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Abstract
Metastasis is responsible for approximately 90% of cancer related deaths, and cellular responses to cues from the environment alter the way cells escape from the primary tumor. Cancer cells modify their shape in response to their environment, but the exact mechanisms and physiological triggers driving tumor cell morphological conversions are poorly understood. This tumor cell migratory plasticity is heavily reliant upon rearrangement of the F-actin cytoskeleton through the mammalian diaphanous (mDia) related formins. Dynamic actin polymerization underlies both spear-like and bleb structures respectively characterizing the mesenchymal and amoeboid motility programs utilized by metastatic cells. mDia2 is a member of the formin family of Rho GTPase effectors that regulate F-actin polymerization. mDia2 assembles non-branched actin filaments required for motility. mDia2 is functionally coupled with its binding partner and negative regulator DIP, regulating cortical actin and inducing membrane blebbing in amoeboid cells. However, a physiological trigger of this mechanism was not known.
We discovered that mDia2 and DIP co-tether to blebs and this linkage is required for bleb formation. DIP controls mesenchymal/amoeboid morphological interconvertability, while the chemokine CXCL12 induces assembly of mDia2:DIP complexes to bleb cortices. These results demonstrate how DIP-directed, mDia2-dependent F-actin dynamics regulate morphological plasticity in motile cancer cells. We also show in breast tumor cells that CXCL12 engages its receptor CXCR4 to induce RhoA activation and the direct association of mDia2 and DIP. We show that CXCR4 and GTP-bound RhoA are required for CXCL12-mediated blebbing. Furthermore, we identify Net1 as the CXCL12-directed RhoGEF required for activating RhoA to drive amoeboid blebbing. Our data demonstrate a novel signaling axis linking CXCR4, its ligand CXCL12 and Net1 with RhoA/mDia2/DIP signaling.
Lastly, we demonstrate the role of mDia2 in tumor formation using a mouse mammary fat pad model. Loss of mDia2 drastically increases tumor burden but does not alter the metastatic potential of these cells. Loss of mDia2 also alters the collagen environment adjacent to and within the tumor, with collagen bundles orienting perpendicular to the tumor edge. These data lend us a more comprehensive understanding of the role that migratory plasticity has on tumor progression and points to the need to target multiple modes of motility when considering anti-motility treatment options.
