An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer

Abstract

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor-positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor-positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.

Figure 1

Detection of PIK3CA and AKT1 mutations. A mass spectrometry tracing of an AKT1 E17K-mutant SNP allele in a hormone receptor–positive breast cancer. wt, wild type.

Figure 2

Effects of PTEN loss and PIK3CA mutation on AKT activation in human breast tumors. PTEN and the two AKT phosphorylation sites (AKTp308 and AKTp473) were quantified using reverse-phase protein lysate array. The quantification data were then log transformed (base 2), mean centered, ordered by increasing PTEN expression level from above down, and plotted in the heat map shown. In the mean centering schema used, red indicates a relatively high level of (phospho)protein expression and green indicates a relatively low level of (phospho)protein expression. The level of PTEN expression is shown in lane 1 and the levels of AKT phosphorylation at Thr³⁰⁸ and Ser⁴⁷³ are shown in lanes 2 and 3, respectively. AKT phosphorylation was expressed as a ratio to total AKT expression for presentation in this figure. Clearly, AKT phosphorylation is strongly inversely correlated with PTEN protein expression. However, there is no clear association between PIK3CA mutation and AKT phosphorylation at either amino acid site. Further, there was no significant difference (P = 0.41) in the frequency of tumors with PIK3CA mutations among those tumors with the highest and lowest quartiles of PTEN expression [17 of 77 (22.1%) and 12 of 77 (15.6%), respectively]. Tumors from one institution batch (i.e., from Clinic Hospital) were used for this analysis.

Figure 3

Effect of PTEN and PIK3CA mutations on AKT activation/phosphorylation at Ser⁴⁷³ in 40 breast cancer cell lines. AKT phosphorylation at Ser⁴⁷³ (AKTp473) was significantly higher in PTEN-mutant cell lines than in PIK3CA-mutant (P = 0.005) or PTEN/PIK3CA wild-type (P = 0.001) cell lines. In contrast, there was no significant difference (P = 0.64) in AKT phosphorylation at Ser⁴⁷³ between PIK3CA-mutant and PTEN/PIK3CA wild-type cell lines. AKTp473 was quantified using RPPA and expressed on the Y axis after logarithmic conversion and mean centering.

Figure 4

The relative sensitivity of 12 hormone receptor–positive breast cancer cell lines to the PI3K inhibitor LY294002. LY294002 was applied to the panel of hormone receptor–positive breast cancer cell lines and the concentration causing 50% growth inhibition (GI₅₀) was determined and presented as relative sensitivity (−log GI₅₀). The cell lines are presented in order of increasing PTEN protein expression (lane A) as determined using RPPA. PTEN-low (P = 0.0007) and PTEN-mutant (P = 0.02) cell lines are significantly more sensitive to growth inhibition by LY294002 than PIK3CA-mutant cell lines. Lane B, AKT phosphorylation at Ser⁴⁷³ determined using RPPA [color coded as in lane A (i.e., green, low; black, mean; and red, high AKT phosphorylation)]. Lane C, cell line mutation status.

Figure 5

Correlations between PIK3CA mutations and patient survival. No significant differences in RFS (A and B) or OS (C and D) times were found after adjuvant tamoxifen therapy between patients with early-stage PIK3CA wild-type hormone receptor–positive breast tumors and those with PIK3CA-mutant breast tumors.