Akt ͉ oncogenic transformation ͉ p110␣ ͉ PIK3CA

There have long been indications of a role for PI3K (phosphatidylinositol 3-kinase) in cancer pathogenesis. Experimental data document a requirement for deregulation of both transcription and translation in PI3K-mediated oncogenic transformation. The recent discoveries of cancer-specific mutations in PIK3CA, the gene that encodes the catalytic subunit p110alpha of PI3K, have heightened the interest in the oncogenic potential of this lipid kinase and have made p110alpha an ideal drug target.

The PIK3CA gene, coding for the catalytic subunit p110␣ of class IA phosphatidylinositol 3-kinases (PI3Ks), is frequently mutated in human cancer. Mutated p110␣ proteins show a gain of enzymatic function in vitro and are oncogenic in cell culture. Here, we show that three prevalent mutants of p110␣, E542K, E545K, and H1047R, are oncogenic in vivo. They induce tumors in the chorioallantoic membrane of the chicken embryo and cause hemangiosarcomas in the animal. These tumors are marked by increased angiogenesis and an activation of the Akt pathway. The target of rapamycin inhibitor RAD001 blocks tumor growth induced by the H1047R p110␣ mutant. The in vivo oncogenicity of PIK3CA mutants in an avian species strongly suggests a critical role for these mutated proteins in human malignancies.T he PIK3CA gene codes for the catalytic subunit p110␣ of class IA phosphatidylinositol 3-kinases (PI3Ks) (reviewed in refs. 1-3). PIK3CA is the cellular homolog of the retroviral v-p3k oncogene and is amplified in ovarian and cervical cancer (4-6). Genomic profiling of human cancers revealed the presence of somatic, heterozygous point mutations in PIK3CA (7-16). These mutations occur in Ϸ30% of all breast and colon cancers and are less frequent in cancers of the brain, stomach, liver, and ovary. The mutations are nonrandomly distributed over the primary structure of p110␣ and cluster to regions in the p85-binding domain, the C2 domain, the helical domain, and the C terminus of the catalytic domain. The sites most frequently affected by mutation are the residues E542 and E545 in the helical domain and H1047 in the catalytic domain. The glutamates at position 542 and 545 are commonly changed to lysines, and H1047 is often replaced by arginine. The cancer-specific point mutations of p110␣ confer a gain of function resulting in increased lipid kinase activity (15,(17)(18)(19). Expression of p110␣ mutants activates the Akt-signaling pathway in the absence of growth factors and induces oncogenic cellular transformation of chicken embryo fibroblasts (CEFs) and of NIH 3T3 cells (17, 18). The transformation by p110␣ mutants is sensitive to rapamycin, suggesting that the target of rapamycin (TOR) and downstream effector molecules of TOR are crucial components of the oncogenic process (18).Here, we provide evidence for the oncogenicity of p110␣ E542K, E545K, and H1047R in vivo and identify the PIK3CA mutants as oncoproteins. These mutants induce angiogenesis and malignant cell growth in the chorioallantoic membrane (CAM) of the chicken embryo and cause hemangiosarcomas in young chickens. The rapamycin derivative RAD001 interferes with H1047R-induced tumor formation, in agreement with observations made in cell culture and in murine tumor systems that depend on increased PI3K function (18,20,21).

Class I phosphoinositide 3-kinase contains four isoforms of the catalytic subunit, p110␣, -␤, -␥, and -␦. At physiological levels of expression, the wild-type p110␣ isoform lacks oncogenic potential, but gain-of-function mutations and overexpression of p110␣ are correlated with oncogenicity. The p110␤, -␥, and -␦ isoforms induce transformation of cultured cells as wild-type proteins. This oncogenic potential requires kinase activity and can be suppressed by the target of rapamycin inhibitor rapamycin. The p110␦ isoform constitutively activates the Akt signaling pathway; p110␥ activates Akt only in the presence of serum. The isoforms differ in their requirements for upstream signaling. The transforming activity of the p110␥ isoform depends on rat sarcoma viral oncogene homolog (Ras) binding; preliminary data suggest the same for p110␤ and indicate Ras-independent oncogenic potential of p110␦. The surprising oncogenic potential of the wild-type non-␣ isoforms of class I phosphoinositide 3-kinase may explain the dearth of cancerspecific mutations in these proteins, because these non-␣ isoforms could contribute to the oncogenic phenotype of the cell by differential expression.Akt

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