Focal Adhesion Kinase Protein Research Articles

Computational insights into allosteric inhibition of focal adhesion kinase: A combined pharmacophore modeling and molecular dynamics approach

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that modulates integrin and growth factor signaling pathways and is implicated in cancer cell migration, proliferation, and survival. Over the past decade various, FAK kinase, FERM, and FAT domain inhibitors have been reported and a few kinase domain inhibitors are under clinical consideration. However, few of them were identified as multikinase inhibitors. In kinase drug design selectivity is always a point of concern, to improve selectivity allosteric inhibitor development is the best choice. The current research utilized a pharmacophore modeling (PM) approach to identify novel allosteric inhibitors of FAK. The all-available allosteric inhibitor bound 3D structures with PDB ids 4EBV, 4EBW, and 4I4F were utilized for the pharmacophore modeling. The validated PM models were utilized to map a database of 770,550 compounds prepared from ZINC, EXIMED, SPECS, ASINEX, and InterBioScreen, aiming to identify potential allosteric inhibitors. The obtained compounds from screening step were forwarded to molecular docking (MD) for the prediction of binding orientation inside the allosteric site and the results were evaluated with the known FAK allosteric inhibitor (REF). Finally, 14 FAK-inhibitor complexes were selected from the docking study and were studied under molecular dynamics simulations (MDS) for 500 ns. The complexes were ranked according to binding free energy (BFE) and those demonstrated higher affinity for allosteric site of FAK than REF inhibitors were selected. The selected complexes were further analyzed for intermolecular interactions and finally, three potential allosteric inhibitor candidates for the inhibition of FAK protein were identified. We believe that identified scaffolds may help in drug development against FAK as an anticancer agent.

PDLIM7 Promotes Tumor Metastasis in Papillary Thyroid Carcinoma via Stabilizing Focal Adhesion Kinase Protein.

Background: As an actin cytoskeleton interactor, PDZ (postsynaptic density 65-discs large-zonula occludens 1) and LIM (abnormal cell lineage 11-isket 1-mechanosensory abnormal 3) domain protein 7 (PDLIM7) was supposed to play an essential role modulating cytoskeleton. Focal adhesions (FAs), which are located at the cytomembrane terminus of actin cytoskeleton, function as a force sensor and can transform the mechanical signal to a biochemical signal. Focal adhesion kinase (FAK) localizes to and regulates signal transduction in FAs, which play an essential role in cell polarity, migration, and invasion. However, whether PDLIM7 is involved in FAs-associated signal transduction and its role in tumor invasion and metastasis remains largely unknown. Methods: A cohort of 80 patients with papillary thyroid carcinoma (PTC) at The Second Affiliated Hospital of Guilin Medical University, as well as 512 PTC samples from The Cancer Genome Atlas thyroid cancer database was utilized to analyze the expression of PDLIM7 and its association with prognosis. Survival data were assessed using Kaplan-Meier curves, whereas clinicopathological characteristics such as age, sex, tumor size, multilocality, extrathyroidal extension, lymph metastases, Hashimoto's thyroiditis, distant metastasis, and TNM stage were considered. Functional assays were performed in vitro and in a xenograft mouse model to assess the role of PDLIM7 in PTC cell lines. The colocalization of PDLIM7 with FAK and integrin alpha V (ITGAV) was determined using immunofluorescence assay and immunoprecipitation assay. Protein expression levels in cell and tissue biopsies were measured through Western blotting and immunohistochemistry. Results: (1) The PDLIM7 protein frequently upregulated in both PTC tissues and cells, and overexpression of PDLIM7 is associated with advanced clinicopathological characteristics. (2) Knockdown of PDLIM7 effectively inhibits cell proliferation, migration, and invasion in PTC cell lines in vitro. (3) Knockdown of PDLIM7 hinders the growth and metastasis of TPC-1 xenografts in vivo. (4) PDLIM7 demonstrates colocalization with both FAK and the FA molecule ITGAV and the knockdown of PDLIM7 resulted in disassembly of FAs and proteosome-dependent degradation of FAK in PTC cell lines. Conclusions: PDLIM7 function as an oncoprotein in PTC to promote metastasis, and a novel underlying mechanism is proposed that PDLIM7 keeps FAK protein from proteosome-dependent degradation.

FEATURES OF RELATIONS OF MELATONIN WITH THE STATE OF INTRACELLULAR REGULATORSOF THE FUNCTIONAL ACTIVITY OF WHOLE BLOOD MONONUCLEAR CELLS IN CORONARY HEART DISEASE

Introduction. Coronary heart disease (CHD), leading among the causes of death in adulthood and old age, is an urgent medical and social problem. The pathogenesis of most forms of coronary heart disease is based on stenosing atherosclerosis of the coronary arteries, which develops against the background of dyslipidemia and arterial hypertension and is accompanied by the activation of immunocompetent cells (ICCs) of the vascular wall with the development of a subclinical inflammatory reaction, as well as the production of pro-inflammatory factors such as interleukins, chemokines, growth factors and etc. In turn, ICC activity is determined by the state of their intracellular molecular cascades, which transmit signals into the cell and ensure its reactivity to various external stimuli, such as mitogens, cytokines, pathogen components, etcIt has been shown that the central nervous system plays an important role in the regulation of ICC activity due to the production of neurohumoral molecules, such as melatonin, endorphin, sero-tonin, etc., which ensure the coordination of immune responses and their control by the central nervous system. The aim of this study was to study the relationship between melatonin production and intracellular factors that regulate the pro-inflammatory activi-ty of whole blood mononuclear cells and their metabolism in patients with coronary artery disease. Material and methods. As part of the cohort study, 58 patients of both sexes with coronary artery disease aged 49 to 67 years and 20 practically healthy individuals of both sexes were examined. In accordance with the purpose of the study, the concentration of focal adhesion protein kinase (FAK), 5'AMP-activated protein kinase (AMPK), AKT1 protein kinase, signal transducers and transcription activators (STAT) was determined in nuclear cy-toplasmic lysates of whole blood mononuclear cells: STAT3, STAT5A and STAT6, c-Jun N-terminal protein kinase 1 and 2 isoforms (JNK), mitogen-activated protein kinase p38 (p38), extracellular growth kinase 1 and 2 isoforms (ERK), Janus kinase type 2 (JAK2), nuclear transcription factor NF -kB, caspase-1, cyclooxygenase-2 (COX-2), p70-S6K1 protein kinase, p53, p27, p21 proteins. In addition, the concentration of cyclic adenosine monophos-phate (cAMP) and cyclic guanosine monophosphate (cGMP) was determined in cell supernatants. Melatonin concentration was determined in blood se-rum. The material for the study was venous blood taken from the cubital vein in the morning from 6.00 to 6.15. Results. The analysis showed that in patients with coronary artery disease, in comparison with practically healthy individuals, in MNCs of whole blood, there was an increased level of protein kinases FAK, AKT, JNK, ERK, p70-S6K1, factor STAT6, protein p21, against which there was a decrease in the content of STAT3, STAT5A, JAK2, transcription factor NF-kB and caspase-1. These changes were accompanied by increased levels of cGMP and cAMP. Against this background, a decrease in the content of factors was revealed in the MNC. A high concentration of melatonin in patients with CAD was as-sociated with a decrease in the content of protein kinases AMPK, AKT, Jak2, ERK1, protein p21, caspase-1, and cAMP in MNCs, which was observed against the background of an increase in the level of protein p27 and nuclear factor NF-kB. The results of the correlation analysis indicate a different na-ture of the relationship between the level of melatonin and such factors as caspase-1, protein kinases ERK, JAK2, as well as the transcription factor NF-kB and p21 protein, depending on the characteristics of melatonin production in patients with coronary artery disease. Conclusions. In patients with coronary artery disease, melatonin exhibits a modulating effect on the energy balance of ICCs and their metabolism, helps limit pro-inflammatory activity by limiting the functional activity of MAPK/SAPK signaling pathways in MNCs.

3D Chiral Self-Assembling Matrixes for Regulating Polarization of Macrophages and Enhance Repair of Myocardial Infarction.

The regulation of inflammatory response at the site of injury and macrophage immunotherapy is critical for tissue repair. Chiral self-assemblies are one of the most ubiquitous life cues, which is closely related to biological functions, life processes, and even the pathogenesis of diseases. However, the role of supramolecular chiral self-assemblies in the regulation of immune functions in the internal environment of tissues has not been fully explored yet. Herein, 3D supramolecular chiral self-assembling matrixes are prepared to regulate the polarization of macrophages and further enhance the repair of myocardial infarction (MI). Experiments studies show that M-type (left-handed) self-assembling matrixes significantly inhibit inflammation and promote damaged myocardium repair by upregulating M2 macrophage polarization and downstream immune signaling compared with P-type (right-handed), and R-type (non-chirality) self-assembling matrixes. Classical molecular dynamics (MD) simulation demonstrates that M-type self-assembling matrixes display higher stereo-affinity to cellular binding, which enhances the clustering of mechanosensitive integrin β1 (Itgβ1) and activates focal adhesion kinase (FAK) and Rho-associated protein kinase (ROCK), as well as downstream PI3K/Akt1/mTOR signaling axes to promote M2 polarization. This study of designing a 3D chiral self-assembling matrixes microenvironment suitable for regulating the polarization of macrophages will provide devise basis for immunotherapy with biomimetic materials.

Pyk2/FAK Signaling Is Upregulated in Recurrent Glioblastoma Tumors in a C57BL/6/GL261 Glioma Implantation Model.

The majority of glioblastomas (GBMs) recur shortly after tumor resection and recurrent tumors differ significantly from newly diagnosed GBMs, phenotypically and genetically. In this study, using a Gl261-C57Bl/6 mouse glioma implantation model, we identified significant upregulation of proline-rich tyrosine kinase Pyk2 and focal adhesion kinase (FAK) phosphorylation levels-pPyk2 (579/580) and pFAK (925)-without significant modifications in total Pyk2 and FAK protein expression in tumors regrown after surgical resection, compared with primary implanted tumors. Previously, we demonstrated that Pyk2 and FAK are involved in the regulation of tumor cell invasion and proliferation and are associated with reduced overall survival. We hypothesized that the use of inhibitors of Pyk2/FAK in the postsurgical period may reduce the growth of recurrent tumors. Using Western blot analysis and confocal immunofluorescence approaches, we demonstrated upregulation of Cyclin D1 and the Ki67 proliferation index in tumors regrown after resection, compared with primary implanted tumors. Treatment with Pyk2/FAK inhibitor PF-562271, administered through oral gavage at 50 mg/kg daily for two weeks beginning 2 days before tumor resection, reversed Pyk2/FAK signaling upregulation in recurrent tumors, reduced tumor volume, and increased animal survival. In conclusion, the use of Pyk2/FAK inhibitors can contribute to a delay in GBM tumor regrowth after surgical resection.

Endothelial basement membrane laminins - new players in mouse and human myoendothelial junctions and shear stress communication

Basement membranes (BMs) are critical but frequently ignored components of the vascular system. Using high-resolution confocal imaging of whole-mount-stained mesenteric arteries, we identify integrins, vinculin, focal adhesion kinase (FAK) and several BM proteins including laminins as novel components of myoendothelial junctions (MEJs), anatomical microdomains that are emerging as regulators of cross-talk between endothelium and smooth muscle cells (SMCs). Electron microscopy revealed multiple layers of the endothelial BM that surround endothelial projections into the smooth muscle layer as structural characteristics of MEJs. The shear-responsive calcium channel TRPV4 is broadly distributed in endothelial cells and occurs in a proportion of MEJs where it localizes to the tips of the endothelial projections that are in contact with the underlying SMCs. In mice lacking the major endothelial laminin isoform, laminin 411 (Lama4−/−), which we have previously shown over-dilate in response to shear and exhibit a compensatory laminin 511 upregulation, localization of TRPV4 at the endothelial-SMC interface in MEJs was increased. Endothelial laminins do not affect TRPV4 expression, rather in vitro electrophysiology studies using human umbilical cord arterial endothelial cells revealed enhanced TRPV4 signalling upon culturing on an RGD-motif containing domain of laminin 511. Hence, integrin-mediated interactions with laminin 511 in MEJ structures unique to resistance arteries modulate TRPV4 localization at the endothelial-smooth muscle interface in MEJs and signalling over this shear-response molecule.

Abstract 2560: Post-translational modification of focal adhesion kinase (FAK) protein increases in breast cancer cells upon exposure to conditioned medium derived from in vitro and in vivo adipocytes

Abstract Under obese conditions, dysregulation of the normal secretion of adipokines from fat cells has been shown to drive cancer progression. However, the role adipokine dysregulation plays in driving metastasis is still unclear. Recent studies have reported the activation of metastasis-associated cell signaling pathways. One such pathway involves Focal Adhesion Kinase (FAK), a non-receptor cytoplasmic protein that acts as a signal transducer between receptor activation by adipokines and downstream effector proteins that control the biological phenomenon of metastasis. Nevertheless, the association between obesity-induced changes in adipocyte secretory factors and the subsequent increase in the transactivation of FAK through phosphorylation is unknown. To elucidate the potential relationship of adipocyte-secreted factors towards activating FAK, breast cancer cells with low (MCF7) and high (MDA-MB-231) metastatic potential were exposed to in vitro and in vivo, murine adipocyte-derived, conditioned medium. For the in vitro adipocyte model, 3T3-L1 MBX cells differentiated to obesity, with fatty acids supplementation, were used. For the in vivo model, adipocyte-secreted factors from organ-cultured visceral fat harvested from C3H/HeJ female mice fed an either high fat (46%) or control (11%) diets, were collected in the medium. Adipocyte secretions from the in vitro and in vivo adipocyte samples were determined using a mouse adipokine proteomic profiler array and ELISA kit. Under obese conditions, both the 3T3-L1 MBX cells and C3H/HeJ visceral fat were found to have significant changes in their adipokine secretions in comparison to controls. Results showed that the breast cancer cells got increased FAK autophosphorylation when exposed to either in vitro or in vivo-derived obese adipocyte-conditioned medium in comparison to the untreated breast cancer cells. Citation Format: Noshin Mubtasim, Caleb Boren, Benjamin Barr, Lauren Gollahon. Post-translational modification of focal adhesion kinase (FAK) protein increases in breast cancer cells upon exposure to conditioned medium derived from in vitro and in vivo adipocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2560.

Roles of focal adhesion proteins in skeleton and diseases.

The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection of internal organs, production of blood cells and regulation of calcium and phosphate metabolism. The prevalence of skeletal diseases and disorders, such as osteoporosis and bone fracture, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, increases with age, causing pain and loss of mobility and creating a huge social and economic burden globally. Focal adhesions (FAs) are macromolecular assemblies that are composed of the extracellular matrix (ECM), integrins, intracellular cytoskeleton and other proteins, including kindlin, talin, vinculin, paxillin, pinch, Src, focal adhesion kinase (FAK) and integrin-linked protein kinase (ILK) and other proteins. FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell-environment communications and modulates important processes, such as cell attachment, spreading, migration, differentiation and mechanotransduction, in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways. This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.

Lipopolyplex-Mediated Co-Delivery of Doxorubicin and FAK siRNA to Enhance Therapeutic Efficiency of Treating Colorectal Cancer

Tumor metastasis is a major concern in cancer therapy. In this context, focal adhesion kinase (FAK) gene overexpression, which mediates cancer cell migration and invasion, has been reported in several human tumors and is considered a potential therapeutic target. However, gene-based treatment has certain limitations, including a lack of stability and low transfection ability. In this study, a biocompatible lipopolyplex was synthesized to overcome the aforementioned limitations. First, polyplexes were prepared using poly(2-Hydroxypropyl methacrylamide-co-methylacrylate-hydrazone-pyridoxal) (P(HPMA-co-MA-hyd-VB6)) copolymers, which bore positive charges at low pH value owing to protonation of pyridoxal groups and facilitated electrostatic interactions with negatively charged FAK siRNA. These polyplexes were then encapsulated into methoxy polyethylene glycol (mPEG)-modified liposomes to form lipopolyplexes. Doxorubicin (DOX) was also loaded into lipopolyplexes for combination therapy with siRNA. Experimental results revealed that lipopolyplexes successfully released DOX at low pH to kill cancer cells and induced siRNA out of endosomes to inhibit the translation of FAK proteins. Furthermore, the efficient accumulation of lipopolyplexes in the tumors led to excellent cancer therapeutic efficacy. Overall, the synthesized lipopolyplex is a suitable nanocarrier for the co-delivery of chemotherapeutic agents and genes to treat cancers.

ALKBH5 activates FAK signaling through m6A demethylation in ITGB1 mRNA and enhances tumor-associated lymphangiogenesis and lymph node metastasis in ovarian cancer.

Background: Lymph node (LN) metastasis is common in patients with epithelial ovarian cancer (EOC) and is associated with poor prognosis. Tumor-associated lymphangiogenesis is the first stage of LN metastasis. Research on lymphangiogenesis and lymph node metastases can help develop new anti-LN-targeted therapies. Aberrant N6-methyladenosine (m6A) modifications have been reported to be linked to LN metastasis in several cancers, however, their role in EOC lymphangiogenesis and LN metastasis remains unclear. Methods: m6A levels in EOC tissues with or without LN metastases were evaluated by dot blot analysis. Real-time polymerase chain reaction (PCR) and immunofluorescence were used to examine the expression of m6A-related enzymes. Additionally, in vitro and in vivo functional studies were performed to discover the importance of the AlkB homolog 5 (ALKBH5) gene in EOC lymphatic metastasis. To identify the downstream target genes regulated by ALKBH5, we performed RNA pulldown, RNA-binding protein immunoprecipitation-quantitative PCR, co-immunoprecipitation, m6A-modified RNA immunoprecipitation-quantitative PCR, and luciferase reporter assays. Results: m6A modification was reduced in ovarian cancers with LN metastases. ALKBH5 overexpression increased tumor-associated lymphangiogenesis and LN metastasis both in vitro and in vivo. ALKBH5 overexpression also reversed the m6A modification in ITGB1 mRNA and suppressed the YTHDF2 protein-mediated m6A-dependent ITGB1 mRNA degradation, which resulted in increased expression of ITGB1 and phosphorylation of the focal adhesion kinase (FAK) and Src proto-oncogene proteins, thereby increasing LN metastasis. Furthermore, hypoxia induced the expression of hypoxia inducible factor 1 subunit alpha, which increased ALKBH5 expression and enhanced LN metastasis in EOC. Conclusions: The ALKBH5/m6A-ITGB1/FAK signalling axis is important in ovarian cancer lymphangiogenesis and LN metastasis. Antibodies that block ITGB1 and FAK kinase-inhibitors are promising anti-metastatic agents.

Bioactive Nonthermal Biocompatible Plasma Enhances Migration on Human Gingival Fibroblasts.

This study hypothesizes that the application of low-dose nonthermal biocompatible dielectric barrier discharge plasma (DBD-NBP) to human gingival fibroblasts (HGFs) will inhibit colony formation but not cell death and induce matrix metalloproteinase (MMP) expression, extracellular matrix (ECM) degradation, and subsequent cell migration, which can result in enhanced wound healing. HGFs treated with plasma for 3min migrate to each other across the gap faster than those in the control and 5-min treatment groups on days 1 and 3. The plasma-treated HGFs show significantly high expression levels of the cell cycle arrest-related p21 gene and enhanced MMP activity. Focal adhesion kinase (FAK) mediated attenuation of wound healing or actin cytoskeleton rearrangement, and plasma-mediated reversal of this attenuation support the migratory effect of DBD-NBP. Further, this work performs computer simulations to investigate the effect of oxidation on the stability and conformation of the catalytic kinase domain (KD) of FAK. It is found that the oxidation of highly reactive amino acids (AAs) Cys427, Met442, Cys559, Met571, Met617, and Met643 changes the conformation and increases the structural flexibility of the FAK protein and thus modulates its function and activity. Low-dose DBD-NBP-induces host cell cycle arrest, ECM breakdown, and subsequent migration, thus contributing to the enhanced wound healing process.

IN SILICO EVIDENCE OF THE EFFICACY OF PHYTOCOMPONENTS AS COMPETITIVE INHIBITORS AGAINST A TOBACCO SMOKE CARCINOGEN IN NON-SMALL CELL LUNG CANCER

Cigarette smoke is a major risk factor in the development of Non-small cell lung cancer (NSCLC). Nicotine- derived nitrosamine ketone (NNK) is an important carcinogen present in cigarette smoke that induces tumor metastasis by activation of a protein kinase cascade, including Focal adhesion kinase (FAK). The present investigation was aimed at employing molecular docking studies to establish binding interactions and comparison of phytocomponents from different herbs and selected Rosmarinic acid derivatives against the target protein FAK with the purpose of blocking the binding of Nicotine-derived nitrosamine ketone (NNK). Molegro virtual docker (MMV) and Hex (version 8.0.0) software was used as docking tool for investigating the interaction of the protein active site residues with the phytocomponents. Molegro molecular viewer (MMV) and Biovia Discovery studio 2017 R2 visualizer were used to identify the amino acid interactions between phytocomponents and protein and also to calculate binding scores. The molecular interaction of NNK with the target protein was also done to obtain the binding energy of this carcinogen with the protein. Rosmarinic acid derivatives manifested the highest docking score with the target protein FAK. In comparison, NNK showed weaker binding score with FAK. In addition the derivatives showed common interacting amino acids. Hence, docking studies indicated that phytocomponents and certain derivatives could effectively bind to the same active site of FAK and block its interaction with NNK. Thus, in nicotine exposed NSCLC patients, Rosmarinic acid derivatives may work as effective competitive binders.

FOCAL ADHESION KINASE INHIBITION DECREASES CELL VIABILITY AND INDUCES APOPTOSIS IN SET-2 CELLS

Myeloproliferative Neoplasms (MPN) are diseases characterized by accumulation of myeloid cells. Polycythemia vera (PV), Essential Thrombocythemia (TE) and Primary Myelofibrosis (MF) are MPN BCR-ABL negative and mutations in JAK2, MPL and CALR genes are involved in their pathophysiology. For some PV and PMF patients, Ruxolitinib, a JAK inhibitor, has been used, leading to an improvement of constitutional symptoms. However, the allele burden is not completely reduced, and bone marrow transplantation is still the only curative option. Therefore, more studies are necessary to elucidate the mechanisms involved in these diseases. The Focal Adhesion Kinase (FAK) is a tyrosine kinase which participates in adhesion and migration and also has been described as participating in proliferation, cell survival and apoptosis process mainly by activating SRC kinase. FAK has been described as overexpressed in many types of cancer, including acute myeloid leukemia. Moreover, FAK seems to participate in inflammatory process. The aims of this study was to verify the participation of FAK protein in cell viability and apoptosis of SET-2 cells. With this purpose, SET-2 cells, a JAK2 V617F positive cell line, were treated with the FAK inhibitor PF-562,271 at 5 μM and 10 μM for 72h and cell viability and apoptosis were evaluated. The concentration that inhibits 50% of cell viability (IC50) was also determined. DMSO was used as negative control and Doxorrubicin 10 μM (DOXO) as positive control. SET-2 cells were also treated with Ruxolitinib at 300 nM for 72h. The cell viability and IC50 value for PF-562,271 were evaluated using MTT assay and cleaved PARP and cleaved Caspase 3 expression were detected by Western Blotting to verify apoptosis. P-SRC and SRC detection was performed to verify whether FAK inhibition inhibited SRC phosphorylation. The IC50 determined was 3,215μM. PF-562,271 reduced SET-2 cells viability by 70,2% and 85,4% at 5μM and 10μM, respectively. Ruxolitinib reduced cell viability by 88,4% at 300nM, compared to DMSO. It was possible to observe that cleaved PARP and cleaved CASP3 expression was increased in SET-2 cells treated with PF-562,271 at 5μM and 10μM compared to DMSO and also compared to Ruxolitinib 300nM. Therefore, it was possible to verify that FAK participate in cell viability and apoptosis of SET-2 cells. Considering that many studies have demonstrated that in MPN occurs modifications in bone marrow microenvironment, involving inflammation, resistance to apoptosis and excess of proliferation, FAK may participate in the deregulation of this process, justifying more studies regarding signaling pathways involving FAK in MPN. FAPEMIG (APQ02185/16); CT_INFRA2013/FINEP; ACMMV receives master's scholarship from FAPEMIG.

Cryptotanshinone inhibits ovarian tumor growth and metastasis by degrading c-Myc and attenuating the FAK signaling pathway.

Cryptotanshinone (CT), a natural compound derived from Salvia miltiorrhiza Bunge that is also known as the traditional Chinese medicine Danshen, exhibits antitumor activity in various cancers. However, it remains unclear whether CT has a potential therapeutic benefit against ovarian cancers. The aim of this study was to test the efficacy of CT in ovarian cancer cells in vitro and using a xenograft model in NSG mice orthotopically implanted with HEY A8 human ovarian cancer cells and to explore the molecular mechanism(s) underlying CT’s antitumor effects. We found that CT inhibited the proliferation, migration, and invasion of OVCAR3 and HEY A8 cells, while sensitizing the cell responses to the chemotherapy drugs paclitaxel and cisplatin. CT also suppressed ovarian tumor growth and metastasis in immunocompromised mice orthotopically inoculated with HEY A8 cells. Mechanistically, CT degraded the protein encoded by the oncogene c-Myc by promoting its ubiquitination and disrupting the interaction with its partner protein Max. CT also attenuated signaling via the nuclear focal adhesion kinase (FAK) pathway and degraded FAK protein in both cell lines. Knockdown of c-Myc using lentiviral CRISPR/Cas9 nickase resulted in reduction of FAK expression, which phenocopies the effects of CT and the c-Myc/Max inhibitor 10058-F4. Taken together, our studies demonstrate that CT inhibits primary ovarian tumor growth and metastasis by degrading c-Myc and FAK and attenuating the FAK signaling pathway.

Alcohol promotes epithelial mesenchymal transformation-mediated premetastatic niche formation of colorectal cancer by activating interaction between laminin-γ2 and integrin-β1.

BACKGROUNDColorectal cancer (CRC) is a common malignant tumor. Alcohol consumption is positively correlated with CRC malignant metastasis; however, the mechanism is unclear. The interaction between laminin-γ2 (LAMC2) and integrin-β1 (ITGB1) plays a role in premetastatic niche signaling, which may induce epithelial mesenchymal transformation (EMT) and lead to metastasis.AIMTo investigate the effects of alcohol on CRC metastasis from the molecular mechanism of the premetastatic niche.METHODSThe interaction between LAMC2 and ITGB1 was measured by Duolink assay, and the expression levels of LAMC2, ITGB1 and focal adhesion kinase (FAK), snail, fibronectin, N-cadherin and special AT-rich sequence binding protein 1 (SATB1) were measured by quantitative real-time polymerase chain reaction, immunohistochemistry and western blotting. Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 levels were measured via enzyme-linked immunosorbent assay, histopathological assessment via hematoxylin eosin staining, and determination of aberrant crypt foci via methylene blue.RESULTSThe lymph node metastasis rate was higher in the alcohol group than non-alcohol group. There was a significant increase in interaction signals between LAMC2 and ITGB1, and an increase in phosphorylate-FAK/FAK, snail, fibronectin, N-cadherin and SATB1, whereas E-cadherin was reduced in the alcohol group compared to the non-alcohol group in both animal and clinical samples. Serum IL-1β, TNF-α and IL-6 were higher in alcohol group than in non-alcohol group. Alcohol may promote CRC metastasis by influencing the molecular mechanism of the premetastatic niche.CONCLUSIONOur study suggests that alcohol promotes EMT-mediated premetastatic niche formation of CRC by activating the early interaction between LAMC2 and ITGB1 and lead to CRC metastasis.

RIOK3 promotes pancreatic ductal adenocarcinoma cell invasion and metastasis by stabilizing FAK

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, characterized by a high metastatic burden. RIO Kinase 3 (RIOK3) has been shown to promote invasion and metastasis of PDAC by cytoskeleton remodeling, but the exact mechanism is still unknown. In this study, we analyzed transcriptome sequencing data from RIOK3 stable knockdown PANC-1 cells and TCGA-PDAC data and discovered that RIOK3 was substantially related to focal adhesion signaling in PDAC. Additionally, silencing RIOK3 dramatically decreased Focal Adhesion Kinase (FAK) protein expression and phosphorylation (Tyr397 and Tyr925 sites). Immunoprecipitation assay verified the interaction of RIOK3 and FAK. Furthermore, RIOK3 considerably increased the protein stability of FAK protein but not FAK-Y925F protein. The biological function of RIOK3 in increasing PDAC cell invasion and migration was shown to be dependent on FAK activation. Moreover, we discovered that RIOK3 mutations were mainly characterized by amplification. RIOK3 mRNA was found to be significantly elevated in PDAC tissues and was associated with a poor prognosis. Furthermore, RIOK3 mRNA was significantly upregulated in later T-stage, pre-existing lymph node metastases, and later pathological stage samples. Overall, our study found that RIOK3 promotes PDAC cell invasion and metastasis by stabilizing FAK protein expression and upregulating its phosphorylation. This also provides a new target for therapeutic modalities targeting FAK.

The molecular mechanism of baicalein repressing progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway

BackgroundBaicalein (BAI) has a significant anti-cancerous function in the treatment of gastric cancer (GC). Focal adhesion kinase (FAK) is a key regulatory molecule in integrin and growth factor receptor mediated signaling. MicroRNA-7 (miR-7), has been considered as a potential tumor suppressor in a variety of cancers. However, the possible mechanisms by which BAI inhibiting progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway remain unclear. PurposeTo investigate the molecular mechanism and effects of BAI inhibiting progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway. MethodsGastric cancer cell lines with FAK knockdown and overexpression were constructed by lentivirus transfection. After BAI treatment, the effects of FAK protein on proliferation, metastasis and angiogenesis of gastric cancer cells were detected by MTT, EdU, colony formation, wound healing, transwell and Matrigel tube formation assays. In vivo experiment was performed by xenograft model. Immunofluorescence and western blot assay were used to detect the effects of FAK protein on the expression levels of EMT markers and PI3K/AKT signaling pathway related proteins. qRT-PCR and luciferase reporter assay were used to clarify the targeting relationship between miR-7 and FAK. ResultsBAI can regulate FAK to affect proliferation, metastasis and angiogenesis of gastric cancer cells through PI3K/AKT signaling pathway. qRT-PCR showed BAI can upregulated the expression of miR-7 and luciferase reporter assay showed the targeting relationship between miR-7 and FAK. Additionally, miR-7 mediates cell proliferation, metastasis and angiogenesis by directly targeting FAK 3′UTR to inhibit FAK expression. ConclusionBAI repressing progression of gastric cancer mediating miR-7/FAK/AKT signaling pathway.

LED Light-Induced ROS Differentially Regulates Focal Adhesion Kinase Activity in HaCaT Cell Viability.

In this study, changes in cell signaling mechanisms in skin cells induced by various wavelengths and intensities of light-emitting diodes (LED) were investigated, focusing on the activity of focal adhesion kinase (FAK) in particular. We examined the effect of LED irradiation on cell survival, the generation of intracellular reactive oxygen species (ROS), and the activity of various cell-signaling proteins. Red LED light increased cell viability at all intensities, whereas strong green and blue LED light reduced cell viability, and this effect was reversed by NAC or DPI treatment. Red LED light caused an increase in ROS formation according to the increase in the intensity of the LED light, and green and blue LED lights led to sharp increases in ROS formation. In the initial reaction to LEDs, red LED light only increased the phosphorylation of FAK and extracellular-signal regulated protein kinase (ERK), whereas green and blue LED lights increased the phosphorylation of inhibitory-κB Kinase α (IKKα), c-jun N-terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC, and DPI treatments. Even after 24 h of LED irradiation, the activity of FAK and ERK appeared in cells treated with red LED light but did not appear in cells treated with green and blue LED lights. Furthermore, the activity of caspase-3 was confirmed along with cell detachment. Therefore, our results suggest that red LED light induced mitogenic effects via low levels of ROS–FAK–ERK, while green and blue LED lights induced cytotoxic effects via cellular stress and apoptosis signaling resulting from high levels of ROS.

MiR-15p-5p Mediates the Coordination of ICAM-1 and FAK to Promote Endothelial Cell Proliferation and Migration.

Intercellular adhesion molecule-1 (ICAM-1) in endothelial cells is critical for neutrophil adhesion and transmigration across the endothelium. Focal adhesion kinase (FAK), which controls the turnover of focal adhesion to regulate cell adhesion and migration, plays a role in the resolution of inflammation. However, the coordinated involvement of ICAM-1 and FAK during endothelial inflammation has yet to be elucidated. This study reports that, as part of an inflammatory response, ICAM-1 controls FAK expression in endothelial cells via the microRNA miR-15b-5p. Induction of lung injury by lipopolysaccharide (LPS) resulted in higher levels of FAK expression in inflammatory tissues, while in ICAM-1 knockout mice, FAK expression was reduced in the lungs. FAK expression was also reduced in endothelial cells following ICAM-1 siRNA downregulation. Furthermore, ICAM-1 inhibited miR-15b-5p expression while increasing FAK mRNA and protein expression via binding of miR-15b-5p to the 3' untranslated region (UTR) of FAK. ICAM-1 inhibited miR-15b-5p promoter activity and hence reduced miR-15b-5p expression. FAK increased endothelial cell proliferation and migration, whereas miR-15b-5p inhibited cell proliferation and migration. These findings indicate that the inflammatory molecule ICAM-1 regulates FAK expression via miR-15b-5p levels, which in turn controls endothelial cell proliferation and migration.

Biomechanical microenvironment in peripheral nerve regeneration: from pathophysiological understanding to tissue engineering development.

Peripheral nerve injury (PNI) caused by trauma, chronic disease and other factors may lead to partial or complete loss of sensory, motor and autonomic functions, as well as neuropathic pain. Biological activities are always accompanied by mechanical stimulation, and biomechanical microenvironmental homeostasis plays a complicated role in tissue repair and regeneration. Recent studies have focused on the effects of biomechanical microenvironment on peripheral nervous system development and function maintenance, as well as neural regrowth following PNI. For example, biomechanical factors-induced cluster gene expression changes contribute to formation of peripheral nerve structure and maintenance of physiological function. In addition, extracellular matrix and cell responses to biomechanical microenvironment alterations after PNI directly trigger a series of cascades for the well-organized peripheral nerve regeneration (PNR) process, where cell adhesion molecules, cytoskeletons and mechanically gated ion channels serve as mechanosensitive units, mechanical effector including focal adhesion kinase (FAK) and yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) as mechanotransduction elements. With the rapid development of tissue engineering techniques, a substantial number of PNR strategies such as aligned nerve guidance conduits, three-dimensional topological designs and piezoelectric scaffolds emerge expected to improve the neural biomechanical microenvironment in case of PNI. These tissue engineering nerve grafts display optimized mechanical properties and outstanding mechanomodulatory effects, but a few bottlenecks restrict their application scenes. In this review, the current understanding in biomechanical microenvironment homeostasis associated with peripheral nerve function and PNR is integrated, where we proposed the importance of balances of mechanosensitive elements, cytoskeletal structures, mechanotransduction cascades, and extracellular matrix components; a wide variety of promising tissue engineering strategies based on biomechanical modulation are introduced with some suggestions and prospects for future directions.