Hic-5 expression is a major indicator of cancer cell morphology, migration, and plasticity in three-dimensional matrices.

The focal adhesion proteins Hic-5 and paxillin have been previously identified as key regulators of MDA-MB-231 breast cancer cell migration and morphologic mesenchymal-amoeboid plasticity in three-dimensional (3D) extracellular matrices (ECMs). However, their respective roles in other cancer cell types have not been evaluated. Herein, utilizing 3D cell-derived matrices and fibronectin-coated one-dimensional substrates, we show that across a variety of cancer cell lines, the level of Hic-5 expression serves as the major indicator of the cells primary morphology, plasticity, and in vitro invasiveness.

Paxillin inhibits HDAC6 to regulate microtubule acetylation, Golgi structure, and polarized migration.

Polarized cell migration is essential for normal organism development and is also a critical component of cancer cell invasion and disease progression. Directional cell motility requires the coordination of dynamic cell-extracellular matrix interactions as well as repositioning of the Golgi apparatus, both of which can be controlled by the microtubule (MT) cytoskeleton. In this paper, we have identified a new and conserved role for the focal adhesion scaffold protein paxillin in regulating the posttranslational modification of the MT cytoskeleton through an inhibitory interaction with the α-tubulin deacetylase HDAC6.

LIM domains target actin regulators paxillin and zyxin to sites of stress fiber strain.

Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber.

Diverse roles for the paxillin family of proteins in cancer.

The paxillin family of intracellular scaffold proteins includes paxillin, Hic-5, and leupaxin, and all have been identified as key regulators of the cellular migration machinery in both 2- and 3-dimensional microenvironments. Herein, we provide insight into the roles of these proteins during tumorigenesis and metastasis, highlighting their functions in cancer initiation as well as tumor cell dissemination and survival. Furthermore, we speculate on the potential of paxillin family proteins as both future prognostic and therapeutic targets.

CdGAP regulates cell migration and adhesion dynamics in two-and three-dimensional matrix environments.

CdGAP is a Rac1/Cdc42 specific GTPase activating protein (GAP) that localizes to cell-matrix adhesions through an interaction with the adhesion scaffold α-parvin/actopaxin to regulate lamellipodia formation and cell spreading. Herein, we demonstrate, using a combination of siRNA-mediated silencing and overexpression, that cdGAP negatively regulates directed and random migration by controlling adhesion maturation and dynamics through the regulation of both adhesion assembly and disassembly. Interestingly, cdGAP was also localized to adhesions formed in three-dimensional (3D) matrix environments and cdGAP depletion promoted cancer cell migration and invasion through 3D matrices.

Paxillin and Hic-5 interaction with vinculin is differentially regulated by Rac1 and RhoA.

Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases Rac1 and RhoA are indispensable for cell migration as they regulate cell protrusion, cell-extracellular matrix (ECM) interactions and force transduction. However, the consequences of their activity at a molecular level within the cell remain undetermined.

Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis.

Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin , and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs).

Emerging role of paxillin-PKL in regulation of cell adhesion, polarity and migration.

Cell adhesion and motility is of fundamental importance during development, normal physiology and pathologic conditions such as tumor metastasis. Focal adhesion proteins and their dynamic interactions play a critical role in the regulation of directed cell migration upon exposure to extracellular guidance cues. Using a combination of pharmacological inhibitors, knockout and knockdown cells and mutant protein expression, we recently reported that following adhesion and growth factor stimulation the dynamic interaction between paxillin and PKL(GIT2) is regulated by Src/FAK-dependent phosphorylation of PKL and that this interaction is necessary for the coordination of Rho family GTPase signaling controlling front-rear cell polarity and thus directional migration.

Paxillin-kinase-linker tyrosine phosphorylation regulates directional cell migration.

Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to platelet-derived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation.

An integrin-alpha4-14-3-3zeta-paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration.

Alpha4 integrins are used by leukocytes and neural crest derivatives for adhesion and migration during embryogenesis, immune responses and tumour invasion. The pro-migratory activity of alpha4 integrin is mediated in part through the direct binding of the cytoplasmic domain to paxillin. Here, using intermolecular FRET and biochemical analyses, we report a novel interaction of the alpha4 integrin cytoplasmic domain with 14-3-3zeta.

Paxillin comes of age.

Paxillin is a multi-domain scaffold protein that localizes to the intracellular surface of sites of cell adhesion to the extracellular matrix. Through the interactions of its multiple protein-binding modules, many of which are regulated by phosphorylation, paxillin serves as a platform for the recruitment of numerous regulatory and structural proteins that together control the dynamic changes in cell adhesion, cytoskeletal reorganization and gene expression that are necessary for cell migration and survival. In particular, paxillin plays a central role in coordinating the spatial and temporal action of the Rho family of small GTPases, which regulate the actin cytoskeleton, by recruiting an array of GTPase activator, suppressor and effector proteins to cell adhesions.

Quantification of integrin receptor agonism by fluorescence lifetime imaging.

Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET).

Integrin-syndecan cooperation governs the assembly of signalling complexes during cell spreading.

Cell adhesion to fibronectin (FN) triggers the formation and maturation of adhesion complexes by modulating the activity of the Rho family of GTPases. Cells plated onto a ligand of integrin alpha5beta1 spread but fail to form focal adhesions or fully organize actin into bundled stress fibres unless co-stimulated with a ligand of syndecan 4. Engagement of syndecan 4 in such pre-spread cells recapitulates the Rac1 and RhoA activation profiles observed during spreading on whole FN.