Generation and Characterization of Trastuzumab/Pertuzumab-Resistant HER2-Positive Breast Cancer Cell Lines

Abstract

The combination of trastuzumab and pertuzumab as first-line therapy in patients with HER2-positive breast cancer has shown significant clinical benefits compared to trastuzumab alone. However, despite initial therapeutic success, most patients eventually progress, and tumors develop acquired resistance and invariably relapse. Therefore, there is an urgent need to improve our understanding of the mechanisms governing resistance in order to develop targeted therapeutic strategies with improved efficacy. We generated four novel HER2-positive cell lines via prolonged exposure to trastuzumab and pertuzumab and determined their resistance rates. Long-term resistance was confirmed by a significant increase in the colony-forming capacity of the derived cells. We authenticated the molecular identity of the new lines via both immunohistochemistry for the clinical phenotype and molecular profiling of point mutations. HER2 overexpression was confirmed in all resistant cell lines, and acquisition of resistance to trastuzumab and pertuzumab did not translate into differences in ER, PR, and HER2 receptor expression. In contrast, changes in the expression and activity of other HER family members, particularly HER4, were observed. In the same vein, analyses of the receptor and effector kinase status of different cellular pathways revealed that the MAPK pathway may be involved in the acquisition of resistance to trastuzumab and pertuzumab. Finally, proteomic analysis confirmed a significant change in the abundance patterns of more than 600 proteins with implications in key biological processes, such as ribosome formation, mitochondrial activity, and metabolism, which could be relevant mechanisms in the generation of resistance in HER2-positive breast cancer. We concluded that these resistant BCCLs may be a valuable tool to better understand the mechanisms of acquisition of resistance to trastuzumab and pertuzumab-based anti-HER2 therapy.

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“…Among other HER2-targeted therapies, pertuzumab is a monoclonal antibody whose action mechanism is complementary to trastuzumab, inhibiting ligand-dependent HER2–HER3 dimerization and reducing signaling via intracellular pathways such as phosphatidylinositol 3-kinase (PI3K/Akt) [ 57 ]. The combination of pertuzumab and trastuzumab is so able to block not only HER2-HER3 heterodimers, but also EGFR/HER2 ones, resulting in a wider inhibition of the ErbB-stimulated intracellular pathways.…”