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.

Annexin A6 (AnxA6) is a predominantly intracellular calcium-dependent membrane-binding multifunctional protein that is also detected extracellularly and in small extracellular vesicles (exosomes). We previously demonstrated that lapatinib resistance in triple-negative breast cancer (TNBC) cells is associated with AnxA6 upregulation and accumulation of cholesterol in late endosomes. Here, we investigated the fate of AnxA6 and cholesterol in lapatinib-resistant (LAP-R) cells and whether extracellular AnxA6 influences TNBC cell survival. We demonstrate that reduced expression of AnxA6 in LAP-R cells decreased the secretion of MCP-1/CCL2, CCL8/IL-8, DKK1, TSP-1, and OPN by antibody arrays. The secretion of exosomes was also markedly reduced in AnxA6-depleted LAP-R cells, while AnxA6 upregulation stimulated the release of MCP-1 and exosomes. Compared to the respective controls, exosome-associated AnxA6, Rab7, and cholesterol levels were increased in exosomes isolated from AnxA6-expressing LAP-R cells. Mechanistically, we demonstrated by co-immunoprecipitation, GST pulldown, and proximity ligation assays that AnxA6 interacts with SNAP23, a component of the membrane fusion machinery. Finally, blocking extracellular AnxA6 with neutralizing antibodies reduced the viability of AnxA6-low TNBC cells but had little effect on AnxA6-high cells. These findings suggest that extracellular AnxA6 is critical for the survival of highly proliferative AnxA6-low basal-like breast cancer cells and that AnxA6 influences TNBC progression by facilitating the secretion of pro-inflammatory cytokines and cholesterol-enriched exosomes.