Effect Of Focal Adhesion Kinase Research Articles

Effect of Focal Adhesion Kinase and Vinculin Expression on Migration Parameters of Normal and Tumor Epitheliocytes

Effect of Focal Adhesion Kinase and Vinculin Expression on Migration Parameters of Normal and Tumor Epitheliocytes

Abstract PR-004: Inhibition of focal adhesion kinase (FAK) improves pancreatic ductal adenocarcinoma’s response to immunotherapy by targeting cancer stem cells (CSCs)

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate less than 10%. Current therapies consisting of cytotoxic chemotherapeutic agents are not effective in most patients with metastatic PDAC, and the impact of new strategies, including immunotherapies, has not been established. Previous studies have shown that inhibition of focal adhesion kinase (FAK) modulates the PDAC immunosuppressive tumor microenvironment (TME), and sensitizes tumors to immune checkpoint blockade. However, the role of FAK in regulating tumor cell intrinsic resistance to immunotherapy is not well understood. We and others previously demonstrated FAK regulates PDAC cancer stem cells (CSCs) activity including self-renewal, tumor initiation, and drug resistance. These CSCs are responsible for metastatic dissemination and resistance to multiple therapies, including immunotherapy. In this study, we evaluated the sensitivity of PDAC cells, including CSCs to cytotoxic T cells, and we also analyzed the impact of FAK inhibition on the immunogenicity and the response to cytotoxic T cell activity of PDAC CSCs. Methods: To understand how FAK regulates tumor intrinsic resistance to antigen-specific cytotoxic T cells, we utilized OT-I T cells (OT-I cells) that specifically recognize a peptide fragment of ovalbumin (ova) within the context of MHC-I. Mouse KPC pancreatic cancer cell lines expressing ova were generated and co-cultured with cytotoxic CD8+ OT-I cells in vitro. We analyzed the effect of FAK in regulating bulk cells sensitivity to antigen-specific cytotoxic T cells and clonogenic growth of CSCs by flow cytometry, MTT assay and colony formation assay using CRISPR-Cas9 and small molecule FAK inhibitors. We also analyzed the expression of surface proteins that mediate interactions with T cells (class I MHC) in bulk cells and CSCs after FAK inhibition. Results: Loss of FAK activity decreased CSCs activity (self-renewal, colony formation) in PDAC mouse cell lines, which is consistent with our previous data. Using antigen-specific OT-I co-culture model, we found that CSCs are resistant to cytotoxic T cells anti-tumor activity compared to bulk tumor cells. We also found inhibition of FAK activity not only enhances PDAC cells to cytotoxic T cell activity, but also sensitized CSCs to T cell killing. Studies in both human and mouse PDAC cell lines demonstrated decreased MHC-I expression in CSCs compared to the bulk tumor cells which could be reversed by FAK inhibition. Conclusion: We have demonstrated that CSCs are resistant to antigen-specific cytotoxic T cells, and FAK inhibition in CSCs can sensitize these cells to anti-tumor T cell activity that is associated with changes in the expression of factors involved in antigen presentation. Our study provides novel insights into the unique immunogenicity of CSCs and the role of CSCs in regulating tumor intrinsic immunotherapy resistance in PDAC. Our findings also lead to the identification of FAK as a novel therapeutic target for CSCs to improve immunotherapy efficacy in PDAC. Citation Format: Yezi Zhu, Lyndsey Sandow, William Matsui. Inhibition of focal adhesion kinase (FAK) improves pancreatic ductal adenocarcinoma’s response to immunotherapy by targeting cancer stem cells (CSCs) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-004.

Metabolic radiolabeling and in vivo PET imaging of cytotoxic T lymphocytes to guide combination adoptive cell transfer cancer therapy

BackgroundAdoptive T cell transfer-based immunotherapy yields unsatisfactory results in the treatment of solid tumors, partially owing to limited tumor infiltration and the immunosuppressive microenvironment in solid tumors. Therefore, strategies for the noninvasive tracking of adoptive T cells are critical for monitoring tumor infiltration and for guiding the development of novel combination therapies.MethodsWe developed a radiolabeling method for cytotoxic T lymphocytes (CTLs) that comprises metabolically labeling the cell surface glycans with azidosugars and then covalently conjugating them with 64Cu-1,4,7-triazacyclononanetriacetic acid-dibenzo-cyclooctyne (64Cu-NOTA-DBCO) using bioorthogonal chemistry. 64Cu-labeled control-CTLs and ovalbumin-specific CTLs (OVA-CTLs) were tracked using positron emission tomography (PET) in B16-OVA tumor-bearing mice. We also investigated the effects of focal adhesion kinase (FAK) inhibition on the antitumor efficacy of OVA-CTLs using a poly(lactic-co-glycolic) acid (PLGA)-encapsulated nanodrug (PLGA-FAKi).ResultsCTLs can be stably radiolabeled with 64Cu with a minimal effect on cell viability. PET imaging of 64Cu-OVA-CTLs enables noninvasive mapping of their in vivo behavior. Moreover, 64Cu-OVA-CTLs PET imaging revealed that PLGA-FAKi induced a significant increase in OVA-CTL infiltration into tumors, suggesting the potential for a combined therapy comprising OVA-CTLs and PLGA-FAKi. Further combination therapy studies confirmed that the PLGA-FAKi nanodrug markedly improved the antitumor effects of adoptive OVA-CTLs transfer by multiple mechanisms.ConclusionThese findings demonstrated that metabolic radiolabeling followed by PET imaging can be used to sensitively profile the early-stage migration and tumor-targeting efficiency of adoptive T cells in vivo. This strategy presents opportunities for predicting the efficacy of cell-based adoptive therapies and for guiding combination regimens.Graphic

Ephrin B2 mediates high glucose induced endothelial-to-mesenchymal transition in human aortic endothelial cells

Previous study revealed that high glucose (HG) induced endothelial cell (EC) damage via endothelial-to-mesenchymal transition (EndMT). Recent studies suggested the role of Ephrin B2 in mediate ECs damage. However, the underlying mechanism remains unclear. The aim of the present study was to investigate whether Ephrin B2 mediates HG-induced EndMT in human aortic ECs (HAECs) and to determine the possible downstream signaling effector. Primary HAECs were exposed to normal glucose (NG, 5.5 mM), HG (30 mM) and HG+Ephrin B2 small interfering RNA (siRNA), respectively. The pathological changes were investigated by light microscope and confocal microscopy. To study the effects of focal adhesion kinase (FAK) activation on Ephrin B2 in HAECs, cells were incubated with FAK siRNA in HG group. The expression of EndMT-related markers (CD31 and FSP1), Ephrin B2 and FAK were detected by qRT-PCR and western blot. The results showed that HG significantly inhibited the expression of CD31 and increased FSP1 compared with NG group. Moreover, Ephrin B2 was increased after HG incubation. Ephrin B2 siRNA attenuated HG-induced expression of EndMT-related markers. Furthermore, HG increased the expression of FAK and phosphorylated FAK (pho-FAK) in HAECs. In contrast, blocking Ephrin B2 could partially attenuate HG-induced FAK activation. And FAK siRNA further inhibited the EndMT-related markers in HAECs treated with HG. HG-induced EndMT in HAECs might be partially mediated by Ephrin B2 and the downstream FAK pathway.

Enhancement of FAK alleviates ventilator-induced alveolar epithelial cell injury

Mechanical ventilation induces lung injury by damaging alveolar epithelial cells (AECs), but the pathogenesis remains unknown. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is involved in cell growth and intracellular signal transduction pathways. This study explored the potential role of FAK in AECs during lung injury induced by mechanical ventilation. High-volume mechanical ventilation (HMV) was used to create a mouse lung injury model, which was validated by analysis of lung weight, bronchoalveolar lavage fluid and histological investigation. The expression of FAK and Akt in AECs were evaluated. In addition, recombinant FAK was administered to mice via the tail vein, and then the extent of lung injury was assessed. Mouse AECs were cultured in vitro, and FAK expression in cells under stretch was investigated. The effects of FAK on cell proliferation, migration and apoptosis were investigated. The results showed that HMV decreased FAK expression in AECs of mice, while FAK supplementation attenuated lung injury, reduced protein levels/cell counts in the bronchoalveolar lavage fluid and decreased histological lung injury and oedema. The protective effect of FAK promoted AEC proliferation and migration and prevented cells from undergoing apoptosis, which restored the integrity of the alveoli through Akt pathway. Therefore, the decrease in FAK expression by HMV is essential for injury to epithelial cells and the disruption of alveolar integrity. FAK supplementation can reduce AEC injury associated with HMV.

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.

FAK alleviates radiation-induced rectal injury by decreasing apoptosis

Radiation-induced rectal injury is closely related with radiotherapy efficiency. Here, we investigated the effect of focal adhesion kinase (FAK) in radiation-induced rectal injury. Peripheral blood samples of patients with rectal cancer were collected prior to radiotherapy. Differentially expressed genes and copy number variations (CNVs) were analyzed by microarray analysis. The CTCAE v3.0 toxicity grades were used to assess acute rectal injury. The radiosensitivity of human intestinal epithelial crypt (HIEC) cells were assayed by colony formation, mitochondrial membrane potential, flow cytometry and western blotting. The rectums of C57BL/6 mice were X-irradiated locally with a single dose of 15 Gy. The effect of FAK on radiation-induced injury was investigated by hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). FAK mRNA level was inversely correlated with rectal injury severity in patient samples. A CNV amplification located on chromosome 8 was closely related with FAK. Further functional assays revealed increased levels of γH2AX expression and apoptosis-related proteins in FAK-silenced HIEC cells. The ratio of TUNEL, cl-caspase-3, cyto-c and bax/bcl-2 expression in the rectum mucosa treated with a FAK inhibitor increased significantly. These results demonstrated that FAK reduced radiation-induced rectal injury by decreasing apoptosis.

Abstract 5889: Cancer associated fibroblasts regulate cancer stem cell functions in pancreatic ductal adenocarcinoma

Abstract Introduction: Despite recent advancements in treatment, the outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal due to high relapse rate and metastatic dissemination. PDAC cancer stem cells (CSCs) have been shown to play a role in these processes and are associated with worse median survival in patients. Several cell-intrinsic mechanisms of CSC regulation have been identified in PDAC, however the role of the altered tumor microenvironment remains largely unknown. We focusd on the impact of cancer associated fibroblasts (CAFs) on CSCs properties in PDAC. Methods: Stromal fibroblast lines were established from surgically resected PDAC tissues and were marked by specific fluorophore. Following co-culture with CAFs, tumor cells from PDAC cell lines or low passage xenografts derived from primary surgical specimens cells were isolated and evaluated for colony formation and migration. The effect of Focal Adhesion Kinase (FAK) on CAF-mediated clonogenic growth was evaluated using the small molecule FAK inhibitor PF573228 or overexpression of full-length of FAK in PDAC cells. Results: Following co-culture with CAFs, the clonogenic growth of PDAC cell lines increased by 1.3-2.5 fold compared to cells incubated alone (p ≤ 0.05). Furthermore, CAFs enhanced colony formation of cells from patient derived xenografts by at least 4 fold (p ≤ 0.001). To examine the effect of CAFs on PDAC self-renewal, tumor colonies were harvested and replated in methylcellulose. Although cells were not further exposed to CAFs, secondary colony formation of cells originally co-cultured with CAFs increased by > 1.3 fold. We also found that the frequency of ALDH+ PDAC CSCs increased by 12-19 fold compared to control cells. CSCs are also highly migratory and express factors suggestive of the epithelial to mesenchymal transition (EMT) suggesting that they play an important role in metastatic disease. Following co-culture, the migration of PDAC cells increased by 1.2-2.1 fold (p ≤ 0.05) with increased expression of the EMT associated genes SNAIL1, SNAIL2, ZEB1, ZEB2, N-cadherin, and Vimentin. PDAC cells induced CAF activation as evidenced by enhanced αSMA expression. Since activated CAFs are known to secret extracellular matrix proteins, we examined the activation of FAK downstream of integrin signaling and found increased phosphorylation of FAK at Y397. To determine the importance of FAK activation on the interaction between CAFs and PDAC cells, cells were treated with the FAK inhibitor PF573228 that significantly inhibited tumor colony formation. In contrast, the overexpression of FAK-FL further increased CAF-mediated PDAC clonogenic growth. Conclusion: CAFs enhance PDAC CSCs including clonogenic growth, migration, and self-renewal. These effects are partially mediated by FAK, and FAK inhibitors may represent a novel strategy to modulate the interaction of PDAC cells and the tumor microenviroment. Citation Format: Asma Begum, Ross McMillan, Yu-tai Chang, Vesselin Penchev, NV Rajeshkumar, Anirban Maitra, Michael G. Goggins, James R. Eshelman, Christopher Wolfgang, Zeshaan A. Rasheed, William H. Matsui. Cancer associated fibroblasts regulate cancer stem cell functions in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5889. doi:10.1158/1538-7445.AM2017-5889

Focal adhesion kinase activity is required for actomyosin contractility-based invasion of cells into dense 3D matrices

The focal adhesion kinase (FAK) regulates the dynamics of integrin-based cell adhesions important for motility. FAK’s activity regulation is involved in stress-sensing and focal-adhesion turnover. The effect of FAK on 3D migration and cellular mechanics is unclear. We analyzed FAK knock-out mouse embryonic fibroblasts and cells expressing a kinase-dead FAK mutant, R454-FAK, in comparison to FAK wild-type cells. FAK knock-out and FAKR454/R454 cells invade dense 3D matrices less efficiently. These results are supported by FAK knock-down in wild-type fibroblasts and MDA-MB-231 human breast cancer cells showing reduced invasiveness. Pharmacological interventions indicate that in 3D matrices, cells deficient in FAK or kinase-activity behave similarly to wild-type cells treated with inhibitors of Src-activity or actomyosin-contractility. Using magnetic tweezers experiments, FAKR454/R454 cells are shown to be softer and exhibit impaired adhesion to fibronectin and collagen, which is consistent with their reduced 3D invasiveness. In line with this, FAKR454/R454 cells cannot contract the matrix in contrast to FAK wild-type cells. Finally, our findings demonstrate that active FAK facilitates 3D matrix invasion through increased cellular stiffness and transmission of actomyosin-dependent contractile force in dense 3D extracellular matrices.

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.

Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia.

BackgroundAcute lymphoblastic leukemia (ALL) is an aggressive malignant disorder of lymphoid progenitor cells in both children and adults. Although improvements in contemporary therapy and development of new treatment strategies have led to dramatic increases in the cure rate in children with ALL, the relapse rate remains high and the prognosis of relapsed childhood ALL is poor. Molecularly targeted therapies have emerged as the leading treatments in cancer therapy. Multi-cytotoxic drug regimens have achieved success, yet many studies addressing targeted therapies have focused on only one single agent. In this study, we attempted to investigate whether the effect of the mammalian target of rapamycin (mTOR) inhibitor rapamycin is synergistic with the effect of focal adhesion kinase (FAK) down-regulation in the treatment of ALL.MethodsThe effect of rapamycin combined with FAK down-regulation on cell proliferation, the cell cycle, and apoptosis was investigated in the human precursor B acute lymphoblastic leukemia cells REH and on survival time and leukemia progression in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model.ResultsWhen combined with FAK down-regulation, rapamycin-induced suppression of cell proliferation, G0/G1 cell cycle arrest, and apoptosis were significantly enhanced. In addition, REH cell-injected NOD/SCID mice treated with rapamycin and a short-hairpin RNA (shRNA) to down-regulate FAK had significantly longer survival times and slower leukemia progression compared with mice injected with REH-empty vector cells and treated with rapamycin. Moreover, the B-cell CLL/lymphoma-2 (BCL-2) gene family was shown to be involved in the enhancement, by combined treatment, of REH cell apoptosis.ConclusionsFAK down-regulation enhanced the in vitro and in vivo inhibitory effects of rapamycin on REH cell growth, indicating that the simultaneous targeting of mTOR- and FAK-related pathways might offer a novel and powerful strategy for treating ALL.

The effects of focal adhesion kinase on the motility, proliferation and apoptosis of Caco2 and SMMC-7721 cells.

Focal adhesion kinase (FAK) plays important roles in cancer development. However, the significance of FAK expression in colorectal carcinoma and hepatocellular carcinoma has not been clarified. This study aims to explore the roles FAK played in the progression of colorectal carcinoma and hepatocellular carcinoma. RNAi method was used to inhibit the expression of FAK in Caco2 and SMMC-7721 cells. Reverse transcriptase polymerase chain reaction analysis and Western blot analysis were used to examine mRNA and protein expression of FAK. Then, the proliferation, motility and apoptosis of both types of cells were detected using MTT assay, wound healing/transwell assay and nuclear staining assay. The microstructure changes (F-actin, β-tubulin and lamin B1) of SMMC-7721 cells were visualized by immunofluorescence. FAK was overexpressed in both cell lines and down-regulation of FAK resulted in suppression of cell proliferation, inhibition of cell migration and invasion. The apoptosis of cells was increased significantly following the FAK expression inhibition. Moreover, actin polymerization, β-tubulin and lamin B1 expression of cells were significantly decreased. The results highlight the role of FAK in the progression of cancers. These findings suggest FAK serve as a potential therapeutic target for cancer therapy.

Protective Effect of Focal Adhesion Kinase against Skeletal Muscle Reperfusion Injury after Acute Limb Ischemia

In cardiac muscle, ischemia reperfusion (IR) injury is attenuated by mitochondrial function, which may be upregulated by focal adhesion kinase (FAK). The aim of this study was to determine whether increased FAK levels reduced rhabdomyolysis in skeletal muscle too. In a translational in vivo experiment, rat lower limbs were subjected to 4 hours of ischemia followed by 24 or 72 hours of reperfusion. FAK expression was stimulated 7 days before (via somatic transfection with pCMV-driven FAK expression plasmid) and outcomes were measured against non-transfected and empty transfected controls. Slow oxidative (i.e., mitochondria-rich) and fast glycolytic (i.e., mitochondria-poor) type muscles were analyzed separately regarding rhabdomyolysis, apoptosis, and inflammation. Severity of IR injury was assessed using paired non-ischemic controls. After 24 hours of reperfusion, marked rhabdomyolysis was found in non-transfected and empty plasmid-transfected fast-type glycolytic muscle, tibialis anterior. Prior transfection enhanced FAK concentration significantly (p = 0.01). Concomitantly, levels of BAX, promoting mitochondrial transition pores, were reduced sixfold (p = 0.02) together with a blunted inflammation (p = 0.01) and reduced rhabdomyolysis (p = 0.003). Slow oxidative muscle, m. soleus, reacted differently: although apoptosis was detectable after IR, rhabdomyolysis did not appear before 72 hours of reperfusion; and FAK levels were not enhanced in ischemic muscle despite transfection (p = 0.66). IR-induced skeletal muscle rhabdomyolysis is a fiber type-specific phenomenon that appears to be modulated by mitochondria reserves. Stimulation of FAK may exploit these reserves constituting a potential therapeutic approach to reduce tissue loss following acute limb IR in fast-type muscle.

Commentary on ‘Protective Effect of Focal Adhesion Kinase Against Skeletal Muscle Reperfusion Injury After Acute Limb Ischemia’

Commentary on ‘Protective Effect of Focal Adhesion Kinase Against Skeletal Muscle Reperfusion Injury After Acute Limb Ischemia’

Protective Effect of Focal Adhesion Kinase against Skeletal Muscle Reperfusion Injury after Acute Limb Ischemia

In cardiac muscle, ischemia reperfusion (IR) injury is attenuated by mitochondrial function, which may be upregulated by focal adhesion kinase (FAK). The aim of this study was to determine whether increased FAK levels reduced rhabdomyolysis in skeletal muscle too. In a translational invivo experiment, rat lower limbs were subjected to 4 hours of ischemia followed by 24 or 72 hours of reperfusion. FAK expression was stimulated 7 days before (via somatic transfection with pCMV-driven FAK expression plasmid) and outcomes were measured against non-transfected and empty transfected controls. Slow oxidative (i.e., mitochondria-rich) and fast glycolytic (i.e., mitochondria-poor) type muscles were analyzed separately regarding rhabdomyolysis, apoptosis, and inflammation. Severity of IR injury was assessed using paired non-ischemic controls. After 24 hours of reperfusion, marked rhabdomyolysis was found in non-transfected and empty plasmid-transfected fast-type glycolytic muscle, tibialis anterior. Prior transfection enhanced FAK concentration significantly (p= 0.01). Concomitantly, levels of BAX, promoting mitochondrial transition pores, were reduced sixfold (p= 0.02) together with a blunted inflammation (p= 0.01) and reduced rhabdomyolysis (p= 0.003). Slow oxidative muscle, m. soleus, reacted differently: although apoptosis was detectable after IR, rhabdomyolysis did not appear before 72 hours of reperfusion; and FAK levels were not enhanced in ischemic muscle despite transfection (p= 0.66). IR-induced skeletal muscle rhabdomyolysis is a fiber type-specific phenomenon that appears to be modulated by mitochondria reserves. Stimulation of FAK may exploit these reserves constituting a potential therapeutic approach to reduce tissue loss following acute limb IR in fast-type muscle.

Focal adhesion kinase (FAK) siRNA inhibits human hypertrophic scar by suppressing integrin α, TGF‐β and α‐SMA

The effect of focal adhesion kinase (FAK) on suppressing scarring and the potential molecular mechanism underlying it has been investigated. Ten samples of human hypertrophic scars (HS) tissue cultured in vitro were transfected with FAK siRNA mediated by liposome. Quantitative real-time PCR was used to detect the expression of integrin α, transforming growth factor-β (TGF-β), FAK and α-smooth muscle actin (α-SMA) after transfection. MTT assay was used as a measure of fibroblast proliferation. Flow cytometry and (3)H-proincorporation technique gave measurements of the cell cycle and the quantity of collagen synthesis, respectively. Expression of FAK was effectively blocked, accompanied by decreasing expression of integrin α, TGF-β and α-SMA in hypertrophic scars fibroblast (HSFB) cells. One to 4 h after transfection with FAK siRNA, proliferation of HSFB cells was strongly inhibited (P < 0.01), reaching a maximum at 48 h. The proportion of G1 cells was higher and the proportion of the S and G2 cells lower after transfection. The amount of collagen synthesis in HSFB cells decreased when HSFB cells were transfected for 48 h. RNA interference targeting the FAK gene can block the two abnormal signal transduction pathways mediated by the integrin and TGF-β receptors that are responsible for hyperplasia and contracture of the scar, making FAK iRNA therapy a potentially effective approach in HS treatment.

Nanotopography Modulates Mechanotransduction of Stem Cells and Induces Differentiation through Focal Adhesion Kinase

Regulated biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration and embryogenesis in the stem cell niche. Tissue homeostasis involves the interaction of multipotent cells with nanoscaled topographical features in their ECM to regulate aspects of cell behavior. Synthetic nanostructures can drive specific cell differentiation, but the sensing mechanisms for nanocues remain poorly understood. Here, we report that nanotopography-induced human mesenchymal stem cell (hMSC) differentiation through cell mechanotransduction is modulated by the integrin-activated focal adhesion kinase (FAK). On nanogratings with 250 nm line width on polydimethylsiloxane, hMSCs developed aligned stress fibers and showed an upregulation of neurogenic and myogenic differentiation markers. The observed cellular focal adhesions within these cells were also significantly smaller and more elongated on the nanogratings compared to microgratings or unpatterned control. In addition, our mechanistic study confirmed that this regulation was dependent upon actomyosin contractility, suggesting a direct force-dependent mechanism. The topography-induced differentiation was observed on different ECM compositions but the response was not indicative of a direct ECM-induced hMSC differentiation pathway. FAK phosphorylation was required for topography-induced hMSC differentiation while FAK overexpression overruled the topographical cues in determining cell lineage bias. The results indicated that FAK activity had a direct impact on topography-induced gene expression, and that this effect of FAK was independent of cell shape. These findings suggest that hMSC sense and transduce nanotopographical signals through focal adhesions and actomyosin cytoskeleton contractility to induce differential gene expression.

Abstract 4013: S100A4 promotes cell migration and growth through focal adhesion kinase and Src mediated dual signaling pathways in pancreatic cancer cells.

Abstract BACKGROUD: Pancreatic cancer is one of the deadliest types of cancer. Activation and expression of focal adhesion kinase (FAK) and Src kinase is abnormally upregulated in pancreatic cancers. The results of clinical trials targeting either Src or FAK by inhibitors are largely unknown. S100A4 is a calcium-binding protein and aberrant S100A4 expression is an independent biomarker for poor outcomes of pancreatic cancer; however, the involved mechanism is unclear. METHODS: The effects of inhibitors on FAK and Src activation, and on cell migration and growth, were examined in MiaPaca2 and S2VP10 cell lines. To determine whether FAK and Src depend on each other or independently regulate pancreatic cancer cell phenotypes, the effects of FAK or Src mutant expression were determined. The role of S100A4 on FAK and Src activation was evaluated through loss/gain approaches. The association of S100A4 expression with FAK and Src activation was determined by immunostaining on human pancreatic cancer tissue sections. RESULTS: FAK and Src kinase inhibitors blocked activation in a dose dependent manner in both MiaPaca2 and S2VP10 cells. However, it required more inhibitor to inhibit either FAK or Src in S2VP10 cells. Blocking FAK (or Src) does not completely inhibit Src (or FAK) activation. This is confirmed by the finding that FAK mutant expression completely inhibited FAK activation, but not Src activation. Similarly, Src mutant expression did not inhibit FAK activation. Downregulation of S100A4 expression significantly decreased FAK and Src activation. Importantly, increased S100A4 expression is associated with increased FAK and Src activation in pancreatic cancer. CONCLUSIONS: These data support that the Src-FAK axis forms a dual signaling pathway, and each of them contributes independently to the aggressive pancreatic cancer cell phenotype. Also, these data support that S100A4 promotes the Src-FAK dual signaling pathway which possibly leads to a feed-forward loop facilitating pancreatic tumor progression. Citation Format: Guoqiang Cai, Elizabeth H. Kerr, Meng Hu, Youfeng Yang, Donald J. Buchsbaum, William E. Grizzle, Qiang Ding. S100A4 promotes cell migration and growth through focal adhesion kinase and Src mediated dual signaling pathways in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4013. doi:10.1158/1538-7445.AM2013-4013

Periodic Mechanical Stress Stimulates the FAK Mitogenic Signal in Rat Chondrocytes Through ERK1/2 Activity

Background/Aims: The biological effects of periodic mechanical stress on chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying chondrocyte mechanosensing and signaling in response to periodic mechanical stress remain to be determined. In the current study, we examined the effects of focal adhesion kinase (FAK) signaling on periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Methods and Results: Periodic mechanical stress significantly induced sustained phosphorylation of FAK at Tyr³⁹⁷ and Tyr^576/577. Reduction of FAK with targeted shRNA via transfection of NH₂-terminal tyrosine phosphorylation-deficient FAK mutant Y397F or Y576F-Y577F abolished periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis, accompanied by attenuated ERK1/2 phosphorylation. However, activation of Src, PLCγ1 and Rac1 was not prevented upon FAK suppression. Furthermore, pretreatment with the Src-selective inhibitor, PP2, and shRNA targeted to Src or suppression of Rac1 with its selective inhibitor, NSC23766, blocked FAK phosphorylation at Tyr,^576/577 but not Tyr,³⁹⁷ under periodic mechanical stress. Interestingly, FAK phosphorylation neither at Tyr³⁹⁷ nor at Tyr^576/577 was affected by PLCγ1 depletion when periodic mechanical stress was applied. In addition, Tyr³⁹⁷ and Tyr^576/577 phosphorylation levels were reduced upon pretreatment with a blocking antibody against integrin β1 under conditions of periodic mechanical stress. Conclusion: Our findings collectively suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through at least two pathways, integrin β1-Src-Rac1-FAK(Tyr^576/577)-ERK1/2 and integrin β1-FAK (Tyr³⁹⁷)-ERK1/2.

Nanog Increases Focal Adhesion Kinase (FAK) Promoter Activity and Expression and Directly Binds to FAK Protein to Be Phosphorylated

Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.