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Abstract A16: Molecular mechanisms of EGFR therapy resistance in breast cancer cells

The purpose of this study is to investigate molecular mechanisms of epidermal growth factor receptor (EGFR) therapy resistance using a syngeneic pair of gefitinib-sensitive and gefitinib-resistant breast cancer cells. Signaling from EGFRs is usually dysregulated in breast, as well as other cancers, where it is commonly associated with an aggressive phenotype and poor outcome. The initial success of targeted therapies for EGFR1 and HER2 has been marred by de novo or acquired resistance. Gefitinib, a small molecule tyrosine kinase inhibitor with high specificity towards EGFR, was proven to be effective for the treatment of non-small cell lung carcinoma (NSCLC). However, clinical trials of gefitinib as a breast cancer therapeutic have failed due to lack of response, probably due to resistance, and adverse toxic effects. We have developed a Gefitinib resistant variant of the gefitinib sensitive human breast cancer cell line SKBR3 (SKBR3 G.R.), with the objective of studying the involvement of micro (mi)-RNAs in therapy resistance. A partial screening of several miRNAs previously shown to be involved in therapy resistance was performed and miRNA (miR-)200a was identified as a downregulated miRNA in the SKBR3 G.R. cells. Members of the miR-200 family play a critical role in the suppression of epithelial-to-mesenchymal transition (EMT) by repressing key mRNAs involved in this process. Additionally, the miR-200 family can regulate tumor cell adhesion, migration, invasion and metastasis; and has been associated with EGFR inhibitor resistance. Herein, we have analyzed the protein expression of EGFR, phospho-EGFR, and miR-200 family targets Zeb1, β-catenin, and the ErbB receptor inhibitor-1 (ERRFI-1 or Mig6) by western blot analysis in SKBR3 and SKBR3 G.R. breast cancer cells. Our results show a significant increase in the expression of EGFR, Zeb1, and β-catenin; whereas, phospho-EGFR and Mig6 expression was significantly downregulated in SKBR3 G.R. cells when compared to parental cells. Therefore, Mig6 expression may not be regulated via miR-200 activity in these cells. Downregulation of Mig6 may explain the increased EGFR levels due to its role in EGFR internalization and subsequent degradation. Since SKBR3 cells are mesenchymal, they do not express E-cadherin, another Zeb1 target. Our results suggest that in cancer cells that have undergone EMT, decreased miR-200a levels, and thus, increased Zeb1 and β-catenin, a potent pro-cancer transcriptional activator in the absence of an E-cadherin axis, is a potential mechanism of Gefitinib resistance in aggressive breast cancer. This study was supported by NIH/NIGMS SC3GM094824 to SD, the UCC Pilot Project Program, and the UCC Postdoctoral Program sponsored by the PR Science, Technology and Research Trust.