Understanding the Kinomic Contributions to Tyrosine Kinase Inhibitor Resistance in Triple Negative Breast Cancer
Abstract
Resistance to tyrosine kinase inhibitors (TKIs) presents a growing challenge in the development of therapeutic targets for cancers such as triple negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Potential targets in TNBC include the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met, however, targeted anti-EGFR and anti-c-Met therapies have faced challenges in clinical trials due to acquired resistance. We hypothesize that response versus resistance of triple negative breast cancer to the tyrosine kinase inhibitors erlotinib and cabozantinib is mediated by compensatory changes in the kinome and phosphoproteome. To test this, we (1) assessed the sensitivity of MDA-MB-468 and MDA-MB-231 TNBC cell lines to erlotinib and cabozantinib, (2) developed an integrated mass spectrometry proteomics approach to infer changes in kinase activity at kinome-level, and (3) identified mediators of resistance in developed erlotinib- and cabozantinib-resistant TNBC cell lines. First, we observed that MDA-MB-468 cells were sensitive to the anti-proliferative, anti-migratory and anti-invasive effects of erlotinib, whereas MDA-MB-231 cells were sensitive to cabozantinib. Secondly, we used SILAC (Stable Isotope Labeled Amino Acids in Cell Culture)-labeled MDA-MB-468 cells in quantitative mass spectrometry-based kinomics and phosphoproteomics to assess the effects of erlotinib in TKI-responsive cells. Using a modified KSEA analysis, we inferred that erlotinib decreases activity of ERK1/2. Finally, we observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion and activation of EGFR or c-Met downstream signaling (respectively). Using the integrated proteomics approach, we identified upregulation of CDK1, CDK7 and CK2A1 activity in cabozantinib-resistant cells and upregulation in AKT1, CK2A1 and ERK1 activity in erlotinib-resistant cells. Functional validation revealed inhibitor synergy between erlotinib and AKT inhibitor VIII. Using the integrated proteomics approach, we identified several potential kinase mediators of cabozantinib-resistance and confirmed the contribution of AKT1 to erlotinib-resistance in TNBC resistant cell lines. The resistance mediators identified in this study can be used to develop combination therapeutics to apply in further in vivo and clinical studies of resistance to EGFR and c-Met inhibitors in triple negative breast cancer.