α-actinin1 and 4 tyrosine phosphorylation is critical for stress fiber establishment, maintenance and focal adhesion maturation.

In polarized, migrating cells, stress fibers are a highly dynamic network of contractile acto-myosin structures composed of bundles of actin filaments held together by actin cross-linking proteins such as α-actinins. As such, α-actinins influence actin cytoskeleton organization and dynamics and focal adhesion maturation. In response to environmental signals, α-actinins are tyrosine phosphorylated and this affects their binding to actin stress fibers; however, the cellular role of α-actinin tyrosine phosphorylation remains largely unknown. We found that non-muscle α-actinin1/4 are critical for the establishment of dorsal stress fibers and maintenance of transverse arc stress fibers. Analysis of cells genetically depleted of α-actinin1 and 4 reveals two distinct modes for focal adhesion maturation. An α-actinin1 or 4 dependent mode that uses dorsal stress fiber precursors as a template for establishing focal adhesions and their maturation, and an α-actinin-independent manner that uses transverse arc precursors to establish focal adhesions at both ends. Focal adhesions formed in the absence of α-actinins are delayed in their maturation, exhibit altered morphology, have decreased amounts of Zyxin and VASP, and reduced adhesiveness to extracellular matrix. Further rescue experiments demonstrate that the tyrosine phosphorylation of α-actinin1 at Y12 and α-actinin4 at Y265 is critical for dorsal stress fiber establishment, transverse arc maintenance and focal adhesion maturation.

Mena binds α5 integrin directly and modulates α5ß1 function.

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell-cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5ß1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue "LERER" repeats. In fibroblasts, the Mena-α5 complex was required for "outside-in" α5ß1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5ß1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.

Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function.

Entry of tumor cells into the blood stream is a critical step in cancer metastasis. Although significant progress has been made in visualizing tumor cell motility in vivo, the underlying mechanism of cancer cell intravasation remains largely unknown. We developed a microfluidic-based assay to recreate the tumor-vascular interface in three-dimensions, allowing for high resolution, real-time imaging, and precise quantification of endothelial barrier function. Studies are aimed at testing the hypothesis that carcinoma cell intravasation is regulated by biochemical factors from the interacting cells and cellular interactions with macrophages. We developed a method to measure spatially resolved endothelial permeability and show that signaling with macrophages via secretion of tumor necrosis factor alpha results in endothelial barrier impairment. Under these conditions intravasation rates were increased as validated with live imaging. To further investigate tumor-endothelial (TC-EC) signaling, we used highly invasive fibrosarcoma cells and quantified tumor cell migration dynamics and TC-EC interactions under control and perturbed (with tumor necrosis factor alpha) barrier conditions. We found that endothelial barrier impairment was associated with a higher number and faster dynamics of TC-EC interactions, in agreement with our carcinoma intravasation results. Taken together our results provide evidence that the endothelium poses a barrier to tumor cell intravasation that can be regulated by factors present in the tumor microenvironment.

2D protrusion but not motility predicts growth factor-induced cancer cell migration in 3D collagen.

Growth factor-induced migration is a critical step in the dissemination and metastasis of solid tumors. Although differences in properties characterizing cell migration on two-dimensional (2D) substrata versus within three-dimensional (3D) matrices have been noted for particular growth factor stimuli, the 2D approach remains in more common use as an efficient surrogate, especially for high-throughput experiments. We therefore were motivated to investigate which migration properties measured in various 2D assays might be reflective of 3D migratory behavioral responses. We used human triple-negative breast cancer lines stimulated by a panel of receptor tyrosine kinase ligands relevant to mammary carcinoma progression. Whereas 2D migration properties did not correlate well with 3D behavior across multiple growth factors, we found that increased membrane protrusion elicited by growth factor stimulation did relate robustly to enhanced 3D migration properties of the MDA-MB-231 and MDA-MB-157 lines. Interestingly, we observed this to be a more reliable relationship than cognate receptor expression or activation levels across these and two additional mammary tumor lines.

Quantitative analysis of gradient sensing: towards building predictive models of chemotaxis in cancer.

Chemotaxis of tumor cells in response to a gradient of extracellular ligand is an important step in cancer metastasis. The heterogeneity of chemotactic responses in cancer has not been widely addressed by experimental or mathematical modeling techniques. However, recent advancements in chemoattractant presentation, fluorescent-based signaling probes, and phenotypic analysis paradigms provide rich sources for building data-driven relational models that describe tumor cell chemotaxis in response to a wide variety of stimuli. Here we present gradient sensing, and the resulting chemotactic behavior, in a 'cue-signal-response' framework and suggest methods for utilizing recently reported experimental methods in data-driven modeling ventures.

Signaling network state predicts twist-mediated effects on breast cell migration across diverse growth factor contexts.

Epithelial-mesenchymal transition (EMT), whether in developmental morphogenesis or malignant transformation, prominently involves modified cell motility behavior. Although major advances have transpired in understanding the molecular pathways regulating the process of EMT induction per se by certain environmental stimuli, an important outstanding question is how the activities of signaling pathways governing motility yield the diverse movement behaviors characteristic of pre-induction versus postinduction states across a broad landscape of growth factor contexts. For the particular case of EMT induction in human mammary cells by ectopic expression of the transcription factor Twist, we found the migration responses to a panel of growth factors (EGF, HRG, IGF, HGF) dramatically disparate between confluent pre-Twist epithelial cells and sparsely distributed post-Twist mesenchymal cells-but that a computational model quantitatively integrating multiple key signaling node activities could nonetheless account for this full range of behavior. Moreover, motility in both conditions was successfully predicted a priori for an additional growth factor (PDGF) treatment. Although this signaling network state model could comprehend motility behavior globally, modulation of the network interactions underlying the altered pathway activities was identified by ascertaining differences in quantitative topological influences among the nodes between the two conditions.

Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer.

We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena(INV)) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena(INV) increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena(INV) is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena(INV) and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.

Prediction of sphingosine 1-phosphate-stimulated endothelial cell migration rates using biochemical measurements.

The ability to predict endothelial cell migration rates may aid in the design of biomaterials that endothelialize following implantation. However, the complexity of the signaling response to migration-promoting stimuli such as sphingosine 1-phosphate (S1P) makes such predictions quite challenging. A number of signaling pathways impact S1P-mediated cell migration, including the Akt and Src pathways, which both affect activation of the small GTPase Rac. Rac activation promotes the formation of lamellipodia, and thus should be intimately linked to cell migration rates. In immortalized endothelial cells, expression of proteins that inhibit Akt, Src, and Rac (PTEN, CSK, and beta2-chimaerin, respectively) was decreased using RNA interference, resulting in increases in the basal level of activation of Akt, Src, and Rac. Cells were scrape-wounded and stimulated with 1 microM S1P. The timecourse of Akt, Src, and Rac activation was followed over 2 h in the perturbed cells, while migration into the scrape wound was measured over 6 h. Rac activation at 120 min post-stimulation was highly correlated with the mean migration rate of cells, but only in cells stimulated with S1P. Using partial least squares regression, the migration rate of cells into the scrape wound was found to be highly correlated with the magnitude of the early Akt peak (e.g., 5-15 min post-stimulation). These results demonstrated that biochemical measurements might be useful in predicting rates of endothelial cell migration.

RAS mutations affect tumor necrosis factor-induced apoptosis in colon carcinoma cells via ERK-modulatory negative and positive feedback circuits along with non-ERK pathway effects.

More than 40% of colon cancers have a mutation in K-RAS or N-RAS, GTPases that operate as central hubs for multiple key signaling pathways within the cell. Utilizing an isogenic panel of colon carcinoma cells with K-RAS or N-RAS variations, we observed differences in tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. When the dynamics of phosphorylated ERK response to TNFalpha were examined, K-RAS mutant cells showed lower activation whereas N-RAS mutant cells exhibited prolonged duration. These divergent trends were partially explained by differential induction of two ERK-modulatory circuits: negative feedback mediated by dual-specificity phosphatase 5 and positive feedback by autocrine transforming growth factor-alpha. Moreover, in the various RAS mutant colon carcinoma lines, the transforming growth factor-alpha autocrine loop differentially elicited a further downstream chemokine (CXCL1/CXCL8) autocrine loop, with the two loops having opposite effects on apoptosis. Although the apoptotic responses of the RAS mutant panel to TNFalpha treatment showed significant dependence on the respective phosphorylated ERK dynamics, successful prediction across the various cell lines required contextual information concerning additional pathways including IKK and p38. A quantitative computational model based on weighted linear combinations of these pathway activities successfully predicted not only the spectrum of cell death responses but also the corresponding chemokine production responses. Our findings indicate that diverse RAS mutations yield differential cell behavioral responses to inflammatory cytokine exposure by means of (a) differential effects on ERK activity via multiple feedback circuit mechanisms, and (b) differential effects on other key signaling pathways contextually modulating ERK-related dependence.

Endothelial cell migration in human plasma is enhanced by a narrow range of added sphingosine 1-phosphate: implications for biomaterials design.

Sphingosine 1-phosphate (S1P) promotes endothelial cell migration in vitro and may potentially impact the endothelialization of implanted biomaterials. However, the effects of S1P on endothelial cells (EC) in flowing blood could be negligible due to preactivation of signaling cascades. We previously developed biomaterials that release S1P and wished to determine through in vitro experiments the extent to which EC respond to S1P added to human platelet poor plasma. We found that addition of 200 nM S1P to platelet poor plasma significantly increased cell migration in two migration models. A lower concentration of S1P added to plasma (100 nM) did not increase endothelial cell migration rates, while the cell migration response was saturated above 200 nM S1P. Expression of the main S1P receptor in EC, S1P(1), was elevated in plasma compared to low serum medium, but addition of VEGF or fluid flow elicited a further increase in S1P(1) mRNA, consistent with the synergistic effects observed between S1P, VEGF, and fluid flow. Thus, sustained delivery of S1P from biomaterials might only enhance endothelial cell migration if the concentration of S1P at the surface of the material stimulated adjacent EC to the same extent as approximately 200 nM S1P added to plasma.

CD46-induced immunomodulatory CD4+ T cells express the adhesion molecule and chemokine receptor pattern of intestinal T cells.

Tissue homing of activated T cells is typically mediated through their specific integrin and chemokine receptor repertoire. Activation of human primary CD4(+) T cells in the presence of CD46 cross-linking induces the development of a distinct immunomodulatory T cell population characterized by high IL-10/granzyme B production. How these regulatory T cells (Tregs) migrate/home to specific tissue sites is not understood. In this study, we determined the adhesion protein and chemokine receptor expression pattern on human CD3/CD46-activated peripheral blood CD4(+) T cells. CD3/CD46-activated, but not CD3/CD28-activated, T cells up-regulate the integrin alpha(4)beta(7). The interaction of alpha(4)beta(7) with its ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) mediates homing or retention of T cells to the intestine. CD3/CD46-activated Tregs adhere to/roll on MAdCAM-1-expressing HeLa cells, similar to T cells isolated from the human lamina propria (LP). This interaction is inhibited by silencing MAdCAM-1 expression in HeLa cells or by the addition of blocking Abs to beta(7). CD46 activation of T cells also induced the expression of the surface-bound cytokine LIGHT and the chemokine receptor CCR9, both marker constitutively expressed by gut LP-resident T cells. In addition, we found that approximately 10% of the CD4(+) T lymphocytes isolated from the LP of patients undergoing bariatric surgery contain T cells that spontaneously secrete a cytokine pattern consistent with that from CD46-activated T cells. These data suggest that CD46-induced Tregs might play a role in intestinal immune homeostasis where they could dampen unwanted effector T cell responses through local IL-10/granzyme B production.

Endothelial cell migration on RGD-peptide-containing PEG hydrogels in the presence of sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) is a potent chemokinetic agent for endothelial cells that is released by activated platelets. We previously developed Arg-Gly-Asp (RGD)-containing polyethylene glycol biomaterials for the controlled delivery of S1P to promote endothelialization. Here, we studied the effects of cell adhesion strength on S1P-stimulated endothelial cell migration in the presence of arterial levels of fluid shear stress, since an upward shift in optimal cell adhesion strengths may be beneficial for promoting long-term cell adhesion to materials. Two RGD peptides with different integrin-binding specificities were added to the polyethylene glycol hydrogels. A linear RGD bound primarily to beta(3) integrins, whereas a cyclic RGD bound through both beta(1) and beta(3) integrins. We observed increased focal adhesion formation and better long-term adhesion in flow with endothelial cells on linear RGD peptide, versus cyclic RGD, even though initial adhesion strengths were higher for cells on cyclic RGD. Addition of 100 nM S1P increased cell speed and random motility coefficients on both RGD peptides, with the largest increases found on cyclic RGD. For both peptides, much of the increase in cell migration speed was found for smaller cells (<1522 microm(2) projected area), although the large increases on cyclic RGD were also due to medium-sized cells (2288-3519 microm(2)). Overall, a compromise between high cell migration rates and long-term adhesion will be important in the design of materials that endothelialize after implantation.

Delivery of sphingosine 1-phosphate from poly(ethylene glycol) hydrogels.

While protein growth factors promote therapeutic angiogenesis, delivery of lipid factors such as sphingosine 1-phosphate (S1P) may provide better stabilization of newly formed vessels. We developed a biomaterial for the controlled delivery of S1P, a bioactive lipid released from activated platelets. Multiarm poly(ethylene glycol)-vinyl sulfone was cross-linked with albumin, a lipid-transporting protein, to form hydrogels. The rate of S1P release from the materials followed Fickian kinetics and was dependent upon the presence of lipid carriers in the release solution. Delivery of S1P from RGD-modified hydrogels increased the cell migration speed of endothelial cells growing on the materials. The materials also induced angiogenesis in the chorioallantoic membrane assay. Our data demonstrate that the storage and release of lipid factors provides a new route for the induction of angiogenesis by artificial materials.