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The E2F4/p130 repressor complex cooperates with oncogenic ΔNp73α to promote cell survival in human papillomavirus 38 E6/E7-transformed keratinocytes and in cancer cells

ABSTRACT: 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 ΔNp73α 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 ΔNp73α 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 ΔNp73α 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 ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73α isoforms (α, β, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-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 ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162816 | GEO | 2021/06/08

REPOSITORIES: GEO

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Project description:The p53 homologue, p63, is critical for normal epidermal development. While overexpression of deltaNp63alpha has been reported in squamous cell cancers, the contribution of p63 to cancer pathogenesis remains unclear. We previously demonstrated that overexpressed deltaNp63alpha aberrantly maintains proliferation of primary epidermal keratinocytes under conditions that normally induce growth arrest and differentiation. To identify target genes impacted by dysregulated deltaNp63alpha that may contribute to squamous cancer development and progression, microarray analyses were performed. Herein we report that elevated deltaNp63alpha differentially regulates genes in primary mouse keratinocytes involved in a variety of cellular functions, including cell cycle regulation, differentiation, skin barrier formation and function, apoptosis, host defense/inflammation, adhesion, migration and invasion. Of note, multiple protease inhibitor mRNAs were coordinately downregulated under both proliferating and differentiating culture conditions. These downregulated genes include two serine protease inhibitors: maspin (serpinB5) and plasminogen activator inhibitor-2 (PAI-2; serpinB2), as well as a tissue inhibitor of metalloproteinase-3 (TIMP-3). Correspondingly, secreted levels of TIMP-3 and PAI-2 protein declined in the presence of dysregulated deltaNp63alpha, while secreted maspin protein levels remained stable. Intracellular maspin protein expression decreased in response to overexpressed deltaNp63alpha, as did intracellular PAI-2. Unlike PAI-2 and maspin, TIMP-3 protein was not detected intracellularly in control or deltaNp63alpha-overexpressing keratinocytes, supporting a solely extracellular function for TIMP-3. Electrophoretic mobility shift assays using a p53/p63 consensus DNA binding sequence from the maspin promoter revealed binding of an endogenous transcription factor(s) to the consensus sequence in normal keratinocytes that was disrupted by overexpressed deltaNp63alpha. This binding was also interrupted by the addition of a p73 antibody, but not antibodies to p63 or p53, and was absent in samples derived from p73(-/-) keratinocytes, confirming p73 as a constituent of the endogenous transcription factor complex. These data suggest protease inhibitors as novel targets of dysregulated deltaNp63alpha in cancer pathogenesis. Keratinocytes were transduced with adenoviruses encoding deltaNp63alpha. Keratinocytes were either maintained in low calcium conditions (0.05 mM for 24 hours) (n=6) or high calcium conditions (0.12 mM for 24 hours) (n=6). Control samples consisted of keratinocytes transduced with adenoviruses encoding beta-galactosidase also treated with low calcium (n=6) and high calcium (n=6) conditions. Twelve technical repeat microarray experiments were conducted, pairing high-calcium deltaNp63alpha samples with high-calcium beta-galactosidase samples (n=6) and low-calcium deltaNp63alpha samples with low-calcium beta-galactosidase samples (n=6). Six of the technical repeats were conducted as reverse-fluoro experiments.

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The E2F4/p130 repressor complex cooperates with oncogenic ΔNp73α to promote cell survival in human papillomavirus 38 E6/E7-transformed keratinocytes and in cancer cells

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