PI3K isoform dependence of PTEN-deficient tumors can be altered by the genetic context

Abstract

There has been increasing interest in the use of isoform-selective inhibitors of phosphatidylinositide-3-kinase (PI3K) in cancer therapy. Using conditional deletion of the p110 catalytic isoforms of PI3K to predict sensitivity of cancer types to such inhibitors, we and others have demonstrated that tumors deficient of the phosphatase and tensin homolog (PTEN) are often dependent on the p110β isoform of PI3K. Because human cancers usually arise due to multiple genetic events, determining whether other genetic alterations might alter the p110 isoform requirements of PTEN-null tumors becomes a critical question. To investigate further the roles of p110 isoforms in PTEN-deficient tumors, we used a mouse model of ovarian endometrioid adenocarcinoma driven by concomitant activation of the rat sarcoma protein Kras, which is known to activate p110α, and loss of PTEN. In this model, ablation of p110β had no effect on tumor growth, whereas p110α ablation blocked tumor formation. Because ablation of PTEN alone is often p110β dependent, we wondered if the same held true in the ovary. Because PTEN loss alone in the ovary did not result in tumor formation, we tested PI3K isoform dependence in ovarian surface epithelium (OSE) cells deficient in both PTEN and p53. These cells were indeed p110β dependent, whereas OSEs expressing activated Kras with or without PTEN loss were p110α dependent. Furthermore, isoform-selective inhibitors showed a similar pattern of the isoform dependence in established Kras(G12D)/PTEN-deficient tumors. Taken together, our data suggest that, whereas in some tissues PTEN-null tumors appear to inherently depend on p110β, the p110 isoform reliance of PTEN-deficient tumors may be altered by concurrent mutations that activate p110α.

Keywords: PI3K inhibitors; genetically engineered mouse model; ovarian cancer.

Fig. 1.

p110α but not p110β is required for tumor formation in an ovarian tumor model driven by concurrent Pten loss and Kras^G12D expression. (A) Adenovirus expressing Cre recombinase (Ad-Cre) was injected into the ovarian bursa of female mice carrying the indicated genotypes, inducing expression of oncogenic Kras and deletion of Pten (PK), alone or with p110α (PKA) or p110β (PKB) ablation. (B) Ovaries were isolated at different time points after intrabursal injection of Ad-Cre and sent for pathology evaluation. Depicted are typical examples of H&E stainings of complete mouse ovaries. Arrows show hyperplastic lesions in the surface epithelium of the ovary. (C) Histopathological evaluation of ovaries with the indicated genotypes.

Fig. 2.

Immunohistochemical analysis of ovaries after Ad-Cre injection. Immunohistochemical staining of PK, PKA, and PKB ovaries at 10–14 wk after Cre injection with the indicated antibodies. Expression of oncogenic Kras and deletion of Pten (PK), additional deletion of p110α (PKA), and additional deletion of p110β (PKB).

Fig. 3.

The presence of Kras^G12D shifts the PI3K isoform dependence of Pten-null induced tumorigenesis from p110β to p110α. (A) Genotyping and (B) Western blot (WB) analysis of ovarian surface epithelial (OSE) cells with the following genotypes: Expression of oncogenic Kras (K), deletion of Pten (P), combination of both lesions (PK), additional deletion of p110α (PKA, KA, and PA), additional deletion of p110β (PKB, KB, and PB). (C) OSE cells with the indicated genotypes were seeded at the same densities, grown for the indicated times in medium without FBS or growth factors, and stained with crystal violet. Shown are averages and SDs from two independent experiments performed in triplicates. (D–F) OSE cells with the indicated genotypes were injected s.c. into the flank of NCrNu recipient mice and their tumor sizes measured with a caliper. Shown are single tumor sizes with mean values and SEMs (Left) and typical H&E stainings of sections from tumors (Right). (G) WB analysis of ovarian surface epithelial (OSE) cells with the following genotypes: deletion of Pten and trp53 (PP), additional deletion of p110α (PPA), and additional deletion of p110β (PPB). (H) OSE cells with the indicated genotypes were injected s.c. into the flank of NCrNu recipient mice and their tumor sizes measured with a caliper. Shown are single tumor sizes with mean values and SEMs.

Fig. 4.

Pharmacological inhibition of p110α effectively blocks tumorigenic growth of OSE-PK cells. (A) Ovarian surface epithelial (OSE) cells from mice with expression of oncogenic Kras and deletion of Pten (PK) were injected s.c. into the flank of NCrNu recipient mice. Treatment with pharmacological inhibitors (BYL719, 45 mg/kg once daily p.o. and KIN193, 20 mg/kg once daily i.p.) was started 1 d after injection. After 3 wk, mice were killed and tumor sizes were measured with a caliper. Shown are single tumor sizes with mean values and SEMs. *P < 0.001; **P < 0.0001. (B) Tumors from Fig. 4A were homogenized, lyzed, and analyzed by Western blot using the indicated antibodies. Shown is one representative Western blot (Left); quantifications represent mean values and SEMs from eight independent tumors per group (Right). *P < 0.05. (C and D) Ovarian surface epithelial (OSE) cells from mice with deletion of Pten and trp53 (PP) (C) or expression of oncogenic Kras and deletion of Pten (PK) (D) were injected s.c. into the flank of NCrNu recipient mice and treated as described in A. **P < 0.0001, n.s., not statistically significant.