Exon 9 and exon 20 mutations in PIK3CA confer resistance to HER2 inhibitors in HER2-overexpressing breast cancer cells.
Authors: Rexer BN , Chakrabarty A , Rinehart C , Chang J , Engelman J , Arteaga CL 
Published: December 2010

Abstract
Abstract #4054
The anti-tumor effect of HER2 antagonists in HER2-dependent breast cancer cells has been proposed to rely on inhibition of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Mutations in the p110α catalytic subunit of PI3K occur in up to 40% of breast cancers and activation of this pathway has been implicated in resistance to the HER2 antibody trastuzumab (T). PIK3CA mutations cluster in two regions in the helical (E542K, E545K; exon 9) and catalytic (H1047R; exon 20) domains. We studied the role of these mutants in resistance to HER2 inhibitors in breast cancer cells with HER2 amplification. Two lines with endogenous H1047R p110 (PI3K), SUM190 and HCC1954, and SKBR3, SUM225, and BT474 cells stably transduced with retroviral vectors encoding HA-tagged wild-type, E545K, or H1047R p110, were studied for their response to the HER2 tyrosine kinase inhibitor lapatinib ditosylate (L; GW-572016) and T. In monolayer and 3D cell proliferation assays, partial resistance to L and T was conferred by either mutation compared to WT p110. After prolonged L treatment, Ser473 phosphorylation of Akt recovered in all cells with endogenous or ectopic p110 mutants in spite of continued inhibition of Y1248 P-HER2 and Y1289 P-HER3 by L. Further, BT474 cells with either p110 mutant could be passaged in the continued presence of L. Immunoprecipitation of p85, the regulatory subunit of PI3K, showed that PI3K association with HER3 was abrogated by T and L. RNAi of HER3 in MCF10A/HER2/PI3KE545K and MCF10A/HER2/PI3KH1047R cells markedly but not completely inhibited growth suggesting that the PI3K mutants may still depend on HER3 for full activation. We hypothesized that in cells with mutant PI3K, the mutants coexist in a pool with WT enzyme, and that mutant and WT p110α are bound to p85 in variable proportions. Experiments to measure whether the ratio of mutant to WT p110 bound to p85, assayed by mass spectrometry, will increase in the PI3K-mutant cells with acquired resistance to L are in progress. To test the role of these mutants on resistance to anti-HER2 therapies in vivo, athymic mice bearing BT474 xenografts with ectopic WT, E545K, or H1047R p110 are undergoing treatment with L and T. Finally, we analyzed mutations of PIK3CA in a cohort of 40 patients with locally advanced HER2+ breast cancer treated with weekly single-agent T for 3 weeks, followed by T with docetaxel for a total of 12 weeks before surgery (Mohsin et al. JCO 23:2460, 2005). Sequential core biopsies at weeks 1 and 3 after initiation of T were taken. Eight of 40 tumors (20%) expressed mutant PI3K, 2 in exon 9 and 6 in exon 20. PIK3CA mutations did not correlate with change in Ki67 (p=0.97) or cleaved caspase 3 (p=0.51) in weeks 1 or 3 of treatment nor with pathologic complete response (p=0.21). These data suggest that, if mutant PI3K confers relative resistance to T, a shorter time to recurrence may be a more robust endpoint as the initial cellular or clinical response may not be a good indicator of this resistance.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4054.