Abstract
Tumor-associated macrophages (TAMs) are important mediators of triple-negative breast cancer (TNBC) progression, yet the molecular mechanisms driving this process remain incompletely defined. In this study, we identified MLK4, a member of the MAP3K family, as a regulator of TAM-driven oncogenic processes in TNBC. Using a co-culture of TNBC cells with macrophages, we demonstrated that high MLK4 expression in TNBC is essential for macrophage-induced cancer cell proliferation, extracellular matrix (ECM) remodeling, migration, and invasion. Mechanistically, we showed that the cross-talk between TAMs and TNBC cells drives tumor aggressiveness via an MLK4-dependent mechanism by enhancing NF-κB activation and downstream matrix metalloproteinases (MMPs) expression. We also identified the most prominently upregulated factors, including CXCL1 and IL-8, during the co-culture of macrophages and TNBC cells. We further showed that MLK4 expression correlates with increased macrophage infiltration in TNBC patient samples, indicating its potential role in shaping the immunosuppressive tumor microenvironment. Summarizing, our findings uncover a paracrine signaling involving CXCL1 and MLK4-NF-κB-MMPs axis, which mediates the interactions between TAMs and TNBC cells, enhancing proliferation, mesenchymal transition, ECM remodeling and cancer invasion. This work elucidates a new mechanism of macrophage-induced tumor progression and highlights MLK4 as a promising therapeutic target for disrupting cancer cells-macrophage reciprocal communication in TNBC.
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Data availability
All mRNA-seq data have been deposited at GEO DataSets GSE304746.
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Acknowledgements
We would like to thank Dr. Dorota Adamska and Dr. Krzysztof Goryca (CeNT University of Warsaw) for assistance in the mRNA-seq experiment. We thank Julia Ostrowska and Dr. Salwador Cyranowski (the Laboratory of Cytometry, Nencki Institute of Experimental Biology, Polish Academy of Sciences) for assistance in generating flow cytometry data and analysis. We thank Prof. Dominika Nowis, Prof. Monika Kaczmarek, Dr. Karolina Szczepanowska, Dr. Małgorzata Bajor, Prof. Tomasz Rygiel and all the lab members for their insightful comments and sharing reagents. The research was supported by the Polish National Science Centre (2018/31/D/NZ5/01274, 2021/42/E/NZ5/00227 and 2023/49/B/NZ5/02604 grants to AAM), The Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund (“Regenerative Mechanisms for Health” project MAB/2017/2, carried out within the International Research Agendas programme). NGS was performed thanks to the Genomics Core Facility CeNT UW (RRID: SCR_022718), using the NovaSeq 6000 platform financed by the Polish Ministry of Science and Higher Education (decision no. 6817/IA/SP/2018 of 2018-04-10). For the purpose of Open Access, the author has applied a CC-BY public copyright license to any Author Accepted Manuscript (AAM) version arising from this submission. The graphs were created using GraphPad Prism 7 software. Some figures in this article were created with Biorender.com.
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AMB designed the study, optimized and performed experiments, analyzed the data and wrote the manuscript. DM analyzed the mRNA-seq data. KK, AM, VNT, and PJ performed some experiments and analyzed the data. ML performed CIBERSORTx analysis. AAM designed and supervised the study, provided funding, performed experiments, analyzed the data and wrote the manuscript. All authors have read and approved the final version of the article, including the authorship list.
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Mazan-Bury, A., Mehlich, D., Karpińska, K. et al. MLK4 orchestrates macrophage-induced triple-negative breast cancer invasion and ECM remodeling via enhanced paracrine signaling and NF-κB-MMP axis activation. Cell Death Dis (2026). https://doi.org/10.1038/s41419-026-08689-y
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DOI: https://doi.org/10.1038/s41419-026-08689-y
