Arp2/3 is a protein complex that nucleates actin filament assembly in the lamellipodium in adherent cells crawling on planar 2-dimensional (2D) substrates. However, in physiopathological situations, cell migration typically occurs within a 3-dimensional (3D) environment, and little is known about the role of Arp2/3 and associated proteins in 3D cell migration. Using time resolved live-cell imaging and HT1080, a fibrosarcoma cell line commonly used to study cell migration, we find that the Arp2/3 complex and associated proteins N-WASP, WAVE1, cortactin, and Cdc42 regulate 3D cell migration. We report that this regulation is caused by formation of multigeneration dendritic protrusions, which mediate traction forces on the surrounding matrix and effective cell migration. The primary protrusions emanating directly from the cell body and prolonging the nucleus forms independent of Arp2/3 and dependent on focal adhesion proteins FAK, talin, and p130Cas. The Arp2/3 complex, N-WASP, WAVE1, cortactin, and Cdc42 regulate the secondary protrusions branching off from the primary protrusions. In 3D matrices, fibrosarcoma cells as well as migrating breast, pancreatic, and prostate cancer cells do not display lamellipodial structures. This study characterizes the unique topology of protrusions made by cells in a 3D matrix and show that these dendritic protrusions play a critical role in 3D cell motility and matrix deformation. The relative contribution of these proteins to 3D migration is significantly different from their role in 2D migration.

Inflammatory breast cancer (IBC) is a unique and deadly disease with unknown drivers. We hypothesized the inflammatory environment contributes to the IBC phenotype. We used an in vitro co-culture system to investigate interactions between normal and polarized macrophages (RAW 264.7 cell line), bone-marrow derived mesenchymal stem cells (MSCs), and IBC cells (SUM 149 and MDA-IBC3). We used an in vivo model that reproduces the IBC phenotype by co-injecting IBC cells with MSCs into the mammary fat pad. Mice were then treated with a macrophage recruitment inhibitor, anti-CSF1. MSC and macrophages grown in co-culture produced higher levels of pro-tumor properties such as enhanced migration and elevated IL-6 secretion. IBC cells co-cultured with educated MSCs also displayed enhanced invasion and mammosphere formation and blocked by anti-IL-6 and statin treatment. The treatment of mice co-injected with IBC cells and MSCs with anti-CSF1 inhibited tumor associated macrophages and inhibited pSTAT3 expression in tumor cells. Anti-CSF1 treated mice also exhibited reduced tumor growth, skin invasion, and local recurrence. Herein we demonstrate reciprocal tumor interactions through IL-6 with cells found in the IBC microenvironment. Our results suggest IL-6 is a mediator of these tumor promoting influences and is important for the IBC induced migration of MSCs.

Microtubules have long been implicated to play an integral role in metastatic disease, for which a critical step is the local invasion of tumor cells into the 3-dimensional (3D) collagen-rich stromal matrix. Here we show that cell migration of human cancer cells uses the dynamic formation of highly branched protrusions that are composed of a microtubule core surrounded by cortical actin, a cytoskeletal organization that is absent in cells on 2-dimensional (2D) substrates. Microtubule plus-end tracking protein End-binding 1 and motor protein dynein subunits light intermediate chain 2 and heavy chain 1, which do not regulate 2D migration, critically modulate 3D migration by affecting RhoA and thus regulate protrusion branching through differential assembly dynamics of microtubules. An important consequence of this observation is that the commonly used cancer drug paclitaxel is 100-fold more effective at blocking migration in a 3D matrix than on a 2D matrix. This work reveals the central role that microtubule dynamics plays in powering cell migration in a more pathologically relevant setting and suggests further testing of therapeutics targeting microtubules to mitigate migration.—Jayatilaka, H., Giri, A., Karl, M., Aifuwa, I., Trenton, N. J., Phillip, J. M., Khatau, S., Wirtz, D. EB1 and cytoplasmic dynein mediate protrusion dynamics for efficient 3-dimensional cell migration.

Purpose We previously reported using statins was correlated with improved metastasis free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple negative breast cancer (TNBC) cells. Experimental Design TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in 8 public breast cancer gene expression data sets including 1,479 patients. Results Simvastatin increased G1/S phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pretreatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Conclusions Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.

IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks.