Focal Adhesion Kinase Protein Research Articles

The RNA-editing enzyme ADAR promotes lung adenocarcinoma migration and invasion by stabilizing FAK.

Large-scale, genome-wide studies report that RNA binding proteins are altered in cancers, but it is unclear how these proteins control tumor progression. We found that the RNA-editing protein ADAR (adenosine deaminase acting on double-stranded RNA) acted as a facilitator of lung adenocarcinoma (LUAD) progression through its ability to stabilize transcripts encoding focal adhesion kinase (FAK). In samples from 802 stage I LUAD patients, increased abundance of ADAR at both the mRNA and protein level correlated with tumor recurrence. Knocking down ADAR in LUAD cells suppressed their mesenchymal properties, migration, and invasion in culture. Analysis of gene expression patterns in LUAD cells identified ADAR-associated enrichment of a subset of genes involved in cell migration pathways; among these, FAK is the most notable gene whose expression was increased in the presence of ADAR. Molecular analyses revealed that ADAR posttranscriptionally increased FAK protein abundance by binding to the FAK transcript and editing a specific intronic site that resulted in the increased stabilization of FAK mRNA. Pharmacological inhibition of FAK blocked ADAR-induced invasiveness of LUAD cells, suggesting a potential therapeutic application for LUAD that has a high abundance of ADAR.

Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition.

Renal fibrosis and anaemia are two of the most relevant events in chronic kidney disease. Fibrosis is characterized by the accumulation of extracellular matrix proteins in the glomeruli and tubular interstitium. Anaemia is the consequence of a decrease in erythropoietin production in fibrotic kidneys. This work analyses the possibility that the accumulation of abnormal collagens in kidney interstitium could be one of the mechanisms responsible for erythropoietin decreased synthesis. In renal interstitial fibroblast grown on collagen I, erythropoietin mRNA expression and HIF‐2α protein decreased, whereas focal adhesion kinase protein (FAK) phosphorylation and proteasome activity increased, compared to cells grown on collagen IV. Proteasome inhibition or FAK inactivation in cells plated on collagen I restored erythropoietin and HIF‐2α expression. FAK inhibition also decreased the collagen I‐dependent proteasome activation. In a model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction in mice, increased collagen I protein content and an almost complete disappearance of erythropoietin mRNA expression were observed in the ureteral ligated kidney with respect to the contralateral control. Interestingly, erythropoietin synthesis was recovered in obstructed mice treated with proteasome inhibitor. These data suggest that reduced kidney erythropoietin synthesis could be caused by the accumulation of abnormal extracellular matrix proteins.

Focal Adhesion Kinase as a Potential Target in AML and MDS.

Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics (P = 2 × 10-4) and relapse (P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3-ITD (P = 0.0024) or RAS (P = 0.05) mutations and strongly correlated with p-SRC and integrinβ3 levels. FAK protein levels were significantly higher in CD34+ (P = 5.42 × 10-20) and CD34+CD38- MDS (P = 7.62 × 10-9) cells compared with normal CD34+ cells. MDS patients with higher FAK in CD34+ cells tended to have better overall survival (P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia-stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133-44. ©2017 AACR.

MerTK Does Not Mediate Phagocytosis of Staphylococcus aureus but Attenuates Inflammation Induced by Staphylococcal Lipoteichoic Acid Through Blocking NF-κB Activation.

Mer receptor tyrosine kinase (MerTK) expressed in macrophages is essential for phagocytosis of apoptotic cells. Here, we investigate whether MerTK is involved in the phagocytosis of Staphylococcus aureus (S. aureus) and regulation of staphylococcal lipoteichoic acid (LTA)-induced inflammatory response in macrophages. We found that stimulating RAW264.7 macrophages with S. aureus activated multiple signaling pathways including toll-like receptor 2 (TLR2), scavenger receptor A (SR-A), and MerTK. Meanwhile, S. aureus stimulation also induced activation of proteins focal adhesion kinase (FAK) and Rac1, which are related to phagocytosis. Pretreatment with a specific Mer-blocking antibody significantly inhibited S. aureus-induced phosphorylation of MerTK, while it had no effect on S. aureus-induced activation of FAK and Rac1. Moreover, by confocal laser microscope, we observed that the antibody blockade of MerTK had little impact on the phagocytosis of S. aureus by RAW264.7 macrophages. Additionally, pretreatment with this antibody further promoted LTA-induced phosphorylation of nuclear factor κB (NF-κB) p65 subunit and production of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, and macrophage inflammatory protein-2 (MIP-2). Collectively, these results suggest that MerTK does not play an essential role in the phagocytosis of S. aureus but attenuates inflammation induced by staphylococcal LTA through blocking NF-κB activation.

TGF-β1 Pretreatment Improves the Function of Mesenchymal Stem Cells in the Wound Bed.

The wound healing process initiates after injury to a tissue and involves a series of orchestrated events to minimize the invasion of foreign matters such as bacteria and efficiently regenerate the damaged tissue. A variety of cells must be recruited to the tissue during wound healing. However, this process is severely disrupted in patients suffering from chronic illness, including diabetes, leading to impaired healing or non-healing wounds. Current avenues of treatment include negative-pressure therapy, wound debridement, growth factor replacement, and cell-based therapies. Among these therapies, mesenchymal stem cells (MSCs) delivery to the wound holds a very high promise due to the innate abilities of MSCs that include immunogenicity, plasticity, and self-renewal. Bone marrow derived MSCs have been shown to promote more rapid wound healing by increased cytokine production in diabetic mice. However, the lack of understanding of the mechanical and chemical interaction of the transplanted MSCs with the factors present in the regenerative niches limits their efficacy in the wound bed. In this study, we sought to understand how the changes in MSC biochemical and biophysical properties can affect their function in vitro and in vivo. We demonstrate that pretreatment of MSCs with the mechano-stimulatory soluble factor transforming growth factor (TGF-β1), which is highly expressed in injury sites, improves wound closure in a syngeneic murine wound model. This improved wound closure correlated with increased invasion into the wound bed. In vitro studies demonstrated that TGF-β1 pretreatment expedited wound closure by increasing adhesion, traction force, and migration even after removal of the stimulus. Furthermore, this response was mediated by the cytoskeletal protein focal adhesion kinase. Taken together, this study suggests that defined chemical stimuli can benefit site specific adaptability of MSCs to improve their function and therapeutic usefulness.

Effects of Alcohol on Postoperative Adhesion Formation in Ischemic Myocardium and Pericardium

Postoperative formation of adhesions increases risk of complications during cardiac reoperations. We previously demonstrated that swine supplemented with vodka had a significant reduction in adhesions at sternotomy after previous thoracotomy. This follow-up study was conducted to determine reproducibility and the mechanism for adhesion reduction in swine supplemented with ethanol. An ameroid constrictor was placed in the left circumflex in 14 male Yorkshire swine to induce chronic myocardial ischemia through left minithoracotomy. Animals were supplemented postoperatively with ethanol (45 g ETOH, n= 7) or sucrose (80 g SUC, n= 7) for 7 weeks, followed by a reoperative median sternotomy. The ETOH group had significantly fewer adhesions, thinner pericardial thickness, decreased intramyocardial fibrosis, and decreased myocardial collagen deposition compared with the SUC group. In the myocardium, ETOH animals had decreased expression of proadhesion proteins focal adhesion kinase, paxillin, integrin-β1, transforming growth factor-β1 and phosphorylated SMAD and increased expression of adhesion breakdown proteins matrix metalloproteinase (MMP) 1, MMP2, MMP3, and MMP9 compared with SUC animals. However in the pericardium, ETOH animals had increased expression of proadhesion proteins focal adhesion kinase, paxillin, phosphorylated paxillin, vinculin, integrin-β1, tumor necrosis factor-α, transforming growth factor-β, and phosphorylated SMAD3, and decreased expression of adhesion breakdown proteins MMP1, MMP3, MMP9, and plasmin compared with SUC animals. Alcohol supplementation substantially reduced postoperative pericardial adhesion formation, attenuated pericardial thickening, and reduced myocardial fibrosis in response to chronic ischemia. Alcohol supplementation modulates adhesion protein and MMP/tissue inhibitor of metalloproteinase expression, favoring a profile associated with reduced pericardial adhesions. These results suggest that the myocardium is the driving factor in reducing pericardial adhesions and mediating the postoperative healing process.

Dual roles of protein tyrosine phosphatase kappa in coordinating angiogenesis induced by pro-angiogenic factors.

A potential role may be played by receptor-type protein tyrosine phosphatase kappa (PTPRK) in angiogenesis due to its critical function in coordinating intracellular signal transduction from various receptors reliant on tyrosine phosphorylation. In the present study, we investigated the involvement of PTPRK in the cellular functions of vascular endothelial cells (HECV) and its role in angiogenesis using in vitro assays and a PTPRK knockdown vascular endothelial cell model. PTPRK knockdown in HECV cells (HECVPTPRKkd) resulted in a decrease of cell proliferation and cell-matrix adhesion; however, increased cell spreading and motility were seen. Reduced focal adhesion kinase (FAK) and paxillin protein levels were seen in the PTPRK knockdown cells which may contribute to the inhibitory effect on adhesion. HECVPTPRKkd cells were more responsive to the treatment of fibroblast growth factor (FGF) in their migration compared with the untreated control and cells treated with VEGF. Moreover, elevated c-Src and Akt1 were seen in the PTPRK knockdown cells. The FGF-promoted cell migration was remarkably suppressed by an addition of PLCγ inhibitor compared with other small inhibitors. Knockdown of PTPRK suppressed the ability of HECV cells to form tubules and also impaired the tubule formation that was induced by FGF and conditioned medium of cancer cells. Taken together, it suggests that PTPRK plays dual roles in coordinating angiogenesis. It plays a positive role in cell proliferation, adhesion and tubule formation, but suppresses cell migration, in particular, the FGF-promoted migration. PTPRK bears potential to be targeted for the prevention of tumour associated angiogenesis.

FAK signalling controls insulin sensitivity through regulation of adipocyte survival

Focal adhesion kinase (FAK) plays a central role in integrin signalling, which regulates growth and survival of tumours. Here we show that FAK protein levels are increased in adipose tissue of insulin-resistant obese mice and humans. Disruption of adipocyte FAK in mice or in 3T3 L1 cells decreases adipocyte survival. Adipocyte-specific FAK knockout mice display impaired adipose tissue expansion and insulin resistance on prolonged metabolic stress from a high-fat diet or when crossed on an obese db/db or ob/ob genetic background. Treatment of these mice with a PPARγ agonist does not restore adiposity or improve insulin sensitivity. In contrast, inhibition of apoptosis, either genetically or pharmacologically, attenuates adipocyte death, restores normal adiposity and improves insulin sensitivity. Together, these results demonstrate that FAK is required for adipocyte survival and maintenance of insulin sensitivity, particularly in the context of adipose tissue expansion as a result of caloric excess.

Focal Adhesion Kinase Regulatory Interactions Quantified by TIRF-FRET

Focal adhesion kinase related non-kinase (FRNK) is an inhibitor of focal adhesion kinase (FAK)-dependent cell growth, survival, and migration pathways. In vascular smooth muscle cells, both FAK and FRNK are upregulated after vascular injury, suggesting balanced stimulation and inhibition of growth during recovery. Here we propose a novel mechanism by which FRNK can inhibit FAK: direct binding of FRNK to FAK forming an inhibitory complex within focal adhesions. This hypothesis is supported by co-immunoprecipitation of FAK and FRNK. In addition, total internal reflection fluorescence (TIRF)-mode fluorescence resonance energy transfer (FRET) measurements suggest that FRNK binds directly to FAK within focal adhesions of live vascular smooth muscle cells. We hypothesized that this interaction might be regulated by serine phosphorylation of FRNK S217, a site we found to be basally highly phosphorylated at S217 by extracellular signal-regulated kinase 1/2 (ERK). To determine the significance of S217 phosphorylation, we generated a non-phosphorylatable mutant FRNK by mutating S217 to alanine. S217A-FRNK exhibited significantly higher amounts of FRET compared to wild type FRNK, suggesting increased interaction with FAK. Progressive acceptor photobleaching analysis revealed that the FRNK-FAK complex consisted of FRNK and multiple FAK proteins in a high order hetero-oligomer. To determine the functional role of the proposed regulatory site we performed an apoptosis assay using flow cytometry. S217A FRNK was a much more potent inducer of apoptosis in vascular smooth muscle cells compared to wild type FRNK. Additionally, we determined that S217A FRNK was a more potent inhibitor of FAK signaling by examining FAK Y397 phosphorylation. We conclude that dephosphorylation of FRNK at S217 during response to injury results in increased FRNK-FAK complex formation, thereby inhibiting FAK signaling.

Addressing the Functional Determinants of FAK during Ciliogenesis in Multiciliated Cells

We previously identified focal adhesion kinase (FAK) as an important regulator of ciliogenesis in multiciliated cells. FAK and other focal adhesion (FA) proteins associate with the basal bodies and their striated rootlets and form complexes named ciliary adhesions (CAs). CAs display similarities with FAs but are established in an integrin independent fashion and are responsible for anchoring basal bodies to the actin cytoskeleton during ciliogenesis as well as in mature multiciliated cells. FAK down-regulation leads to aberrant ciliogenesis due to impaired association between the basal bodies and the actin cytoskeleton, suggesting that FAK is an important regulator of the CA complex. However, the mechanism through which FAK functions in the complex is not clear, and in this study we examined the role of this protein in both ciliogenesis and ciliary function. We show that localization of FAK at CAs depends on interactions taking place at the amino-terminal (FERM) and carboxyl-terminal (FAT) domains and that both domains are required for proper ciliogenesis and ciliary function. Furthermore, we show that an interaction with another CA protein, paxillin, is essential for correct localization of FAK in multiciliated cells. This interaction is indispensable for both ciliogenesis and ciliary function. Finally, we provide evidence that despite the fact that FAK is in the active, open conformation at CAs, its kinase activity is dispensable for ciliogenesis and ciliary function revealing that FAK plays a scaffolding role in multiciliated cells. Overall these data show that the role of FAK at CAs displays similarities but also important differences compared with its role at FAs.

P1.07-028 Dual Role of the Focal Adhesion Kinase in Small-Cell Lung Cancer

Small cell-lung cancer (SCLC) is a devastating illness with five-year overall survival as low as 5%. The molecular steps leading to SCLC development and progression are still poorly understood and this has translated into the absence of targeted therapies. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase which regulates integrin and growth factor signaling pathways involved in cell proliferation, survival, migration, and invasion. FAK is overexpressed and/or activated in many cancers, including SCLC. We hypothesized that FAK overexpression/activation in SCLC contributes to its aggressive behavior and that FAK may represent a therapeutic target in SCLC. Two SCLC cell lines growing in suspension (NCI-H82, NCI-H146), and adherent SCLC cell lines (NCI-H196, NCI-H446) were treated with PF-228. NCI-H446 and H82 cells were stably transfected with FAK shRNA and/or FRNK using lentivirus vector. Cell proliferation was evaluated by WST-1 assay; cell cycle by flow cytometry with propidium iodide and bromodeoxyuridine; apoptosis by caspase 3 staining in flow cytometry and by cleaved PARPp85 Western blotting (WB); motility by wound healing assay; and invasion by Boyden chambers. FAK expression/activity was evaluated by WB. While PF-228 did not modify total FAK expression, it decreased FAK phosphorylation (Y397). Inhibition of FAK activity by PF-228 decreased cell proliferation, DNA synthesis, induced cell cycle arrest in G2/M phases, and increased apoptosis in dose-dependently. PF-228 also decreased motility in the adherent H196-H446 cells. To confirm the specificity of the antitumoral effects of PF-228, we stably transfected SCLC cells with FAK shRNA and FRNK and then analyzed the phenotypic changes induced by these approaches. Knockdown of total FAK protein by transfection of FAK shRNA inhibited FAK activity, but did not have any effect on cell proliferation, DNA synthesis, and cell cycle. However, reintroduction of FRNK in cells stably transfected with FAK shRNA inhibited cell proliferation and DNA synthesis. Expression of FRNK decreased cell proliferation and DNA synthesis in SCLC cells. Inhibition of FAK activity by PF-228 in SCLC cell lines demonstrates that FAK activity is required for cell proliferation, cycle progression, survival, and motility, suggesting that FAK inhibition may represent a suitable therapeutic target for SCLC. Inhibition of FAK by a genomic approach suggests that FAK has a dual role in SCLC biology, namely (i) a pro-tumoral effect related to the kinase domain, which induces downstream signals (ii) an anti-tumoral effect mediated by the non-kinase C-terminal domain (FRNK domain), which keeps inactive other pro-tumoral effectors.

Inhibition of FAK Exerts Anti-Leukemic Activity and Potentiates ABT-199-Induced Apoptosis in AML

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates cell adhesion, proliferation, stem cell functions, and cell-microenvironment communications. It is activated and/or overexpressed in many malignant cells and promotes tumor progression and metastasis. Several small molecule FAK inhibitors have been developed and some of them have reached clinical trials in solid tumors. High FAK expression was found to be associated with enhanced blast migration, increased cellularity, and poor prognosis in AML, indicating that FAK could be a potential therapeutic target in AML.We showed previously that VS-4718, a potent and selective FAK inhibitor, effectively decreased viable cell number, and also induced cell death in leukemia cell lines with variable potencies in vitro, even in AML cells co-cultured with mesenchymal stromal cells (MSCs) (ASH 2015).To further examine the effect of VS-4718 in vivo, we transplanted Molm14-GFP/Luc cells into NSGS (NOD-SCID IL2Rgnull-3/GM/SF, NSG-SGM3) mice, and treated the mice with VS-4718 (75 mg/kg) twice a day via oral gavage. We found that VS-4718 as a single agent exerted anti-leukemia activity as assessed by in vivo imaging for leukemia burden, human CD45 positivity in mouse peripheral blood, and histological staining of mouse tissues. VS-4718 treated mice survived significantly longer than the untreated controls (medium survival 27 vs 20 days, P = 0.0003).FAK activates multiple signaling pathways and supports tumor cell survival. We found that inhibition of FAK with VS-4718 in Molm14 cells reduced the expression of MCL-1. The BCL-2 antagonist ABT-199 is being tested clinically for the treatment of hematological malignancies. However, as a single agent, ABT-199-treated cells can acquire drug resistance by upregulating MCL-1 and BCL-XL after treatment. We therefore hypothesized that combination of VS-4718 and ABT-199 would be more effective in inducing cell death and reversing the resistance of AML cells exposed to ABT-199 alone. In vitro studies showed that VS-4718 significantly improved the potency of ABT-199 in AML cell lines (ABT-199 EC50 at 24 h: 880.3 nM and 14.5 nM in the presence of 0.4 mM VS-4718, respectively, in Molm14 cells), and the combination of VS-4718 and ABT-199 also synergistically killed primary AML cells even when co-cultured with MSCs in the majority of samples examined, while largely sparing normal BM CD34+ cells.Furthermore, the upregulation of MCL-1 in ABT-199-treated AML cells was antagonized by combining ABT-199 with VS-4718. BCL-XL is known to be regulated by STAT5. The activation of STAT5, which can be regulated by FAK, is considered to be significant in maintaining MCL-1 expression in FLT3-ITD AML cells. We observed that treatment with VS-4718 decreased the level of p-STAT5 as well as MCL-1 and BCL-XL in Molm14 cells harboring FLT3-ITD mutation.These results suggest a novel therapeutic strategy for targeting FAK and BCL-2 family proteins for the treatment of AML. DisclosuresPachter:Verastem, Inc: Employment. Weaver:Verastem, Inc: Employment. Carter:PRISM Pharma/Eisai: Research Funding.

RLj-RGD3, a Novel Recombinant Toxin Protein from Lampetra japonica, Protects against Cerebral Reperfusion Injury Following Middle Cerebral Artery Occlusion Involving the Integrin-PI3K/Akt Pathway in Rats.

BackgroundThe RGD-toxin protein Lj-RGD3 is a naturally occurring 118 amino acid peptide that can be obtained from the salivary gland of the Lampetra japonica fish. This unique peptide contains 3 RGD (Arg-Gly-Asp) motifs in its primary structure. Lj-RGD3 is available in recombinant form (rLj-RGD3) and can be produced in large quantities using DNA recombination techniques. The pharmacology of the three RGD motif-containing peptides has not been studied. This study investigated the protective effects of rLj-RGD3, a novel polypeptide, against ischemia/reperfusion-induced damage to the brain caused by middle cerebral artery occlusion (MCAO) in a rat stroke model. We also explored the mechanism by which rLj-RGD3 acts by measuring protein and mRNA expression levels, with an emphasis on the FAK and integrin-PI3K/Akt anti-apoptosis pathways.MethodsrLj-RGD3 was obtained from the buccal secretions of Lampetra japonica using gene recombination technology. Sprague Dawley (SD) rats were randomly divided into the following seven groups: a sham group; a vehicle-treated (VT) group; 100.0 μg·kg-1, 50.0 μg·kg-1 and 25.0 μg·kg-1 dose rLj-RGD3 groups; and two positive controls, including 1.5 mg·kg-1 Edaravone (ED) and 100.0 μg·kg-1 Eptifibatide (EP). MCAO was induced using a model consisting of 2 h of ischemia and 24 h of reperfusion. Behavioral changes were observed in the normal and operation groups after focal cerebral ischemia/reperfusion was applied. In addition, behavioral scores were evaluated at 4 and 24 h after reperfusion. Brain infarct volumes were determined based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining. Moreover, neuronal apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays. We determined the expression levels of focal adhesion kinase (FAK), phosphatidyl inositol 3-kinase (PI3K), protein kinase B (Akt, PKB), caspase-3 and Bcl-2 in the brain using western blot analysis and RT-PCR assays. The research protocol was approved by the Institutional Ethics Committee of Dalian Medical University.ResultsThe behavioral scores and cerebral infarct volumes of the rLj-RGD3 groups were markedly lower at 4 and 24 h/RF. The rLj-RGD3 protein significantly ameliorated pathological changes in the brain and reduced the number of apoptotic neurons. Furthermore, the FAK and PI3K/Akt pathways were activated. rLj-RGD3 significantly increased the expression of FAK, p-FAK and Bcl-2 proteins. In contrast, caspase-3 expression was inhibited.Conclusion/SignificanceWe conclude that recombinant Lampetra japonica RGD-peptide (rLj-RGD3) exerts a protective effect against cerebral ischemia/reperfusion injury in the brain. In addition, the mechanism of this protection is associated with the activation of the integrin-PI3K/Akt pathway. These results provide a theoretical foundation and an experimental basis for using RGD peptides as novel drugs for treating ischemic cerebral vascular diseases in addition to promoting the research and development of marine biotechnology drugs.

Crystal Structure Determination and Molecular Docking Studies of 4- (5-Phenyl Pyrazin-2-Yl)-4h-1,2,4 Triazole-3-Thiol with Focal Adhesion Kinase Inhibitors

The main objective of the present work is to determine crystal structure of the ligand by x-ray methods and to perform molecular docking studies of the ligand 4- Phenyl -5-Pyrazinyl-3-mercapto 1,2,4 Triazole with protein focal adhesion kinase (FAK) domain using the software, Autodock and pymol. Macromolecular modeling by docking studies provides the most detailed view possible of drug receptor interaction. It has created a new rational approach to drug design, where the structure of drug is designed, based on its fit to three dimensional structures of receptor site, rather than basing it on analogies to other active structures. The above titled compound is binding with FAK protein. This may act as inhibitor to FAK and can be used for anticancer therapy target.

Focal adhesion kinase (FAK) and mechanical stimulation negatively regulate the transition of airway smooth muscle tissues to a synthetic phenotype.

The effects of mechanical forces and focal adhesion kinase (FAK) in regulating the inflammatory responses of airway smooth muscle (ASM) tissues to stimulation with interleukin (IL)-13 were investigated. Canine tracheal tissues were subjected to different mechanical loads in vitro, and the effects of mechanical load on eotaxin secretion and inflammatory signaling pathways in response to IL-13 were determined. Eotaxin secretion by tissues in response to IL-13 was significantly inhibited in muscles maintained at a higher (+) load compared with those at a lower (-) load as assessed by ELISA, and Akt activation was also reduced in the higher (+) loaded tissues. Conversely the (+) mechanical load increased activation of the focal adhesion proteins FAK and paxillin in the tissues. The role of FAK in regulating the mechanosensitive responses was assessed by overexpressing FAK-related nonkinase in the tissues, by expressing the FAK kinase-dead mutant FAK Y397F, or by treating tissues with the FAK inhibitor PF-573228. FAK inactivation potentiated Akt activity and increased eotaxin secretion in response to IL-13. FAK inhibition also suppressed the mechanosensitivity of Akt activation and eotaxin secretion. In addition, FAK inactivation suppressed smooth muscle myosin heavy chain expression induced by the higher (+) mechanical load. The results demonstrate that the imposition of a higher mechanical load on airway smooth muscle stimulates FAK activation, which promotes the expression of the differentiated contractile phenotype and suppresses the synthetic phenotype and the inflammatory responses of the muscle tissue.

Transcriptome analysis in aged murine hematopoietic stem cells highlights an axon guidance receptor as a novel regulator of hematopoietic stem cell function and aging

Hematopoietic stem cell (HSC) aging is associated with functional decline, yet the molecular changes contributing to this remain unclear. Previous studies have compared gene expression in pooled young and old murine HSCs but there is little consensus among them (3 genes). With the addition of our transcriptome analysis from individual C57BL/6 mice, only one gene, Neogenin, is consistently reported as upregulated with age. Surprisingly, this gene has not been investigated in HSC function or aging. Although frequently described as an axon guidance receptor due to its role in axon migration, it is also implicated in neuronal and embryonic stem cell differentiation. In this study we investigate the function of Neogenin in HSCs. We confirm increased Neogenin expression in long-term hematopoietic stem cells (LT-HSCs) compared to progenitors and validate its protein expression on their cell surface. Neogenin ligands Repulsive Guidance Molecules RGMA and RGMB as well as Netrins Netrin-1 and Netrin-4 are expressed by bone marrow mesenchymal stem cells and osteoblasts, suggesting the presence of Neogenin signalling in the HSC niche. Using intracellular FACS to measure protein at the single cell level we show that the degree of Neogenin expression is positively correlated to another differentially expressed gene, Stat3. Neogenin stimulation by RGMB increases Stat3 gene expression within 1 hour (p < 0.05) and phosphorylation (Tyr705) within 5 minutes (p< 0.0001). Interestingly, aged HSCS show increased basal phosphorylation of Stat3 (p< 0.0001). Neogenin stimulation also activates Focal Adhesion Kinase and Cell division control protein 42 (Cdc42), both of which are associated with HSC aging. With the use of live-cell imaging we monitored chemotaxis of bone marrow cells in response to RGMA. At 500ng/ml bone marrow cells migrate away from RGMA, similar to what has been described in developing axons. It has been reported that old HSCs have defects in homing, increased mobilization and are differentially localized in the bone marrow. Based on our migration data, it is possible that upregulation of Neogenin affects HSC homing and/or localization. Future studies repeat migration experiments with purified LT-HSCS, investigate differential expression of Neogenin ligands in young and old stromal cells and the functional consequence of knock-down of Neogenin in young and old HSCs. In summary, our data reveal multifunctional receptor Neogenin as a novel regulator of HSCs able to affect gene expression, pathway activation and migration.

Effects of combining erlotinib and RNA-interfered downregulation of focal adhesion kinase expression on gastric cancer.

ObjectiveTo investigate the synergistic effects of combining erlotinib and RNA-interference downregulation of focal adhesion kinase (FAK) expression on the proliferation, apoptosis, invasion and migration of the human gastric adenocarcinoma cell line AGS.MethodsCells were divided into five experimental groups: Group A, nontransfected control; Group B, transfected with empty vector; Group C, transfected with FAK-shRNA; Group D, erlotinib treatment; Group E, combination erlotinib treatment and transfected with FAK-shRNA. FAK protein levels were confirmed via Western blotting. Cell proliferation (CCK-8 assay, apoptosis (flow cytometry), cell invasion (transwell assay) and migration (scratch assay) were evaluated.ResultsRNA interference significantly decreased FAK protein levels. Cell proliferation, invasion and migration were significantly lower in Groups C, D and E compared with Group A, and significantly lower in Group E than in Groups C and D.ConclusionsRNA interference effectively silences FAK expression and inhibits malignant cell proliferation and invasion in gastric cancer cells. The effect of FAK inhibition is increased by co-treatment with erlotinib.

Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts.

Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p < 0.05) and induced damage to the cell morphology in a dose- and time-dependent manner. Further research demonstrated that FAK directly interacted with TSC2 (Tuberous sclerosis 2) tuberin domain through its C-terminus, which contains the complete FAT domain. In conclusion, our results indicated that FAK may be widely expressed in Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs.

Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer.

Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio‐ and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre‐treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease‐free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence.

PIAS1-FAK Interaction Promotes the Survival and Progression of Non-Small Cell Lung Cancer

The sequence of genomic alterations acquired by cancer cells during tumor progression and metastasis is poorly understood. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates cytoskeleton remodeling, mitogenic signaling and cell survival. FAK has previously been reported to undergo nuclear localization during cell migration, cell differentiation and apoptosis. However, the mechanism behind FAK nuclear accumulation and its contribution to tumor progression has remained elusive. We report that amplification of FAK and the SUMO E3 ligase PIAS1 gene loci frequently co-occur in non-small cell lung cancer (NSCLC) cells, and that both gene products are enriched in a subset of primary NSCLCs. We demonstrate that endogenous FAK and PIAS1 proteins interact in the cytoplasm and the cell nucleus of NSCLC cells. Ectopic expression of PIAS1 promotes proteolytic cleavage of the FAK C-terminus, focal adhesion maturation and FAK nuclear localization. Silencing of PIAS1 deregulates focal adhesion turnover, increases susceptibility to apoptosis in vitro and impairs tumor xenograft formation in vivo. Nuclear FAK in turn stimulates gene transcription favoring DNA repair, cell metabolism and cytoskeleton regulation. Consistently, ablation of FAK by CRISPR/Cas9 editing, results in basal DNA damage, susceptibility to ionizing radiation and impaired oxidative phosphorylation. Our findings provide insight into a mechanism regulating FAK cytoplasm-nuclear distribution and demonstrate that FAK activity in the nucleus promotes NSCLC survival and progression by increasing cell-ECM interaction and DNA repair regulation.