Focal Adhesion Sites Research Articles

Mechanistic Insight in Surface Nanotopography Driven Cellular Migration.

Cellular migration plays a vital role in many physiological processes. To elucidate the role of surface nanotopography on the downstream signaling pathways underlying cell migration, model surfaces having well-defined hill-like surface nanotopography and uniform surface chemistry were designed and implemented using plasma polymerization and covalent attachment of nanoparticles of predetermined size. A scratch wound assay, immunostaining, and gene expression of focal adhesion (FA) proteins were performed to determine the influence of surface nanotopography on cell migration. The results of this study demonstrate that the gap closure between cell monolayers is faster on surfaces having greater nanoscale topography. The phenomenon is predominantly driven by cell migration and was independent from cell proliferation. Qualitative and quantitative assessment of proteins involved in the signaling pathways underlying cell migration showed significant modulation by surface nanotopography. Specifically, focal adhesion sites decreased with the increase in surface nanotopography scale while the expression of FA proteins increased. This implies that nanotopography mediated modulation of cell migration is directly governed by the recruitment of receptor and adapter proteins responsible for cell-surface interaction. The results of this study indicate that biomaterial devices and constructs having rationally designed surface nanotopography and chemistry could be utilized to regulate wound healing and tissue regeneration.

A polarized nucleus-cytoskeleton-ECM connection in migrating cardioblasts controls heart tube formation in Drosophila.

The formation of the cardiac tube is a remarkable example of complex morphogenetic processes conserved from invertebrates to humans. It involves coordinated collective migration of contralateral rows of cardiac cells. The molecular processes underlying the specification of cardioblasts (CBs) prior to migration are well established and significant advances have been made in understanding the process of lumen formation. However, the mechanisms of collective cardiac cells migration remain elusive. Here, we have identified CAP and MSP300 as novel actors involved during CB migration. They both exhibit highly similar temporal and spatial expression patterns in Drosophila migrating cardiac cells, and are necessary for the correct number and alignment of CBs, a prerequisite for the coordination of their collective migration. Our data suggest that CAP and MSP300 are part of a protein complex linking focal adhesion sites to nuclei via the actin cytoskeleton that maintains post-mitotic state and correct alignment of CBs.

CCM3 is a gatekeeper in focal adhesions regulating mechanotransduction and YAP/TAZ signalling.

The YAP/TAZ transcriptional programme is not only a well-established driver of cancer progression and metastasis but also an important stimulator of tissue regeneration. Here we identified Cerebral cavernous malformations 3 (CCM3) as a regulator of mechanical cue-driven YAP/TAZ signalling, controlling both tumour progression and stem cell differentiation. We demonstrate that CCM3 localizes to focal adhesion sites in cancer-associated fibroblasts, where it regulates mechanotransduction and YAP/TAZ activation. Mechanistically, CCM3 and focal adhesion kinase (FAK) mutually compete for binding to paxillin to fine-tune FAK/Src/paxillin-driven mechanotransduction and YAP/TAZ activation. In mouse models of breast cancer, specific loss of CCM3 in cancer-associated fibroblasts leads to exacerbated tissue remodelling and force transmission to the matrix, resulting in reciprocal YAP/TAZ activation in the neighbouring tumour cells and dissemination of metastasis to distant organs. Similarly, CCM3 regulates the differentiation of mesenchymal stromal/stem cells. In conclusion, CCM3 is a gatekeeper in focal adhesions that controls mechanotransduction and YAP/TAZ signalling.

Bottom-up reconstitution of focal adhesion complexes.

Focal adhesions (FA) are large macromolecular assemblies relevant for various cellular and pathological events such as migration, polarization, and metastatic cancer formation. At FA sites at the migrating periphery of a cell, hundreds of players gather and form a network to respond to extra cellular stimuli transmitted by the integrin receptor, the most upstream component within a cell, initiating the FA signaling pathway. Numerous cellular experiments have been performed to understand the FA architecture and functions; however, their intricate network formation hampers unraveling the precise molecular actions of individual players. Here, in vitro bottom‐up reconstitution presents an advantageous approach for elucidating the FA machinery and the hierarchical crosstalk of involved cellular players.

Differential cellular interactions and responses to ultrathin micropatterned graphene oxide arrays with or without ordered in turn RGD peptide films

Owing to the surface interactions at the biomaterial-cell interface inevitable for improved cellular functions, a variety of efforts has been made for the development of the physical and chemical strategies to synchronize and adjust cellular behaviors by utilizing the micro/nanopatterned structures. As one of the most recent emerging nanomaterials, graphene oxide (GO) or reduced GO has increasingly utilized as biocompatible scaffolds, antibacterial films, and biosensor platforms. Here, we report a simple fabrication method for the alternately patterned arrays of the arginine-glycine-aspartic acid (RGD) peptide and GO (RGD/GO) on a single substrate of glass via a sequential process of self-assembly. The responses of fibroblasts to the nanoscopic topographical cues and their preference were explored on the well-separated GO microstructures with or without micropatterned RGD stripes. When separately cultured on those two different arrays of GO and RGD/GO micropatterns, the cells expressed noticeable discrimination in cellular behaviors such as the adhesion, localized population and alignment, and proliferation, according to the presence and absence of alternate RGD patterns. It was also revealed that the GO surface exhibits preferable interactions with cells through the developed focal adhesion sites to the RGD surface. Conclusively, our findings suggest that alternate micropatterns of RGD peptide and graphene family nanomaterials (i.e., GO) may not only be employed for an analyzing device to precisely control the cell migration/motility but also applied in a biosensor interface for electrochemical detection and analysis of cellular responsiveness under a specific external stimulus.

The ECM Modulator ITIH5 Affects Cell Adhesion, Motility and Chemotherapeutic Response of Basal/Squamous-Like (BASQ) Bladder Cancer Cells.

This study aims at characterizing the role of the putative tumor suppressor ITIH5 in basal-type bladder cancers (BLCA). By sub-classifying TCGA BLCA data, we revealed predominant loss of ITIH5 expression in the basal/squamous-like (BASQ) subtype. ITIH5 expression inversely correlated with basal-type makers such as KRT6A and CD44. Interestingly, Kaplan–Meier analyses showed longer recurrence-free survival in combination with strong CD44 expression, which is thought to mediate ITIH-hyaluronan (HA) binding functions. In vitro, stable ITIH5 overexpression in two basal-type BLCA cell lines showing differential CD44 expression levels, i.e., with (SCaBER) and without squamous features (HT1376), demonstrated clear inhibition of cell and colony growth of BASQ-type SCaBER cells. ITIH5 further enhanced HA-associated cell-matrix attachment, indicated by altered size and number of focal adhesion sites resulting in reduced cell migration capacities. Transcriptomic analyses revealed enrichment of pathways and processes involved in ECM organization, differentiation and cell signaling. Finally, we provide evidence that ITIH5 increase sensitivity of SCaBER cells to chemotherapeutical agents (cisplatin and gemcitabine), whereas responsiveness of HT1376 cells was not affected by ITIH5 expression. Thus, we gain further insights into the putative role of ITIH5 as tumor suppressor highlighting an impact on drug response potentially via the HA-CD44 axis in BASQ-type BLCA.

Fabrication and characterization of highly ordered nanotubes of anodic aluminum oxide

Investigated the electrochemical synthesis and characterized of a nanometer scale porous anodic aluminum oxide (AAO) membrane with a mean pore diameter about of 80-100 nm. The anodizing process done by varying the anodizing temperature from 20 °C to 25 °C. The membranes exhibit interesting properties such as controllable pore diameters, periodicity and density distribution. These properties can preselect by adjusting the controlling parameters of a temperature-controlled two-step anodization process. The surface features of the nanometer scale membrane such as pore density, pore diameter and interpore distance were quantified using scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM and AFM investigations revealed the presence of focal adhesion sites over the surface of the porous membranes. The positive outcomes of the study, indicates that AAO membranes can used for applications in the future.

The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites

The extracellular matrix (ECM) plays an important role in maintaining tissue homeostasis and poses a significant physical barrier to in vivo cell migration. Accordingly, as a means of enhancing tissue invasion, tumor cells use matrix metalloproteinases to degrade ECM proteins. However, the in vivo ECM is comprised not only of proteins but also of a variety of nonprotein components. Hyaluronan (HA), one of the most abundant nonprotein components of the interstitial ECM, forms a gel-like antiadhesive barrier that is impenetrable to particulate matter and cells. Mechanisms by which tumor cells penetrate the HA barrier have not been addressed. Here, we demonstrate that transmembrane protein 2 (TMEM2), the only known transmembrane hyaluronidase, is the predominant mediator of contact-dependent HA degradation and subsequent integrin-mediated cell–substrate adhesion. We show that a variety of tumor cells are able to eliminate substrate-bound HA in a tightly localized pattern corresponding to the distribution of focal adhesions (FAs) and stress fibers. This FA-targeted HA degradation is mediated by TMEM2, which itself is localized at site of FAs. TMEM2 depletion inhibits the ability of tumor cells to attach and migrate in an HA-rich environment. Importantly, TMEM2 directly binds at least two integrins via interaction between extracellular domains. Our findings demonstrate a critical role for TMEM2-mediated HA degradation in the adhesion and migration of cells on HA-rich ECM substrates and provide novel insight into the early phase of FA formation.

The Art of Mast Cell Adhesion.

Cell adhesion is one of the basic phenomena occurring in a living organism, affecting many other processes such as proliferation, differentiation, migration, or cell viability. Mast cells (MCs) are important elements involved in defending the host against various pathogens and regulating inflammatory processes. Due to numerous mediators, they are contributing to the modulation of many basic cellular processes in a variety of cells, including the expression and functioning of different adhesive molecules. They also express themselves many adhesive proteins, including ICAM-1, ICAM-3, VCAM-1, integrins, L-selectin, E-cadherin, and N-cadherin. These molecules enable MCs to interact with other cells and components of the extracellular matrix (ECM), creating structures such as adherens junctions and focal adhesion sites, and triggering a signaling cascade. A thorough understanding of these cellular mechanisms can create a better understanding of MC biology and reveal new goals for MC targeted therapy. This review will focus on the current knowledge of adhesion mechanisms with the involvement of MCs. It also provides insight into the influence of MCs or MC-derived mediators on the adhesion molecule expression in different cells.

Fluorescence Correlation Spectroscopy Reveals Interaction of Some Microdomain-Associated Lipids with Cellular Focal Adhesion Sites.

Cells adhere to the extracellular matrix at distinct anchoring points, mostly focal adhesions. These are rich in immobile transmembrane- and cytoskeletal-associated proteins, some of which are known to interact with lipids of the plasma membrane. To investigate their effect on lipid mobility and molecular interactions, fluorescently labeled lipids were incorporated into the plasma membranes of primary myofibroblasts using fusogenic liposomes. With fluorescence correlation spectroscopy, we tested mobilities of labeled microdomain-associated lipids such as sphingomyelin (SM), ganglioside (GM1), and cholesterol as well as of a microdomain-excluded phospholipid (PC) and a lipid-like molecule (DiIC18(7)) in focal adhesions (FAs) and in neighboring non-adherent membrane areas. We found significantly slower diffusion of SM and GM1 inside FAs but no effect on cholesterol, PC, and DiIC18(7). These data were compared to the molecular behavior in Lo/Ld-phase separated giant unilamellar vesicles, which served as a model system for microdomain containing lipid membranes. In contrast to the model system, lipid mobility changes in FAs were molecularly selective, and no particle enrichment occurred. Our findings suggest that lipid behavior in FAs cannot be described by Lo/Ld-phase separation. The observed slow-down of some molecules in FAs is potentially due to transient binding between lipids and some molecular constituent(s).

Multilayer platform to model the bioactivity of hyaluronic acid in gastric cancer

Hyaluronic acid (HA) has a key role in cancer progression. The HA's molecular weight (Mw) is altered in this pathological state: increased concentration of shorter fragments due to the overexpressed hyaluronidases and ROS. Aiming to mimic this microenvironment, we developed a Layer-by-Layer (LbL) platform presenting HA of different Mws, namely 6.4, 752 and 1500 kDa, to study the influence of HA Mw on the formation of focal adhesion sites (FAs), and the involvement of paxillin and CD44 in this process. High paxillin expression and formation of FAs, via CD44, is observed for MKN45 cells seeded on LbLs presenting HA 6.4 kDa, with the activation of the ERK1/2 pathway, responsible for cell motility and tumour progression. In contrast, activation of p38 pathway, usually related with cancer latency, is observed for cells seeded on LbLs with high Mw HA, i.e. 1500 kDa. Overall, we demonstrate the suitability of the developed platform to study cancer invasiveness.

Early endocytosis as a key to understanding mechanisms of plasma membrane tension regulation in filamentous fungi.

Two main systems regulate plasma membrane tension (PMT) and provide a close connection between the protoplast and the cell wall in fungi: turgor pressure and the actin cytoskeleton. These systems work together with the plasma membrane focal adhesion to the cell wall and their contribution to fungal cell organization and physiology has been partially studied. However, it remains controversial in model filamentous ascomycetes and oomycetes and even less investigated in filamentous basidiomycetes. Early endocytosis can be used to research the mechanisms regulating PMT since the dynamics of early endocytosis is largely dependent on this tension. This study examined the effects of actin polymerization inhibitors and hyperosmotic shock on early endocytosis and cell morphology in two filamentous basidiomycetes. The main obtained results are: (i) the depolymerisation of F-actin leads to the fast formation of endocytic pits while inhibiting of their scission from the plasma membrane and (ii) the moderate hyperosmotic shock does not affect the dynamics of early endocytosis. These and our other results have allowed suggesting a curtain model for the regulation of PMT in basidiomycetes. According to the proposed curtain model, the PMT in many non-apical cells of hyphae is more often regulated not by turgor pressure but by a system of actin driver cables that are associated with the proteins of the focal adhesion sites. The change in PMT occurs similar to the movement of a curtain along the curtain rod using the curtain drivers. This model addresses the fundamental properties of the fungal structure and physiology. It requires confirmation including the currently technically unavailable high-quality labelling of the actin cytoskeleton of the basidiomycetes.

A genomic island in Brucella involved in the adhesion to host cells: Identification of a new adhesin and a translocation factor.

Adhesion to host cells is the first step in the virulence cycle of any pathogen. In Gram-negative bacteria, adhesion is mediated, among other virulence factors such as the lipopolysaccharides, by specific outer-membrane proteins generally termed adhesins that belong to a wide variety of families and have different evolutionary origins. In Brucella, a widespread zoonotic pathogen of animal and human health concern, adhesion is central as it may determine the intracellular fate of the bacterium, an essential stage in its pathogenesis. In the present paper, we further characterised a genomic locus that we have previously reported encodes an adhesin (BigA) with a bacterial immunoglobulin-like domain (BIg-like). We found that this region encodes a second adhesin, which we have named BigB; and PalA, a periplasmic protein necessary for the proper display in the outer membrane of BigA and BigB. Deletion of bigB or palA diminishes the adhesion of the bacterium and overexpression of BigB dramatically increases it. Incubation of cells with the recombinant BIg-like domain of BigB induced important cytoskeletal rearrangements and affected the focal adhesion sites indicating that the adhesin targets cell-cell or cell-matrix proteins. We additionally show that PalA has a periplasmic localisation and is completely necessary for the proper display of BigA and BigB, probably avoiding their aggregation and facilitating their transport to the outer membrane. Our results indicate that this genomic island is entirely devoted to the adhesion of Brucella to host cells.

Abstract 4183: Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator

Abstract Background and objectives: Despite the outstanding antitumor activity of halichondrins in mice, the limited supply from the natural sources has prevented drug development using intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) under good manufacturing practice (GMP) conditions. E7130 is not only a novel microtubule dynamics inhibitor, but also a novel tumor-microenvironment ameliorator. E7130 can increase intratumoral CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts (CAFs) at pharmacologically relevant compound concentrations. Here, we analyzed the molecular mechanisms of the α-SMA-positive CAF reduction with E7130. Methods: The expression levels of α-SMA, ECM proteins, ER-TR7 (pan-fibroblast marker), and Ki67 were examined using E7130-treated tumors. In vitro co-culture experiments, normal human fibroblasts were co-cultured with human cancer cells. To examine the effects of E7130 on the TGF-β-induced CAF activation, normal human fibroblasts were treated with TGF-β (final concentration of 1 ng/mL) and E7130 at the several concentrations, and gene expression analysis and immunocytochemical analysis were conducted. Results: E7130 reduced α-SMA-positive CAF and malignant ECM proteins without changing total fibroblast number in tumors. The in vitro co-culture system revealed that TGF-β from cancer cells activate normal human fibroblast and increased α-SMA expression. In addition, treatment with E7130 interfered with α-SMA induction by TGF-β in fibroblasts without growth inhibitory activity. We further found that E7130 inhibited TGF-β-induced PI3K/AKT/mTOR pathway, which resulted in the reduction of α-SMA expression in fibroblasts. Moreover, TGF-β treatment enhanced β-tubulin expression and focal adhesions formation, which were diminished by co-treatment with E7130 in fibroblasts. Many signaling complexes are assembled in focal adhesion sites, and those complexes dispatch several downstream signals, including those involved in the PI3K/AKT/mTOR pathway. Conclusions: Drug discovery using halichondrins was based on their outstanding in vivo antitumor (inhibitory) activity in mice and bone metastasis in mouse melanoma model first reported in 1996. From these results, we hypothesized that halichondrins are not simple microtubule-targeted compounds in tumor cell, and have developed E7130, which harbors unique tumor microenvironment ameliorating effects. Here we proved that E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process without killing the fibroblast by disrupting microtubule network formation, which is important for focal adhesion assembly and thereby the downstream activation of the PI3K/AKT/mTOR pathway. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4183.

The G protein-coupled oestrogen receptor, GPER1, mediates direct anti-inflammatory effects of oestrogens in human cholinergic neurones from the nucleus basalis of Meynert.

It has been well established, particularly in animal models, that oestrogens exert neuroprotective effects in brain areas linked to cognitive processes. A key protective role could reside in the capacity of oestrogen to modulate the inflammatory response. However, the direct neuroprotective actions of oestrogens on neurones are complex and remain to be fully clarified. In the present study, we took advantage of a previously characterised primary culture of human cholinergic neurones (hfNBM) from the foetal nucleus basalis of Meynert, which is known to regulate hippocampal and neocortical learning and memory circuits, aiming to investigate the direct effects of oestrogens under inflammatory conditions. Exposure of cells to tumour necrosis factor (TNF)α (10ngmL-1 ) determined the activation of an inflammatory response, as demonstrated by nuclear factor-kappa B p65 nuclear translocation and cyclooxygenase-2 mRNA expression. These effects were inhibited by treatment with either 17β-oestradiol (E2 ) (10nmolL-1 ) or G1 (100nmolL-1 ), the selective agonist of the G protein-coupled oestrogen receptor (GPER1). Interestingly, the GPER1 antagonist G15 abolished the effects of E2 in TNFα-treated cells, whereas the ERα/ERβ inhibitor tamoxifen did not. Electrophysiological measurements in hfNBMs revealed a depolarising effect caused by E2 that was specifically blocked by tamoxifen and not by G15. Conversely, G1 specifically hyperpolarised the cell membrane and also increased both inward and outward currents elicited by a depolarising stimulus, suggesting a modulatory action on hfNBM excitability by GPER1 activation. Interestingly, pretreating cells with TNFα completely blocked the effects of G1 on membrane properties and also significantly reduced GPER1 mRNA expression. In addition, we found a peculiar subcellular localisation of GPER1 to focal adhesion sites that implicates new possible mechanisms of action of GPER1 in the neuronal perception of mechanical stimuli. The results obtained in the present study indicate a modulatory functional role of GPER1 with respect to mediating the oestrogen neuroprotective effect against inflammation in brain cholinergic neurones and, accordingly, may help to identify protective strategies for preventing cognitive impairments.

Mitomycin C modulates intracellular matrix metalloproteinase-9 expression and affects corneal fibroblast migration

Mitomycin C (MMC) is often used to prevent postoperative corneal haze and subconjunctival fibrosis in ocular surgery. It also affects the motility and viability of the residual ocular cells, including corneal stromal cells. Extracellular matrix metalloproteinase-9 (MMP-9) contributes to the promotion of cell movement in macrophage and cancer cells, but the intracellular role of MMP-9 remained unclear. Herein, we illustrated the novel role of intracellular MMP-9 in MMC-suppressed cell migration using isolated human corneal fibroblasts (HCFs). In HCFs, MMC enhanced intracellular MMP-9 at transcriptional and protein levels. Using co-immunoprecipitation analysis, we confirmed that MMC enhanced the association between intracellular MMP-9 and inactive FAK/paxillin (PXN) complexes, i.e. PXN without phospho-tyrosine 118 (pY118) and FAK without phospho-tyrosine 397 (pY397). To verify the role of intracellular MMP-9 in migration, its gene was directly isolated from HCFs and highly expressed in HCFs by a lentivirus-based pseudovirus system with encephalomyocarditis virus (EMCV)-driven enhanced green fluorescent protein (GFP) as the MMP-9-IG-versus IG-expressing cells. Compared with the IG-expressing cells, higher intracellular MMP-9 expression in the MMP-9-IG-expressing HCFs proliferated and migrated more slowly. Phosphorylation of FAK at Y397 and PXN at both Y31 and Y118 were significantly less in the MMP-9-IG-expressing HCFs. These suggested that MMC-upregulated intracellular MMP-9 clutched inactive FAK/PXN complexes at focal adhesion sites to form a new “inactive” trimer, prohibited FAK/PXN complexes phosphorylation and retarded corneal fibroblast migration.

Chiral geometry regulates stem cell fate and activity

Geometry sensing of cells inevitably involves cytoskeletal remodeling and the activation of biochemical signaling, which control multiple aspects of cell behaviors, such as proliferation, differentiation and migration. A variety of size-, shape- and geometry-dependent cell behaviors have been revealed, but the role of geometric chirality in regulating cellular behaviors and the underlying biophysical mechanisms remain elusive. Here, we report an intriguing mechanotransduction of stem cells on chiral geometries that human mesenchymal stem cells (hMSCs) prefer to migrate towards dextral geometry with nearly 30% relative advantage in migration speed, referred to as “chirotaxis”. We also found that cell adhesion, proliferation, and differentiation of hMSCs are greatly enhanced for cells cultured on dextral geometry than those on sinistral geometry, by triggering transcription factor AP-1 complex through p38/MAPK signaling that regulates hMSCs fate and activity. We demonstrated that the cytoskeletal network consisting of transverse and radial stress fibers exhibits a strengthening/offsetting effect on dextral/sinistral geometry through focal adhesion sites, and consequently, cell's cytoskeletal contractility on the dextral geometry is nearly 80% higher. These findings highlight the importance of geometric chirality as an extracellular cue in regulating stem cell's behaviors through cell-material interactions.

TUBB4B Downregulation Is Critical for Increasing Migration of Metastatic Colon Cancer Cells.

Tumor metastasis, the major problem for clinical oncology in colon cancer treatment, is linked with an epithelial-mesenchymal transition (EMT). The observed cellular transformation in this process is manifested by cell elongation, enhanced cell migration and invasion ability, coordinated by cytoskeleton reorganization. In the present study, we examined the role of tubulin-β4 (TUBB4B) downregulation that occurs during EMT in colon cancer cells, in the modulation of the function of microtubules. Based on biochemical and behavioral analysis (transmigration) we posit that the decrease of the TUBB4B level is critical for microtubule-vimentin interaction and contributes to the maintenance of polarity in migrating cells. The microscopic studies revealed that TUBB4B decrease is accompanied by cell elongation and increased number of matured focal adhesion sites, which is a characteristic of the cell metastatic stage. We also demonstrated faster polymerization of microtubules in cells with a lower level of TUBB4B. Simultaneous TUBB3 upregulation, reported during EMT, acts additively in this process. Our studies suggest that the protein level of TUBB4B could be used as a marker for detection of the preinvasive stages of the colon cancer cells. We also concluded that chemotherapy enriched to increase TUBB4B level and/or to stabilize microtubule polymerization might more effectively prevent metastasis in colon cancer development.

SAT-574 Elucidating the Role of Nuclear Focal Adhesion Kinase (FAK) in Thyroid Cancer

Background: Advanced papillary (PTC) and Anaplastic Thyroid Cancer (ATC) are the leading causes of endocrine cancer deaths. Thus there is a critical need to identify novel targeted therapies for these patients. Focal Adhesion Kinase (FAK) and Src are overexpressed in thyroid cancer and represent promising therapeutic targets. However, Src inhibition has had limited success in the clinic due to development of resistance. A possible mechanism of resistance may be through FAK which is a nonreceptor tyrosine kinase that functions as a kinase and scaffold/adaptor protein to regulate growth, survival, migration, and invasion. FAK is thought to be primarily autophosphorylated at tyrosine 397 (Y397) in response to integrin or growth factor receptor signaling, which results in the recruitment of Src and downstream signaling pathways. FAK has been found to go to the nucleus where it causes increased survival, tumor growth, and immune evasion. Hypothesis: We hypothesized that localization of FAK to the nucleus is a mechanism of resistance to Src inhibition, and that dual inhibition of FAK and Src will overcome resistance to Src inhibition and lead to decreased invasion in thyroid cancer. Results: We found that BRAF-mutant thyroid cancer cells resistant to the Src inhibitor, dasatinib (DasRes cells) exhibited increased tumor volume when injected orthotopically into the thyroid gland of nude mice. Immunohistochemical of pY397 FAK showed an increased level of active nuclear FAK in the DasRes tumors compared to the control tumors. Interestingly, we found that combined FAK and Src inhibition led to decreased levels of active and total nuclear FAK in DasRes cells, which correlated with decreased invasion and growth in DasRes BRAF-mutant cells compared to either single agent. Given the decrease in invasion in DasRes cells in response to combined FAK and Src inhibition, we then asked if loss of FAK localization to the nucleus leads to decreased invasion. To address this hypothesis, we transduced empty vector (EV), WT FAK, or a FAK Nuclear localization mutant into BRAF-mutant thyroid cancer cells with Crispr-Cas9 Knockout (KO) of FAK. Interestingly, we found that preventing FAK from localizing to the nucleus caused increased invasion and decreased proliferation compared to KO FAK thyroid cells transduced with WT FAK. Conclusions: Taken together, this data indicates that a mechanism of resistance to Src inhibition could be through FAK nuclear localization and that inhibiting both FAK and Src leads to decreased levels of nuclear FAK, invasion, and growth. However, localization of FAK to the nucleus appears to be responsible for proliferation while cytosolic FAK contributes to invasion. Thus this data provides a rationale for targeting FAK and Src in thyroid cancer to inhibit invasion of FAK at focal adhesion sites and to inhibit proliferation through decreased nuclear FAK.

Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response

Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5β1 and αvβ3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvβ3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.