European-Asian (EAs), including sublineages European (E) and Asian (As), African 1 (AFR1), African 2 (AFR2) and North American/Asian-American (NA/AA), including the sublineages North American, Asian-American 1 and Asian-American 2 [12], [13]. Epidemiological studies have shown an association between cervical cancer and variation of HPV16 and reported that certain HPV16 E6 variants might be more oncogenic than the prototype and thus carried a higher risk for the development of invasive cervical disease [11], [14], [15]. Moreover, higher prevalence of HPV16 As variant in invasive cervical cancer is also noted in Asian population [16], [17], [18].

Several studies reported that the E6 variants of HPV16 differed in their abilities to abrogate serum/calcium-dependent differentiation and to induce p53 degradation in vitro [19]. An E6 variant of the HPV16 AA lineage was reported to promote proliferation, immortalisation, transformation and migration of primary human foreskin keratinocytes (PHFKs) more efficiently than the E6 of the HPV16 prototype [20], [21]. HPV16 E6AA variant and European variant (E350G) exhibited higher efficiency in colony formation than the E6 prototype did, though they showed similar abilities in degrading p53. Furthermore, several host genes were differentially up-, or down-regulated in PHFKs expressing those E6 variants [22]. This may account for differences in oncogenic potential among HPV16 variants.

In this study, we hypothesized that the E6D25E, an As variant of HPV16, may have higher oncogenic potential than the E6 prototype by inducing distinct expression patterns of cellular proteins. Therefore we compared biological and biochemical activities of the E6D25E and the E6 prototype in normal human cervical keratinocytes. The protein expression profile of cells expressing E6D25E and E6 prototype was investigated by two-dimensional polyacrylamide gel electrophoresis and the cellular proteins differentially expressed were identified by mass spectrometry. Protein reactome pathways analysis indicated that tumor rejection antigen (gp96) 1 variant, specifically a down-regulated in the E6D25E expressing cells, was involved in Toll-like signaling pathway [23]. Keratin 7 (K7), which was up-regulated, is involved in transformation of squamocolumnar junction cells [24]. These expression profile might associate with impairing innate immune response and transformation during HPV infection. Such property of E6D25E may account for its higher oncogenic potential of E6D25E than E6 prototype.