The E2F4/p130 repressor complex cooperates with oncogenic ?Np73a to promote cell survival in human papillomavirus 38 E6/E7-transformed keratinocytes and in cancer cells

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ?Np73a isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses such as Epstein–Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ?Np73a mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ?Np73a associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ?Np73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ?Np73a isoforms (a, ß, ?, d, e, ?, ?, ?, and ?1). We show that the ?Np73a-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ?Np73a, indicating that the interaction with ?Np73a rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. Overall design: Gene expression analysis in presence or absence of the E2F4/E2F5 transcription factors in 38HK cell line. 38HK cells are transiently transfected with Scramble siRNA (3 control samples) or E2F4+E2F5 siRNAs (3 knockdown samples) using Lipofectamine 2000 reagent. Cells are collected 48 hours post-transfection and the level of proteins evaluated by Western-blot. Total RNA samples are extracted from 38HK cells (around 10e6 cells per sample) using the RNeasy Mini kit form QIAGEN. Samples are quantified by Qubit.