Abstract
The tumor microenvironment (TME) promotes tumor cell invasion and metastasis. An important step in the shift to a pro-cancerous microenvironment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are present in a majority of solid tumors and can directly promote tumor cell motility via cytokine, chemokine and growth factor secretion into the TME. The exact effects that the TME has upon cytoskeletal regulation in motile tumor cells remain enigmatic. The conserved formin family of cytoskeleton regulating proteins plays an essential role in the assembly and/or bundling of unbranched actin filaments. Mammalian Diaphanous-related formin 2 (mDia2/DIAPH3/Drf3/Dia) assembles a dynamic F-actin cytoskeleton that underlies tumor cell migration and invasion. We therefore sought to understand whether CAF-derived chemokines impact breast tumor cell motility through modification of the formin-assembled F-actin cytoskeleton. In MDA-MB-231 cells, conditioned media (CM) from WS19T CAFs, a human breast tumor-adjacent CAF line, significantly and robustly increased wound closure and invasion relative to normal human mammary fibroblast (HMF)-CM. WS19T-CM also promoted proteasome-mediated mDia2 degradation in MDA-MB-231 cells relative to control HMF-CM and WS21T CAF-CM, a breast CAF cell line that failed to promote robust MDA-MB-231 migration. Cytokine array analysis of CM identified up-regulated secreted factors in WS19T relative to control WS21T CM. We identified CXCL12 as a CM factor influencing loss of mDia2 protein while increasing MDA-MB-231 cell migration. Our data suggest a mechanism whereby CAFs promote tumor cell migration and invasion through CXCL12 secretion to regulate the mDia2-directed cytoskeleton in breast tumor cells.
Highlights
90% of cancer-related deaths are due to advanced metastatic disease [1]
We first assessed if factors present in WS19T carcinoma-associated fibroblasts (CAFs)-conditioned conditioned media (CM) altered tumor cell motility in wound healing assays
When WS19T-CM was applied to the U251 glioblastoma and OVCA429 ovarian cancer cell lines in wound closure assays (S2 Fig), we did not observe significant increases in motility suggesting that breast tumor CAF-secreted factors might be tissue-specific or different concentration requirements drive progression in different tissues of origin
Summary
90% of cancer-related deaths are due to advanced metastatic disease [1]. Soluble factors from breast cancer cells upregulated the oncoprotein YAP in adjacent fibroblasts which in turn increased actin cytoskeleton contractility and created a self-sustaining, activated CAF phenotype with enhanced secretion of pro-tumorigenic factors [12]. As mDia formins nucleate and polymerize actin, force is generated by these newly synthesized filaments and this force deforms the cell membrane, creating protrusive structures underlying cell migration and invasion [18]. Functional inhibition of mDia through association with its negative regulator, Dia-interacting protein (DIP), caused non-apoptotic blebbing, a hallmark of amoeboid motility in breast tumor cells [42]. We demonstrated conditioned media (CM) from WS19T breast tumor-adjacent CAFs significantly increases MDA-MB-231 breast tumor cell migration and invasion, and is correlated with significant loss of mDia protein expression through a proteasomal-dependent mechanism. We determined by membrane-based cytokine array that stromal-secreted CXCL12 is a significantly upregulated component of CAF-CM that underlies mDia loss in MDA-MB-231 cells and the resultant increase in cell migration
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