Activation of Focal Adhesion Kinase and Src Mediates Acquired Sorafenib Resistance in A549 Human Lung Adenocarcinoma Xenografts.

Despite encouraging clinical results with sorafenib monotherapy in patients with KRAS-mutant non-small-cell lung cancer (NSCLC), the overall survival benefit of this drug is limited by the inevitable development of acquired resistance. The exact mechanism underlying acquired sorafenib resistance in KRAS-mutant NSCLC is unclear. In this study, the mechanism of acquired sorafenib resistance was explored using a biologically relevant xenograft model, which was established by using the A549 human lung adenocarcinoma cell line and an in vivo-derived, sorafenib-resistant A549 subline (A549/SRFres). Results from the initial study demonstrated that sorafenib treatment significantly decreased E-cadherin (P < 0.05) levels but significantly increased matrix metallopeptidase 9 (MMP9) levels (P < 0.01) in A549/SRFres tumors, whereas expression levels of phospho-protein kinase B (AKT), phospho-focal adhesion kinase (FAK), and phospho-Src were elevated in sorafenib-treated A549 and A549/SRFres tumors. We next examined whether concomitant dasatinib treatment could overcome acquired sorafenib resistance by blocking the FAK/Src escape route that mediates resistance. Despite the observed in vitro synergy between sorafenib and dasatinib, the in vivo antitumor effect of half-dose sorafenib-dasatinib combination therapy was inferior to that of the full-dose sorafenib treatment. Although the sorafenib-dasatinib combination effectively inhibited Src and AKT phosphorylation, it did not block the Y576/577-FAK phosphorylation, nor did it decrease vimentin protein expression; unexpectedly, it increased Y397-FAK phosphorylation and MMP9 protein expression in tumors. These results suggest that acquired sorafenib resistance in KRAS-mutant A549 xenografts involves the compensatory activation of FAK and Src, and Src inhibition alone is insufficient to diminish sorafenib-promoted epithelial-mesenchymal transition process and invasive potentials in tumors.

The effects of CEP-37440, an inhibitor of focal adhesion kinase, in vitro and in vivo on inflammatory breast cancer cells.

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC. METHODS: Cell proliferation assays were performed in the presence of several concentrations of CEP-37440 using IBC and triple-negative breast cancer non-IBC cell lines. In vitro, we studied the expression of total FAK1, phospho-FAK1 (Tyr 397), total ALK and phospho-ALK (Tyr 1604). In vivo, we tested CEP-37440 using FC-IBC02, SUM149, and SUM190 IBC xenograft mouse models. RESULTS: CEP-37440 at low concentration decreased the proliferation of the IBC cell lines FC-IBC02, SUM190, and KPL4, while not affecting the proliferation of normal breast epithelial cells. At higher concentration, CEP-37440 was also able to inhibit the proliferation of the IBC cell line MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468; the IBC cell line SUM149 showed a slight response to the drug. CEP-37440 decreased the cell proliferation of FC-IBC02, SUM190, and KPL4 by blocking the autophosphorylation kinase activity of FAK1 (Tyr 397). None of the cells evaluated expressed ALK. In vivo, after 7 weeks of CEP-37440 treatment, the SUM190, FC-IBC02, and SUM149 breast tumor xenografts were smaller in mice treated with 55 mg/kg bid CEP-37440 compared to the controls; the tumor growth inhibition (TGI) was 79.7 %, 33 %, and 23 %, respectively. None of the FC-IBC02 breast xenografts mice treated with CEP-37440 developed brain metastasis while 20 % of the mice in the control group developed brain metastasis. Expression array analyses in FC-IBC02 cells showed that CEP-37440 affects the expression of genes related to apoptosis, interferon signaling, and cytokines. CONCLUSIONS: CEP-37440 is effective against some IBC cells that express phospho-FAK1 (Tyr 397), and its antiproliferative activity is related to its ability to decrease phospho-FAK1. Our results suggest that combinational therapies could be more effective than using CEP-37440 as a single agent.

Molecular mechanism of fluoroquinolones modulation on corneal fibroblast motility.

Topical fluoroquinolones are widely used to prevent ocular infections after ophthalmic surgery. However, they have been shown to affect the corneal cell motility, whose mechanism remains indefinite. The purpose of this study was to investigate how fluoroquinolones affect corneal stromal cell motility. Human corneal fibroblasts (HCFs) were incubated in ciprofloxacin (CIP), levofloxacin (LEV), or moxifloxacin (MOX) at 0, 10, 50, and 100 µg/ml for up to 3 days. Effect of CIP, LEV, or MOX on HCF migration was monitored using migration assay. HCF viability was determined by WST-1 assay. Expression of focal adhesion kinase (FAK), paxillin (PXN), and their phosphorylated forms were analyzed by immunoblotting. Binding affinity between FAK and PXN was determined by co-immunoprecipitation. Our results revealed that CIP and MOX, but not LEV, noticeably retarded HCF migration. HCF proliferation was significantly reduced by CIP (38.2%), LEV (29.5%), and MOX (21.3%), respectively (p = 0.002). CIP and MOX suppressed the phosphorylation of PXN at tyrosines (10.2 ± 4.3%, p < 0.001; 11.7 ± 2.4%, p < 0.001, respectively), including tyrosine 118 (33.3 ± 5.2%, p < 0.001; 34.0 ± 4.4%, p < 0.001, respectively). CIP and MOX diminished the binding affinity between FAK and PXN (8.2 ± 1.8%, p < 0.001; 9.0 ± 4.5%, p < 0.001, respectively). Nevertheless, tyrosine dephosphorylation and FAK dissociation of PXN were not found in LEV-treated HCFs. None of these fluoroquinolones affect phosphorylation of FAK-Y397. We conclude that CIP and MOX, but not LEV, might delay corneal fibroblast migration via interfering with recruitment of PXN to focal adhesions and dephosphorylation of PXN at the tyrosines.

Schwann cells promote endothelial cell migration.

Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.

VEGF Enhances the Migration of MSCs in Neural Differentiation by Regulating Focal Adhesion Turnover.

Mesenchymal stem cells (MSCs) hold great promise in neural regeneration, due to their intrinsic neuronal potential and migratory tropism to damaged nervous tissues. However, the chemotactic signals mediating the migration of MSCs remain poorly understood. Here, we investigated the regulatory roles for focal adhesion kinase (FAK) and Rac1 in vascular endothelial growth factor (VEGF)-stimulated migration of MSCs in neural differentiation. We found that MSCs in various differentiation states show significant different chemotactic responses to VEGF and cells in 24-h preinduction state possess the highest migration speed and efficiency. FAK, as the downstream signaling molecule, is involved in the VEGF-induced migration by regulating the assembly and distribution of focal adhesions (FAs) and reorganization of F-actin. The features of FAs and cytoskeletons and the ability of lamellipodia formation are closely related to the neural differentiation states of MSCs. VEGF promotes FA formation with an asymmetric distribution of FAs and induces the activation of Y397-FAK and Y31/118-paxillin of undifferentiated and 24-h preinduced MSCs in a time-dependent manner. Inhibition of FAK by PF-228 or expressing FAK-Y397F mutant impairs the dynamics of FAs in MSCs during VEGF-induced migration. Furthermore, Rac1 regulates FA formation in a FAK-dependent manner. Overexpression of constitutive activated mutants of Rac1 increases the number of FAs in undifferentiated and 24-h preinduced MSCs, while VEGF-induced increase of FA formation is decreased by inhibiting FAK by PF-228. Collectively, these results demonstrate that FAK and Rac1 signalings coordinately regulate the dynamics of FAs during VEGF-induced migration of MSCs in varying neural differentiation states.

Short term exposure of beta cells to low concentrations of interleukin-1ß improves insulin secretion through focal adhesion and actin remodeling and regulation of gene expression.

Type 2 diabetes involves defective insulin secretion with islet inflammation governed in part by IL-1ß. Prolonged exposure of islets to high concentrations of IL-1ß (>24 h, 20 ng/ml) impairs beta cell function and survival. Conversely, exposure to lower concentrations of IL-1ß for >24 h improves these same parameters. The impact on insulin secretion of shorter exposure times to IL-1ß and the underlying molecular mechanisms are poorly understood and were the focus of this study. Treatment of rat primary beta cells, as well as rat or human whole islets, with 0.1 ng/ml IL-1ß for 2 h increased glucose-stimulated (but not basal) insulin secretion, whereas 20 ng/ml was without effect. Similar differential effects of IL-1ß depending on concentration were observed after 15 min of KCl stimulation but were prevented by diazoxide. Studies on sorted rat beta cells indicated that the enhancement of stimulated secretion by 0.1 ng/ml IL-1ß was mediated by the NF-κB pathway and c-JUN/JNK pathway acting in parallel to elicit focal adhesion remodeling and the phosphorylation of paxillin independently of upstream regulation by focal adhesion kinase. Because the beneficial effect of IL-1ß was dependent in part upon transcription, gene expression was analyzed by RNAseq. There were 18 genes regulated uniquely by 0.1 but not 20 ng/ml IL-1ß, which are mostly involved in transcription and apoptosis. These results indicate that 2 h of exposure of beta cells to a low but not a high concentration of IL-1ß enhances glucose-stimulated insulin secretion through focal adhesion and actin remodeling, as well as modulation of gene expression.

Protective effect of focal adhesion kinase against skeletal muscle reperfusion injury after acute limb ischemia.

OBJECTIVES: 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. MATERIAL AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Cisplatin at sub-toxic levels mediates integrin switch in lung cancer cells.

BACKGROUND: Resistance to chemotherapeutic agents, as well as enhanced metastasis, have been frequently reported in lung cancer. MATERIALS AND METHODS: Cytotoxicity and proliferative effects of cisplatin on H460 lung cancer cells were evaluated by the MTT assay. Migration capacity was evaluated by the wound healing assay. The number of filopodia per cell were detected by rhodamine-phalloidin staining assay. The changes of protein levels of integrins, and migration-related proteins in response to cisplatin at sub-toxic concentrations were determined by western blotting. RESULTS: Herein we demonstrate for the first time that exposure to low concentrations of cisplatin results in increase of cell motility with the alteration of integrin expression. Cisplatin-treated cells exhibited a significant increase in the number of filopodia per cell in correlation with enhanced migration. Migration regulatory proteins, namely activated forms of focal-adhesion kinase (FAK) and ATP-dependent tyrosine kinase (AKT), were found to significantly be up-regulated in cisplatin-treated cells in comparison to those of the non-treated control. Active Rho A-GTP and Rac-GTP were found to be increased in accordance with activation of FAK/AKT signals. Furthermore, we found that such migration enhancement may be in part due to the integrin switch mediated by cisplatin treatment. Cisplatin induced a dramatic alteration in the integrin expression pattern by up-regulating integrin α4, αv, ß1, and ß5 which were previously reported to increase cell motility, while it had no effect on integrin α5, and ß3. CONCLUSION: As the integrin switch is a hallmark of highly aggressive cancer, these findings may provide insights for better understanding of cancer cell adaptation after exposure to cisplatin.

Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

OBJECTIVE: To investigate how epithelial mechanotransduction pathways impact wound repair. BACKGROUND: Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. METHODS: A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. RESULTS: Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. CONCLUSIONS: Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

The prognostic significance of focal adhesion kinase expression in stage I non-small-cell lung cancer.

INTRODUCTION: Focal adhesion kinase (FAK) plays a significant role in cancer cell survival signaling and is overexpressed in various malignancies, including lung cancer. Previous studies suggest that FAK overexpression is an independent factor predicting poor prognosis in non-small-cell lung cancer (NSCLC). The aim of this study is to confirm these findings specifically in stage I NSCLC. METHODS: A retrospective tissue microarray (TMA) analysis of FAK protein expression by immunohistochemistry was performed in 157 surgically resected stage I NSCLC specimen and in the corresponding matched normal lung tissue. The FAK 4.47 monoclonal antibody was used for FAK immunostaining. The scoring system of triplicate tumor cores included intensity of staining plus extent of staining for a composite score that ranged from 0 to 6. The association between FAK score and survival was evaluated. RESULTS: There were 103 stage IA and 54 stage IB patients, with mean follow-up of 5.5 years. Normal lung alveoli and interstitial tissue had mean FAK score of 0 (median score 0, range 0 to 2). Tumor samples had mean FAK score 3.1 (median score 3.5, range 0-6), with 57% of the samples having FAK score ≥ 3. Continuous FAK score was not associated with demographic data, tumor histology, or grade, nor survival in this cohort of stage I NSCLC patients. CONCLUSIONS: FAK is expressed in more than 50% of stage I NSCLC lung cancer but not in normal lung alveoli and interstitial tissue. FAK expression is not associated with survival outcome in this North American cohort.

Tumour-associated endothelial-FAK correlated with molecular sub-type and prognostic factors in invasive breast cancer.

BACKGROUND: Breast cancer is a heterogeneous disease that can be classified into one of 4 main molecular sub-types: luminal A, luminal B, Her2 over-expressing and basal-like (BL). These tumour sub-types require different treatments and have different risks of disease progression. BL cancers can be considered a sub-group of Triple negative (TN) cancers since they lack estrogen (ER), progesterone (PR) and Her2 expression. No targeted treatment currently exists for TN/BL cancers. Thus it is important to identify potential therapeutic targets and describe their relationship with established prognostic factors. Focal adhesion kinase (FAK) is upregulated in several human cancers and also plays a functional role in tumour angiogenesis. However, the association between breast cancer sub-types and tumour endothelial-FAK expression is unknown. METHODS: Using immunofluorescence, we quantified FAK expression in tumour endothelial and tumour cell compartments in 149 invasive breast carcinomas and correlated expression with clinical, pathological and molecular parameters. RESULTS: Low endothelial-FAK expression was independently associated with luminal A tumours at univariate (p < 0.001) and multivariate (p = 0.001) analysis. There was a positive correlation between FAK expression in the vascular and tumour cell compartments (Spearman's correlation co-efficient = 0.394, p < 0.001). Additionally, endothelial and tumour cell FAK expression were significantly increased in TN tumours (p = 0.043 and p = 0.033 respectively), in tumours with negative ER and PR status, and in high grade tumours at univariate analysis. CONCLUSION: Our findings establish a relationship between endothelial-FAK expression levels and the molecular sub-type of invasive breast cancer, and suggest that endothelial-FAK expression is potentially more clinically relevant than tumour cell FAK expression in breast cancer.

Focal adhesion protein expression in human diffuse large B-cell lymphoma.

AIMS: Focal adhesions have been associated with poor prognosis in multiple cancer types, but their prognostic value in diffuse large B-cell lymphoma (DLBCL) has not been evaluated. The aim of this study was to investigate the expression patterns and the prognostic value of the focal adhesion proteins FAK, Pyk2, p130Cas and HEF1 in DLBCL. METHODS AND RESULTS: Focal adhesion protein expression was examined using immunohistochemistry in normal lymphoid tissues and in 60 DLBCL patient samples. Kaplan-Meier survival and Cox regression analysis were performed to evaluate the correlation of focal adhesion protein expression with patient prognosis. FAK, Pyk2, p130Cas and HEF1 expression was mostly found in the germinal centres of normal human lymphoid tissues. When assessed in DLBCL samples, FAK, Pyk2, p130Cas and HEF1 were highly expressed in 45%, 34%, 42% and 45% of the samples, respectively. Multivariate Cox analysis revealed that decreased FAK expression was a significant independent predictor of poorer disease outcome. CONCLUSIONS: FAK expression is an independent prognostic factor in DLBCL. Our results suggest that the addition of FAK immunostaining to the current immunohistochemical algorithms may facilitate risk stratification of DLBCL patients.

Osteoactivin induces transdifferentiation of C2C12 myoblasts into osteoblasts.

Osteoactivin (OA) is a novel osteogenic factor important for osteoblast differentiation and function. Previous studies showed that OA stimulates matrix mineralization and transcription of osteoblast specific genes required for differentiation. OA plays a role in wound healing and its expression was shown to increase in post fracture calluses. OA expression was reported in muscle as OA is upregulated in cases of denervation and unloading stress. The regulatory mechanisms of OA in muscle and bone have not yet been determined. In this study, we examined whether OA plays a role in transdifferentiation of C2C12 myoblast into osteoblasts. Infected C2C12 with a retroviral vector overexpressing OA under the CMV promoter were able to transdifferentiate from myoblasts into osteoblasts. Immunofluorescence analysis showed that skeletal muscle marker MF-20 was severely downregulated in cells overexpressing OA and contained significantly less myotubes compared to uninfected control. C2C12 myoblasts overexpressing OA showed an increase in expression of bone specific markers such as alkaline phosphatase and alizarin red staining, and also showed an increase in Runx2 protein expression. We also detected increased levels of phosphorylated focal adhesion kinase (FAK) in C2C12 myoblasts overexpressing OA compared to control. Taken together, our results suggest that OA is able to induce transdifferentiation of myoblasts into osteoblasts through increasing levels of phosphorylated FAK.

Inhibition of focal adhesion kinase induces apoptosis in human osteosarcoma SAOS-2 cells.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase protein, acts as an early modulator of integrin signaling cascade, regulating basic cellular functions. In transformed cells, unopposed FAK signaling has been considered to promote tumor growth, progression, and metastasis. The aim of this study was to assess the role of focal adhesion kinase in human osteosarcoma SAOS-2 cells. SAOS-2 cells were transfected with PGPU6/GFP/shNC, and PGPU6/GFP/FAK-334 (shRNA-334), respectively. Expression of FAK was detected by real-time PCR and western blots. MTT assay was used to examine changes in cell proliferation. Cell apoptosis was analyzed by flow cytometry. The expression of caspase-3,-7,-9 was measured by Western blots. The expression of FAK in SAOS-2 cells significantly decreased in shRNA-334 group contrast to the control group (P < 0.01). Cells proliferation was inhibited by shRNA-334 and shRNA-334 + cisplatin, and the effects were clearly enhanced when cells treated with the anticancer agents. The level of cell apoptosis in shRNA-334 and shRNA-334 + cisplatin group was higher than in the control group (P < 0.01). The current data support evidence that down-regulation of FAK could induce SAOS-2 apoptosis through the caspase-dependent cell death pathway. Inhibition of the kinases may be important for therapies designed to enhance the apoptosis in osteosarcoma.

Benzo-[a]-pyrene induces FAK activation and cell migration in MDA-MB-231 breast cancer cells.

Benzo-[a]-pyrene (B[a]P) is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant. It is a mammary carcinogen in rodents and contributes to the development of human breast cancer. However, the signal transduction pathways induced by B[a]P and its role in breast cancer progression have not been studied in detail. Here, we demonstrate that B[a]P induces cell migration through a lipoxygenase- and Src-dependent pathway, as well as the activation of focal adhesion kinase, Src, and the extracellular signal-regulated kinase 2 in MDA-MB-231 breast cancer cells. However, B[a]P is not able to promote migration in the mammary nontumorigenic epithelial cells MCF12A. Moreover, B[a]P promotes an increase of αvß3 integrin-cell surface levels and an increase of metalloproteinase (MMP)-2 and MMP-9 secretions. In summary, our findings demonstrate that B[a]P induces the activation of signal transduction pathways and biological processes involved in the invasion/metastasis process in MDA-MB-231 breast cancer cells.

Upregulation of focal adhesion kinase by 14-3-3ε via NFκB activation in hepatocellular carcinoma.

Focal adhesion kinase (FAK) is implicated in cancer cell survival, proliferation and migration. Expression of FAK expression is elevated and associated with tumor progression and metastasis in various tumors, including hepatocellular carcinoma (HCC). Increased 14-3-3ε expression is shown to be a potential prognostic factor to predict higher risk of distant metastasis and worse overall survival in HCC. The aim of this study is to investigate whether FAK is associated or regulated by 14-3-3ε to modulate tumor progression in HCC. In this study, 114 primary HCC tumors including 34 matched metastatic tumors were subjected to immunohistochemistry analysis of FAK and 14-3-3ε expression. Overexpression of FAK was significantly associated with increased risk of extrahepatic metastasis (p=0.027) and reduced 5-year overall survival rate (p=0.017). A significant correlation of FAK and 14-3-3ε expression was observed in primary tumor (p < 0.001) and also metastatic tumors. Furthermore, overexpression of 14-3-3 ε induced FAK expression and promoter activity which were determined by Western blotting analysis and luciferase-reporter assay. Moreover, 14-3-3ε enhanced NFκB activation and increased nuclear translocation of NFκB. Results from chromatin immunoprecipitation assay revealed that 14-3-3ε induced NFκB binding on FAK promoter region. These findings suggest that FAK expression is correlated with and upregulated by 14-3-3ε via activation of NFκB. Target to suppress or inactivate FAK alone, or combine with 14-3-3ε is thus considered as the potential therapeutic strategy for preventing HCC tumor progression.

Focal adhesion kinase splice variants maintain primitive acute myeloid leukemia cells through altered Wnt signaling.

Focal adhesion kinase (FAK) activity contributes to many advanced cancer phenotypes, but little is known about its role in human acute myeloid leukemia (AML). Here, we show that FAK splice variants are abnormally expressed in the primitive leukemic cells of poor prognosis AML patients. In the CD34(+) 38(-) 123(+) long-term culture-initiating cell-enriched leukemic cells of these patients, FAK upregulates expression of Frizzled-4 and phosphorylates Pyk2 to enable the required association of Pyk2 with the Wnt5a/Frizzled-4/LRP5 endocytosis complex and downstream activation of ß-catenin, thereby replacing the Wnt3a-controlled canonical pathway used by normal hematopoietic stem cells. Transduction of primitive normal human hematopoietic cells with FAK splice variants induces a marked increase in their clonogenic activity and signaling via the Wnt5a-controlled canonical pathway. Targeting FAK or ß-catenin efficiently eradicates primitive leukemic cells in vitro suggesting that FAK could be a useful therapeutic target for improved treatment of poor prognosis AML cases.