Activating mutations of the estrogen receptor confer chemotherapy resistance in breast cancer through the activation of JNK pathway

Introduction: Endocrine therapy is the mainstay treatment for estrogen receptor-α (ER) + metastatic breast cancer (BC) patients. However, all patients eventually develop resistance to these therapies. Recently, our lab discovered activating mutations, Y537S and D538G, in the ER ligand-binding domain (LBD) that confer resistance in nearly 40% of patients. We and others noted that the disease in LBD-ER patients is characterized by increased liver metastasis and a worse prognosis. Patients who develop endocrine resistance are treated with chemotherapy, and chemo-resistance is a major obstacle in these patients. We hypothesized that LBD-ER mutations confer resistance to chemotherapy, leading to worse prognosis. Therefore, our aim was to elucidate whether LBD mutations confer resistance to chemotherapy, growth advantage in liver microenvironment and elucidate the mechanism of action.

Methods: MCF-7 cells stably-expressing WT or the LBD mutations were used. Cell proliferation and viability were determined using MTT, methylene blue and colony assays. mRNA expression was performed using qRT-PCR, protein expression by Western-blot and transcriptional activity by a gene-reporter assay. Apoptosis studied by cleaved-PARP and Annexin/ DAPI analysis. Tumor growth was evaluated in an orthotropic model of BC.

Results:hemotherapeutic drugs doxorubicin and paclitaxel induced less apoptosis in LBD- cells, proliferated better, compared to WT-ER cells. In vivo, we found that the D538G-ER cells exhibited liver tropism while Y537S-ER cells mainly metastasized to the lungs and lymph nodes. Recent studies suggest that the JNK pathway may confer resistance to chemotherapy and that c-Jun overexpression is linked to breast cancer liver metastasis. Indeed, D538G-cells exhibited elevated JNK pathway activity evidenced by p-JNK and down-stream transcriptional activity, and activity further increased following doxorubicin treatment. In accordance, JNK inhibition further elevated apoptosis in D538G cells.

Conclusion: Our results indicate a new role to LBD-ER, beyond resistance to endocrine therapy, namely, resistance to chemotherapy. We revealed that in D538G-ER the resistance is mediated through the activation of the JNK pathway. Importantly, D538G confers predilection to liver metastasis. These results may aid in overcoming resistance in these D538G-patients by targeting the JNK pathway.