Knock in of the AKT1 E17K mutation in human breast epithelial cells does not recapitulate oncogenic PIK3CA mutations

Abstract

An oncogenic mutation (G49A:E17K) in the AKT1 gene has been described recently in human breast, colon, and ovarian cancers. The low frequency of this mutation and perhaps other selective pressures have prevented the isolation of human cancer cell lines that harbor this mutation thereby limiting functional analysis. Here, we create a physiologic in vitro model to study the effects of this mutation by using somatic cell gene targeting using the nontumorigenic human breast epithelial cell line, MCF10A. Surprisingly, knock in of E17K into the AKT1 gene had minimal phenotypic consequences and importantly, did not recapitulate the biochemical and growth characteristics seen with somatic cell knock in of PIK3CA hotspot mutations. These results suggest that mutations in critical genes within the PI3-kinase (PI3K) pathway are not functionally equivalent, and that other cooperative genetic events may be necessary to achieve oncogenic PI3K pathway activation in cancers that contain the AKT1 E17K mutation.

Figure 1. Resequencing for the AKT1 G49A:E17K mutation in primary human breast cancers and knock in of the AKT1 G49A:E17K mutation in MCF10A human breast epithelial cells

(a) Representative sequencing traces from gDNA templates of matched tumor and normal tissue. (b) Design of the targeting construct. The AKT1 G49A:E17K mutation is on the 3′ homology arm, which includes all of exon 3. “Neo” signifies a cassette containing a short synthetic intron, splice acceptor, internal ribosome entry site, neomycin resistance gene, and polyadenylation signal. (c) Sequence analysis of gDNA and cDNA from parental MCF10A cells and targeted subclones.

Figure 2. AKT1 and PIK3CA mutants differentially affect PI3K and MAPK signaling

Immunoblot analysis of parental MCF10A cells and their isogenic AKT1 and PIK3CA knock in derivatives under (a) EGF-free conditions and (b) physiologic 0.2 ng/mL EGF growth conditions.

Figure 3. AKT1 mutation does not affect EGF independent growth or acinar morphogenesis

(a) Equal numbers of parental MCF10A cells and derivatives with knock in of wild type or mutant AKT1, or exon 9 or exon 20 PIK3CA mutations were seeded in 6-well plates in EGF-free growth medium with 1% charcoal dextran treated FBS. Media was replaced daily. Plates were stained with crystal violet after 6 days. A representative experiment is shown. (b) MCF10A cells and knock in derivatives, as well as Kras^V12 overexpressing derivatives, were seeded on growth factor-reduced Matrigel in medium containing 20 ng/mL EGF, (Bar = 100 μM).

Figure 4. AKT1 mutation does not alter the proliferative response to estrogen and tamoxifen

(a) Establishment of ER expressing MCF10A AKT1 E17K mutant cells. Immunoblot analysis of ER and GAPDH expression in MCF7, ERIN (parental MCF10A cells transfected with ER), TERIAKI (AKT1 E17K knock in MCF10A cells transfected with ER), and untransfected AKT1 E17K knock in cells. (b) Proliferative response to estrogen and tamoxifen. ERIN and TERIAKI cells were seeded in EGF-free, phenol red-free growth medium supplemented with vehicle, 17 β-estradiol (E2), tamoxifen (Tam), or both. Medium was replaced daily. Flasks were stained with crystal violet after six days in culture.

Figure 5. PIK3CA mutants, but not AKT1 mutants, display increased sensitivity to rapamycin and the PI3K inhibitor LY294002

Cells were seeded in triplicate wells on day 0 in medium containing 0.2 ng/ml EGF. Drugs or vehicle (DMSO) were added on days 1 and 4. Cells were harvested and counted on day 6. Values are shown as percentage of vehicle control cell number and represent averages of three independent experiments with standard deviations. * p=NS compared to MCF10A. ** p < 0.02 compared to MCF10A. ***p < 0.005 compared to MCF10A.