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Proc Natl Acad Sci U S A. Jan 31, 2006; 103(5): 1475–1479.
Published online Jan 23, 2006. doi:  10.1073/pnas.0510857103
PMCID: PMC1360603
Medical Sciences

Cancer-specific mutations in PIK3CA are oncogenic in vivo

Abstract

The PIK3CA gene, coding for the catalytic subunit p110α of class IA phosphatidylinositol 3-kinases (PI3Ks), is frequently mutated in human cancer. Mutated p110α proteins show a gain of enzymatic function in vitro and are oncogenic in cell culture. Here, we show that three prevalent mutants of p110α, 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 p110α mutant. The in vivo oncogenicity of PIK3CA mutants in an avian species strongly suggests a critical role for these mutated proteins in human malignancies.

Keywords: chorioallantoic membrane, hemangiosarcoma, phosphatidylinositol 3-kinase, p110

The PIK3CA gene codes for the catalytic subunit p110α 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 ≈30% 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 p110α 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 p110α confer a gain of function resulting in increased lipid kinase activity (15, 17-19). Expression of p110α 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 p110α 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 p110α 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).

Results

p110α Mutant Proteins Induce Neoplastic Cell Growth in the Chicken CAM. To explore the oncogenic effects of cancer-specific p110α mutations in vivo, we used the CAM of the chicken embryo as an in vivo tumor model. The CAM is a vascularized membrane located underneath the shell membrane, engulfing the chicken embryo, and is commonly used to measure angiogenesis and oncogenesis (22, 23). We inoculated the CAMs of 9-day-old chicken embryos with CEFs transformed by p110α mutant proteins E542K, E545K, and H1047R. CEFs stably transfected with wild-type p110α or empty replication-competent retroviral avian sarcoma-leukosis virus long-term repeat with splice acceptor (RCAS) vector served as nontransforming controls, and cells expressing the highly oncogenic protein myr-p110α, which contains an N-terminal myristylation signal, were used as a positive control (24). CEFs transfected with RCAS constructs release infectious viruses that harbor the RCAS genome plus insert and thus spread expression of the RCAS construct to neighboring cells. CAMs treated with E542K, E545K, and H1047R display increased vascularization and the formation of neoplastic nodules (Fig. 1). Areas that show abnormal cell growth are marked by strongly elevated angiogenesis. The positive control myr-p110α induces angiogenesis and neoplastic cell growth similar to the p110α mutants, in agreement with a previous report (23). Histological analysis of the H1047R tumor reveals hemangiosarcoma-like characteristics that closely resemble those observed in tumors induced by myr-p110α (Fig. 2). Large areas of polymorphic cells and multiple enlarged blood channels with a complete disruption of endothelial linings are common features. The tumor sections are dotted with frequent metaphases, and some of these are highly abnormal. The areas of hyperplasia on CAMs inoculated with the E542K or E545K mutants do not show hemangiosarcoma-like features in hematoxylin- and eosin-stained sections but nevertheless represent foci of abnormal cell growth (data not shown). In contrast, cells transfected with empty RCAS or wild-type p110α fail to induce angiogenesis or aberrant cell growth on the CAM, in agreement with previous observations in cell culture, suggesting that mere overexpression of p110α is insufficient for oncogenesis (18, 24).

Fig. 1.
Neoplastic cell growth and angiogenesis induced by p110α mutants in the CAM of the chicken embryo. CAMs of 9-day-old chicken embryos were each inoculated with 106 CEFs stably expressing p110α mutant proteins, myr-p110α, wild-type ...
Fig. 2.
Histology of tumors in the CAM. Three-micrometer cuts of membranes shown in Fig. 1 were stained with hematoxylin and eosin. Micrographs were taken by using the ×4 objective (Left) and the ×40 objective (Right). The corresponding area of ...

p110α Mutant Proteins Cause Tumors in the Animal. Data from in vitro studies and CAM assays indicate that the mutant proteins p110α E542K, E545K, and H1047R promote aberrant cell growth and may therefore cause tumors in the animal. To test this possibility, we s.c. injected CEFs transformed by the p110α mutants into the wing web of newly hatched chickens and monitored the animals for the following 27 days. Cells transfected with myr-p110α or empty RCAS vector were used as controls. Expression and the integrity of p110α, as well as retroviral Gag proteins on the day of injection were verified by Western blot analysis (Fig. 3B). Animals injected with any of the three p110α mutants developed tumors at the site of injection (Fig. 3A). Control animals treated with cells expressing the empty vector RCAS remained negative, and all animals injected with myr-p110α-expressing cells developed tumors. The growth rates of mutant-induced tumors varied, with the p110α H1047R protein as the most potent carcinogen. The mutants also differed in their frequencies of tumor induction, depending on whether the mutation occurred in the helical or in the catalytic domain of the p110α protein. Eighty percent of animals treated with H1047R (four of five) developed tumors, whereas E542K or E545K caused tumors at a frequency of 50% (four of eight). The difference in tumor incidence and growth cannot be explained by expression levels of individual p110α proteins, because cells used for injections show similar expression levels of E542K, E545K, and H1047R (Fig. 3B). These results suggest that the H1047R mutation in the catalytic domain is more potent than the E542K and E545K mutations in the helical domain, in accordance with data obtained from CAM assays and focus-formation assays (18). Histological analysis showed that all tumors induced by p110α mutant proteins were highly hemorrhagic and contained areas of polymorphic and neoplastic cells, similar to tumors caused by myr-p110α and to tumors of the CAM (Fig. 4). All tumors also show a high degree of angiogenesis. The H1047R-induced tumors classify as hemangiosarcomas; tumors caused by the helical domain mutations E542K and E545K were diagnosed as either hemangiomas or hemangiosarcomas.

Fig. 3.
Tumor growth induced by p110α mutants in chickens. (A) CEFs (106) stably transfected with p110α mutants, myr-p110α, or empty RCAS were injected into the wing web of newly hatched chickens (day 0). Tumor growth was monitored for ...
Fig. 4.
Histology of a H1047R tumor. Tumor tissue and the corresponding tissue of an animal that was injected with RCAS-transfected cells were dissected and fixed in paraffin. Three-micrometer sections were stained with hematoxylin and eosin and photographed ...

Activation of Akt Signaling in Mutant-Induced Tumors. Previous studies demonstrated that somatic mutations in PIK3CA constitute gain-of-function mutations, transmitting signals to downstream effector molecules independently of growth factors (18). To investigate whether tumors derived from H1047R show an activation of the PI3K pathway, we extracted total protein from tumors and probed for Akt and glycogen synthase kinase 3β (GSK-3β) with phospho-specific antibodies (Fig. 5). All tumors tested showed increased levels of phosphorylated Akt relative to the corresponding control tissues from the untreated wing of the same animal. Phosphorylation of Akt tightly correlates with elevated expression of p110α H1047R (data not shown). In most samples, GSK-3β is hyperphosphorylated at the Akt-specific target site, providing further evidence for an up-regulation of Akt signaling. These in vivo data are in agreement with results from experiments in cell culture and establish the oncogenicity of the p110α mutants and their activating effect on the Akt-signaling pathway.

Fig. 5.
Activation of Akt signaling in tumors induced by H1047R. Tumors of animals injected with H1047R-expressing cells (T) and corresponding control tissue of the untreated wing (C) were used for the extraction of protein lysates. Fifty micrograms of total ...

Inhibition of H1047R-Dependent Tumor Growth by RAD001. Cancers induced by deregulation of the PI3K pathway depend on the TOR kinase and are exquisitely sensitive to the TOR inhibitor rapamycin and its derivatives (20, 21). Tissue culture experiments demonstrated that cellular transformation induced by p110α mutants is similarly inhibited by rapamycin (18). Therefore, TOR may also be necessary in mutant-induced tumor formation in the animal. To test this hypothesis, we determined tumor growth in the presence of the rapamycin derivative RAD001 [everolimus, 40-O-(2-hydroxyethyl)rapamycin] (Novartis Pharma, Basel). We injected CEFs expressing p110α H1047R into 10 2-day-old chickens and treated 5 animals with RAD001 and 5 animals with placebo. RAD001 or placebo was administered orally at 10 mg/kg per day by gavage feeding and did not cause any toxicity, because animals were healthy and continually gained in size and body weight. Medication began on the day of injection and continued for the following 17 days. Animals on placebo rapidly developed tumors, and the onset as well as the growth rate of tumors was comparable to the ones in the previous experiments (Figs. (Figs.3A3A and and6A).6A). Tumor formation in animals treated with RAD001, however, was largely suppressed. During RAD001 treatment, three of five animals developed small colorless nodules that showed no signs of angiogenesis. Upon removal of the drug, these nodules quickly became hemorrhagic and rapidly increased in size, suggesting that continuous administration of RAD001 is required to inhibit tumor growth and angiogenesis. Because the incidence of tumor development induced by p110α H1047R is not 100%, we medicated a second group of animals, all of which had developed ≈3-mm tumors before therapy (Fig. 6B). Similar to the data shown in Fig. 6A, RAD001 treatment led to an initial regression of tumors with a concomitant loss of excess vascularization. Prolonged RAD001 treatment resulted in a stable disease with no increase or decrease of H1047R-induced tumors. Once therapy was terminated, however, all tumors became hemorrhagic and resumed growth at a pace comparable to animals on placebo.

Fig. 6.
Inhibition of H1047R-induced tumor growth by RAD001 therapy. (A) Ten 2-day-old chickens were injected with 106 CEF stably expressing H1047R (day 0) and medicated daily with 10 mg/kg RAD001 or placebo. On day 17, RAD001 treatment was terminated. (B) A ...

Discussion

The mutants of p110α tested in this study are cancer-specific, show enzymatic gain of function, and induce oncogenic transformation in cell culture (18). The data presented here extend the oncogenic potential of these mutants to animals. The E542K, E545K, and H1047R mutants map to mutational hot spots of the PIK3CA gene. This nonrandom distribution suggests that the mutations confer a selective advantage to the cell and is in accord with the interpretation that they contribute to the neoplastic phenotype of the cancer cell. The in vivo oncogenicity of the mutants adds strong support to this idea.

A mutant protein becomes an attractive target for cancer therapy when it combines the following four properties: (i) The occurrence of the protein is restricted to cancer tissue. (ii)Ithas a specific enzymatic activity. (iii) The enzymatic activity shows a gain of function compared with the wild-type enzyme. (iv) The mutant protein plays a causative role in the disease process. All four qualities are found in the p110α mutants. In these respects, the p110α mutants are similar to other exceptional cancer targets such as BCR-ABL, a fusion protein kinase that plays a causative role in chronic myeloid leukemia and has been targeted by the small molecule inhibitor imatinib with unequalled success in the clinic (25, 26).

The molecular mechanisms by which the mutants gain enzymatic function and hence acquire oncogenic potential are not known. The mutations could affect binding to the enzyme substrate or to a regulatory protein, or they could enhance the stability of the protein or affect the release from an intramolecular inhibitory interaction. The mutations could induce major conformational changes or locally confined structural alterations. Structural analysis and studies with small molecule inhibitors will provide answers to these questions.

Overexpression of the wild-type p110α protein from the retroviral vector RCAS can lead to oncogenic transformation in cell culture (18, 24). These rare oncogenic events derive from the use of the RCAS vector that expresses the p110α insert from actively replicating retroviral genomes. In the course of viral replication, the insert can become spontaneously fused to viral Gag sequences, generating a chimeric protein that, by virtue of its Gag region, has a constitutive membrane address and strong oncogenic potential. In all transformed cell foci induced by wild-type p110α, the lipid kinase is fused to viral Gag (18, 24). This tendency of the RCAS vector to generate oncogenic fusion proteins raises the possibility that the tumors developing after injection of mutant p110α-producing cells are caused not by the mutants themselves but by RCAS-generated Gag-p110α fusions. We have probed several tumor lysates for Gag fusions yet were unable to detect such chimeric proteins (data not shown). These results show that tumor induction by p110α mutants is not an artifact of the vector system and that the mutant proteins are oncogenic on their own. This conclusion is also supported by the rapidity of tumor induction, which is comparable to that of the highly oncogenic myristylated p110α and unlike the protracted process of oncogenic transformation associated with Gag-p110α fusions.

The tumors induced by mutant p110α proteins on the CAM and in the animal are highly hemorrhagic, and this phenotype appears to be linked to the activation of the Akt/TOR signaling pathway. We have shown previously that PI3K-Akt signaling is essential for angiogenesis (23). The growth of the tumors is effectively inhibited by the rapamycin-like inhibitor RAD001, suggesting an essential role for TOR in tumorigenesis. RAD001 treatment also leads to a complete regression of the hemorrhagic phenotype. This result is in accord with observations in mouse models, demonstrating that tumor development in these animals is suppressed by RAD001 with a concomitant decrease in angiogenesis (27). The angiogenic activity of p110α mutants may be mediated, at least in part, by hypoxia-inducible factor 1α (HIF-1α), which transcriptionally stimulates the expression of vascular endothelial growth factor (VEGF) (23). HIF-1α is regulated by TOR, and, therefore, suppression of angiogenesis by RAD001 could be a consequence of HIF-1α inactivation through inhibition of TOR (28, 29).

Avian cells appear to be highly sensitive to oncogenic transformation induced by gain-of-function PI3K, and this quality makes them exceptionally suitable for investigations on small molecule inhibitors of PI3K. There are evidently species-specific differences in the sensitivity to oncogenic signals. Human cells are notoriously difficult to transform in culture, requiring multiple genetic changes (30). However, the basic growth-regulatory machineries of all vertebrate cells appear to be identical. The induction of tumors by PI3K mutants in an avian species therefore reflects an oncogenic potential that is probably also active in human cells but for full expression may require additional factors.

Materials and Methods

Cell Culture and DNA Transfection. Fertilized chicken eggs (White Leghorn) were obtained from SPAFAS (Preston, CT). Primary CEFs were prepared as described in ref. 31. For stable transfections, 3 × 106 CEFs were transfected with 5 μg of RCAS vectors by DMSO shock (32). The RCAS constructs encoding chicken p110α, myr-p110α, E542K, E545K, and H1047R are described elsewhere (18, 24). Expression of p110α proteins was verified by Western blot analysis before in vivo studies.

CAM Assays. CAM assays were performed by following a published protocol (22, 23). Briefly, fertilized chicken eggs (White Leghorn) obtained from McIntyre Poultry & Fertile Eggs (Lakeside, CA) were incubated at 37.5°C with 70% humidity for 9 days. Eggs were candled to confirm embryo growth and to locate vascularized areas of the CAM. Two holes were drilled, one into the air-sack at the bottom of the eggs, another one at the lateral end above the blood vessels, penetrating through the outer shell membrane without injuring the CAM. The CAM was dropped by applying suction to the air-sack, exposing an ≈3-cm× 4-cm area of the CAM. CAMs were inoculated with 106 CEFs stably transfected with RCAS constructs encoding E542K, E545K, H1047R, myr-p110α, wild-type p110α, or empty vector. Eggs were sealed and incubated for another 9 days. Exposed areas of CAMs were dissected, washed with PBS, photographed, and prepared for histology. Three-micrometer sections of tissues embedded in paraffin were stained by hematoxylin and eosin by following standard protocols.

Animal Studies. Left wing webs of 1- to 2-day-old chickens (White Leghorn) were injected with 106 CEF stably transfected with empty RCAS or RCAS constructs encoding p110α proteins. Right wing webs were left untreated. Tumor growth was monitored 2-4 times a week. Animals with tumors larger than 10 mm in diameter were killed. Tumors and control tissues were isolated, documented by photography, and either used for the preparation of protein lysates or fixed in paraffin for histology. RAD001 solution (2% wt/wt) and placebo were obtained from Novartis Pharma. RAD001 was administered orally by gavage feeding at 10 mg/kg per day in 5% glucose. Placebo was given accordingly in 5% glucose. During medication, the body weight of animals was determined daily to evaluate potential side effects.

Western Blot Analysis. CEFs were grown to confluence, rinsed with PBS, and lysed in protein lysis buffer containing 1% Nonidet P-40, 10% glycerol, 20 mM Tris (pH 7.9), 150 mM NaCl, 1 mM MgCl2, 1 mM PMSF, 50 mM NaF, 1 mM DTT, 50 mM β-glycerophosphate, 1 mM Na3VO4, and 1× protease inhibitor mixture (Roche, Gipf-Oberfrick, Switzerland). Thirty micrograms of total protein was separated on a denaturing Tris-acetate/3-8% polyacrylamide gradient gel (Invitrogen), transferred onto a nitrocellulose membrane, and probed with polyclonal anti-p110α antibodies (Santa Cruz Biotechnology) and monoclonal anti-Gagp19 antibodies, kindly provided by Volker Vogt (Cornell University, Ithaca, NY). Blots were probed with monoclonal anti-tubulin immunoglobulins (ICN) to confirm equal loading. For the generation of tumor protein lysates, tumors were trimmed and ground in erythrocyte lysis buffer (ELB) containing 154 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA by using the large pestle of a Kontes tissue grinder. Samples were incubated on ice for 15 min to ensure lysis of erythrocytes and centrifuged for 10 min at 390 × g. Pellets were washed with ELB and centrifuged as described above. Finally, tissue pellets were suspended in tumor lysis buffer (1% Triton X-100/20 mM Tris, pH 7.9/150 mM NaCl/2 mM EDTA/1 mM Na3VO4/40 mM NaF/1× protease inhibitor mixture) and ground thoroughly, completely homogenizing the tissue. Protein lysates were incubated on ice for 30 min and cleared from debris by centrifugation at 16,450 × g for 15 min. Preparation of lysates from tissues of the corresponding control wings was performed accordingly. Fifty micrograms each of tumor protein lysates and control lysates were subjected to Western blot analyses by either Tris-acetate SDS/3-8% PAGE or Tris-glycine SDS/10% PAGE and probed with the following primary antibodies from Cell Signaling Technology (Beverly, MA): anti-Akt, anti-phospho-Akt (Ser-473), anti-GSK-3β, and anti-phospho-GSK-3β (Ser-9).

Acknowledgments

We thank D. Mikolon for instructions about the CAM assay, L. Hamaguchi-Ueno for technical assistance in cell culture, J. Everitt and her team at the vivarium, K. Clingerman and S. Spray for a tutorial on gavage feeding, and K. Osborn for his expert opinion on tumor pathology. This work was supported by grants from the National Cancer Institute. This manuscript is no. 17856-MEM of The Scripps Research Institute.

Notes

Author contributions: A.G.B. and P.K.V. designed research; A.G.B. and S.K. performed research; A.G.B., S.K., and P.K.V. analyzed data; and P.K.V. wrote the paper.

Conflict of interest statement: No conflicts declared.

Abbreviations: CAM, chorioallantoic membrane; CEF, chicken embryo fibroblast; GSK-3β, glycogen synthase kinase 3β; PI3K, phosphatidylinositol 3-kinase; RCAS, replication-competent retroviral avian sarcoma-leukosis virus long-term repeat with splice acceptor; TOR, target of rapamycin.

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