Cancer-specific mutations in PIK3CA are oncogenic in vivo

Abstract

The PIK3CA gene, coding for the catalytic subunit p110alpha of class IA phosphatidylinositol 3-kinases (PI3Ks), is frequently mutated in human cancer. Mutated p110alpha proteins show a gain of enzymatic function in vitro and are oncogenic in cell culture. Here, we show that three prevalent mutants of p110alpha, 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 p110alpha mutant. The in vivo oncogenicity of PIK3CA mutants in an avian species strongly suggests a critical role for these mutated proteins in human malignancies.

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 p110α, or the empty vector RCAS. Eggs were sealed and incubated for another 9 days. CAMs were dissected and prepared for macroscopic photography using a binocular microscope.

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 the ×40 image is indicated by a box in the ×4 image. a, nucleated avian erythrocytes; b, malignant cell formation; c, smooth muscle cells of a normal blood vessel. Arrowheads indicate cell nuclei in metaphases.

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 27 days. Values represent the mean of four animals (H1047R, myr-p110α, and two each of E542K and E545K) or three animals (RCAS). Error bars are shown (bars facing up for H1047R and E542K/E545K, bars facing down for myr-p110α). The survey of H1047R tumors was terminated on day 15 because of rapid tumor growth. (B) Cells used in A on the day of injection were analyzed by Western blotting for expression of p110α and viral Gag proteins. The ^* indicates nonspecific interactions with anti-Gag^p19 antibodies.

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 by using the ×4 objective. (Insets) ×40 magnifications. a, nucleated erythrocytes; b, polymorphic neoplastic cells; c, normal blood vessel.

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 protein was separated by denaturing gel electrophoresis and probed with antibodies directed to phospho-Akt (Ser-473), total Akt, phospho-GSK-3β (Ser-9), and total GSK-3β. Tag numbers 104, 105, 157, and 115 identified animals used for protein extraction.

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 second group of 10 animals was injected with 10⁶ CEFs expressing H1047R (day 0). Medication began after 8 days, when tumors in all animals were visible. RAD001 or placebo was administered daily at 10 mg/kg for the following 11 days. The gray shaded areas indicate the time frame of therapy. Two animals injected with cells expressing RCAS did not develop tumors and were used as negative controls (data not shown in the graph).