Advanced metastatic breast cancers overexpress epidermal growth factor receptor (EGFR 1-4) family members and do not respond to standard anti-estrogen therapy. Therefore, a number of targeted therapies have been developed against this family of receptors. However, disease recurrence and resistance to treatments remains a problem in the clinical setting. Therefore, it is critical to develop strategies to overcome therapy resistance in breast cancer. The purpose of this dissertation was to determine the mechanisms of anti-EGFR/HER2 therapy resistance in breast cancer, and test new strategies to overcome such therapy resistance.

To this end, we created syngeneic therapy resistant cell lines by exposing EGFR1 and human epidermal growth factor receptor 2 (HER2) positive therapy sensitive SKBR3 human breast cancer cells to different concentrations of the EGFR1 and HER2 targeted therapeutics gefitinib (Gef) and lapatinib (Lap). Three resistant syngeneic cell variants were created from the SKBR3 parental cell line; SKBR3 Gef R. 0.1microM, SKBR3 Gef R. 0.5microM and SKBR3 Lap R. 0.1microM. Sensitivity of these paired cell variants was determined by exposing them to a range of drug concentrations to obtain the IC50s for cell viability. Results showed that therapy resistant cells had a clinically relevant fold resistance. To determine if the treatments still inhibit the phosphorylation of EGFR1 and HER2 in therapy sensitive and resistant cell lines, we performed western blots for total expression and phosphorylation of the receptors. Similar to the sensitive cells, EGFR and HER2 activation via autophosphorylation remained inhibited in the therapy resistant variants constantly exposed to the treatments, except in cells resistant to high concentrations of gefitinib, suggesting bypass mechanisms of therapy resistance. Lapatinib is known to promote apoptosis in HER2 overexpressing breast cancer cells, therefore, we tested caspase 3/7 activity in therapy sensitive and resistant cells with and without treatment. Apoptosis assays showed that the SKBR3 parental cell line had higher caspase 3/7 activity when treated with lapatinib. However, when the SKBR3 Lap R. 0.1microM cell line was exposed to treatment, the cells did not respond to the treatments with caspase 3/7 activation. To determine if the therapy resistant cells include a higher percentage of stem cell-like cells, mammosphere assays were performed. Both, gefitinib and lapatinib treatments, reduced mammosphere formation in the parental therapy sensitive, but not in the therapy resistant cell lines, indicating more cancer stem cell-like activity in the resistant variants. Since signaling molecules downstream of EGFR family have been shown to contribute to therapy resistance, we tested for the expression and phosphorylation of AKT and mitogen activated protein kinase (MAPK). Results showed no changes in expression and phosphorylation (activation) of AKT and MAPK in the therapy resistant variants compared to the therapy sensitive cells. Interestingly, expression and activation of the small Rho GTPase Rac were elevated in the therapy resistant variants compared to the therapy sensitive cells. Therefore, the potential of the Rac inhibitor EHop-016 to overcome therapy resistance was tested, and found to reduce the viability of therapy sensitive, as well as gefitinib and lapatinib resistant cell variants, by ~ 80%. Our data demonstrate that targeting Rac to chemosensitize anti-EGFR/HER2 therapy resistance in breast cancer could be a potential strategy to overcome therapy resistance.

On the other hand, differentially expressed non-coding RNAs (ncRNAs) in resistant and sensitive cells were identified from quantitative RT-PCR analysis. Dysregulation of microRNAs (miRNAs) has been proposed as a possible mechanism of resistance to different treatments, including tyrosine kinase inhibitors (TKIs). Results revealed a downregulation of miR-200 and upregulation of miR-221/222 families, which are known to regulate Epithelial-to-mesenchymal transition (EMT), a hallmark of therapy resistance. Parallel with the downregulation of miR-200a, its targets zinc finger E-box binding homeobox (Zeb)1/2, and transcriptional activator beta-catenin were upregulated in the therapy resistant cells. Furthermore, as expected, the miR-221/222 targets, tumor suppressors phosphatase and tensin homolog deleted in chrosmosome 10 (PTEN) and the transcriptional repressor of Zeb2, transcriptional repressor GATA binding 1 (TRPS1), were decreased in the therapy resistant cells. Ectopic miR-200a expression in cells sensitive and resistant to lapatinib showed a decrease in cell viability, as shown by the decrease in IC50, when treated with lapatinib, suggesting an increase in response to the treatment. Inhibition of miR-221 or miR-222 improved the response to both gefitinib and lapatinib in all cell lines. Therefore, overexpression of miR-200a reduces the malignancy of SKBR3 parental cell lines, and sensitizes lapatinib resistant cells to lapatinib therapy. Moreover, inhibition of miR-221 or miR-222 also reduces cancer malignancy and therapy resistance to both lapatinib and gefitinib.