Pharmacological Inhibition Of Focal Adhesion Kinase Research Articles

Apigenin and Rutaecarpine reduce the burden of cellular senescence in bone marrow stromal stem cells.

Osteoporosis is a systemic age-related disease characterized by reduced bone mass and microstructure deterioration, leading to increased risk of bone fragility fractures. Osteoporosis is a worldwide major health care problem and there is a need for preventive approaches. Apigenin and Rutaecarpine are plant-derived antioxidants identified through functional screen of a natural product library (143 compounds) as enhancers of osteoblastic differentiation of human bone marrow stromal stem cells (hBMSCs). Global gene expression profiling and Western blot analysis revealed activation of several intra-cellular signaling pathways including focal adhesion kinase (FAK) and TGFβ. Pharmacological inhibition of FAK using PF-573228 (5 μM) and TGFβ using SB505124 (1μM), diminished Apigenin- and Rutaecarpine-induced osteoblast differentiation. In vitro treatment with Apigenin and Rutaecarpine, of primary hBMSCs obtained from elderly female patients enhanced osteoblast differentiation compared with primary hBMSCs obtained from young female donors. Ex-vivo treatment with Apigenin and Rutaecarpine of organotypic embryonic chick-femur culture significantly increased bone volume and cortical thickness compared to control as estimated by μCT-scanning. Our data revealed that Apigenin and Rutaecarpine enhance osteoblastic differentiation, bone formation, and reduce the age-related effects of hBMSCs. Therefore, Apigenin and Rutaecarpine cellular treatment represent a potential strategy for maintaining hBMSCs health during aging and osteoporosis.

Nuclear FAK in endothelium: An intrinsic inhibitor of NF-κB activation in atherosclerosis

Background and aimsHyperlipidemia leads to the accumulation of oxidized low-density lipoprotein (oxLDL) within the vessel wall where it causes chronic inflammation in endothelial cells (ECs) and drives atherosclerotic lesions. Although focal adhesion kinase (FAK) is critical in proinflammatory NF-κB activation in ECs, it is unknown if hyperlipidemia alters FAK-mediated NF-κB activity in vivo to affect atherosclerosis progression. MethodsWe investigated changes in EC FAK and NF-κB activation using Apoe−/− mice fed a western diet (WD). Both pharmacological FAK inhibition and EC-specific FAK inhibited mouse models were utilized. FAK and NF-κB localization and activity were also analyzed in human atherosclerotic samples. ResultsECs of hyperlipidemic mice clearly showed much higher levels of FAK activation in the cytoplasm, which was associated with increased NF-κB activation compared to normal diet (ND) group. On the contrary, FAK is mostly localized in the nucleus and inactive in ECs under healthy conditions with a low NF-κB activity. Both pharmacological and EC-specific genetic FAK inhibition in WD fed Apoe−/− mice exhibited a significant decrease in FAK activity and cytoplasmic localization, NF-κB activation, macrophage recruitment, and atherosclerotic lesions compared to the vehicle or FAK wild-type groups. Analyses of human atherosclerotic specimens revealed a positive correlation between increased active cytoplasmic FAK within ECs and NF-κB activation in the lesions. ConclusionsHyperlipidemic conditions activate NF-κB pathway by increasing EC FAK activity and cytoplasmic localization in mice and human atherosclerotic samples. As FAK inhibition can efficiently reduce vascular inflammation and atherosclerotic lesions in mice by reversing EC FAK localization and NF-κB activation, these findings support a potential use for FAK inhibitors in treating atherosclerosis.

FAK mediates LPS-induced inflammatory lung injury through interacting TAK1 and activating TAK1-NFκB pathway

Acute lung injury (ALI), characterized by inflammatory damage, is a major clinical challenge. Developing specific treatment options for ALI requires the identification of novel targetable signaling pathways. Recent studies reported that endotoxin lipopolysaccharide (LPS) induced a TLR4-dependent activation of focal adhesion kinase (FAK) in colorectal adenocarcinoma cells, suggesting that FAK may be involved in LPS-induced inflammatory responses. Here, we investigated the involvement and mechanism of FAK in mediating LPS-induced inflammation and ALI. We show that LPS phosphorylates FAK in macrophages. Either FAK inhibitor, site-directly mutation, or siRNA knockdown of FAK significantly suppresses LPS-induced inflammatory cytokine production in macrophages. FAK inhibition also blocked LPS-induced activation of MAPKs and NFκB. Mechanistically, we demonstrate that activated FAK directly interacts with transforming growth factor-β-activated kinase-1 (TAK1), an upstream kinase of MAPKs and NFκB, and then phosphorylates TAK1 at Ser412. In a mouse model of LPS-induced ALI, pharmacological inhibition of FAK suppressed FAK/TAK activation and inflammatory response in lung tissues. These activities resulted in the preservation of lung tissues in LPS-challenged mice and increased survival during LPS-induced septic shock. Collectively, our results illustrate a novel FAK-TAK1-NFκB signaling axis in LPS-induced inflammation and ALI, and support FAK as a potential target for the treatment of ALI.

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor–induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

Abstract P488: FAK Inhibition Reduces Hemangioma Proliferation By Blocking YAP Mediated HBEGF Expression

Hemangiomas are vascular tumors that develop from proliferative endothelial cells (EC). While primarily occurring on the skin of the head, neck, back and chest, hemangiomas can occur within any vascular beds including brain, liver and heart, which can lead to life-threatening complications. Non-surgical treatments are limited, and there are few phases I and II trials underway. Therefore, better understanding of what drives hemangioma proliferation and new therapeutic targets are needed. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that acts as a primary nexus of extracellular signals from integrins and growth factor receptors that sustain EC migration, proliferation, and survival. We observed that genetic and pharmacological inhibition of FAK significantly blocked vascular endothelial growth factor (VEGF)/ fibroblast growth factor (FGF)-induced Matrigel plug angiogenesis in vivo. To identify angiogenic factors regulated by FAK catalytic activity, we used an angiogenesis array in human umbilical vein ECs (HUVECs) and found that FAK inhibition reduced major regulators of angiogenesis including heparin-binding epidermal growth factor (HB-EGF). Further, using immunoblotting and RT-qPCR, we confirmed that FAK inhibition significantly reduced HB-EGF protein and mRNA expression in various mouse, human EC lines and human infantile hemangioma cell line, resulting in reduced EC proliferation. Importantly, HB-EGF mRNA expression was also reduced within in vivo Matrigel plugs of FAK inhibitor treated mice. Mechanistically, FAK binds to and phosphorylates YAP, which enhances HB-EGF expression. Loss of YAP tyrosine phosphorylation following FAK inhibition decreased YAP nuclear localization and protein stability. Since FAK inhibitors are undergoing clinical trials to treat cancer, these results suggest that FAK inhibitors could also prove beneficial in blocking EC proliferation associated with hemangioma.

FAKing a way to resistance

Cancer The number of cancer therapies available for clinical use is larger than ever before, but unfortunately, resistance arises for every type of therapy deployed. Many of these mechanisms are based on the acquisition of genetic alterations, but nongenetic resistance can also occur. Marin-Bejar et al. examined the patterns of treatment resistance in melanoma and identified recurrent resistance driven by the emergence of undifferentiated stem-like neural crest cells. These stem-like cells were characterized by activation of focal adhesion kinase (FAK) signaling, which in turn could be targeted by the pharmacological inhibition of FAK. Cancer Cell 10.1016/j.ccell.2021.05.015 (2021).

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Src Mediates Epigallocatechin-3-O-Gallate-Elicited Acid Sphingomyelinase Activation.

Epigallocatechin-3-O-gallate (EGCG) is one of the major bioactive compounds known to be present in green tea. We previously reported that EGCG shows selective toxicity through activation of the protein kinase B (Akt)/cyclic guanosine monophosphate (cGMP)/acid sphingomyelinase (ASM) axis via targeting its receptor 67-kDa laminin receptor (67LR), which is overexpressed in cancer. However, little is known about upstream mechanisms of EGCG-elicited ASM activation. In this study we show that the proto-oncogene tyrosine-protein kinase Src, also known as c-src, plays a crucial role in the anticancer effect of EGCG. We showed that EGCG elicits phosphorylation of Src at Tyr 416, a crucial phosphorylation site for its activity, and that the pharmacological inhibition of Src impedes the upstream events in EGCG-induced cell death signaling including upregulation of Akt activity, increase in cGMP levels, and activation of ASM. Moreover, focal adhesion kinase (FAK), which is involved in the phosphorylation of Src, is colocalized with 67LR. EGCG treatment enhanced interaction of FAK and 67LR. Consistent with these findings, pharmacological inhibition of FAK significantly neutralized EGCG-induced upregulation of Akt activity and activation of ASM. Taken together, FAK/Src play crucial roles in the upstream signaling of EGCG.

Abstract IA08: Regulation of YAP by tyrosine phosphorylation of core Hippo pathway components: Lessons learned from the GNAQ oncogene and FAK

Abstract Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. In prior studies we have focused on uveal melanoma, the most frequent eye malignancy in adults that is caused by constitutively active mutations in the GNAQ oncogenes, which encode Gαq and Gα11 heterotrimeric G protein α subunits. We found that GNAQ promotes the activation of YAP1 by a mechanism involving the activation of RhoA by the direct association of Gαq to TRIO, a Rho-GEF, and the consequent stimulation of acting polymerization and the release of YAP from its complex with AMOT. We also showed that YAP1 is essential for uveal melanoma cell growth, thus representing a potential therapeutic target for this malignancy. However, no effective and safe YAP1 inhibitors are currently available. Using a novel integrated bioinformatics pipeline, we recently found that PTK2, encoding Focal Adhesion Kinase (FAK), represents a candidate synthetic lethal gene with GNAQ activation (Feng et al., Cancer Cell 2019). We found that Gαq activates FAK through the TRIO-RhoA noncanonical Gαq-signaling, and that genetic ablation or pharmacologic inhibition of FAK inhibits uveal melanoma growth. Surprisingly, analysis of the FAK-regulated transcriptome indicated that GNAQ stimulates YAP1 through FAK. Dissection of the underlying mechanism revealed that FAK regulates YAP1 by tyrosine phosphorylation of MOB1, thereby disrupting MOB1/LATS association and core Hippo pathway signaling. By coupling the power of an unbiased computational pipeline to the unique genetic landscape of uveal melanoma, we uncovered a molecular framework regulating YAP1 and identified FAK as a druggable signaling hub downstream from GNAQ in uveal melanoma. Our findings established FAK as a potential therapeutic target for uveal melanoma and other diseases that may involve FAK activation and unrestrained YAP1 function. Further studies on how multiple growth promoting and oncogenic tyrosine kinase signaling networks may converge to phosphorylate core Hippo pathway components to activate YAP1 in cancer will be also discussed. Citation Format: J. Silvio Gutkind. Regulation of YAP by tyrosine phosphorylation of core Hippo pathway components: Lessons learned from the GNAQ oncogene and FAK [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA08.

Endothelial focal adhesion kinase contributes to intestinal microvascular leakage during ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury triggers a series of inflammatory responses in the microcirculation characterized by vascular leakage and leukocyte activation leading to organ dysfunction. In this study, we examine whether focal adhesion kinase (FAK) contributes to endothelial barrier dysfunction in response to I/R in splanchnic tissues. Using an in vivo model of intestinal I/R injury, we showed that the increase of microvascular leakage induced by I/R injury was greatly attenuated in endothelial specific, tamoxifen inducible, FAK knockout animals as well as wild type animals given pharmacological inhibition of FAK with PF‐573228. Using murine intestinal endothelial cells, we performed electric cell‐substrate impedance sensing (ECIS) assays with or without FAK inhibitor in the presence of H2O2. Results showed that FAK inhibition significantly attenuated endothelial barrier dysfunction induced by H2O2. Western blot analysis showed that H2O2 promoted FAK phosphorylation in a time course similar to that of endothelial barrier dysfunction. Confocal imaging analysis demonstrated that H2O2 increases the disruption of junctional proteins. The effect was blocked by FAK inhibitor. Taken together, the results suggested that FAK plays an important role in mediating intestinal vascular leakage during I/R injury.Support or Funding InformationNIH HL120954, VA BX000799

Resveratrol inhibits adipocyte differentiation and cellular senescence of human bone marrow stromal stem cells.

Bone marrow adipose tissue (BMAT) is a unique adipose depot originating from bone marrow stromal stem cells (BMSCs) and regulates bone homeostasis and energy metabolism. An increased BMAT volume is observed in several conditions e.g. obesity, type 2 diabetes, osteoporosis and is known to be associated with bone fragility and increased risk for fracture. Therapeutic approaches to decrease the accumulation of BMAT are clinically relevant. In a screening experiment of natural compounds, we identified Resveratrol (RSV), a plant-derived antioxidant mediating biological effects via sirtuin- related mechanisms, to exert significant effects of BMAT formation. Thus, we examined in details the effects RSV on adipocytic and osteoblastic differentiation of tolermerized human BMSCs (hBMSC-TERT). RSV (1.0μM) enhanced osteoblastic differentiation and inhibited adipocytic differentiation of hBMSC-TERT when compared with control and Sirtinol (Sirtuin inhibitor). Global gene expression profiling and western blot analysis revealed activation of a number of signaling pathways including focal adhesion kinase (FAK). Pharmacological inhibition of FAK using (PF-573228) and AKT inhibitor (LY-294002) (5μM), diminished RSV-induced osteoblast differentiation. In addition, RSV reduced the levels of senescence-associated secretory phenotype (SASP), gene markers associated with senescence (P53, P16, and P21), intracellular ROS levels and increased gene expression of enzymes protecting cells from oxidative damage (HMOX1 and SOD3). In vitro treatment of primary hBMSCs from aged patients characterized with high adipocytic and low osteoblastic differentiation ability with RSV, significantly enhanced osteoblast and decreased adipocyte formation when compared to hBMSCs from young donors. RSV targets hBMSCs and inhibits adipogenic differentiation and senescence-associated phenotype and thus a potential agent for treating conditions of increased BMAT formation.

Sensitization of HT29 colorectal cancer cells to vemurafenib in three-dimensional collagen cultures.

The extracellular matrix to which cancer cells adhere affects cellular sensitivity to anticancer drugs. We sought to examine the changes in sensitivity of colorectal cancer cells carrying the BRAF V600E mutation to vemurafenib cultured in three-dimensional (3D) collagen-I gels, while also identifying the signaling pathways involved in these changes. HT29 colorectal cancer cells were cultured in conventional tissue culture (TC) plastic plates or in collagen-I gels. The HT29 cells demonstrated approximately 10-fold higher sensitivity to vemurafenib in 3D-collagen-I gels compared with those cultured on conventional TC plastic plates. Furthermore, in cells cultured on TC plastic, vemurafenib was found to augment tyrosine phosphorylation of focal adhesion kinase (FAK), while 3D-cultured cells expressed lower levels of FAK and vemurafenib did not affect its tyrosine phosphorylation, suggesting that FAK contributes to vemurafenib resistance. However, pharmacological inhibition of FAK did not sensitize the cells to vemurafenib. Also, the level of tyrosine-phosphorylated epidermal growth factor receptor (EGFR)/ERBB2 family proteins was found to be lower in cells cultured in 3D-collagen gel compared with those in cells cultured on TC plastic. Afatinib, an inhibitor of the EGFR/ERBB family of kinases, sensitized the cells to higher concentrations of vemurafenib, implying their participation invemurafenib resistance. Adhesion to collagen-I gel but not to the collagen-I-coated plastic surface sensitized the cells, suggesting that the rigidity of the media rather than adherence to collagen-I may be important for cellular sensitivity to vemurafenib.

The effects of focal adhesion kinase and platelet-derived growth factor receptor beta inhibition in a patient-derived xenograft model of primary and metastatic Wilms tumor.

Aggressive therapies for patients with metastatic Wilms tumor (WT) with subsequent severe late effects warrant the search for novel therapies. The role of focal adhesion kinase (FAK), a non-receptor tyrosine kinase important in pediatric solid tumor development and progression, has not been examined in metastatic WT. Using a novel patient-derived xenograft (PDX) of a primary and matched, isogenic, metastatic WT, the hypothesis of the current study was that FAK would contribute to metastatic WT and small molecule inhibition would decrease tumor growth. Immunohistochemical staining, immunoblotting, cell viability and proliferation assays, cell cycle analysis, and cellular motility and attachment-independent growth assays were performed. FAK was present and phosphorylated in both WT PDXs and in the human samples from which they were derived. FAK inhibition decreased cellular survival, proliferation, and cell cycle progression in both PDXs but only significantly decreased migration, invasion, and attachment-independent growth in the primary WT PDX. Kinomic profiling revealed that platelet-derived growth factor receptor beta (PDGFRβ) may be affected by FAK inhibition in WT. Pharmacologic inhibition of FAK and PDGFRβ was synergistic in primary WT PDX cells. These findings broaden the knowledge of metastatic WT and support further investigations on the potential use of FAK and PDGFRβ inhibitors.

A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK

Activating mutations in GNAQ/GNA11, encoding Gαq G proteins, are initiating oncogenic events in uveal melanoma (UM). However, there are no effective therapies for UM. Using an integrated bioinformatics pipeline, we found that PTK2, encoding focal adhesion kinase (FAK), represents a candidate synthetic lethal gene with GNAQ activation. We show that Gαq activates FAK through TRIO-RhoA non-canonical Gαq-signaling, and genetic ablation or pharmacological inhibition of FAK inhibits UM growth. Analysis of the FAK-regulated transcriptome demonstrated that GNAQ stimulates YAP through FAK. Dissection of the underlying mechanism revealed that FAK regulates YAP by tyrosine phosphorylation of MOB1, inhibiting core Hippo signaling. Our findings establish FAK as a potential therapeutic target for UM and other Gαq-driven pathophysiologies that involve unrestrained YAP function.

Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy.

Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.

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.

Targeting FAK scaffold functions inhibits human renal cell carcinoma growth.

Human conventional renal cell carcinoma (CCC) remains resistant to current therapies. Focal Adhesion Kinase (FAK) is upregulated in many epithelial tumors and clearly implicated in nearly all facets of cancer. However, only few reports have assessed whether FAK may be associated with renal tumorigenesis. In this study, we investigated the potential role of FAK in the growth of human CCC using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as normal/tumoral renal tissue pairs. FAK was found constitutively expressed in human CCC both in culture cells and freshly harvested tumors obtained from patients. We showed that CCC cell growth was dramatically reduced in FAK-depleted cells or after FAK inhibition with various inhibitors and this effect was obtained through inhibition of cell proliferation and induction of cell apoptosis. Additionally, our results indicated that FAK knockdown decreased CCC cell migration and invasion. More importantly, depletion or pharmacological inhibition of FAK substantially inhibited tumor growth in vivo. Interestingly, investigations of the molecular mechanism revealed loss of FAK phosphorylation during renal tumorigenesis impacting multiple signaling pathways. Taken together, our findings reveal a previously uncharacterized role of FAK in CCC whereby FAK exerts oncogenic properties through a non canonical signaling pathway involving its scaffolding kinase-independent properties. Therefore, targeting the FAK scaffold may represent a promising approach for developing innovative and highly specific therapies in human CCC.

Junctional complex and focal adhesion rearrangement mediates pulmonary endothelial barrier enhancement by FTY720 S-phosphonate

RationaleModulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC). Methods/ResultsIn human lung EC, Tys significantly increased peripheral redistribution of adherens junction proteins VE-cadherin and β-catenin and tight junction protein ZO-1. Inhibition of VE-cadherin with blocking antibody significantly attenuated Tys-induced transendothelial resistance (TER) elevation, while ZO-1 siRNA partially inhibited this elevation. Tys significantly increased focal adhesion formation and phosphorylation of focal adhesion kinase (FAK). Pharmacologic inhibition of FAK significantly attenuated Tys-induced TER elevation. Tys significantly increased phosphorylation and peripheral redistribution of the actin-binding protein, cortactin, while cortactin siRNA partially attenuated Tys-induced TER elevation. Although Tys significantly increased phosphorylation of Akt and GSK3β, neither PI3 kinase nor GSK3β inhibition altered Tys-induced TER elevation. Tys significantly increased Rac1 activity, while inhibition of Rac1 activity significantly attenuated Tys-induced VE-cadherin redistribution and TER elevation. ConclusionJunctional complex, focal adhesion rearrangement and Rac1 activation play critical roles in Tys-mediated barrier protection in pulmonary EC. These results provide mechanistic insights into the effects of this potential ALI therapy.

Pharmacological Inhibition of Focal Adhesion Kinase Attenuates Cardiac Fibrosis in Mice Cardiac Fibroblast and Post-Myocardial-Infarction Models

Background: To investigate the role of focal adhesion kinase (FAK)-mediated signaling in hypoxia-induced cardiac fibroblasts (CFs) differentiation and cardiac fibrosis post-myocardial infarction (MI) on a mice model. Methods: CFs of neonatal C57BL/6 mice were treated under normoxic, hypoxic, or hypoxic+PP2 (known as a Src kinase family inhibitor) conditions. Gene expressions of FAK, alpha-smooth muscle actin (α-SMA) and collagen type I alpha 1 (Col1α1), or α-SMA and vimentin levels were performed by RT-PCR and immunofluorescence staining, respectively. Thirty mice were surgically treated into Sham (n=7) and MI (n=23) groups; and FAK inhibitor PF-562271 was given to six survivor MI mice (as PF group, from 15 survivors). Heart function and collagenous tissues were examined by echocardiography, as well as by Masson‘s trichrome and Sirius red staining, respectively. Type I collagen, FAK protein, mTOR, ERK1/2, AKT, P70S6K and phospho-FAK levels were also analyzed. Results: FAK inhibition with PP2 significantly decreased CFs differentiation and collagen synthesis under hypoxia treatment. In vivo, PF-562271 treatment resulted in fibrosis attenuation; however, deteriorated heart function of MI mice could not be significantly improved. PF-562271 may affect phospho-mTOR (p<0.05), phospho-ERK1/2 (p<0.01), phospho-AKT (p<0.001) and phospho-P70S6K (p<0.05) to exert its benefits. FAK can be activated either under hypoxia in CFs or MI in a mouse model to promote fibrosis. However, pharmacological inhibition of FAK can attenuate fibrosis response. Conclusion: This study provides novel evidence that FAK inhibition may become a promising pharmaceutical strategy to attenuate fibrosis post-MI.

Abstract 752: Genetic and pharmacological FAK inhibition disrupt a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

Abstract Ovarian cancer spreads via cell shedding and growth within malignant ascites. Effective targeted therapies have not been developed for ovarian cancer. Ascites contains an abundance of matrix proteins, and spheroids maintain integrin receptor expression. Through databases analyses we find that elevated osteopontin (OPN), β5 integrin, and focal adhesion kinase (FAK) mRNA levels are associated with decreased overall survival of serous ovarian cancer patients treated with platinum and taxol. In ovarian tumor tissue arrays, increased FAK activation (FAK Y397 phosphorylation) correlated with elevated tumor grade in parallel with increased in β5 integin and OPN levels. FAK is a cytoplasmic tyrosine kinase that remains active in spheroids, and treatment of seven ovarian carcinoma cell lines with sub-micromolar levels of FAK inhibitor (PND-1186) identified sensitive (HEY and OVCAR8), intermediate (OVCAR3, ID8-IP, and IGROV1-IP), and resistant (SKOV3-IP and OVCAR10) cells to blockage of growth under anchorage-independent conditions. Genetic or pharmacological FAK inhibition within ID8-IP or HEY cells selectively prevents anchorage-independent growth in culture and tumor growth in mice with corresponding reductions in β5 integrin and OPN expression. β5 knockdown reduced HEY growth in soft agar, tumor growth in mice, FAK Y397 phosphorylation, and OPN expression in spheroids. Although FAK inhibitor resistant ovarian carcinoma cells (SKOV3-IP and OVCAR10) were associated with anchorage-independent Akt S473 phosphorylation, membrane-targeted and activated Akt expression in sensitive cells (HEY and OVCAR8) resulted in only a partial rescue of FAK inhibitor-associated growth block. These results support the hypothesis that OPN, αvβ5 integrins, and FAK may function as a signaling axis promoting ovarian tumor progression. Although Akt signaling pathway activation is a common event in serous ovarian cancer, our results suggest that this may not impart complete resistance to FAK inhibitor treatment. Supported by NIH CA102310 Citation Format: Isabelle Tancioni, Sean Uryu, Florian Sulzmaier, Nina Shah, Christine Lawson, Nichol L.G. Miller, Christine Jean, Xiao Lei Chen, Kristy K. Ward, David D. Schlaepfer. Genetic and pharmacological FAK inhibition disrupt a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 752. doi:10.1158/1538-7445.AM2014-752