The phosphatidylinositol 3-kinase (PI3K) isoform dependence of tumor formation is determined by the genetic mode of PI3K pathway activation rather than by tissue type

Abstract

Previous work has shown that prostate cancer in a Pten-null murine model is dependent on the p110β isoform of phosphatidylinositol 3-kinase (PI3K), while breast cancer driven by either polyoma middle T antigen (MT) or HER2 is p110α dependent. Whether these differences in isoform dependence arise from tissue specificity or from the nature of the oncogenic signal activating the PI3K pathway is important, given increasing interest in using isoform-specific PI3K inhibitors in cancer therapy. To approach this question, we studied the PI3K isoform dependence of our recently constructed prostate cancer model driven by MT. Since MT activates a number of signaling pathways, we first confirmed that the MT-driven prostate cancer model was actually dependent on PI3K. A newly generated transgenic prostate line expressing an MT allele (Y315F) known to be defective for PI3K binding displayed a markedly reduced ability to drive tumor formation. We next selectively ablated expression of either p110α or p110β in mice in which wild-type MT was expressed in the prostate. We found that tumor formation driven by MT was significantly delayed by the loss of p110α expression, while ablation of p110β had no effect. Since the tumor formation driven by MT is p110α dependent in the prostate as well as in the mammary gland, our data suggest that PI3K isoform dependence is driven by the mode of PI3K pathway activation rather than by tissue type.

Importance: Middle T antigen (MT), the oncogene of polyomavirus, can drive tumor formation in a variety of cell types and tissues. Interestingly, MT has no intrinsic enzymatic activity but instead functions by binding and activating cellular signaling proteins. One of the most important of these is the lipid kinase PI3K, which was first studied in MT immunoprecipitates. Ubiquitously expressed PI3K comes in two major isoforms: p110α and p110β. Previous work in animal models showed that p110α was the key isoform in breast tumors driven by oncogenes, including MT and HER2, while p110β was key in prostate tumors driven by Pten loss. We asked the simple question of whether a prostate tumor driven by MT depends on p110α, which would suggest that the mode of activation determines p110 isoform dependence, or p110β, which would suggest that tissue type determines isoform dependence. The clear answer is that MT depends on p110α in both the prostate and breast.

FIG 1

The PI3K binding site on MT is essential for efficient transformation in a polyomavirus MT-driven prostate cancer model. (A) Hematoxylin-eosin staining of formalin-fixed prostate tissue from mice with the indicated genotypes at different ages. (B) Relative wet weight of ventral and dorsolateral prostate lobes from mice with the indicated genotypes at different ages. Shown are mean values and standard errors of the means for at least 5 mice per genotype. MT, polyomavirus middle T antigen; MT Y315F, p85 binding-defective MT; ns, not statistically significant; *, P < 0.05; **, P < 0.001.

FIG 2

PI3K signaling is essential for efficient transformation in a polyomavirus MT-driven prostate cancer model. (A, B) Lipid kinase assay (A) and Western blot analysis (B) of prostate tissue from mice with the indicated genotypes at 30 to 34 weeks. Shown are the results of one representative experiment (left) and quantifications from 3 independent experiments with standard errors of the means (right). For kinase assay quantifications, the PIP signal was normalized to the IP efficiency (IP signal/input signal in the Western blot). (C) Immunohistochemistry of formalin-fixed prostate tissue from mice with the indicated genotypes at 30 to 34 weeks.

FIG 3

Ablation of the PI3K catalytic subunit p110α delays MT-driven transformation in the prostate. (A) Hematoxylin-eosin staining of formalin-fixed prostate tissue from mice with the indicated genotypes at different ages. Arrowheads, PIN; arrows, normal epithelium. (B) Relative wet weight of ventral and dorsolateral prostate lobes from mice with the indicated genotypes at different ages. Shown are mean values and standard errors of the mean for at least 5 mice per genotype. ns, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

FIG 4

Reduced PI3K signaling due to ablation of the catalytic subunit p110α is essential to inhibit MT-driven transformation in the prostate. (A, B) Lipid kinase assay (A) and Western blot analysis (B) of prostate tissue from mice with the indicated genotypes at 24 to 28 weeks. Shown are the results of one representative experiment (left) and quantifications from 3 independent experiments with standard errors of the means (right). For kinase assay quantifications, the lipid kinase signal was normalized to the IP efficiency (IP signal/input signal in the Western blot). (C) Immunohistochemistry staining of p-Akt in formalin-fixed prostate tissue sections from 28-week-old mice with the indicated genotypes.

FIG 5

Different mechanisms for delayed PIN lesions in MT-driven transformation. (A) Hematoxylin-eosin staining of formalin-fixed prostate tissue from mice with the indicated genotypes at 56 weeks. (B) Immunohistochemistry of p-Akt and Ki67 in formalin-fixed prostate tissue sections from MT/Cre/p110α^L/L mice at different ages. (C, D) MT-associated lipid kinase activity (left) and Western blot analysis (middle) of prostate tissue from 3 different mice with the indicated genotypes per time point. (Right) Quantifications for 6 samples per time point. w, weeks. The lipid kinase signal was normalized to the IP efficiency (IP signal/input signal in the Western blot). n.s., not statistically significant; **, P < 0.01. (E) Genotyping of total prostate tissue lysates from mice with the indicated genotypes at 50 weeks. Upper band, floxed allele; lower band, wild-type allele. Cre excision should lead to the complete disappearance of the floxed allele (deleted allele not detectable with the primers used).