Abstract P3-03-04: Sensitivity to lapatinib differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA and relies on production of PIP3
Authors: Garay JP , Korkola JE , Gray JW 
Published: March 2017

Abstract
Introduction: HER2 is amplified in nearly 25% of all primary breast cancers. Lapatinib is a targeted therapy that inhibits overactive HER2 signaling but invariably resistance to this targeted therapy occurs in a substantial number of patients. The PI3K-AKT axis is the major pathway downstream of HER2 signaling. Activated PI3-kinase phosphorylates the membrane lipid PIP2 resulting in PIP3. PIP3 is as a docking site for pleckstrin homology (PH) domain proteins, such as the AKT. AKT influences a variety of pathways inside the cell involving cell growth, regulation of apoptosis, glucose metabolism, and others. Mutations in the gene PIK3CA deregulate this signaling axis. In HER2 amplified cancers, co-occurrence of PIK3CA mutations have been reported in approximately 20% of cases. Hotspot mutations of PIK3CA translate to changes in either the helical domain (E545K) or kinase domain (H1047R) of the protein and these two hotspots comprise over 80% of all reported oncogenic mutations across all tumor types. Crystallographic studies have shown conformational differences between the two hotspot mutations in PIK3CA, yet it is unclear if functional differences exist between the two mutations.
Methods: We generated isogenic knockin mutants of the helical domain (E545K) and kinase domain (H1047R) of PIK3CA in the HER2-amplified breast cancer cell line SK-BR-3. Mutant and parental cell lines were subjected to drug sensitivity assays measured by cell growth during prolonged exposure to drug. We investigated changes of relevant intracellular signaling pathways via western blot analysis. Additionally, we used immunofluorescence of PIP3 and confocal microscopy to visualize cellular differences in the production of this signaling molecule.
Results: Our results demonstrate a distinction between the helical domain (E545K) and kinase domain (H1047R) mutations of PIK3CA. Mutations in the helical domain do not confer resistance to lapatinib while mutations in the kinase domain do. This is a result of sustained AKT signaling even in the presence of high dose lapatinib in cells with the kinase domain mutation. We also show the PTEN loss phenocopies this phenomenon. Finally, we show that kinase domain mutations allow the protein to generate significantly higher levels of PIP3 which is the necessary molecule for downstream signaling through AKT but helical domain mutations do not.
Conclusion: This phenotypic disparity between helical and kinase domain mutations of PIK3CA has important clinical implications. It is possible to imagine that in a heterogeneous tumor in which some cells are wildtype and some cells carry this mutation for PIK3CA treatment with lapatinib will select for cells with the mutation conferring a growth advantage. Our results show that only H1047R mutant cells demonstrate lapatinib resistance and this is achieved via sustained AKT signaling through continual production of PIP3. Altogether, we demonstrate a mechanism of de novo resistance to HER2-targeted therapy in breast cancer.
Citation Format: Garay JP, Korkola JE, Gray JW. Sensitivity to lapatinib differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA and relies on production of PIP3 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-04.