The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation

Youcef Ben Khalifa,Mathieu Daynac,Meng-Kwang Marcus Tan,Françoise Thierry,Sébastien Teissier,Quang Tien Phan,Wei Qi Wong,Paul Lambert

doi:10.1371/journal.ppat.1002256

Youcef Ben Khalifa, Mathieu Daynac + Show 6 more

Open Access

https://doi.org/10.1371/journal.ppat.1002256

Copy DOI

Abstract

Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis.

Highlights

Infection of the anogenital mucosal epithelium with high risk Human Papilloma Virus (HPV) is linked to 99% of cervical carcinomas [1]
We showed that the stability of the TAp63b isoform is downmodulated by E6, functionally linking repression of p63 target genes to expression of the E6 oncogene in cervical carcinoma cells
Levels of mRNA for the DN and TA isoforms of p63 were measured by Real Time PCR in HeLa and Caski cancer cell lines, compared to the non HPV-associated keratinocyte cell lines HaCaT and N-Tert

Summary

Introduction

Infection of the anogenital mucosal epithelium with high risk Human Papilloma Virus (HPV) is linked to 99% of cervical carcinomas [1]. Cell lines derived from these cervical carcinomas remain associated with HPV and contain part of the viral genome integrated in the cellular genome. Not all viral genes are retained in this integration; the E6 and E7 oncogenes remain, while the open reading frames encoding viral proteins E1 and E2, necessary for viral DNA replication, are disrupted [2,3]. We have previously used the HPV18-associated HeLa cell line to study transcriptional modulation of viral and cellular genes following repression of the E6 and E7 oncogenes, and found that a large number of cellular genes were modulated via E6 and E7 [4,5]. In particular we are interested in the potential effect of HPV E6 and E7 on other less well defined members of the p53 family. While p53 is a tumor suppressor and does not obviously participate in embryonic development, p63 and p73 on the contrary are strongly linked to embryonic development in mice [10,11]

Methods

Results

Conclusion