ΔNp63α regulates keratinocyte proliferation by controlling PTEN expression and localization

Abstract

DNp63a, implicated as an oncogene, is upregulated by activated Akt, part of a well-known cell survival pathway. Inhibition of Akt activation by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and the presence of putative p63-binding sites in the pten promoter led us to investigate whether DNp63a regulates PTEN expression. Knockdown of DNp63a led to increases in PTEN levels and loss of activated Akt, while overexpression of DNp63a decreased PTEN levels and elevated active Akt. The repression of PTEN by DNp63a occurs independently of p53 status, as loss of DNp63a increases PTEN expression in cell lines with and without functional p53. In addition, decreased levels of DNp63a resulted in an increase in nuclear PTEN. Conversely, in vivo nuclear PTEN was absent in the proliferative basal layer of the epidermis where DNp63a expression is highest. Additionally, we show that in keratinocytes a balance between DNp63a and PTEN regulates Akt activation and maintains normal proliferation rates. This balance is disrupted in non-melanoma skin cancers through increased DNp63a levels, and could enhance proliferation and subsequent neoplastic development. Our studies show that DNp63a negatively regulates PTEN, thereby providing a feedback loop between PTEN, Akt and DNp63a, which has an integral role in skin cancer development. Cell Death and Differentiation (2011) 18, 1924-1933 doi:10.1038/cdd.2011; published online 3 June 2011The p53 transcription factor family consists of the tumor suppressor p53 and the homologous p63 and p73. Unlike p53, p63 is essential for normal epidermal stratification and the proliferative potential of the epithelial stem cells. 1,2 p63 exists as various isoforms with contrasting functions. 2 The TA isoforms (TAp63a, TAp63b and TAp63g) have a full-length N-terminal transactivation domain, whereas the DN isoforms (DNp63a, DNp63b and DNp63g) have a short but distinct transactivation domain. All isoforms have a DNA-binding domain that shares high homology with p53 that allows p63 proteins to bind to p53 DNA-binding sites. 3,4 Similar to p53, the TA isoforms of p63 and p73 can promote apoptosis and growth arrest through the induction of antiproliferative genes. In contrast, the DN isoforms have been shown to induce pro-survival genes and inhibit anti-proliferative genes. [4][5][6] Several studies indicate that DNp63a, the predominant isoform in adult tissue, may function as an oncogene as it can exert a dominant-negative effect over p53 and the TAp63 and TAp73 isoforms. 2 Additionally, DNp63a is frequently overexpressed in a variety of squamous cell (SCC) and basal cell carcinomas (BCCs). 7,8 The survival factor Akt can increase DNp63a levels and in turn, DNp63a protects against UV-B-induced apoptosis via Akt activation. 9,10 However, the mechanism behind the positive feedback loop between DNp63a and Akt has not been described. Akt activation can be negated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN dephosphorylates phosphatidylinositol 3,4,5-trisphosphate, thereby...

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes

“…Cells were grown, treated and either lysed and analyzed by western blot or immunofluorescence as described previously. 21 For all figures, except Figure S2, PTEN was detected with mouse monoclonal anti-PTEN 6H2.1 antibody in PBS-NGS (1:50 for H1299 cells or 1:25 for all other cells from Cascade Bioscience). Confirmation of PTEN localization by immunofluorescence was shown in Figure S2 with rabbit monoclonal anti-PTEN (#9559) at a dilution of 1:25 (Cell Signaling).…”

“…Cells were transfected with non-silencing control or PTEN specific siRNA using a previously described protocol, excepting the use of Lipofectamine RNAiMax instead of Oligofectamine (Life Technologies). 21 The two Akt inhibitors, MK2206 and Perifosine, were purchased from SelleckChem.…”

Molecular dynamics of the full-length p53 monomer

“…[121][122][123][124][125][126] While the DBD domain is highly conserved among vertebrates and invertebrates, the C terminus varies, resulting in a change from dimeric structure to a tetramer in the vertebrates. [127][128][129] The more ancient members of the family include p73, involved in cancer, 130 neurodevelopment, 131,132 and aging, 133 and p63, involved in epidermal development, 119,[134][135][136] cancer, [137][138][139][140][141] reproduction, 142 and heart development. 143 Understanding the structural restrain of its structure is pivotal to understand the function of p53 [144][145][146] as well as its potential therapeutic exploitation.…”

p53/p63/p73 in the Epidermis in Health and Disease

“…As a transcriptional repressor, p63 maintains proliferation of basal epidermal keratinocytes by directly repressing expression of antiproliferative target genes, including 14-3-3s, p16/Ink4a, p19/Arf, p21, and PTEN (Westfall et al 2003;Watt et al 2008;Su et al 2009a;Leonard et al 2011;Ferone et al 2012). In addition to cellcycle-associated genes, p63 inhibits the expression of nonkeratinocyte genes (i.e., mesodermal genes), as well as a number of other genes such as distinct components of the Notch (Notch1, Hes1) and bone morphogenetic protein (BMP) signaling pathways (Smad7) in epidermal keratinocytes (Nguyen et al 2006;De Rosa et al 2009;Shalom-Feuerstein et al 2011).…”