Kim JS, Lee C, Bonifant C, Ressom H, Waldman TActivation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA. Mol Cell Biol 27: 662-677

Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, 3970 Reservoir Road NW, NRB E304, Washington, DC 20057, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 02/2007; 27(2):662-77. DOI: 10.1128/MCB.00537-06
Source: PubMed


In an effort to identify genes whose expression is regulated by activated phosphatidylinositol 3-kinase (PI3K) signaling, we performed microarray analysis and subsequent quantitative reverse transcription-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN(-/-) cells via an Akt1-dependent and p14(ARF)-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescence-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic, but not wild-type, PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on phosphatidylinositol (3,4,5)-triphosphate-induced mitogenesis during human cancer pathogenesis.

Full-text preview

Available from:
  • Source
    • "All pBabe constructs carried puromycin resistance and were delivered by cell infection, and cells were selected as described previously (Freije et al., 2012). Lentiviral constructs were control plKO1 (CT; Sigma-Aldrich), control GFP pLVTHM (CTGFP), and three constructs expressing shRNA specific against p53: the GFP expressing vector pLVUH-shp53 (shP53) and two non-GFP constructs pLKO1-p53-shRNA-427 (shP53-427; Addgene; Kim et al., 2007) and pLKO1-p53-shRNA-941 (shP53-941; Addgene; Kim et al., 2007). More details can be found in Supplemental Experimental Procedures. "
    [Show abstract] [Hide abstract] ABSTRACT: Tumor suppressor p53 is a major cellular guardian of genome integrity, and its inactivation is the most frequent genetic alteration in cancer, rising up to 80% in squamous cell carcinoma (SCC). By adapting the small hairpin RNA (shRNA) technology, we inactivated endogenous p53 in primary epithelial cells from the epidermis of human skin. We show that either loss of endogenous p53 or overexpression of a temperature-sensitive dominant-negative conformation triggers a self-protective differentiation response, resulting in cell stratification and expulsion. These effects follow DNA damage and exit from mitosis without cell division. p53 preserves the proliferative potential of the stem cell compartment and limits the power of proto-oncogene MYC to drive cell cycle stress and differentiation. The results provide insight into the role of p53 in self-renewal homeostasis and help explain why p53 mutations do not initiate skin cancer but increase the likelihood that cancer cells will appear. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Nov 2014 · Cell Reports
  • Source
    • "Through application of homologous targeting strategies a panel of isogenic MCF10a sub-lines have been derived expressing single onco-mutant alleles or unable to express a specific open-reading frame. Examples include PTEN−/−, p110αH1047R/WT and p110αE545K/WT [78], [79], [80], [81]. "
    [Show abstract] [Hide abstract] ABSTRACT: We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/-) cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/-) MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN.
    Full-text · Article · Oct 2013 · PLoS ONE
  • Source
    • "To maximise the possibility of identifying SLs specific to PTEN, we carried out triplicate screens in a pair of isogenically matched HCT116-derived wild-type (PTEN+/+) and PTEN−/− colorectal tumour cell lines [5]. PTEN deficiency in this model was achieved by targeting a truncating mutation to both copies of PTEN at exon 2, with the resultant mutant alleles encoding an ostensibly dysfunctional PTEN mutant protein consisting of only the 24 N-terminal amino-acids [6], [7]. "
    [Show abstract] [Hide abstract] ABSTRACT: PTEN (Phosphatase and tensin homolog) is a tumour suppressor gene commonly defective in human cancer, and is thus a potentially important therapeutic target. Targeting tumour suppressor loss-of-function is possible by exploiting the genetic concept of synthetic lethality (SL). By combining the use of isogenic models of PTEN deficiency with high-throughput RNA interference (RNAi) screening, we have identified Nemo-Like Kinase (NLK) inhibition as being synthetically lethal with PTEN deficiency. This SL is likely mediated by the transcription factor FOXO1 (Forkhead box O1), an NLK substrate, as the selectivity of NLK gene silencing for PTEN deficient cells can be reversed by FOXO1 knockdown. In addition, we provide evidence that PTEN defective cells targeted by NLK gene depletion undergo senescence, suggesting that NLK function is critical for the continued proliferation of PTEN deficient cells. Taken together, these data provide new insight into the potential of targeting of NLK to treat a range of tumourigenic conditions characterised by PTEN deficiency.
    Full-text · Article · Oct 2012 · PLoS ONE
Show more