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. 2013 Oct 1;19(19):5413-22.
doi: 10.1158/1078-0432.CCR-13-0884. Epub 2013 Jul 25.

PIK3CA and AKT1 mutations have distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model system

Julia A Beaver  1 , John P Gustin, Kyung H Yi, Anandita Rajpurohit, Matthew Thomas, Samuel F Gilbert, D Marc Rosen, Ben Ho Park, Josh Lauring
Affiliations

PIK3CA and AKT1 mutations have distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model system

Julia A Beaver et al. Clin Cancer Res. .

Abstract

Purpose: Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown.

Experimental design: We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to "correct" PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background.

Results: Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206.

Conclusions: AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies.

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Conflict of interest statement

Conflict of interest statement: B.H.P. is a paid consultant for GlaxoSmithKline and is a paid member for the scientific advisory board of Horizon Discovery, Ltd. J.L. and B.H.P. are entitled to receive royalty payments for commercial use of cell lines described in this work under a licensing agreement between Johns Hopkins University and Horizon Discovery, Ltd.

Figures

Figure 1
Figure 1. Establishment of MCF-7PIK3CAWT cells and PIK3CA WT/AKT1 E17K derivatives
Sequencing of genomic DNA and cDNA is shown. Parental MCF-7 cells have two mutant PIK3CA E545K alleles and one wild type allele. The cell line designated “Het” has one allele with targeted correction to wild type and one remaining mutant allele. A second round of gene targeting was performed to obtain the PIK3CA WT cells. The PIK3CA WT cells were used to knock in the AKT1 E17K mutation.
Figure 2
Figure 2. PI3-kinase pathway signaling in the MCF-7 cell line panel
Cells were grown in medium containing 5% FBS. Equal amounts of protein were loaded on SDS-PAGE gels and blotted with the antibodies shown. The full list of antibodies is included in Supplementary Table 1.
Figure 3
Figure 3. Growth characteristics of MCF-7 cells with different PI3K pathway genotypes
A–C. Proliferation in vitro in 0.5% charcoal/dextran-treated FBS medium (CD-FBS) (A), 1 nM ?-estradiol in 0.5% CD-FBS (B), and 5% FBS medium (C). Cells were seeded at 5–10,000 cells/well in 12-well plates in triplicate and counted every 2–3 days. D. Colony formation in soft agar. Cells were seeded at 30,000 cells/well in triplicate in 0.4% agar atop a layer of 0.6% agar, and colonies were stained and counted after 21 days. E. In vivo growth of xenografts in estrogen-supplemented nude mice. 10 mice per group were inoculated with one million cells of each genotype in Matrigel. Tumors were measured weekly. In all cases error bars represent standard deviations.
Figure 4
Figure 4. Differential sensitivity of PIK3CA and AKT1 mutant cells to targeted pathway inhibitors
MCF-7 derived cells of the indicated genotypes were treated with increasing concentrations of the class I-specific PI3K inhibitor GDC-0941 (A), the allosteric AKT inhibitor MK-2206 (B), and the dual PI3K-mTOR inhibitor NVP-BEZ235 (C). Viable cell number was determined with a luminescence assay. Results are depicted as viable cell number relative to untreated controls. Experiments were performed in triplicate and repeated three times. Averages and standard errors of all three experiments are shown.
Figure 5
Figure 5. PI3K pathway signaling in GDC-0941 and MK-2206 treated MCF-7 derivatives with PIK3CA or AKT1 mutations
Cells were grown in medium containing 5% FBS and treated with vehicle or increasing concentrations of GDC-0941 (0 nM, 50 nM, 100 nM, and 400 nM) or MK-2206 (0 nM, 100 nM, 250 nM, 1000 nM). After 24 hours of drug treatment, lysates were prepared and equal amounts of protein were loaded onto SDS-PAGE gels and blotted with the indicated antibodies.
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