Knockdown Of Focal Adhesion Kinase Research Articles

Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer

IntroductionOsimertinib is a standard treatment for patients with EGFR-mutant NSCLC. Although some osimertinib resistance mechanisms have been identified, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non-genetic mechanisms underlying osimertinib resistance. MethodsWe established two osimertinib-resistant cell lines from EGFR mutation-positive PC-9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3. ResultsPhosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these up-regulated phosphoproteins. The small-interfering RNA–mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific small-interfering RNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with EGFR-mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib. ConclusionsPhosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular-targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC.

Combination of FAK inhibitor and cytokine-induced killer cell therapy: An alternative therapeutic strategy for patients with triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by the loss of expression of several biomarkers, which limits treatment strategies for the disease. In recent years, immunotherapy has shown promising results in the treatment of various tumors. Emerging evidence demonstrated that TNBC is an immune-activated cancer, suggesting that immunotherapy could be a feasible treatment option for TNBC. Cytokine-induced killer (CIK) cell therapy is considered as a potential treatment for cancer treatment. However, it is still not approved as a standard treatment in the clinical setting. Our previous study demonstrated that focal adhesion kinase (FAK) plays important role in regulating the sensitivity of TNBC cells to CIK cells. In this study, we further verify the role of FAK in regulating the immune response in vivo. Our in vitro study indicated that knockdown of FAK in TNBC cells or treat with the FAK inhibitor followed by co-culture with CIK cells induced more cell death than CIK cells treatment only. RNA-seq analysis indicated that suppression of FAK could affect several immune-related gene expressions in TNBC cells that affects the immune response in the tumor microenvironment of TNBC cells. The combination of FAK inhibitor and CIK cells significantly suppressed tumor growth than the treatment of FAK inhibitor or CIK cells alone in vivo. Our findings provide new insights into the cytotoxic effect of CIK cell therapy in TNBC treatment and indicate that the combination of CIK cell therapy with FAK inhibitors may be an alternative therapeutic strategy for patients with TNBC.

High Mobility Group Box 1 as an Autocrine Chemoattractant for Oligodendrocyte Lineage Cells in White Matter Stroke.

The migration of oligodendrocyte precursor cells (OPC) is a key process of remyelination, which is essential for the treatment of white matter stroke. This study aimed to investigate the role of HMGB1 (high mobility group box 1), a damage-associated molecular pattern released from dying oligodendrocytes, as an autocrine chemoattractant that promotes OPC migration. The migratory capacity of primary cultured OPCs was measured using the Boyden chamber assay. The downstream pathway of HMGB1-mediated OPC migration was specified by siRNA-induced knockdown or pharmacological blockade of TLR2 (toll-like receptor 2), RAGE (receptor for advanced glycation end product), Src, ERK1/2 (extracellular signal-regulated kinase1/2), and FAK (focal adhesion kinase). Conditioned media were collected from oxygen-glucose deprivation-treated oligodendrocytes, and the impact on OPC migration was assessed. Lesion size and number of intralesional Olig2(+) cells were analyzed in an in vivo model of white matter stroke with N5-(1-iminoethyl)-L-ornithine (L-NIO). HMGB1 treatment promoted OPC migration. HMGB1 antagonism reversed such effects to untreated levels. Among the candidates for the downstream signal of HMGB1-mediated migration, the knockdown of TLR2 rather than that of RAGE attenuated the migration-promoting effect of HMGB1. Further specification of the HMGB1-TLR2 axis revealed that the phosphorylation of ERK1/2 and its downstream molecule FAK, rather than of Src, was decreased in TLR2-knockdown OPCs, and pharmacological inhibition of ERK1/2 and FAK led to decreased OPC migration. Oxygen-glucose deprivation-conditioned media promoted OPC migration, suggesting the autocrine chemoattractant function of HMGB1. In vivo, TLR2(-/-)-mice showed lesser intralesional Olig2(+) cells compared to wild-type controls in response to L-NIO induced ischemic injury regardless of HMGB1 administration. HMGB1, through the TLR2-ERK1/2-FAK axis, functions as an autocrine chemoattractant to promote OPC migration, which is an initial and indispensable step in remyelination. Thus, a novel treatment strategy for white matter stroke based on the HMGB1-TLR2 axis in the oligodendrocyte lineage could be feasible.

High Mobility Group Box 1 as an Autocrine Chemoattractant for Oligodendrocyte Lineage Cells in White Matter Stroke.

The migration of oligodendrocyte precursor cells (OPC) is a key process of remyelination, which is essential for the treatment of white matter stroke. This study aimed to investigate the role of HMGB1 (high mobility group box 1), a damage-associated molecular pattern released from dying oligodendrocytes, as an autocrine chemoattractant that promotes OPC migration. The migratory capacity of primary cultured OPCs was measured using the Boyden chamber assay. The downstream pathway of HMGB1-mediated OPC migration was specified by siRNA-induced knockdown or pharmacological blockade of TLR2 (toll-like receptor 2), RAGE (receptor for advanced glycation end product), Src, ERK1/2 (extracellular signal-regulated kinase1/2), and FAK (focal adhesion kinase). Conditioned media were collected from oxygen-glucose deprivation-treated oligodendrocytes, and the impact on OPC migration was assessed. Lesion size and number of intralesional Olig2(+) cells were analyzed in an in vivo model of white matter stroke with N5-(1-iminoethyl)-L-ornithine (L-NIO). HMGB1 treatment promoted OPC migration. HMGB1 antagonism reversed such effects to untreated levels. Among the candidates for the downstream signal of HMGB1-mediated migration, the knockdown of TLR2 rather than that of RAGE attenuated the migration-promoting effect of HMGB1. Further specification of the HMGB1-TLR2 axis revealed that the phosphorylation of ERK1/2 and its downstream molecule FAK, rather than of Src, was decreased in TLR2-knockdown OPCs, and pharmacological inhibition of ERK1/2 and FAK led to decreased OPC migration. Oxygen-glucose deprivation-conditioned media promoted OPC migration, suggesting the autocrine chemoattractant function of HMGB1. In vivo, TLR2(-/-)-mice showed lesser intralesional Olig2(+) cells compared to wild-type controls in response to L-NIO induced ischemic injury regardless of HMGB1 administration. HMGB1, through the TLR2-ERK1/2-FAK axis, functions as an autocrine chemoattractant to promote OPC migration, which is an initial and indispensable step in remyelination. Thus, a novel treatment strategy for white matter stroke based on the HMGB1-TLR2 axis in the oligodendrocyte lineage could be feasible.

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.

Abstract 2413: αEGFR-E-P125A antibody-endostatin fusion protein reduces vasculogenic mimicry and metastasis by inhibiting FAK, integrin, and EGFR signaling

Abstract Triple-Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used either alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). Our laboratory previously developed a novel targeted therapy, αEGFR-E-P125A, an antibody-endostatin fusion protein, by linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and decreases metastatic tumor growth (lung metastasis) in both the MDA-MB-231-4175 and MDA-MB 468 TNBC xenograft models. Preliminary studies of the mechanism of αEGFR-E-P125A action indicate that αEGFR-E-P125A downregulates VM through combined inhibition of EGFR and integrin signaling. Phospho-array analysis of αEGFR-E-P125A treated MDA-MB-231-4175 cells demonstrated downregulation of phosphorylation of EGFR at Y1069, a site of aberrant EGFR signaling which promotes TNBC motility. The phospho-array also demonstrated inhibition of focal adhesion kinase (FAK) phosphorylation at Y397. FAK signaling occurs downstream of β1 and β3 integrins, and the Y397 site is implicated in the regulation of endothelial and TNBC motility, VM, and metastatic behavior. We specifically inhibited FAK at the Y397 site using a small molecule inhibitor, PF-573228, and demonstrated that FAK inhibition at Y397 alone inhibited the TNBC VM in vitro. shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM formation in vitro. Immunoprecipitation (IP) of EGFR detected the presence of β1 integrin, and IP of β1 integrin detected EGFR in both MDA-MB-231-4175 and MDA-MB-468 TNBC cells treated with αEGFR-E-P125A. αEGFR-E-P125A treatment increased levels of bound β1 integrin following IP of EGFR relative to untreated or Cetuximab treated TNBC cells. These results indicate that αEGFR-E-P125A is binding to both EGFR and β1 integrin simultaneously, suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and β1 integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita P. Sankar, Hyun Mi Cho, Hava Gil-Henn, Sundaram Ramakrishnan, Rathin Das, Christian Elledge, Yu Zhang, Seung-Uon Shin, Joseph D. Rosenblatt. αEGFR-E-P125A antibody-endostatin fusion protein reduces vasculogenic mimicry and metastasis by inhibiting FAK, integrin, and EGFR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2413.

CPNE8 Promotes Gastric Cancer Metastasis by Modulating Focal Adhesion Pathway and Tumor Microenvironment.

Little is known about the oncogenic role or biological function of copine Ⅷ (CPNE8) in gastric cancer (GC). Based on TCGA database, we screened for CPNE8 and analyzed the expression of CPNE8 in GC. The correlations between CPNE8 and clinical features were analyzed using TCGA and GEO databases. The prognostic value of CPNE8 was assessed using Cox analysis and Kaplan-Meier curves. The results showed that increased expression of CPNE8 was positively correlated with metastasis and can be considered an independent prognostic risk factor for poor survival. We found that CPNE8 can promote cell proliferation, migration, and invasiveness in GC using in vitro and in vivo experiments. Our study demonstrated that CPNE8 promotes tumor progression via regulation of focal adhesion, and these effects can be rescued by focal adhesion kinase (FAK) inhibitor GSK2256098 or knockdown of FAK. In addition, CPNE8 was correlated significantly with the infiltration of cancer-associated fibroblasts and immune cells, as demonstrated by various algorithms, and high CPNE8 expression predicted poor efficacy of immune checkpoint therapy. Our findings suggest that CPNE8 modulates focal adhesion and tumor microenvironment to promote GC progression and invasiveness and could serve as a novel prognostic biomarker in GC.

Abstract LT015: Overcoming chemotherapy resistance in triple negative breast cancer via targeting lysyl oxidase (LOX)

Abstract Chemoresistance is a major obstacle in the treatment of triple negative breast cancer (TNBC), the most aggressive breast cancer subtype. To overcome chemoresistance, we have selected TNBC tumors for chemotherapy resistance in vivo, characterized their transcriptomes by RNA-sequencing and identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key inducer of chemoresistance. LOX overexpression has two distinct effects in hypoxic tumors treated with chemotherapy. As an ECM remodeler, LOX enhances collagen cross-linking and fibronectin assembly, thereby decreasing drug penetration. In addition, LOX exerts a surprising novel effect on transcription, increasing the expression of Integrin Subunit Alpha 5 (ITGA5), the major receptor for fibronectin (FN1), leading to activation of Focal Adhesion Kinase (FAK)/Src signaling and chemoresistance. Inhibition of LOX or ITGA5 with shRNA-mediated knockdown or inhibition of FAK or Src kinases with small molecule inhibitors in combination with doxorubicin greatly enhanced tumor growth inhibition in vivo relative to individual treatments. The role of LOX in chemoresistance has further been demonstrated using chemoresistant TNBC patient-derived xenografts (PDXs) and organoids, treated with doxorubicin alone or in combination with the LOX family inhibitor, BAPN. Notably, higher LOX, ITGA5, or FN1 levels are associated with shorter survival in chemotherapy treated TNBC patients. Currently available LOX inhibitors suffer from lack of specificity and high toxicity. To identify a more potent and selective LOX inhibitor, we performed a high-throughput screen (HTS) of a diversified small-molecule library. HTS resulted in identification of several hits that inhibit LOX enzymatic activity without any cytotoxicity. A lead compound was identified after the screening of hits based on their inhibitory effects on LOX enzymatic activity and the degree of chemosensitization in collagen-embedded cells. We are currently performing structure-activity relationship (SAR) analysis to optimize the lead compound for more potent activity and better drug-like properties. In addition, we are analyzing the mechanisms through which LOX enhances ITGA5 transcription and how the enzymatic activity of LOX contributes to transcriptional regulation. Our study provides a pre-clinical rationale for the development and testing of LOX inhibitors to overcome chemotherapy resistance in TNBC patients. Citation Format: Ozge Saatci, Ozge Akbulut, Abdol-Hossein Rezaeain, Carolyn Banister, Vitali Sikirzhytski, Sercan Aksoy, Aytekin Akyol, Aysegul Uner, Phillip Buckhaults, Mikhail Chernov, Campbell McInnes, Yasser Riazalhosseini, Ozgur Sahin. Overcoming chemotherapy resistance in triple negative breast cancer via targeting lysyl oxidase (LOX) [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT015.

EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK

Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.

Targeted Modulation of FAK/PI3K/PDK1/AKT and FAK/p53 Pathways by Cucurbitacin B for the Antiproliferation Effect Against Human Cholangiocarcinoma Cells.

Inadequate responses to traditional chemotherapeutic agents in cholangiocarcinoma (CCA) emphasize a requirement for new effective compounds for the treatment of this malignancy. This study aimed to investigate the antiproliferative property of cucurbitacin B on KKU-100 CCA cells. The determination of underlying molecular mechanisms was also carried out. The results revealed that cucurbitacin B suppressed growth and replicative ability to form colonies of CCA cells, suggesting the antiproliferative effect of this compound against the cells. Flow cytometry analysis demonstrated that the interfering effect of cucurbitacin B on the CCA cell cycle at the G2/M phase was accountable for its antiproliferation property. Accompanied with cell cycle disruption, cucurbitacin B altered the expression of proteins involved in the G2/M phase transition including downregulation of cyclin A, cyclin D1, and cdc25A, and upregulation of p21. Additional molecular studies demonstrated that cucurbitacin B suppressed the activation of focal adhesion kinase (FAK) which consequently resulted in inhibition of its kinase-dependent and kinase-independent downstream targets contributing to the regulation of cell proliferation including PI3K/PDK1/AKT and p53 proteins. In this study, the transient knockdown of FAK using siRNA was employed to ascertain the role of FAK in CCA cell proliferation. Finally, the effect of cucurbitacin B on upstream receptor tyrosine kinases regulating FAK activation was elucidated. The results showed that the inhibitory effect of cucurbitacin B on FAK activation in CCA cells is mediated via interference of EGFR and HER2 expression. Collectively, cucurbitacin B might be a promising drug for CCA treatment by targeting FAK protein.

Abstract 4589: SRC drives invasion of luminal, but not basal, bladder cancer

Abstract Preventing progression from non-muscle invasive to muscle invasive bladder cancer (BC) is a major clinical priority. This transition is defined by tumor invasion through the bladder epithelium basement membrane into the lamina propria and muscle wall. Inhibition of SRC, a pro-invasion, non-receptor tyrosine kinase that signals as part of a complex with Focal Adhesion Kinase (FAK), may prevent BC progression. We hypothesize that SRC inhibition will disrupt migration and invasion of BC cells. Using The Cancer Genome Atlas (TCGA) mRNA expression data of human bladder tumors, we generated a classifier to subtype tumors as either “luminal” or “basal” and compared SRC expression between the groups. We then sequenced a panel of 30 bladder cancer cell lines. Each cell line was given a subtype call based on our luminal- basal classifier, and levels of SRC mRNA expression were examined. To determine the impact of SRC inhibition on luminal and basal subtypes, we exposed BC cell lines to the SRC-Family Kinase inhibitor, AZD0530. The impact of SRC inhibition on proliferation was measured by MTT assay. Transwell assays were used to examine the effect of SRC inhibition on migration, collagen invasion, and Matrigel invasion. Phosphorylation of SRC downstream targets after AZD0530 treatment was assessed by western blot. The role of SRC target and signaling partner FAK in migration was determined by siRNA knockdown and transwell assay. Luminal human bladder tumors had significantly greater SRC mRNA expression than basal tumors (p<0.001). SRC expression was also enriched in luminal BC cell lines (p<0.05). Inhibiting SRC with AZD0530 had minimal impact on proliferation with no subtype specific effects. Migration and collagen invasion of all luminal cell lines was significantly reduced by AZD0530 treatment (p<0.05). The majority of luminal models capable of invading Matrigel were also inhibited by AZD0530. In contrast, invasion and migration of the majority of basal cell lines was not significantly reduced by SRC inhibition, and was significantly increased in several cases (p<0.05). Both luminal and basal models exhibited reduced phosphorylation of SRC downstream targets when exposed with AZD0530. siRNA knockdown of FAK had no impact on migration in a basal cell line. Enrichment of SRC expression in luminal tumors and significant reductions in luminal cell line invasion after AZD0530 treatment suggest SRC promotes invasion in luminal BC. These results suggest AZD0530, and other SRC-Family Kinase inhibitors, could serve as effective anti-progression agents in luminal BC. Citation Format: Bryan Wehrenberg, Andrea Ochoa, Woonyoung Choi, David McConkey. SRC drives invasion of luminal, but not basal, bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4589.

Kisspeptin regulation of human decidual stromal cells motility via FAK-Src intracellular tyrosine kinases.

Can of Clinical Genetics, Maastricht University Medical Centre, Maastricht kisspeptin and its analogues regulate the motility of human decidual stromal cells and what intracellular signaling pathways are involved? Kisspeptin analogue-mediated cell motility in human decidual stromal cells via the focal adhesion kinase (FAK)-steroid receptor coactivator (Src) pathway suggesting that kisspeptin may modulate embryo implantation and decidual programming in human pregnancy. The extravillous trophoblast invades the maternal decidua during embryo implantation and placentation. The motile behavior and invasive potential of decidual stromal cells regulate embryo implantation and programming of human pregnancy. Human decidual stromal cells were isolated from healthy women undergoing elective termination of a normal pregnancy at 6- to 12-week gestation, after informed consent. Kisspeptin analogues were synthetic peptides. Cell motility was estimated by an invasion and migration assay. Immunoblot analysis was performed to investigate the expression of kisspeptin receptor and the effects of kisspeptin analogues on the phosphorylation of FAK and Src. Small interfering RNAs (siRNAs) were used to knock down the expression of kisspeptin receptor, FAK, Src, matrix metallo-proteinases (MMPs) 2 and 9, and extracellular signal-regulated protein kinase (ERK) 1/2. The kisspeptin receptor was expressed in human decidual stromal cells. Kisspeptin agonist decreased, but antagonist increased, cell motility. Kisspeptin agonist decreased the phosphorylation of FAK and Src tyrosine kinases, whereas antagonist increased it. These effects on phosphorylation were abolished by kisspeptin receptor siRNA. The activation of cell motility by kisspeptin analogues was suppressed by siRNA knockdown of endogenous FAK (decreased 66%), Src (decreased 60%), kisspeptin receptor (decreased 26%), MMP-2 (decreased 36%), MMP-9 (decreased 23%), and ERK 1/2 inhibitor (decreased 27%). Human decidual stromal cells were obtained from women having terminations after 6-12weeks of pregnancy and differences in timing could affect their properties. Kisspeptin acting within the endometrium has a potential modulatory role on embryo implantation and decidual programming of human pregnancy. This work was supported by grant NSC-104-2314-B-182A-146-MY2 (to H.-M.W.) from the Ministry of Science and Technology, Taiwan, and grants CMRPG3E0401 and CMRPG3E0402 (to H.-M.W.). This work was also supported by grants from the Canadian Institutes of Health Research to P.C.K.L. P.C.K.L. is the recipient of a Child & Family Research Institute Distinguished Investigator Award. The authors have no conflicts of interest to disclose. N/A.

FAK and Pyk2 activity promote TNF-\u03b1 and IL-1\u03b2-mediated pro-inflammatory gene expression and vascular inflammation

Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1β (IL-1β) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1β stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1β signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE−/− mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.

The function of FAK/CCDC80/E-cadherin pathway in the regulation of B16F10 cell migration.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in the development and progression of cancer. However, the regulatory role of FAK in cell migration remains unclear. The aim of the present study was to investigate the mechanism underlying the regulation of melanoma cell migration by FAK. The effect of FAK knockdown on gene expression in B16F10 cells was examined by gene chip analysis. The expression levels of coiled-coil domain containing 80 (CCDC80) and epithelial (E)-cadherin were analyzed by reverse transcription quantitative polymerase chain reaction and western blotting. Wound healing and transwell assays were used to monitor B16F10 cell migration. It was identified that the knockdown of FAK increased the expression levels of CCDC80 and E-cadherin, while the overexpression of CCDC80 elevated E-cadherin expression. Concurrently, upregulation of CCDC80 inhibited the migration of B16F10 cells, and downregulation of CCDC80 promoted the migration of B16F10 cells. The clinical data from the Oncomine database also revealed that the mRNA level of FAK was increased while the mRNA levels of CCDC80 and E-cadherin were decreased in patients with melanoma compared with normal controls. Taken together, the results of the present study suggest that the regulation of B16F10 melanoma cell migration by FAK is potentially mediated by CCDC80.

Cigarette smoke-induced EGFR activation promotes epithelial mesenchymal migration of human retinal pigment epithelial cells through regulation of the FAK-mediated Syk/Src pathway.

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is inevitable change of age-related macular degeneration (AMD). Smoking is a major risk factor for the development of EMT in several diseases, including lung cancer. Cigarette smoke-induced stress promotes the production of epidermal growth factor (EGF) in RPE cells. However, the underlying signaling pathways induced by aberrant EGF receptor (EGFR) expression in cigarette smoke-exposed RPE cells remain largely unknown. In the present study, the morphological transformation and production of EMT-associated cytokines were investigated to analyze the effect of smoking on the retina. Furthermore, EGF-treated or cigarette smoke-exposed RPE cells, as well as the downstream targets of EGFR, were investigated to identify the key molecules involved in EMT of cigarette smoke-stimulated RPE cells via immunoblotting. Exposure of RPE cells to cigarette smoke extract (CSE) induced secretion of VEGF and TGF-β1, and increased the expression of EMT markers. CSE-mediated focal adhesion kinase (FAK) activation resulted in the phosphorylation and activation of spleen associated tyrosine kinase (Syk)/Src proto-oncogene, non-receptor tyrosine kinase (Src), leading to migration and invasion of RPE cells. Knockdown of FAK or pharmacological inhibition of Syk/Src abrogated CSE-mediated VEGF and TGF-β1 production and blocked the phosphorylation of Smad2/3 in CSE-stimulated RPE cells. Erlotinib (an EGFR inhibitor) suppressed EGF and CSE-mediated switch from an epithelial to mesenchymal phenotype. Baicalein, an inhibitor of 12/15-lipooxygenase, also efficiently suppressed CSE-induced EMT processes by inhibiting EGFR-associated downstream signaling transduction. The results identified a novel signaling pathway mediated by EGFR in CSE-activated RPE cells, and suggest baicalein as a potential new therapeutic drug for CSE-associated retinopathy.

Attenuation of Focal Adhesion Kinase Reduces Lipopolysaccharide-Induced Inflammation Injury through Inactivation of the Wnt and NF-κB Pathways in A549 Cells.

Overall analysis and understanding of mechanisms are of great importance for treatment of infantile pneumonia due to its high morbidity and mortality worldwide. In this study, we preliminarily explored the function and mechanism of focal adhesion kinase (FAK) in regulation of inflammatory response induced by lipopolysaccharides in A549 cells. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, quantitative reverse transcription polymerase chain reaction, and Western blot analysis were used to explore the correlation of FAK expression with cell apoptosis, viability, and the inflammatory cytokine activity in A549 cells. The results showed that knockdown of FAK enhanced cell viability, suppressed apoptosis, and decreased inflammatory cytokine activity. In addition, downregulation of FAK could activate the Wnt and nuclear factor κB signaling pathways. These findings suggest that FAK might be involved in progression of infantile pneumonia and could be a new therapeutic target for this disease.

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 4125: Targeting metastasis and cancer stem-like cells in triple negative breast cancer through inhibition of focal adhesion kinase

Abstract Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer closely associated with an early pattern of metastasis and lack of targeted therapies. As such the prognosis for TNBC is poor. Whilst targeting signalling pathways known to promote metastasis-supporting behaviours may well provide an important therapeutic opportunity for such cancers, recent evidence points to an association between cancer stem-like cell (CSC) populations and the aggressive behaviours of TNBC, including their chemoresistance. Aberrant activity and/or expression of the non-receptor tyrosine kinase FAK (focal adhesion kinase) is known to contribute to the development and progression of cancer due to its central role as a mediator of cell attachment, migration and survival. More recently, FAK has been suggested to influence anoikis resistance, thus implying its activity may contribute to the phenotype of CSCs. Here we have explored the role of FAK in TNBC cells as a putative therapeutic target to suppress the metastatic phenotype. FAK activity was inhibited in MDA-MB-231 (TNBC) and MCF7 (luminal breast cancer) cells pharmacologically using PF-562,271 (PF271), a competitive-inhibitor of the ATP-kinase domain or through siRNA-mediated knockdown to probe scaffolding function of FAK. Functional assessment of TNBC proliferation, migration and invasion was performed using cell counting and Boyden chamber assays respectively, whilst stem-like behaviours (anchorage-independent growth, self-renewal) were investigated using stem-cell enrichment (mammosphere-based assays). The surface marker profile of CSCs was investigated using FACS, while changes in intracellular signalling pathways in response to FAK inhibition was revealed through Western blotting and immunofluorescent microscopy. MDA-MB-231 and MCF7 cells displayed comparable levels of total and activated (Y397/Y861) FAK which were depleted in response to PF271 in a dose dependent manner: however, TNBC cells were significantly more sensitive. Cellular migration in TNBC cell models occurred in a FAK-dependent manner with both PF271 and FAK siRNA suppressing endogenous and fibroblast-stimulated migration/invasion (p<0.001 versus vehicle control). Inhibition of FAK also caused a significant decrease in the growth rate of TNBC cells. FACS analysis of MDA-MB-231 cell cultures revealed a significant subpopulation of cells exhibiting the CSC surface marker profile CD24-/CD44+. Moreover, TNBC cells were able to form self-renewing mammospheres in non-adherent culture. PF271 treatment or siRNA-mediated knockdown of FAK significantly attenuated mammosphere-forming ability and self-renewal of these cultures. These data point to a role for FAK as a mediator of metastatic behaviour in TNBC cells, whilst also showing the capacity to influence self-renewal of a CSC population. FAK may therefore represent an emerging therapeutic target in such cases. Citation Format: Samuel Jones, Christopher Smith, Stephen Hiscox, Wen G. Jiang. Targeting metastasis and cancer stem-like cells in triple negative breast cancer through inhibition of focal adhesion kinase. [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 4125.

Cbl-b promotes cell detachment via ubiquitination of focal adhesion kinase.

Cancer cell detachment from the primary tumor site represents the first stage of metastasis. Previous studies have identified that cell detachment is triggered by cytoskeletal disruption, which may induce a wide variety of cellular changes. Focal adhesion kinase (FAK) exhibits crucial cellular functions, including regulation of the cytoskeleton. These observations have provided exciting insights into the effect of FAK in cell detachment; however, the involvement of FAK in cell detachment remains controversial. The aim of the present study was to evaluate the effect of FAK and its function in the process of cell detachment. The results revealed that FAK expression was downregulated following trypsin treatment in human gastric, lung, colon and breast cancer cell lines, as well as a human gastric epithelial cell line. Knockdown of FAK enhanced cell detachment in gastric cancer MGC803 cells, indicating that FAK inhibits cell detachment. Further investigation revealed that trypsin induced monoubiquitination of FAK. In addition, the lysosome inhibitor, NH4Cl, decreased trypsin-induced degradation of FAK. Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, was involved in this process, which interacted with FAK, as demonstrated by co-precipitation experiments, and promoted trypsin-induced ubiquitin-lysosome degradation of FAK. These results indicate that Cbl-b promotes cell detachment via ubiquitination of FAK. These findings provide novel insights regarding the effect of FAK and Cbl-b in the process of cancer cell detachment.

Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) induces TGF-β-regulated apoptosis signal in human gingival epithelial cells via fibronectin/integrinβ1/FAK cascade.

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor β (TGF-β). However, the molecular mechanisms by which microbes induce the activation of TGF-β remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF-β receptor (TGF-βR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF-βR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF-β by cooperating with integrin. Accordingly, we hypothesized that Omp29-induced actin rearrangements via FAK activity would enhance the activation of TGF-β, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF-βR/smad2 signalling and decreased active TGF-β protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF-βR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinβ1 expression by siRNA transfection attenuated TGF-βR/smad2 signalling activity and reduction of TGF-β levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinβ1, which is associated with TGF-β signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29-induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinβ1/FAK signalling-dependent TGF-β release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF-βR/smad2 pathway.