Tyrosine Phosphorylation Of Focal Adhesion Kinase Research Articles

Oxidative stress-induced FAK activation contributes to uterine serous carcinoma aggressiveness.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer (EC), characterized by its high propensity for metastases. In fact, while endometrioid endometrial carcinoma (EEC), which accounts for 85% of EC, presents a good prognosis, USC is the most frequently fatal. Herein, we used for the first time a peptide-based tyrosine-kinase-activity profiling approach to quantify the changes in tyrosine kinase activation between USC and EEC. Among the tyrosine kinases highly activated in USC, we identified focal adhesion kinase (FAK). We conducted mechanistic studies using cellular models. In a USC cell line, targeting FAK either by inhibitors PF-573228 and defactinib (VS-6063) or by gene silencing limits 3D cell growth and reduces cell migration. Moreover, results from our studies suggest that oxidative stress is increased in USC tumors compared to EEC ones. Reactive oxygen species (ROS) induce tyrosine phosphorylation of FAK and a concomitant tyrosine phosphorylation of paxillin, a mediator of FAK signal transduction. Mechanistically, by tracking hundreds of individual cells per condition, we show that ROS increased cell distance and migration velocity, highlighting the role of ROS-FAK-PAX signaling in cell migration. Both defactinib and ROS scavenger N-acetylcysteine (NAC) revert this effect, pointing toward ROS as potential culprits for the increase in USC cell motility. A proof of concept of the role of FAK in controlling cell growth was obtained in in vivo experiments using cancer-tissue-originated spheroids (CTOS) and a patient-derived orthotopic xenograft model (orthoxenograft/PDOX). Defactinib reduces cell proliferation and protein oxidation, supporting a pro-tumoral antioxidant role of FAK, whereas antioxidant NAC reverts FAK inhibitor effects. Overall, our data points to ROS-mediated FAK activation in USC as being responsible for the poor prognosis of this tumor type and emphasize the potential of FAK inhibition for USC treatment.

Vacuolin-1 enhances RA-induced differentiation of human myeloblastic leukemia cells: evidence for involvement of a CD11b/FAK/LYN/SLP-76 axis subject to endosomal regulation that drives late differentiation steps

BackgroundRetinoic acid(RA), an embryonic morphogen, regulates cell differentiation. Endocytosis regulates receptor signaling that governs such RA-directed cellular processes. Vacuolin-1 is a small molecule that disrupts endocytosis, motivating interest in its effect on RA-induced differentiation/arrest. In HL-60 myeloblastic-leukemia cells, RA causes differentiation evidenced by a progression of cell-surface and functional markers, CD38, CD11b, and finally reactive oxygen species(ROS) production and G1/0 cell cycle arrest in mature cells.ResultsWe found that Vacuolin-1 enhanced RA-induced CD11b, ROS and G1/0 arrest, albeit not CD38. Enhanced CD11b expression was associated with enhanced activation of Focal Adhesion Kinase(FAK). Adding vacuolin-1 enhanced RA-induced tyrosine phosphorylation of FAK, Src Family Kinases(SFKs), and the adaptor protein, SLP-76, expression of which is known to drive RA-induced differentiation. Depleting CD11b cripples late stages of progressive myeloid differentiation, namely G1/0 arrest and inducible ROS production, but not expression of CD38. Loss of NUMB, a protein that supports early endosome maturation, affected RA-induced ROS and G1/0 arrest, but not CD38 expression.ConclusionHence there appears to be a novel CD11b/FAK/LYN/SLP-76 axis subject to endosome regulation which contributes to later stages of RA-induced differentiation. The effects of vacuolin-1 thus suggest a model where RA-induced differentiation consists of progressive stages driven by expression of sequentially-induced receptors.

Pleckstrin homology domain of phospholipase D2 is a negative regulator of focal adhesion kinase

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK.

Homeopathic Rhus toxicodendron Induces Cell Adhesions in the Mouse Pre-osteoblast Cell Line MC3T3-e1.

Rhus toxicodendron (R. tox) has been used as a homeopathic remedy for the treatment of inflammatory conditions. Previously, we reported that R. tox modulated inflammation in the mouse chondrocyte and pre-osteoblastic MC3T3-e1 cell line. During the inflammatory process, cells adhere to the extracellular matrix (ECM) and then migrate to the inflammation site. We examine here the process of cell adhesion in MC3T3-e1 cells after their stimulation with homeopathic R. tox. For the cell-substrate adhesion assay, the cultured MC3T3-e1 cells were trypsinized, starved for 1 h in serum-free media, and plated onto culture plates coated with fibronectin (FN), 30c R. tox or gelatin, respectively. The cells were allowed to adhere for 20 min incubation and unattached cells were washed out. Adherent cells were measured using the water-soluble tetrazolium salt-8 assay. The intracellular signals after stimulation of R. tox were examined by analyzing the tyrosine phosphorylation of focal adhesion kinase (FAK), Src kinase, and Paxillin using immunoblot assay. Formation of focal adhesion (FA, an integrin-containing multi-protein structure that forms between intracellular actin bundles and the ECM) was analyzed by immunocytochemistry using NIH ImageJ software. Cell adhesion increased after stimulation with R. tox (FN, 20.50%; R. tox, 44.80%; and gelatin, 17.11% vs. uncoated cells [control]). Tyrosine phosphorylation of FAK, Paxillin, and Src increased compared with that of gelatin when stimulated with R. tox. Additionally, R. tox-stimulated cells formed many FAs (number of FAs per cell, 35.82 ± 7.68) compared with gelatin-stimulated cells (number of FAs per cell, 19.80 ± 7.18) and exhibited extensive formation of actin stress fibers anchored by FAs formed at the cell periphery. Homeopathic R. tox promotes the formation of cell adhesions in vitro.

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

SERINE-910 Phosphorylated Focal Adhesion Kinase Expression Predicts Better Overall and Disease-free Survival in Melanoma.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that mediates multiple cellular functions such as survival, invasion, and migration. FAK has been found to be over-expressed in various human cancers, including melanoma. FAK molecule has several tyrosine, serine, and threonine phosphorylation sites which have an important regulatory role. Tyrosine phosphorylation of FAK has been extensively studied, however little is known about the role of serine phosphorylation. We sought to examine the frequency and extent of serine-910 phosphorylated FAK (P-FAKSer910) expression in a spectrum of melanocytic proliferations as well as it's correlation with other histopathologic predictors and its effect on patient's survival. Clinicopathologic features and immunohistochemical expression of P-FAKSer910 were evaluated in 147 melanocytic proliferations: 73 primary melanoma (PM), 19 metastatic melanoma (MetM), 2 melanoma in situ, and 53 melanocytic nevi (MN). Higher cytoplasmic intensity predicted better overall survival (OS) in PM (χ=5.69; P=0.034) and was associated with 48% decrease in death risk (HR, 0.52; 95% CI, 0.28-0.95; P=0.036). In contrast, increased nuclear intensity was significantly associated with better disease-free survival (DFS) when stratified by tumor stage Log-rank test, trend of survival (χ=5.83, P=0.015) and independently on multivariate analysis when subcategorized into 3-tier categories (HR, 0.43; 95% CI, 0.18-0.98; P=0.045). Also, Clark's level and tumor-infiltrating lymphocytes (TILS) were independent predictors of DFS. Cytoplasmic intensity correlated inversely with American Joint Commission on Cancer stage in primary melanoma cases as well with vascularity in both primary and metastatic melanoma. Nuclear intensity independently correlated negatively with angioinvasion and TILS when subcategorized to 3 tier system. We found American Joint Commission on Cancer tumor stage, Clark's level, depth, ulceration, TILS, mitosis, angioinvasion, and tumor vascularity predictors of both DFS and OS. There was no significant difference in cytoplasmic or nuclear expression among the major categories of melanocytic proliferation. In this pilot immunohistochemistry-based study, we found P-FAKSer910 expression in melanoma by cytoplasmic intensity to correlate with better OS while nuclear intensity correlated with better DFS.

Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression

ABSTRACTOncogenic KIT or PDGFRA receptor tyrosine kinase (TK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is the standard of care for patients with metastatic GIST. However, approximately 10% of KIT-positive GIST metastases lose KIT expression at the time of clinical progression during imatinib therapy. In the present report, we performed TK-activation screens, using phosphotyrosine-TK double immunoaffinity purification and mass spectrometry, in GIST in vitro models lacking KIT expression. These studies demonstrated tyrosine-phosphorylated EGFR, AXL, and EPHA2 in four of six KIT-negative GIST lines (GIST62, GIST522, GIST54, GIST226, GIST48B, and GIST430B), and tyrosine-phosphorylated focal adhesion kinase (FAK) in each of the six KIT-negative lines. AXL expression was strong in KIT-negative or -weak clinical GIST samples that were obtained from progressing metastases during imatinib therapy. AXL knockdown inhibited viability in three KIT-negative GIST cell lines (GIST62, GIST54, and GIST522), but not in an AXL-negative, KIT-positive GIST control cell line (GIST430). AXL inhibition by R428, a specific AXL kinase inhibitor, reduced viability in AXL-activated GIST54. AXL knockdown in GIST62, GIST522, and GIST54 was accompanied by an increase in p21, p27, and p53 expression. By contrast, gefitinib-mediated EGFR inhibition, PF562271-mediated FAK inactivation, and shRNA-mediated knockdowns of EPHA2 and FAK had no effect on viability or colony formation of the KIT-negative GISTs. These findings highlight the potential relevance of AXL/p53 signaling as a therapeutic target in a subset of GISTs that have lost KIT oncoprotein expression.

MiR-375 inhibits the hepatocyte growth factor-elicited migration of mesenchymal stem cells by downregulating Akt signaling.

The migration of mesenchymal stem cells (MSCs) is critical for their use in cell-based therapies. Accumulating evidence suggests that microRNAs are important regulators of MSC migration. Here, we report that the expression of miR-375 was downregulated in MSCs treated with hepatocyte growth factor (HGF), which strongly stimulates the migration of these cells. Overexpression of miR-375 decreased the transfilter migration and the migration velocity of MSCs triggered by HGF. In our efforts to determine the mechanism by which miR-375 affects MSC migration, we found that miR-375 significantly inhibited the activation of Akt by downregulating its phosphorylation at T308 and S473, but had no effect on the activity of mitogen-activated protein kinases. Further, we showed that 3'phosphoinositide-dependent protein kinase-1 (PDK1), an upstream kinase necessary for full activation of Akt, was negatively regulated by miR-375 at the protein level. Moreover, miR-375 suppressed the phosphorylation of focal adhesion kinase (FAK) and paxillin, two important regulators of focal adhesion (FA) assembly and turnover, and decreased the number of FAs at cell periphery. Taken together, our results demonstrate that miR-375 inhibits HGF-elicited migration of MSCs through downregulating the expression of PDK1 and suppressing the activation of Akt, as well as influencing the tyrosine phosphorylation of FAK and paxillin and FA periphery distribution.

3D matrix-embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells.

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Others and we previously demonstrated endothelial biology being intertwined with biochemical and structural composition of the subendothelial basement membrane. We now demonstrate that a three-dimensional growing environment significantly shields endothelial cells from cytokine-induced apoptosis. Detailed analysis reveals differences in intracellular signaling pathways in naive endothelial cells and cytokine-stimulated endothelial cells when cells are grown within a three-dimensional collagen-based matrix compared to cells grown on two-dimensional tissue culture plates. Main findings are significantly reduced p53 expression and level of p38-phosphorylation in three-dimensional grown endothelial cells. Despite similar concentrations of focal adhesion kinase, three-dimensional matrix-embedded endothelial cells express significantly less tyrosine-phosphorylated focal adhesion kinase. Pretreatment with antibodies against integrin αv β3 partially reversed the protective effect of three-dimensional matrix-embedding on endothelial apoptosis. Our findings provide detailed insights into the mechanisms of endothelial apoptosis with respect to the spatial matrix environment. These results enhance our understanding of endothelial biology and may otherwise help in the design of tissue-engineered materials. Furthermore, findings on focal adhesion kinase phosphorylation might enhance our understanding of clinical studies with tyrosine kinase inhibitors.

FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle

Aims/hypothesisInsulin-mediated signals and AMP-activated protein kinase (AMPK)-mediated signals are activated in response to physiological conditions that represent energy abundance and shortage, respectively. Focal adhesion kinase (FAK) is implicated in insulin signalling and cancer progression in various non-muscle cell types and plays a regulatory role during skeletal muscle differentiation. The role of FAK in skeletal muscle in relation to insulin stimulation or AMPK activation is unknown. We examined the effects of insulin or AMPK activation on FAK phosphorylation in human skeletal muscle and the direct role of FAK on glucose and lipid metabolism. We hypothesised that insulin treatment and AMPK activation would have opposing effects on FAK phosphorylation and that gene silencing of FAK would alter metabolism.MethodsHuman muscle was treated with insulin or the AMPK-activating compound 5-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to determine FAK phosphorylation and glucose transport. Primary human skeletal muscle cells were used to study the effects of insulin or AICAR treatment on FAK signalling during serum starvation, as well as to determine the metabolic consequences of silencing the FAK gene, PTK2.ResultsAMPK activation reduced tyrosine phosphorylation of FAK in skeletal muscle. AICAR reduced p-FAKY397 in isolated human skeletal muscle and cultured myotubes. Insulin stimulation did not alter FAK phosphorylation. Serum starvation increased AMPK activation, as demonstrated by increased p-ACCS222, concomitant with reduced p-FAKY397. FAK signalling was reduced owing to serum starvation and AICAR treatment as demonstrated by reduced p-paxillinY118. Silencing PTK2 in primary human skeletal muscle cells increased palmitate oxidation and reduced glycogen synthesis.Conclusions/interpretationAMPK regulates FAK signalling in skeletal muscle. Moreover, siRNA-mediated FAK knockdown enhances lipid oxidation while impairing glycogen synthesis in skeletal muscle. Further exploration of the interaction between AMPK and FAK may lead to novel therapeutic strategies for diabetes and other chronic conditions associated with an altered metabolic homeostasis.

Deficiency in VHR/DUSP3, a suppressor of focal adhesion kinase, reveals its role in regulating cell adhesion and migration.

Vaccinia H1-related phosphatase (VHR/DUSP3) is a member of the dual-specificity phosphatase family. Deregulation of VHR is observed in various malignant diseases. We identified focal adhesion kinase (FAK) as a VHR-interacting molecule. Over-expression of VHR decreased tyrosine phosphorylation of FAK and decreasing VHR promoted FAK tyrosine phosphorylation. In vitro assays proved that recombinant VHR directly dephosphorylated FAK and paxillin. VHR-knockout mice did not have obvious abnormality; however, VHR-knockout cells showed decreased expression of integrins and FAK but stronger FAK and paxillin phosphorylation upon attachment to fibronectin. Additionally, VHR-knockout fibroblast and lung epithelial cells had elevated ligand-induced epidermal growth factor receptor (EGFR) phosphorylation. Inducible expression of VHR suppressed directional cell migration, and VHR deficiency resulted in a higher cell migratory ability. VHR-knockout cells have stronger FAK phosphorylation in cell adhesions, long-lasting trailing ends and slower turnover of focal adhesions. These collective data indicate that VHR is a FAK phosphatase and participates in regulating the formation and disassembly of focal adhesions.

FAK phosphorylation plays a central role in thrombin-induced RPE cell migration

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-differentiation and migration is essential for devising effective treatments aimed to the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the disassembly of focal adhesions induced by Focal Adhesion Kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-ζ, as well as by Rho/ROCK, since the inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment.

Focal adhesion kinases crucially regulate TGFβ-induced migration and invasion of bladder cancer cells via Src kinase and E-cadherin.

Focal adhesion kinase (FAK) is a non-receptor protein-tyrosine kinase that is triggered off by special extracellular signals such as some growth factors and integrins. FAK is found in cell–matrix attachment sites and implicated in cell migration, invasion, movement, gene expression, survival and apoptosis. In this study, we aimed to investigate whether FAK plays a role in invasion and migration of bladder cancer cells. Using an FAK-specific small interfering RNA (siRNA) and an FAK inhibitor PF-228, we found that inhibition of FAK tyrosine phosphorylation or knockdown of FAK suppressed invasion and migration of bladder cancer cells. Src is an important mediator of FAK-regulated migratory and invasive activity. Tyrosine phosphorylation of Src and FAK is mutually dependent and plays a key role in transforming growth factor beta (TGFβ)-induced invasion and migration. E-cadherin acts downstream of FAK and is a critical negative regulator in FAK-regulated invasion and migration of bladder cancer cells. These findings imply that FAK is involved in oncogenic signaling of invasion and migration, which can be a novel therapeutic target to treat patients with bladder cancer.

Abstract 3812: FAK inhibition resensitizes platinum-resistant serous ovarian cancer

Abstract Platinum and taxol administration is standard of care chemotherapy for serous ovarian cancer. Tumor recurrence occurs in a high percentage of patients, and this is directly related to poor overall survival. Ovarian cancer stem cell (CSC) resistance to chemotherapy treatment can give rise to tumor recurrence. Focal adhesion kinase (FAK), an intracellular tyrosine kinase, has been linked to mesothelioma and breast CSC survival. Here, we find that FAK activation is elevated in platinum (CP)-resistant ovarian cancer cells and that FAK tyrosine phosphorylation is increased after CP treatment of CP-sensitive ovarian cancer cells. Nanomolar levels of FAK inhibitor (VS-4718) selectively blocked CP-resistant ovarian carcinoma methylcellulose colony growth via cell cycle inhibition but not apoptosis. Oral VS-4718 administration to mice reduces CP-resistant orthotopic tumor burden with a concomitant decrease in tumor-associated aldehyde dehydrogenase (ALDH) activity, a marker of ovarian CSCs. Residual ovarian tumor cells from VS-4718-treated mice exhibit reduced ALDH activity and secondary tumor initiating capacity. CRISPR-mediated FAK knockout or VS-4718 treated ovarian carcinoma cells exhibit diminished Oct-4 transcription factor and ALDH-1A1 CSC-associated protein marker expression. Co-administration of VS-4718 with CP-taxol chemotherapy reduced CP-resistant tumor burden and exhibited additive inhibitory effects on ovarian carcinoma spheroid growth. As we find that CP activates FAK and that FAK activity sustains ovarian carcinoma CSC phenotypes, our results support the testing of FAK inhibitors in combination with CP to prevent recurrent and chemo-resistant ovarian cancer. Citation Format: Lisa M. Bean, Florian J. Sulzmaier, Isabelle Tancioni, Sean Uryu, Christine Jean, Christine Lawson, Xiao Lei Chen, Elizabeth G. Kleinschmidt, Kristen M. Anderson, Edward A. Cordasco, Joshua Axelrod, Vihren N. Kolev, Jonathan A. Pachter, Dwayne G. Stupack, David D. Schlaepfer. FAK inhibition resensitizes platinum-resistant serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3812.

Imatinib and Nilotinib increase glioblastoma cell invasion via Abl-independent stimulation of p130Cas and FAK signalling.

Imatinib was the first targeted tyrosine kinase inhibitor to be approved for clinical use, and remains first-line therapy for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukaemia. We show that treatment of human glioblastoma multiforme (GBM) tumour cells with imatinib and the closely-related drug, nilotinib, strikingly increases tyrosine phosphorylation of p130Cas, focal adhesion kinase (FAK) and the downstream adaptor protein paxillin (PXN), resulting in enhanced cell migration and invasion. Imatinib and nilotinib-induced tyrosine phosphorylation was dependent on expression of p130Cas and FAK activity and was independent of known imatinib targets including Abl, platelet derived growth factor receptor beta (PDGFRβ) and the collagen receptor DDR1. Imatinib and nilotinib treatment increased two dimensional cell migration and three dimensional radial spheroid invasion in collagen. In addition, silencing of p130Cas and inhibition of FAK activity both strongly reduced imatinib and nilotinib stimulated invasion. Importantly, imatinib and nilotinib increased tyrosine phosphorylation of p130Cas, FAK, PXN and radial spheroid invasion in stem cell lines isolated from human glioma biopsies. These findings identify a novel mechanism of action in GBM cells for two well established front line therapies for cancer resulting in enhanced tumour cell motility.

β1,6-branched complex-type N-glycans affect FAK signaling in metastatic melanoma cells

ABSTRACTIntegrin-dependent binding of the cell to extracellular matrix (ECM) is a key activator of the focal adhesion kinase (FAK) signaling pathway. N-glycosylation of integrins affects their interactions with ECM proteins. Using WM266-4 cells with overexpression of β1,6-acetylglucosaminyltransferase V, we showed that β1,6-branched N-glycans increased tyrosine phosphorylation of FAK in metastatic melanoma cells, resulting in enhanced migration on vitronectin (VN). The co-localization of αvβ3 integrin and FAK in focal adhesions of melanoma cells growing on VN indicates their interaction in signal transduction. Melanoma cell migration on VN was mediated by αvβ3 caring overexpressed β1,6-branched structures, important for FAK upregulation.

Inhibition of focal adhesion kinase induces apoptosis in bladder cancer cells via Src and the phosphatidylinositol 3-kinase/Akt pathway.

Focal adhesion kinase (FAK) is a 125-kDa, cytosolic, non-receptor, protein tyrosine kinase localized at focal adhesions that can be activated by multiple inputs and in different manners. FAK is implicated in signaling pathways regulating cell movement, invasion, survival, gene expression and cancer stem cell self-renewal. The aim of the present study was to investigate whether FAK plays a role in the apoptosis of bladder cancer cells. The study employed in situ deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and Annexin V labeling flow cytometry. It was found that both the knockdown of FAK and the suppression of FAK phosphorylation were able to induce apoptosis in bladder cancer cells. Caspase-3 was activated during the apoptosis induced by the suppression of FAK phosphorylation. Src was involved in FAK-regulated apoptosis in bladder cancer cells, while the suppression of Src phosphorylation was able to inhibit FAK tyrosine phosphorylation and induce apoptosis. Furthermore, phosphatidylinositol 3-kinase (PI3K)/Akt signaling was inhibited via the suppression of FAK tyrosine phosphorylation. Conversely, the expression of neither the general nor the tyrosine-phosphorylated FAK was regulated by inhibiting PI3K/Akt, which suggested that PI3K/Akt acted downstream of FAK to regulate apoptosis in bladder cancer cells. These findings indicate the presence of a mechanism of apoptosis involving FAK-mediated oncogenic signaling. FAK may function as an important regulator of extracellular signaling-mediated apoptosis in bladder cancer and be used as a novel therapeutic target in the treatment of the condition.

Myoferlin depletion elevates focal adhesion kinase and paxillin phosphorylation and enhances cell-matrix adhesion in breast cancer cells.

Breast cancer is the second leading cause of malignant death among women. A crucial feature of metastatic cancers is their propensity to lose adhesion to the underlying basement membrane as they transition to a motile phenotype and invade surrounding tissue. Attachment to the extracellular matrix is mediated by a complex of adhesion proteins, including integrins, signaling molecules, actin and actin-binding proteins, and scaffolding proteins. Focal adhesion kinase (FAK) is pivotal for the organization of focal contacts and maturation into focal adhesions, and disruption of this process is a hallmark of early cancer invasive potential. Our recent work has revealed that myoferlin (MYOF) mediates breast tumor cell motility and invasive phenotype. In this study we demonstrate that noninvasive breast cancer cell lines exhibit increased cell-substrate adhesion and that silencing of MYOF using RNAi in the highly invasive human breast cancer cell line MDA-MB-231 also enhances cell-substrate adhesion. In addition, we detected elevated tyrosine phosphorylation of FAK (FAK(Y397)) and paxillin (PAX(Y118)), markers of focal adhesion protein activation. Morphometric analysis of PAX expression revealed that RNAi-mediated depletion of MYOF resulted in larger, more elongated focal adhesions, in contrast to cells transduced with a control virus (MDA-231(LVC) cells), which exhibited smaller focal contacts. Finally, MYOF silencing in MDA-MB-231 cells exhibited a more elaborate ventral cytoskeletal structure near focal adhesions, typified by pronounced actin stress fibers. These data support the hypothesis that MYOF regulates cell adhesions and cell-substrate adhesion strength and may account for the high degree of motility in invasive breast cancer cells.

Lysophosphatidic Acid Stimulates Activation of Focal Adhesion Kinase and Paxillin and Promotes Cell Motility, via LPA1–3, in Human Pancreatic Cancer

Pancreatic cancer is highly metastatic and with poor prognosis. In previous studies, lysophosphatidic acid (LPA) was shown to be a critical component of ascites which promoted the invasion and metastasis of pancreatic cancer. Two focal adhesion proteins, focal adhesion kinase (FAK) and paxillin, were crucially involved in cell migration, cytoskeleton reorganization, and the dynamics of focal adhesion. This study examined the involvement of LPA1-3 in LPA-induced activation of FAK and paxillin, and in cell motility, in pancreatic cancer PANC-1 cells. Reverse transcriptase polymerase chain reaction analysis was used to examine mRNA expression of LPA receptors in PANC-1. Cellular protein expression of FAK and paxillin was analyzed by western blotting. The subcellular location of FAK and paxillin was visualized by immunofluorescence. Cell migration was measured by use of a transwell migration chamber. Three LPA receptors (LPA1, LPA2, and LPA3) were significantly expressed in PANC-1 cells. Treatment with LPA induced both time and dose-dependent tyrosine phosphorylation of FAK and paxillin. LPA also affected translocation of FAK and paxillin from cytoplasm to focal adhesions at the cell periphery and enhanced cell motility of PANC-1. Pretreatment with 3-(4-(4-((1-(2-chlorophenyl)ethoxy)carbonyl amino)-3-methyl-5-isoxazolyl)benzylsulfanyl)propanoic acid (Ki16425), an antagonist of LPA1 and LPA3, before LPA attenuated the LPA-induced tyrosine phosphorylation and redistribution of FAK and paxillin and abrogated LPA-induced cellular migration activity. These results suggest LPA induces activation of FAK and paxillin via LPA1-3, which may contribute to the increased cell motility in human pancreatic cancer PANC-1 cells. Thus, an understanding of the regulation by LPA of cell motility in pancreatic cancer could identify novel targets for therapy.

Fusicoccin A, a Phytotoxic Carbotricyclic Diterpene Glucoside of Fungal Origin, Reduces Proliferation and Invasion of Glioblastoma Cells by Targeting Multiple Tyrosine Kinases

Glioblastoma multiforme (GBM) is a deadly cancer that possesses an intrinsic resistance to pro-apoptotic insults, such as conventional chemotherapy and radiotherapy, and diffusely invades the brain parenchyma, which renders it elusive to total surgical resection. We found that fusicoccin A, a fungal metabolite from Fusicoccum amygdali, decreased the proliferation and migration of human GBM cell lines in vitro, including several cell lines that exhibit varying degrees of resistance to pro-apoptotic stimuli. The data demonstrate that fusicoccin A inhibits GBM cell proliferation by decreasing growth rates and increasing the duration of cell division and also decreases two-dimensional (measured by quantitative video microscopy) and three-dimensional (measured by Boyden chamber assays) migration. These effects of fusicoccin A treatment translated into structural changes in actin cytoskeletal organization and a loss of GBM cell adhesion. Therefore, fusicoccin A exerts cytostatic effects but low cytotoxic effects (as demonstrated by flow cytometry). These cytostatic effects can partly be explained by the fact that fusicoccin inhibits the activities of a dozen kinases, including focal adhesion kinase (FAK), that have been implicated in cell proliferation and migration. Overexpression of FAK, a nonreceptor protein tyrosine kinase, directly correlates with the invasive phenotype of aggressive human gliomas because FAK promotes cell proliferation and migration. Fusicoccin A led to the down-regulation of FAK tyrosine phosphorylation, which occurred in both normoxic and hypoxic GBM cell culture conditions. In conclusion, the current study identifies a novel compound that could be used as a chemical template for generating cytostatic compounds designed to combat GBM.