Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jan;27(2):662-77.
doi: 10.1128/MCB.00537-06. Epub 2006 Oct 23.

Activation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA

Jung-Sik Kim  1 , Carolyn Lee, Challice L Bonifant, Habtom Ressom, Todd Waldman
Affiliations

Activation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA

Jung-Sik Kim et al. Mol Cell Biol. 2007 Jan.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Functional activation of p53 responses in PTEN−/− cells. (A) p53-regulated genes whose expression is upregulated in PTEN−/− cells, as determined by both microarray analysis and qRT-PCR. (B) Formal demonstration of p53 dependence. HCT116 PTEN+/+ and PTEN−/− cells were infected with control (pLKO.1) and p53 shRNA lentiviruses, and the expression levels of the listed p53-regulated genes were measured by qRT-PCR. The y axis represents the difference (n-fold) in gene expression between HCT116 PTEN−/− and PTEN+/+ cells. The extent of p53 depletion as measured by Western blotting is depicted in the inset. As indicated, p53 depletion led to a reduction in the expression of each of the five genes preferentially in HCT116 PTEN−/− cells. The error bars indicate standard deviations.
FIG. 2.
FIG. 2.
Increased expression of p53 and p21 proteins in PTEN−/− cells. (A) p53 and p21 levels in proliferating HCT116 PTEN+/+, PTEN+/−, and PTEN−/− cells. Immunoblotting was performed with the antibodies indicated. (B) p53 levels in the same cell lines after DNA damage. HCT116 cells with the PTEN genotypes indicated were treated with 5 μg/ml etoposide for 24 h. Immunoblotting was performed with the antibodies indicated.
FIG. 3.
FIG. 3.
Reduction in p53 levels by LY294002 in PTEN−/− cells. (A) HCT116 PTEN−/− cells were treated with 0, 30, 60, or 90 μM LY294002 for 24 h. Protein lysates were prepared, and immunoblots were performed with the primary antibodies indicated. (B) A172 cells and (C) NHA were treated with 0, 2, 10, 30, or 60 μM LY294002 for 24 h. Protein lysates were prepared, and immunoblots were performed with the primary antibodies indicated.
FIG. 4.
FIG. 4.
Enhanced p53 stability in PTEN−/− cells. (A) HCT116 PTEN+/+ and PTEN−/− cells were treated with 100 μg/ml cycloheximide (CHX) for the times indicated. Protein lysates were prepared, and immunoblots were performed with the antibodies indicated. (B) Bands were quantified using Scion Image densitometry software (Scion Corporation, Frederick, MD), and half-lives were determined using linear-regression analysis. (C) Protein lysates were prepared from HeLa, HCT116 PTEN+/+, and PTEN−/− cells, and immunoblots were performed with three different p14ARF primary antibodies. (D) Protein lysates from two different HCT116 PTEN+/+ and HCT116 PTEN−/− cell lines were studied by immunoblotting with an Hdm2 antibody.
FIG. 5.
FIG. 5.
Effects of Akt1 depletion on p53 activation in PTEN−/− cells. (A) HCT116 PTEN−/− cells were infected with the lentiviruses indicated, and pooled clones were established and studied by immunoblotting them to document levels of Akt1, p53, and α-tubulin proteins. (B) HCT116 PTEN+/+ and HCT116 PTEN−/− cells were infected with control and Akt1 shRNA lentiviruses, pooled clones were established, and total RNA was prepared and studied by qRT-PCR to document the relative expression levels of p21 and GDF15. The y axis represents the difference in gene expression (n-fold) between HCT116 PTEN−/− and PTEN+/+ cells. As indicated, Akt1 depletion led to a reduction in p21 and GDF15 preferentially in PTEN−/− cells. The error bars represent standard deviations.
FIG. 6.
FIG. 6.
Effects of PTEN depletion in untransformed human cells. BJ-hTERT fibroblasts and RPE-hTERT epithelial cells were infected with the lentiviruses indicated, and pooled clones were prepared as described in Materials and Methods. (A and B) Protein lysates were prepared, and immunoblots were performed with the primary antibodies indicated. p16INK4a levels were undetectable in BJ-hTERT cells. (C) (a and b) pLKO.1- and PTEN-shRNA-infected RPE-hTERT cells were imaged via phase-contrast light microscopy. The scale bars represent 200 μm. (c and d) pLKO.1- and PTEN-shRNA-infected RPE-hTERT cells were double stained for γ-H2AX (green) and DAPI (blue) and imaged by fluorescence microscopy. The scale bar represents 10 μm. (D) RPE-hTERT and BJ-hTERT cells were infected with control or PTEN shRNA lentiviruses, flow cytometry was performed as described in Materials and Methods, and the S-phase fraction was quantified using ModFit software (Verity Software House, Topsham, ME). (E) The sizes of the cells described in panel C were measured using a Multisizer 3 Coulter Counter (Beckman Coulter, Fullerton, CA).
FIG. 7.
FIG. 7.
p53 is required for the cell cycle arrest caused by PTEN depletion. RPE-hTERT cells were infected in combination with lentiviruses encoding PTEN shRNAs, p53 shRNAs, or control shRNAs as indicated. (A) Protein lysates were prepared, and immunoblotting was performed using the primary antibodies indicated. (B) S phase was quantified using ModFit software (Verity Software House, Topsham, ME) after flow cytometry analysis, as described in Materials and Methods. (C) Cells were harvested and counted every 1 or 2 days to document proliferation. (D) Cell size was measured using a Multisizer 3 Coulter Counter (Beckman Coulter, Fullerton, CA).
FIG. 8.
FIG. 8.
Activation of p53 by oncogenic PIK3CA. (A) MCF10A cells stably expressing early-stop (W11STOP), wild-type (WT), and oncogenic FLAG-PIK3CA (H1047R) were harvested. Immunoprecipitation and immunoblotting were performed as described in Materials and Methods with the antibodies indicated. (B) Total RNA was prepared from the indicated pooled clones, and qRT-PCR was employed to measure levels of p21 and GDF15. The error bars represent standard deviations.
FIG. 9.
FIG. 9.
PIK3CA gene targeting. (A) Homologous recombination between the genomic locus and the AAV targeting vector deletes exon 2 and replaces it with a thymidine kinase (TK) Neor gene. The PCR primers used for identification of knockouts are indicated, as are the restriction enzyme cleavage sites and the probe used for Southern blotting-based confirmation of knockouts (KO). (B) Confirmation of PIK3CA targeting by Southern blot analysis. Fragments corresponding to the untargeted allele and the targeted allele are shown. (C) Sequence-based identification of the untargeted allele. PCR products from the untargeted allele were sequenced to determine which allele had been deleted in PIK3CA gene-targeted cells. Asterisks indicate the locations of the PIK3CA mutation A3140G (H1047R), and N denotes heterozygosity. (D) Morphological features of PIK3CA gene-targeted cells: (a) HCT116 parental cells and derivatives with (b) oncogenic PIK3CA deleted and (c) wild-type PIK3CA deleted. The scale bar represents 200 μm.
FIG. 10.
FIG. 10.
Effects of PIK3CA deletion on p53 levels and activity. (A) Protein lysates from HCT116 parental cells and PIK3CA gene-targeted cells were prepared, and immunoblotting was performed using the primary antibodies indicated. (B) Total RNA was prepared from HCT116 parental cells and PIK3CA gene-targeted derivatives, and qRT-PCR was employed to measure the levels of p21 and GDF15. The error bars represent standard deviations.
FIG. 11.
FIG. 11.
Activation of p53 by oncogenic PIK3CA. MCF10A cells stably expressing early-stop (W11STOP), wild-type (WT), and oncogenic (H1047R) PIK3CA and p53 shRNAs or control shRNAs as indicated were plated in soft agar and grown for 2 weeks, and the colonies were stained with 0.005% crystal violet. The colonies were photographed (A) and counted (B) as described in Materials and Methods. The scale bar represents 1.0 mm.
FIG. 12.
FIG. 12.
Model of p53 activation by PIP3 signaling. Inactivation of PTEN or activation of PIK3CA leads to an increase in cellular levels of PIP3 and subsequent activation of Akt. This leads to simultaneous activation of mitogenesis and activation of p53-dependent cellular senescence. Oncogenes are depicted in red and tumor suppressor genes in blue.
See this image and copyright information in PMC

References

    1. Atadja, P., H. Wong, I. Garkavtsev, C. Veilette, and K. Riabowol. 1995. Increased activity of p53 in senescing fibroblasts. Proc. Natl. Acad. Sci. USA 92:8348-8352. - PMC - PubMed
    1. Bachman, K. E., P. Argani, Y. Samuels, N. Silliman, J. Ptak, S. Szabo, H. Konishi, B. Karakas, B. G. Blair, C. Lin, B. A. Peters, V. E. Velculescu, and B. H. Park. 2004. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol. Ther. 3:772-775. - PubMed
    1. Bar, J., N. Lukaschuk, A. Zalcenstein, S. Wilder, R. Seger, and M. Oren. 2005. The PI3K inhibitor LY294002 prevents p53 induction by DNA damage and attenuates chemotherapy-induced apoptosis. Cell Death Differ. 12:1578-1587. - PubMed
    1. Barbieri, C. E., C. E. Barton, and J. A. Pietenpol. 2003. ΔNp63α expression is regulated by the phosphoinositide 3-kinase pathway. J. Biol. Chem. 278:51408-51414. - PubMed
    1. Brummelkamp, T. R., R. Bernards, and R. Agami. 2002. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2:243-247. - PubMed

Publication types

MeSH terms

Substances

Associated data