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. 2005 Jan 18;102(3):802-7.
doi: 10.1073/pnas.0408864102. Epub 2005 Jan 12.

Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic

Sohye Kang  1 , Andreas G Bader, Peter K Vogt
Affiliations

Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic

Sohye Kang et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in genes that encode components of the phosphatidyl-inositol 3-kinase (PI3-kinase) signaling pathway are common in human cancer. The recent discovery of nonrandom somatic mutations in the PIK3CA gene of many human tumors suggests an oncogenic role for the mutated enzyme. We have determined the growth-regulatory and signaling properties of the three most frequently observed PI3-kinase mutations: E542K, E545K, and H1047R. Expressed in chicken embryo fibroblasts, all three mutants induce oncogenic transformation with high efficiency. This transforming ability is correlated with elevated catalytic activity in in vitro kinase assays. The mutant-transformed cells show constitutive phosphorylation of Akt, of p70 S6 kinase, and of the 4E-binding protein 1. Phosphorylation of S6 kinase and of 4E-binding protein 1 is regulated by the target of rapamycin (TOR) kinase and affects rates of protein synthesis. The inhibitor of TOR, rapamycin, strongly interferes with cellular transformation induced by the PI3-kinase mutants, suggesting that the TOR and its downstream targets are essential components of the transformation process. The oncogenic transforming activity makes the mutated PI3-kinase proteins promising targets for small molecule inhibitors that could be developed into effective and highly specific anticancer drugs.

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Figures

Fig. 1.
Fig. 1.
Cellular transformation induced by mutant PI3-kinase (PI3K). CEF were transfected with RCAS vectors encoding PI3-kinase mutants or wt as indicated by using various amounts of DNA. The cultures were overlaid with nutrient agar and fixed and stained with crystal violet on day 10.
Fig. 2.
Fig. 2.
Characterization of single transformed cell foci. Individual foci of transformed CEF transfected with either mutant or wt PI3-kinase (PI3K) constructs were isolated and grown until they reached confluency in 100-mm plates. After cell lysis, 20 μg of the lysates were separated on a 3-8% gradient SDS/polyacrylamide gel, and the transferred blot was probed with antibodies as indicated.
Fig. 3.
Fig. 3.
In vitro PI3-kinase activity of mutant proteins. (A) PI3-kinase (PI3K) activity was measured with the immunoprecipitated lysates of CEF that were transfected with WT, mutant (E542K, E545K, H1047R), or myristylated p110α expression construct. PIP, phosphoinositol phosphate; Ori, origin. (B) Expression of different p110α proteins. Twenty micrograms of the indicated cell lysates used for the measurement of the PI3-kinase activity were separated on a 3-8% gradient SDS/polyacrylamide gel, and the transferred blot was probed with anti-p110α antibody. Myristylated p110α expression construct contains a deletion of the first 72 aa, as noted in Materials and Methods.
Fig. 4.
Fig. 4.
Constitutive activation of Akt signaling by mutant p110α. CEF that were transfected with wt or the mutant p110α were serum starved for 42 h and subsequently stimulated with PDGF for 15 min. Cells were then lysed, and the proteins were separated on a 4-20% gradient SDS/polyacrylamide gel. The transferred blot was probed with antibodies directed against total proteins or antibodies recognizing p-S473 Akt, p-T389 S6K, or p-S65 4E-BP1. Anti-4E-BP1 antibody detects both phosphorylated (*) and nonphosphorylated forms of the protein.
Fig. 5.
Fig. 5.
Rapamycin inhibits transformation induced by mutant p110α. CEF were inoculated (100 μl) with 100-fold dilutions of retroviral vectors expressing the indicated mutant p110α or with the PR-A strain of Rous sarcoma virus expressing the v-Src oncoprotein. The cells were overlaid with nutrient agar supplemented with 5 ng/ml rapamycin or solvent only. After 14 days, the cultures were fixed and stained.
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