Focal Adhesion Kinase Autophosphorylation Research Articles

Mitoxantrone Targets the ATP-binding Site of FAK, Binds the FAK Kinase Domain and Decreases FAK, Pyk-2, c-Src, and IGF-1R In Vitro Kinase Activities

Focal Adhesion Kinase (FAK) is a non-receptor kinase that is overexpressed in many types of tumors and plays a key role in cell adhesion, spreading, motility, proliferation, invasion, angiogenesis, and survival. Recently, FAK has been proposed as a target for cancer therapy, and we performed computer modeling and screening of the National Cancer Institute (NCI) small molecule compounds database to target the ATP-binding site of FAK, K454. More than 140,000 small molecule compounds were docked into the crystal structure of the kinase domain of FAK in 100 different orientations using DOCK5.1 that identified small molecule compounds, targeting the K454 site, called A-compounds. To find the therapeutic efficacy of these compounds, we examined the effect of twenty small molecule compounds on cell viability by MTT assays in different cancer cell lines. One compound, A18 (1,4-bis(diethylamino)-5,8- dihydroxy anthraquinon) was a mitoxantrone derivative and significantly decreased viability in most of the cells comparable to the to the level of FAK kinase inhibitors TAE-226 (Novartis, Inc) and PF-573,228 (Pfizer). The A18 compound specifically blocked autophosphorylation of FAK like TAE-226 and PF-228. ForteBio Octet Binding assay demonstrated that mitoxantrone (1,4-dihydroxy- 5,8-bis[2-(2-hydroxyethylamino) ethylamino] anthracene-9,10-dione directly binds the FAK-kinase domain. In addition, mitoxantrone significantly decreased the viability of breast cancer cells in a dose-dependent manner and inhibited the kinase activity of FAK and Y56/577 FAK phosphorylation at 10-20 μM. Mitoxantrone did not affect phosphorylation of EGFR, but decreased Pyk-2, c-Src, and IGF-1R kinase activities. The data demonstrate that mitoxantrone decreases cancer viability, binds FAK-Kinase domain, inhibits its kinase activity, and also inhibits in vitro kinase activities of Pyk-2 and IGF-1R. Thus, this novel function of the mitoxantrone drug can be critical for future development of anti-cancer agents and FAK-targeted therapy research.

Abstract C44: Activation of focal adhesion kinase by direct interaction with β4 integrin in an EGFR-Src-dependent pathway in tumor progression

Abstract Although β4 integrin and FAK (focal adhesion kinase) are individually known to be associated with tumor progression, only recently have they been suggested to function in a signaling axis. Here, we demonstrate in vivo and in vitro physical and functional interactions between β4 integrin and FAK. An 11-amino acid motif of FAK was identified as essential for interaction with the cytodomain of β4 integrin in malignant cells but not in less malignant cells, implicating the role of this interaction in malignancies. Furthermore, this interaction occurs in an adhesion-dependent manner and results in autophosphorylation of FAK's Tyr-397. EGF signaling and Src activity were indispensable for this interaction. Finally, disruption of the β4 integrin/FAK complexes reduced cell proliferation, migration and invasion as well as tumorigenesis, in concurrence with the decreases in phospho-Akt and phospho-p38. Together, our data elucidate an underlying mechanism for the regulation of tumor progression by the novel β4 integrin/FAK complexes through a physical interaction that is EGF/Src dependent. The significance of this finding is highlighted by often co-overexpression of both integrin β4 and FAK in patients' cancerous tissues with colon cancer revealed by immunohistochemistry analyses. Based on the structural prediction for the complex of integrin β4 and FAK, it may lead us to gain more insight on the anticancer therapies of aggressive cancers. Citation Format: Yu-Ling Tai, Tang-Long Shen. Activation of focal adhesion kinase by direct interaction with β4 integrin in an EGFR-Src-dependent pathway in tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C44.

Pharmacologic Blockade of FAK Autophosphorylation Decreases Human Glioblastoma Tumor Growth and Synergizes with Temozolomide

Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15 to 18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report, we tested the FAK autophosphorylation inhibitor, Y15, in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose- and time-dependent manner, caused apoptosis, and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small-molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide.

AMP-activated protein kinase inhibits vascular smooth muscle cell proliferation and migration and vascular remodeling following injury

Vascular smooth muscle cell (VSMC) activation promotes a synthetic phenotype that underlies many vessel growth disorders. In this regard it has been suggested that the metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) has significant antigrowth and antimetastatic properties and may serve as a viable therapeutic target. In the current study we hypothesized that AMPK reduces neointima formation following balloon injury and that this occurs through reduction in VSMC proliferation and migration. Data reveal that local or systemic dosing with the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) significantly increased AMPK activity in vivo and inhibited neointima formation in rat carotid arteries 2 wk after injury. In primary VSMCs, AICAR inhibited migration and induced cytostatic growth arrest through increased protein phosphatase 2A-mediated inhibition of mitosis-promoting cyclin B. AICAR also significantly enhanced AMPK-specific T278 phosphorylation of the actin anticapping vasodilator-activated serum phosphoprotein, increased G- to F-actin ratios and stress fiber formation, and abrogated PDGF-stimulated S397 autophosphorylation of focal adhesion kinase, promigratory cytoplasmic accumulation of paxillin, and extracellular matrix proteolysis by matrix metalloproteinase-9. Together, these results provide compelling evidence that AMPK serves to inhibit vascular smooth muscle migration and proliferation through regulation of cytoskeletal/focal adhesion/ECM stability, increasing our knowledge of this important metabolic regulator and providing support for its continued investigation in the treatment of vascular growth disorders.

Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction

Disruption of epithelial barrier function was identified as one of the pathologic mechanisms in inflammatory bowel diseases (IBD). Epithelial barrier consists of various intercellular junctions, in which the tight junction (TJ) is an important component. However, the regulatory mechanism of tight junction is still not clear. Here we examined the role of focal adhesion kinase (FAK) in the epithelial barrier function on Caco-2 monolayers using a specific FAK inhibitor, PF-573, 228 (PF-228). We found that the decrease of transepithelial resistance and the increase of paracellular permeability were accompanied with the inhibition of autophosphorylation of FAK by PF-228 treatment. In addition, PF-228 inhibited the FAK phosphorylation at Y576/577 on activation loop by Src, suggesting Src-dependent regulation of FAK in Caco-2 monolayers. In an ethanol-induced barrier injury model, PF-228 treatment also inhibited the recovery of transepithelial resistance as well as these phosphorylations of FAK. In a sucrose gradient ultracentrifugation, FAK co-localized with claudin-1, an element of the TJ complex, and they co-migrate after ethanol-induced barrier injury. Immunofluorescence imaging analysis revealed that PF-228 inhibited the FAK redistribution to the cell border and reassembly of TJ proteins in the recovery after ethanol-induced barrier injury. Finally, knockdown of FAK by siRNA resulted in the decrease of transepithelial resistance. These findings reveal that activation of FAK is necessary for maintaining and repairing epithelial barrier in Caco-2 cell monolayer via regulating TJ redistribution.

Protein kinase Cα/β inhibitor Gö6976 promotes PC12 cell adhesion and spreading through membrane recruitment and activation of protein kinase Cδ

Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/β. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1μM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/β signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.

384 - The Role of Antiestrogens in Breast Canser Cells' Invasiveness

ABSTRACT Aim The current study investigates the effect of two selective antagonists of the estrogen receptor (ER), fulvestrant (FL) and tamoxifen (Tam), on the invasive ability of breast cancer cells. ER blockage using anti-estrogens is associated with a small induction of invasiveness. Moreover, matrix metalloproteinases (MMPs) contribute to this procedure by cleaving components of the extracellular matrix, forming a path for the migrating cells. In addition, the focal adhesion kinase (FAK) plays a central role in invasiveness. Methods We used two ER+ breast cancer cell lines, MCF7 and T47D. Cells were stimulated by estradiol (E2) and then treated with FL, Tam and the metabolites endoxifen (End) and 4OH-tamoxifen (4OHT). The invasiveness was evaluated using the matrigel assay and MMPs expression with gelatin zymography assay. Using immunofluoresence, we study the expression and localization of phospho FAK and its correlation with F-actin. Results We found that E2 exerts a protective role in invasiveness. On the contrary, the anti-estrogens, as well as their metabolites reversed the effect of E2, a finding that was more obvious with Tam. The effect of the agents on MMPs expression was more pronounced at 48 h, when Tam and 4OHT were found to reduce the levels of both MMP-2 and MMP-9 compared to FL. Following E2 exposure, the maximum autophosphorylation of FAK (Y397) was observed at 10 min. At the same time point we assessed the effect of our agents, regarding the spatial organization of actin fibers. The concurrent administration of E2 with FL, Tam or End was associated with rearrangement of cytoskeleton, a finding that was not observed in untreated cells, as well as after the use of 4OHT and E2 or E2 alone. Conclusions Our results indicate that the metabolite 4OHT is superior to the pro-drug Tam as well as the pure anti-estrogen FL with respect to invasiveness, MMPs induction and actin rearrangement. Regarding the comparison of Tam with FL, the effect on MMPs and rearrangement was similar. However, FL was found to be slightly superior to Tam concerning invasiveness. Disclosure All authors have declared no conflicts of interest.

Abstract LB-192: The FAK inhibitors VS-4718 and VS-5095 attenuate breast cancer stem cell function in vitro and tumor growth in vivo

Abstract As a key mediator of integrin signaling, focal adhesion kinase (FAK) regulates cellular responses to extracellular matrix interactions. Amplification and overexpression of FAK have been observed in aggressive human cancers including breast cancer. FAK has been implicated in multiple steps in carcinogenesis including tumor growth, metastasis and angiogenesis. We now demonstrate the importance of FAK in breast cancer stem cell function, and the reduction of cancer stem cell function by the selective FAK inhibitors VS-4718 and VS-5095. VS-4718 and VS-5095 are potent and selective FAK inhibitors which were optimized following high throughput screening. Both VS-4718 and VS-5095 block fibronectin-stimulated FAK autophosphorylation of Tyr397 with low nanomolar cellular potency and are highly selective for FAK among a panel of protein kinases. Consistent with their mechanism of action, VS-4718 and VS-5095 showed greater inhibitory potency on the growth of multiple cancer cell lines in 3D matrigel culture as compared to conventional 2D culture. To determine if FAK plays a role in the biology of breast cancer stem cells in addition to its reported function in normal mammary stem cell biology, the effects of these FAK inhibitors were characterized using two different in vitro assays. It was previously demonstrated that immortalized mammary epithelial cells (HMLEs) driven to undergo epithelial to mesenchymal transition (EMT) by knockdown of E-cadherin (HMLE-shECad) exhibit many of the characteristics of cancer stem cells and can be used to identify agents that selectively target cancer stem cells. VS-4718 exhibited 25-fold greater potency against proliferation of mesenchymal HMLE-shECad cells as compared to epithelial HMLE-shGFP control cells, suggesting preferential effects on breast cancer stem cells. Furthermore, pre-treatment of SUM159 triple negative breast cancer cells with VS-5095 in matrigel attenuated secondary tumorsphere formation, suggesting that FAK is important for the self-renewal function of breast cancer stem cells. The role of FAK in breast cancer stem cell renewal was further corroborated by the observation that FAK shRNA inhibited tumorsphere formation by SUM159 cells. The in vivo efficacy of the FAK inhibitor VS-5095 was evaluated in the MDA-MB-231 human triple negative breast cancer xenograft model. By oral administration, VS-5095 induced significant dose-dependent tumor growth inhibition. In summary, these results demonstrate the importance of FAK in the self-renewal of breast cancer stem cells, and support the clinical development of the selective FAK inhibitors VS-4718 and VS-5095 to target breast cancer stem cells for the treatment of triple negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-192. doi:1538-7445.AM2012-LB-192

AMP‐Activated Protein Kinase Reduces Vascular Smooth Muscle Growth by Inhibiting Actin Cytoskeleton Dynamics Necessary for Migration

Abluminal vascular smooth muscle cell (VSMC) migration is a functional underpinning of vasculoproliferative disorders. Upon activation, the dynamic nature of the actin cytoskeleton and rapid focal adhesion turnover provides the structural basis necessary for cell motility. Preliminary data show that activation of the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits migration in rat primary VSMCs. In the current study we tested the hypothesis that AMPK enhances focal adhesion stability and inhibits vasodilator‐stimulated phosphoprotein (VASP) anti‐capping function on microfilament elongation. Treatment of VSMCs with the AMPK activator AICAR (1 mM) significantly (~50%; p<0.05) increased phosphorylated VASP at Thr278, a reported AMPK‐specific site, thus inhibiting microfilament anti‐capping by VASP. This inhibition of VASP was independent of the traditional VASP‐activating kinases PKA and PKG. In addition, AICAR inhibited PDGF‐stimulated autophosphorylation of focal adhesion kinase (FAK) at Ser397, a necessary event for kinase activity and focal adhesion turnover. Inhibition of phospho‐FAK at Ser397 induced focal adhesion stability necessary for cytostasis. These data lend strong support for the notion that AMPK has capacity to promote focal adhesion stability and to inhibit cytoskeletal rearrangement necessary for VSMC migration.

A small molecule focal adhesion kinase (FAK) inhibitor, targeting Y397 site: 1-(2-hydroxyethyl) -3, 5, 7-triaza-1-azoniatricyclo [3.3.1.13,7]decane; bromide effectively inhibits FAK autophosphorylation activity and decreases cancer cell viability, clonogenicity and tumor growth in vivo

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in most solid types of tumors and plays an important role in the survival signaling. Recently, we have developed a novel computer modeling combined with a functional assay approach to target the main autophosphorylation site of FAK (Y397). Using these approaches, we identified 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide, called Y11, a small molecule inhibitor targeting Y397 site of FAK. Y11 significantly and specifically decreased FAK autophosphorylation, directly bound to the N-terminal domain of FAK. In addition, Y11 decreased Y397-FAK autophosphorylation, inhibited viability and clonogenicity of colon SW620 and breast BT474 cancer cells and increased detachment and apoptosis in vitro. Moreover, Y11 significantly decreased tumor growth in the colon cancer cell mouse xenograft model. Finally, tumors from the Y11-treated mice demonstrated decreased Y397-FAK autophosphorylation and activation of poly (ADP ribose) polymerase and caspase-3. Thus, targeting the major autophosphorylation site of FAK with Y11 inhibitor is critical for development of cancer therapeutics and carcinogenesis field.

Dentin Phosphoprotein (DPP) Activates Integrin-mediated Anchorage-dependent Signals in Undifferentiated Mesenchymal Cells

Dentin phosphoprotein (DPP), a major noncollagenous protein of the dentin matrix, is a highly acidic protein that binds Ca(2+) avidly and is thus linked to matrix mineralization. Here, we demonstrate that the RGD domain in DPP can bind to integrins on the cell surface of undifferentiated mesenchymal stem cells and pulp cells. This coupling generates intracellular signals that are channeled along cytoskeletal filaments and activate the non-receptor tyrosine kinase focal adhesion kinase, which plays a key role in signaling at sites of cellular adhesion. The putative focal adhesion kinase autophosphorylation site Tyr(397) is phosphorylated during focal adhesion assembly induced by DPP on the substrate. We further demonstrate that these intracellular signals propagate through the cytoplasm and activate anchorage-dependent ERK signaling. Activated ERK translocates to the nucleus and phosphorylates the transcription factor ELK-1, which in turn coordinates the expression of downstream target genes such as DMP1 and dentin sialoprotein (DSP). These studies suggest a novel paradigm demonstrating that extracellular DPP can induce intracellular signaling that can be propagated to the nucleus and thus alter gene activities.

Copper Deficiency Induced Emphysema Is Associated with Focal Adhesion Kinase Inactivation

BackgroundCopper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK).MethodologyTo examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA).Principal FindingsCopper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.ConclusionsThese data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.

Abstract B244: The small molecule focal adhesion kinase inhibitor Y15 decreases human glioblastoma cell viability, clonogenicity, and tumor growth.

Abstract Introduction: Glioblastomas are the third most frequent cause of cancer-associated deaths due to the cancer cell rapid growth and high invasion and novel therapeutic approaches are needed for this disease. Focal adhesion Kinase (FAK) is known to be highly expressed and activated in glioblastoma and plays significant role in tumorigenesis. Experimental procedures: In this report we tested FAK autophosphorylation inhibitor, Y15 (called also Inhibitor 14) or 1,2,4,5-Benzenetetraamine tetrahydrochloride) that specifically decreased Y397-FAK autophosphorylation in DBTRG and U87 glioblastoma cells. To test Y15 we used the following in vitro assays: viability, clonogenicity, detachment, apoptosis, invasion, immunostaining, Western blotting and microarray analysis of gene expression; and in vivo assay: xenograft tumor growth model. Results: Y15 significantly decreased viability and clonogenicity of DBTRG and U87 cells in a dose-dependent manner. Y15 inhibitor increased detachment, apoptosis and inhibited cell invasion in a dose-dependent manner. In addition, Y15 decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Moreover, we tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarray assay and among 34, 694 genes affected by Y15 there we 8087 and 6555 genes either up- or down-regulated in DBTRG and U87 cells, respectively (p<0.05). Moreover, Y15-treated DBTRG and U87 cells changed expression of 237 common genes more than 1.5 fold, p<0.05 that play role in survival signaling, cell growth and motility. In addition, combination of temozolomide with Y15 significantly decreased viability and clonogenicity in both cell lines. In addition, Y15 inhibitor delivered orally significantly decreased DBTRG xenograft tumor growth. The tumors from Y15-treated mice expressed decreased Y397-FAK autophosphorylation, activated PARP and caspase-3. Conclusion: Inhibition of FAK autophosphorylation with the oral small molecule inhibitor Y15 can be an effective therapy approach to block glioblastoma tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B244.

Abstract C191: Preclinical investigation of the antitumor efficacy in lung cancer of Y15, a novel focal adhesion kinase inhibitor.

Abstract Introduction: Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that associates with a number of cell adhesion components, such as integrins, paxillin and talin, as well as with various signaling proteins such as phosphatidylinositol 3-kinase (PI3K) and the Src family of protein kinases. It thus plays an important role in cell motility, invasion, growth and cell survival. Y15 (1,2,4,5-benzentetraamine tetra hydrochloride) is a small molecule that inhibits FAK autophosphorylation by selectively targeting the Y397 phosphorylation site of FAK. It has shown activity against breast, colon and pancreatic cancers. We herein present data in lung cancer. Methods: Various lung cancer cell lines as well as minimally passaged patient-derived tumor (containing PIK3CA mutation E545K) propagated in vitro were treated with escalating doses of Y15 or DMSO for 72 hours to evaluate single-agent activity. Cell lines were also treated with cytotoxic agents (cisplatin, gemcitabine, pemetrexed, paclitaxel) in combination with escalating doses of Y15. Cellular viability was assessed by trypan blue exclusion assay and colony formation assay. For in vivo experiments, selected cell lines were implanted into nude mice and vehicle versus Y15 treatment, via either intraperitoneal or oral gavage administration, once daily was started when tumors reached a mean volume of 50–60 mm3. Western blot will be performed to investigate alterations in the activity of various signaling proteins. Results: Y15 exhibited time and dose-dependent cell death in 6 out of 9 lung cancer cell lines tested. Activity was demonstrated beginning at 4 uM dose, with almost 100% cell kill demonstrated at 10 uM dose. There was also corresponding dose-dependent loss of clonogenic ability starting at 1 uM dose in sensitive cell lines. In addition, Y15 enhanced paclitaxel-induced cell death. Mice treated with Y15 at 100 mg/kg oral gavage administration showed reduced tumor growth compared to control mice. Conclusion: Y15 demonstrates promising antitumor efficacy against various non-small cell lung cancer cell lines. Further development of this agent in lung cancer is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C191.

Dynamic actin remodeling during epithelial–mesenchymal transition depends on increased moesin expression

Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.

Focal adhesion kinase inhibitors are potent anti-angiogenic agents

Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase and scaffold protein localized to focal adhesions, is uniquely positioned at the convergence point of integrin and receptor tyrosine kinase signal transduction pathways. FAK is overexpressed in many tumor cells, hence various inhibitors targeting its activity have been tested for anti-tumor activity. However, the direct effects of these pharmacologic agents on the endothelial cells of the vasculature have not been examined. Using primary human umbilical vein endothelial cells (HUVEC), we characterized the effects of two FAK inhibitors, PF-573,228 and FAK Inhibitor 14 on essential processes for angiogenesis, such as migration, proliferation, viability and endothelial cell tube formation. We observed that treatment with either FAK Inhibitor 14 or PF-573,228 resulted in reduced HUVEC viability, migration and tube formation in response to vascular endothelial growth factor (VEGF). Furthermore, we found that PF-573,228 had the added ability to induce apoptosis of endothelial cells within 36 h post-drug administration even in the continued presence of VEGF stimulation. FAK inhibitors also resulted in modification of the actin cytoskeleton within HUVEC, with observed increased stress fiber formation in the presence of drug. Given that endothelial cells were sensitive to FAK inhibitors at concentrations well below those reported to inhibit tumor cell migration, we confirmed their ability to inhibit endothelial-derived FAK autophosphorylation and FAK-mediated phosphorylation of recombinant paxillin at these doses. Taken together, our data indicate that small molecule inhibitors of FAK are potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting tumor angiogenesis.

Thrombin-dependent modulation of β1-integrin-mediated signaling up-regulates prolidase and HIF-1α through p-FAK in colorectal cancer cells

Products of prolidase [E.C. 3.4.13.9] activity, proline or hydroxyproline, contribute to up-regulation of hypoxia-inducible factor-1α (HIF-1α). Prolidase activity is regulated by β(1)-integrin signaling. We studied the effects of echistatin (a well-known disintegrin) and thrombin (a serine protease capable of activation of integrin α(2)β(1) receptor) on prolidase activity and expressions of prolidase, α(2)β(1)-integrin receptor, focal adhesion kinase (FAK), MAP-kinases (ERK(1) and ERK(2)), and nuclear HIF-1α in human colon adenocarcinoma (DLD-1) cells. It has been found that treatment of the cells with thrombin contributes to decrease in the expression of prolidase and simultaneously increase in its phosphorylation, resulting in maintenance of the enzyme activity. The phenomenon was accompanied by thrombin-dependent recovery of depressed autophosphorylation of FAK (pY(397)) under the effect of FAK inhibitor (1,2,4,5-benzenetetramine tetrahydrochloride). Although integrin α(2)β(1) receptor expression was not affected by thrombin, the signaling induced by thrombin up-regulated nuclear HIF-1α expression. It was accompanied by increase in the expression of MAP kinases, ERK1 and ERK2. It suggests that integrin-dependent signaling through p-FAK is up-regulated in DLD-1 cells and it may represent potential target for anti-cancer therapy.

EGF‐induced ERK‐activation downstream of FAK requires rac1‐NADPH oxidase

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.

Effect of Focal Adhesion Kinase on the Regulation of Realignment and Tenogenic Differentiation of Human Mesenchymal Stem Cells by Mechanical Stretch

Focal adhesion kinase (FAK) is a focal adhesion-associated protein kinase involved in cell adhesion and spreading. It is recruited as a participant in focal adhesion dynamics between cells and has a role in cell motility, differentiation, and survival. The role of FAK in the differentiation of human mesenchymal stem cells (hMSCs), however, is not well understood, particularly in terms of tenogenic differentiation. In this study, we reported that FAK regulates the mechanical stretch-induced realignment of hMSCs. We showed that FAK can be activated by mechanical stretch and, with a 10 μM PF 573228 (a novel small molecule inhibitor of FAK) treatment, FAK autophosphorylation at Tyr397 is significantly decreased. Moreover, our findings demonstrated that this decrease in FAK autophosphorylation at Tyr397 leads to the attenuation of upregulation of mechanical stretch-induced mRNA expression of tendon-related genes, including type I collagen, type III collagen, tenascin-C, and scleraxis. These results indicate that the FAK signaling molecule plays an important role in regulating cell realignment and tenogenic differentiation of hMSCs when induced by mechanical stretch. Collectively, our findings provide novel insight into the role of FAK in the realignment and mechanotransduction of hMSCs during the process of tenogenic differentiation induced by mechanical stretch.

In vitro osteoblastic differentiation of human mesenchymal stem cells and human dental pulp stem cells on poly‐L‐lysine‐treated titanium‐6‐aluminium‐4‐vanadium

Three-dimensional (3D) titanium-6-aluminium-4-vanadium (Ti6Al4V) is a widely used biomaterial for orthopedic prosthesis and dental implants; thanks to its very high-mechanical strength and resistance to corrosion. Human mesenchymal stem cells (hMSCs) and dental pulp stem cells (hDPSCs) are responsible for bone regeneration following colonization of prosthesis or dental implants. Both hMSCs and hDPSCs have lower ability to colonize this biomaterial in comparison with tissue culture-treated plastic. Both hMSCs and hDPSCs show lack of focal adhesion kinase (FAK) activation when grown on Ti6Al4V. This signal is restored in the presence of poly-L-lysine (poly-L-lys). Poly-L-lys has been used as part of organoapatite or together with zinc and calcium ions. Our results suggest that poly-L-lys alone induces FAK activation through β1-INTEGRIN, because the presence of β1-INTEGRIN blocking antibody avoided FAK autophosphorylation. Presence of poly-L-lys also increases expression of osteoblastic differentiation marker genes in hMSCs and hDPSCs grown on Ti6Al4V.