S2-7: Mechanisms of Action and Biological Significance of HER2 Mutations in HER2−Overexpressing Breast Cancer.
Authors: Boulbes DR , Jin Q , Arold ST , Ladbury JE , Yu D , Esteva FJ 
Published: June 2012

Abstract
Background: Trastuzumab is the most successful HER2−directed therapy in patients with early-stage and advanced HER2 positive breast cancer. Although trastuzumab improves survival in the adjuvant setting, 15–20% of the patients develop metastasis. Lapatinib is currently the only tyrosine kinase inhibitor approved for the treatment of patients with metastatic breast cancer after trastuzumab progression. However, progression eventually occurs and the disease remains incurable for the majority of patients. Recently, somatic mutations in the HER2 kinase domain have been reported in lung adenocarcinomas which result in constitutive phosphorylation of HER2, EGFR and cellular substrates. Activation of these receptors and pathways in lung cancer was associated with tumor progression and trastuzumab resistance, but tumors remained sensitive to a tyrosine kinase inhibitor. Because there are many different types of breast cancer that respond differently to treatments, more approaches are needed to predict which patients will most likely respond to a given therapy.
Material and Methods: Using gene sequencing on 78 HER2 positive breast tumors, our laboratory identified 4 novel missense variants in the kinase domain of HER2. None of the patients whose tumors carried one of these mutations achieved objective response to trastuzumab. Mutants were created using directed mutagenesis, inserted in a lentiviral expression plasmid and stably expressed in different breast cancer or non-tumorigenic cell lines. An ATP-based assay was used for cell survival studies. Migration studies were performed using Boyden-chambers coated with Matrigel. Anchorage-independent colony formation was assessed in soft-agar. Signaling pathways and phosphorylation status were analyzed by Western blot. Localization results were obtained by immunofluorescence and confocal microscopy followed by deconvolution analysis.
Results: Two of the mutants were dramatically under-phosphorylated and presented an altered cellular localization revealed by immunofluorescence studies in both cell lines and patient surgical samples. Also, cells expressing HER2 mutants showed an increased ability to invade Matrigel and migrate and to form colonies in soft-agar, suggesting the induction of a more aggressive behavior. In particular, one of the mutations was strongly associated with resistance to lapatinib treatment in cell survival and soft-agar assays, and higher doses of lapatinib were necessary to inhibit the ERK and AKT pathways. Computational analysis revealed that the mechanism of lapatinib resistance could be explained by a sterical obstruction of the ATP-binding pocket of the protein kinase domain that would impede the binding of lapatinib.
Conclusion: HER2 mutations confer a more aggressive phenotype. A specific mutation directly interacts with Lapatinib binding to HER2 and predicts resistance to Lapatinib in HER2−overexpressing breast cancer cells. Further characterization of novel HER2 mutations may have a direct implication in the development of novel markers for early diagnostics, patient selection and characterization of more appropriate and personalized treatment.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S2-7.