Sakwe, A. M. | Search bioRxiv

bioRxiv2025

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

Background/ObjectivesTriple-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 contribution of mesenchymal-like and epithelial TNBC cells in the persistence of chemotherapy resistant disease remains poorly understood. MethodsMesenchymal-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. ResultsAnalysis of the response of stage IV 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 model mesenchymal-like, and upregulation of CCL2/MCP-1, CTSS and DKK-1 in model epithelial TNBC cells. The inhibitory phosphorylation of GSK-3{beta} (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. ConclusionsThese data reveal that chemotherapy resistance of epithelial and mesenchymal-like TNBC cellular subsets upregulated distinct profiles of proinflammatory and immune cell chemotactic cytokines and modulated the activities of GSK-3{beta}, p90 RSK1/2 and the related MSK1/2. Targeting these factors and/or the associated signaling pathways may help overcome chemotherapy resistance in TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/685128v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1c5481dorg.highwire.dtl.DTLVardef@101a766org.highwire.dtl.DTLVardef@1954523org.highwire.dtl.DTLVardef@ee2ddb_HPS_FORMAT_FIGEXP M_FIG C_FIG 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. This study reveals that Chemotherapy resistance in epithelial and mesenchymal-like TNBC cells is maintained by distinct signatures of proinflammatory and immune cell chemotactic cytokines, and activities of GSK-3{beta} and p90 ribosomal S6 kinases.

cancer biology↗

bioRxiv2024

Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

Pro-inflammatory cytokines are secreted via the classical pathway from secretory vesicles or the non-classical pathway via extracellular vesicles (EVs), that together, play critical roles in triple-negative breast cancer (TNBC) progression. Annexin A6 (AnxA6) is a Ca2+-dependent membrane-binding protein that in TNBC is implicated in cell growth and invasiveness. AnxA6 is associated with EVs, but whether it affects the secretion of proinflammatory cytokines and/or EVs remains to be fully elucidated. To assess if AnxA6 influences the secretion of cytokines and extracellular vesicles, we used cytokine arrays to analyze secreted factors in cleared culture supernatants from control AnxA6 expressing and AnxA6 downregulated MDA-MB-468 TNBC cells. This revealed the diminished secretion of monocyte chemoattractant protein 1 (MCP-1/CCL2), interleukin 8 (IL-8), dickkopf1 (DKK1), throbospondin-1 (TSP-1), and osteopontin (OPN) following AnxA6 downregulation. We also show that the secretion of small EVs is strongly reduced in AnxA6 downregulated cells and that upregulation of AnxA6 promoted the secretion of treatment was also associated with increased EVs associated Rab7, cholesterol, and MCP-1 levels. Moreover, cholesterol content in EVs was significantly higher in AnxA6-expressing cells than in AnxA6 downregulated cells and following chronic lapatinib induced upregulation of AnxA6. Mechanistically, we demonstrate that the secretion of MCP-1 and/or EVs is AnxA6 dependent and that this requires the translocation of AnxA6 to cellular membranes and its interaction with SNAP23. AnxA6 neutralizing antibodies strongly diminished the survival of AnxA6 low TNBC cells but had minimal effects on the survival of TNBC cells expressing relatively high levels of the protein. Together, these data suggest that AnxA6 facilitates the secretion of EVs and proinflammatory cytokines that may be critical for TNBC progression.

cancer biology↗