Contribution of Mesenchymal-like and Epithelial Cellular Subsets to Chemotherapy Resistance in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) tumors are typically heterogeneous, predominantly epithelial tissues with discrete patches of mesenchymal-like TNBC cells that differ in their invasiveness, proliferation potential and response to treatment. However, the impact of mesenchymal-like and epithelial TNBC cells on the persistence of chemotherapy-resistant disease remains poorly understood. Mesenchymal-like and epithelial TNBC cell types were detected by multiplex fluorescent immunohistochemistry using antibodies against vimentin, Ki67, and Annexin A6 (AnxA6). Chemotherapy drug-resistant mesenchymal-like and epithelial TNBC cell populations were established by pulse exposure and stepwise dose escalation and validated by 3D cultures and unbiased antibody arrays. Analysis of the response of TNBC tumors treated with six common chemotherapy regimens resulted in 36% complete response and 64% partial response with residual tumor sizes ranging from 0.5 to 37.0 mm. Treatment of TNBC cells with chemotherapy agents led to distinct resistance signatures including downregulation of survivin and upregulation of M-CSF and CXCL8/IL-8 in the model mesenchymal-like TNBC cells, and upregulation of CCL2/MCP-1, CTSS and DKK-1 in model epithelial TNBC cells. The inhibitory phosphorylation of GSK-3β (p-S9) increased in paclitaxel-resistant epithelial cells but decreased in resistant mesenchymal-like TNBC cells. Finally, chemotherapy resistance also activated p90 ribosomal S6 kinases (RSK1/2) in both cell types, while activation of mitogen- and stress-activated kinases (MSK1/2) was only observed in chemotherapy-resistant epithelial TNBC cells. These data reveal that chemotherapy resistance of epithelial and mesenchymal-like TNBC cellular subsets led to distinct profiles of proinflammatory and immune cell chemotactic cytokines and modulated the activities of GSK-3β, p90 RSK1/2 and the related MSK1/2. Targeting these factors and/or the associated signaling pathways may help overcome chemotherapy resistance in TNBC.