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Simple Summary: Protein tyrosine kinases (PTKs) are classified into two groups: one group includes tyrosine kinases, and the second group includes serine/threonine kinases. The group of tyrosine kinases includes both receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (NRTKs) that function as "on" or "off" switches for many cellular functions. NRTKs are kinase enzymes which are overexpressed and activated in many cancer types and regulate variable cellular functions, including cell growth and progression and their dependent mechanisms and the associated signaling pathways. Thus, targeting NRTKs is of great interest to improve the treatment strategy of different tumor types. Protein tyrosine kinases (PTKs) function as key molecules in the signaling pathways in addition to their impact as a therapeutic target for the treatment of many human diseases, including cancer. PTKs are characterized by their ability to phosphorylate serine, threonine, or tyrosine residues and can thereby rapidly and reversibly alter the function of their protein substrates in the form of significant changes in protein confirmation and affinity for their interaction with protein partners to drive cellular functions under normal and pathological conditions. PTKs are classified into two groups: one of which represents tyrosine kinases, while the other one includes the members of the serine/threonine kinases. The group of tyrosine kinases is subdivided into subgroups: one of them includes the member of receptor tyrosine kinases (RTKs), while the other subgroup includes the member of non-receptor tyrosine kinases (NRTKs). Both these kinase groups function as an "on" or "off" switch in many cellular functions. NRTKs are enzymes which are overexpressed and activated in many cancer types and regulate variable cellular functions in response to extracellular signaling-dependent mechanisms. NRTK-mediated different cellular functions are regulated by kinase-dependent and kinase-independent mechanisms either in the cytoplasm or in the nucleus. Thus, targeting NRTKs is of great interest to improve the treatment strategy of different tumor types. This review deals with the structure and mechanistic role of NRTKs in tumor progression and resistance and their importance as therapeutic targets in tumor therapy. [ABSTRACT FROM AUTHOR]

Kisspeptin (a product of the KISS1 gene and its receptor) plays an important role in obstetrics, gynecology, and cancer cell metastasis and behavior. In hypothalamic-pituitary-gonadal axis and placentation, Kisspeptin/Kisspeptin receptor affects hormone release and represses trophoblast invasion into maternal deciduae. Endometrial cancer is one of the common gynecological cancers and is usually accompanied by metastasis, the risk factor that causes death. Recently, research has demonstrated that Kisspeptin/Kisspeptin receptor expression in aggressive-stage endometrial cancer tissues. However, the detailed mechanism of Kisspeptin/Kisspeptin receptor in regulating the motility of endometrial cancers is not well understood. In this study, we use endometrial cancer cell lines RL95-2, Ishikawa, HEC-1-A, and HEC-1-B as models to explore the molecular mechanism of Kisspeptin on cell motility. First, we discovered that Kisspeptin/Kisspeptin receptor was expressed in endometrial cancer cells, and Kisspeptin significantly regulated the migration and invasion of endometrial cancer cells. Furthermore, we explored the epithelial–mesenchymal transition marker expression and the underlying signals were regulated on Kisspeptin treatment. In conclusion, we suggest that Kisspeptin regulates endometrial cancer cell motility via FAK and Src expression and the ERK1/2, N-Cadherin, E-Cadherin, beta-Catenin, Twist, and matrix metalloproteinase signaling pathways. We expect these molecules could be candidates for the development of new approaches and therapeutic targets. [ABSTRACT FROM AUTHOR]

Previous studies have indicated the efficacy of momordin Ic (MIc), a plant-derived triterpenoid, against several types of cancers, implying its potential for further development. However, comprehensive insights into the molecular mechanisms and targets of MIc in cholangiocarcinoma (CCA) are lacking. This study aimed to investigate the actions of MIc against CCA at the molecular level. Network pharmacology analysis was first employed to predict the mechanisms and targets of MIc. The results unveiled the potential involvement of MIc in apoptosis and cell migration, pinpointing Src and FAK as key targets. Subsequently, cell-based assays, in accordance with FAK/Src-associated metastasis, were conducted, demonstrating the ability of MIc to attenuate the metastatic behaviours of KKU-452 cells. The in vitro results further indicated the capability of MIc to suppress the epithelial-mesenchymal transition (EMT) process, notably by downregulating EMT regulators, including N-cadherin, vimentin, ZEB2 and FOXC1/2 expression. Furthermore, MIc suppressed the activation of the FAK/Src signalling pathway, influencing critical downstream factors such as MMP-9, VEGF, ICAM-1, and c-Myc. Molecular docking simulations also suggested that MIc could interact with FAK and Src domains and restrain kinases from being activated by hindering ATP binding. In conclusion, this study employs a comprehensive approach encompassing network pharmacology analysis, in vitro assays, and molecular docking to unveil the mechanisms and targets of MIc in CCA. MIc mitigates metastatic behaviours and suppresses key pathways, offering a promising avenue for future therapeutic strategies against this aggressive cancer.

Introduction: Osimertinib 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. Methods: We 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. Results: Phosphoproteomic 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. Conclusions: Phosphoproteomic 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.

Cancer is a serious health problem due to the complexity of establishing an effective treatment. The purpose of this work was to evaluate the activity of a triazaspirane as a migration and invasion inhibitor in PC3 prostatic tumor cells through a possible negative regulation of the FAK/Src signal transduction pathway and decreased secretion of metalloproteinases 2 and 9. Molecular docking analysis was performed using Moe 2008.10 software. Migration (wound-healing assay) and invasion (Boyden chamber assay) assays were performed. In addition, the Western blot technique was used to quantify protein expression, and the zymography technique was used to observe the secretion of metalloproteinases. Molecular docking showed interactions in regions of interest of the FAK and Src proteins. Moreover, the biological activity assays demonstrated an inhibitory effect on cell migration and invasion, an important suppression of metalloproteinase secretion, and a decrease in the expression of p-FAK and p-Src proteins in treated PC3 cells. Triazaspirane-type molecules have important inhibitory effects on the mechanisms associated with metastasis in PC3 tumor cells. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) is a multifunctional protein involved in cellular communication, integrating and transducing extracellular signals from cell-surface membrane receptors. It plays a central role intracellularly and extracellularly within the tumor microenvironment. Perturbations in FAK signaling promote tumor occurrence and development, and studies have revealed its biological behavior in tumor cell proliferation, migration, and adhesion. Herein we provide an overview of the complex biology of the FAK family members and their context-dependent nature. Next, with a focus on cancer, we highlight the activities of FAK signaling in different types of cancer and how knowledge of them is being used for screening natural compounds used in herbal medicine to fight tumor development. [ABSTRACT FROM AUTHOR]

Breast cancer is the most commonly diagnosed cancer among women, causing 15% of patient deaths. The metastasis of breast cancer cells is the leading cause of death for patients. Several studies have shown that Discoidin Domain Receptor 1 (DDR1) was highly expressed in breast cancer and could influence tumor cell behaviors. However, the specific role of DDR1 in breast cancer metastasis is still elusive. In this study, we uncovered that DDR1 is significantly increased in breast cancer and inversely correlated with the prognosis of patients. Knockdown of DDR1 suppressed the migration and invasion of breast cancer cells. Additionally, overexpression of DDR1 enhanced the metastatic capacity of cancer cells. Immunoblotting revealed that activation of Src and FAK, which are involved in cancer cell metastasis, were correlated with the expression level of DDR1. Co-immunoprecipitation experiments showed that DDR1 could bind to Src and FAK. Finally, the inhibition of FAK and Src could attenuate DDR1 enhanced migration ability of breast cancer cells. In summary, our study revealed that DDR1 was highly expressed in breast cancer and negatively correlated with the prognosis of breast cancer patients. DDR1 facilitates migration and invasion in breast cancer cells via activation of the Src-FAK signaling. Accordingly, blocking DDR1/Src/FAK axis is a promising therapeutic strategy for breast cancer treatment. [ABSTRACT FROM AUTHOR]

The engagement of cadherins, cell-to-cell adhesion proteins, triggers a dramatic increase in the levels and activity of the Rac/Cdc42 GTPases, through the inhibition of proteasomal degradation. This leads to an increase in transcription and secretion of IL6 family cytokines, activation of their common receptor, gp130, in an autocrine manner and phosphorylation of the signal transducer and activator of transcription-3 (Stat3) on tyrosine-705 by the Jak kinases. Stat3 subsequently dimerizes, migrates to the nucleus and activates the transcription of genes involved in cell division and survival. The Src oncogene also increases Rac levels, leading to secretion of IL6 family cytokines and gp130 activation, which triggers a Stat3-ptyr705 increase. Interestingly, at the same time, Src downregulates cadherins in a quantitative manner, while cadherins are required to preserve gp130 levels for IL6 family signalling. Therefore, a fine balance between Src527F/Rac/IL6 and Src527F/cadherin/gp130 levels is in existence, which is required for Stat3 activation. This further demonstrates the important role of cadherins in the activation of Stat3, through preservation of gp130 function. Conversely, the absence of cadherin engagement correlates with low Stat3 activity: In sparsely growing cells, both gp130 and Stat3-ptyr705 levels are very low, despite the fact that cSrc is active in the FAK (focal adhesion kinase)/cSrc complex, which further indicates that the engagement of cadherins is important for Stat3 activation, not just their presence. Furthermore, the caveolin-1 protein downregulates Stat3 through binding and sequestration of cadherins to the scaffolding domain of caveolin-1. We hypothesize that the cadherins/Rac/gp130 axis may be a conserved pathway to Stat3 activation in a number of systems. This fact could have significant implications in Stat3 biology, as well as in drug testing and development. [ABSTRACT FROM AUTHOR]

Cancer is a serious health problem due to the complexity of establishing an effective treatment. The purpose of this work was to evaluate the activity of a triazaspirane as a migration and invasion inhibitor in PC3 prostatic tumor cells through a possible negative regulation of the FAK/Src signal transduction pathway and decreased secretion of metalloproteinases 2 and 9. Molecular docking analysis was performed using Moe 2008.10 software. Migration (wound-healing assay) and invasion (Boyden chamber assay) assays were performed. In addition, the Western blot technique was used to quantify protein expression, and the zymography technique was used to observe the secretion of metalloproteinases. Molecular docking showed interactions in regions of interest of the FAK and Src proteins. Moreover, the biological activity assays demonstrated an inhibitory effect on cell migration and invasion, an important suppression of metalloproteinase secretion, and a decrease in the expression of p-FAK and p-Src proteins in treated PC3 cells. Triazaspirane-type molecules have important inhibitory effects on the mechanisms associated with metastasis in PC3 tumor cells.

Stimulation of the skin and muscles in the field of acupuncture and moxibustion has been shown to increase local blood flow and metabolism or maintain the body in a sustained healthy state. However, little is known about cellular structural changes in response to the electrical stimulus or how the localization of specific proteins is regulated by such stimulation. Cultured fibroblasts were subject to periodic electrical stimulation for 0 min (the unstimulated control), 2 h, 5 h, and 20 h. After 2 h, cell stress fibers and focal adhesions became enlarged, and the stress fibers exhibited an increase in thickness. Within 20 h of periodic stimulation, both the stress fibers and focal adhesions gradually became larger and thicker. An anti-phosphotyrosine antibody (PY-20) was used to stain the cells after electrical stimulations, which showed the increased staining of focal adhesions. They also exhibited the increased staining of the active form of a focal adhesion kinase (FAK) (pY397) and c-Src (pY418), indicating that electrical stimulation had affected certain proteins associated with signal transduction. An ELISA analysis showed that 20 h of electrical stimulation gradually increased the amount of the active form of c-Src until it was approximately tripled, whereas 5 h of electrical stimulation approximately doubled the amount of the active form of FAK, this being the maximum reached. These findings indicate that electrical stimulation stimulates the activity of c-Src and FAK signaling proteins and alters the structure of the actin cytoskeleton and focal adhesions.

During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

Major depressive disorder is a chronic psychiatric disease with a high prevalence. Brain mechanisms for depression at cellular and molecular levels are far from clear. Increasing evidence from clinical and preclinical studies reveals critical roles of the non-receptor tyrosine kinase (nRTK) superfamily in the pathophysiology, symptomatology, and therapy of depression. To date, several nRTK members from three nRTK subfamilies, i.e., the Src family kinase (SFK), the Janus tyrosine kinase (JAK) and the focal adhesion kinase (FAK) subfamilies, may connect to the intracellular, intranuclear, and synaptic signaling network linking chronic stress to depression- and anxiety-like behavior. These SFK/JAK/FAK nRTKs are abundantly expressed in the prefrontal cortex and hippocampus, two core limbic regions implicated in depression, and are enriched at synaptic sites. In various acute or chronic animal models of depression, the nRTKs were significantly altered (up- or downregulated) in their phosphorylation, expression, subcellular/subsynaptic distribution, and/or function. Stress that precipitates depressive behavior also influenced the interaction of nRTKs with other signaling molecules and downstream substrates, including ionotropic and metabotropic glutamate receptors. The commonly-used antidepressants showed the ability to alter nRTK activity. In sum, the limbic SFK/JAK/FAK nRTKs are sensitive to stress and undergo drastic adaptations in response to chronic depression. These long-lasting adaptations contribute to the remodeling of signaling network or synaptic plasticity critical for the vulnerability to depression and the therapeutic efficacy of antidepressants. [ABSTRACT FROM AUTHOR]

Owing to the crucial role of Src in cancer metastasis, interruption of Src and its signaling has been considered a promising strategy for cancer metastasis treatment. Cucurbitacin B, a dietary triterpenoid, has been shown to possess anti-proliferative and apoptosis-inducing activities in cholangiocarcinoma (CCA) cells via suppressing the activation of FAK which is a main downstream Src effector. We hypothesized that cucurbitacin B might act as a Src suppressant which conferring anti-metastasis effect against CCA cells. To investigate this, the role of Src in regulating metastasis behavior of CCA cells and the effect of cucurbitacin B on Src-mediated metastatic phenotype of these cells were determined. The results showed that activation of Src significantly enhanced the migratory and invasive abilities of CCA cells. Molecular analysis revealed that Src-facilitated metastasis behavior of CCA cells occurred by modifying expression of a wide range of metastasis-related genes in the cells. Consistent with gene expression results, activation of Src significantly induced the protein expression of 2 important metastasis-associated molecules, MMP-9 and VEGF. Cucurbitacin B markedly suppressed activation of Src and its key effector, FAK. As a consequence, the alteration of expression profiles of metastasis-associated genes induced by Src activator in CCA cells was diminished by cucurbitacin B treatment. The compound also down-regulated Src-induced expression of MMP-9 and VEGF proteins in the cells. Moreover, molecular docking analysis revealed that cucurbitacin B could interact with Src kinase domain and possibly restrain the kinase from being activated by hindering the ATP binding. In conclusion, cucurbitacin B exhibited anti-metastatic property in CCA cells via negatively influencing Src and Src-related oncogenic signaling. This compound may therefore be a potential therapeutic drug for further development as an anti-Src agent for treatment of metastatic CCA. [ABSTRACT FROM AUTHOR]

Abstract Background Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood. Methods In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression. Results COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor. Conclusion COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC.

Breast cancer is the most common invasive neoplasia, and the second leading cause of the cancer deaths in women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloprotease secretion, and invasion. We found that leptin activates FAK and Src and induces the localization of FAK to the focal adhesions. Interestingly, leptin promotes the activation of FAK through a Src- and STAT3-dependent canonical pathway. Specific inhibitors of FAK, Src and STAT3 sho wed that the effect exerted by leptin in cell migration in breast cancer cells is dependent on these proteins. Moreover, we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9 and invasion in a FAK and Src-dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.

Focal adhesion kinase (FAK) is a multifunctional protein involved in cellular communication, integrating and transducing extracellular signals from cell-surface membrane receptors. It plays a central role intracellularly and extracellularly within the tumor microenvironment. Perturbations in FAK signaling promote tumor occurrence and development, and studies have revealed its biological behavior in tumor cell proliferation, migration, and adhesion. Herein we provide an overview of the complex biology of the FAK family members and their context-dependent nature. Next, with a focus on cancer, we highlight the activities of FAK signaling in different types of cancer and how knowledge of them is being used for screening natural compounds used in herbal medicine to fight tumor development.

Focal adhesion kinase (FAK), human myofibrillogenesis regulator-1 (MR-1), ephrin receptor type A4 (EphA4), proto-oncogene tyrosine kinase Src (Src), and protein kinase C (PKC) are important markers in proliferation, survival, and migration in some cancers. However, the significance of each is still unclear in different malignancies, including acute myeloid leukemia (AML). Therefore, this study was conducted to investigate their serum levels in Egyptian adult de novo AML patients (n = 70) against healthy volunteers (n = 20). We managed to study the correlation between each pair and to investigate their association with diagnosis, prognosis, and survival. Serum levels were analyzed using enzyme-linked immunosorbent assay (ELISA). We found that FAK, MR-1, Src, and PKC serum levels were significantly higher in AML patients compared to control (p < 0.0001), and this was associated with significantly lower EphA4 level (p < 0.0001). Interestingly, we also observed a significant negative correlation of FAK (p = 0.027), MR-1 (p = 0.003), Src (p = 0.038), and PKC (p = 0.03) with patients' overall survival (OS) while there was a positive significant correlation between EphA4 and OS (p = 0.007). In conclusion, this study suggests that FAK, MR-1, EphA4, Src, and PKC may be used as early diagnostic and prognostic markers with high sensitivity and specificity in AML patients and thus may be incorporated into the patients' early diagnostic and prognostic panels. [ABSTRACT FROM AUTHOR]

Procaspase-8, the zymogen form of the apoptosis-initiator caspase-8, undergoes phosphorylation following integrin-mediated cell attachment to an extracellular matrix substrate. Concordant with cell attachment to fibronectin, a population of procaspase-8 becomes associated with a peripheral insoluble compartment that includes focal complexes and lamellar microfilaments. Phosphorylation of procaspase-8 both impairs its maturation to the proapoptotic form and can promote cell migration. Here we show that the cytoskeletal adaptor protein CrkL promotes caspase-8 recruitment to the peripheral spreading edge of cells, and that the catalytic domain of caspase-8 directly interacts with the SH2 domain of CrkL. We show that the interaction is abolished by shRNA-mediated silencing of Src, in Src-deficient MEFs, and by pharmacologic inhibitors of the kinase. The results provide insight into how tyrosine kinases may act to coordinate the suppression caspase-8 mediated apoptosis, while promoting cell invasion.

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia. • FAK is persistently activated by mild hypothermia and regulates Rbm3 transcription. • Src is activated by FAK and regulates hypothermia-induced Rbm3 transcription. • Transcriptional factor NF-κB acts downstream of FAK/Src signaling under mild hypothermia. • FAK/Src signaling mediates neuroprotective effects of cooling against rotenone. • MAPK signaling pathways are dispensable for hypothermia-induced RBM3 expression. [ABSTRACT FROM AUTHOR]

Major depressive disorder is a chronic psychiatric disease with a high prevalence. Brain mechanisms for depression at cellular and molecular levels are far from clear. Increasing evidence from clinical and preclinical studies reveals critical roles of the non-receptor tyrosine kinase (nRTK) superfamily in the pathophysiology, symptomatology, and therapy of depression. To date, several nRTK members from three nRTK subfamilies, i.e., the Src family kinase (SFK), the Janus tyrosine kinase (JAK) and the focal adhesion kinase (FAK) subfamilies, may connect to the intracellular, intranuclear, and synaptic signaling network linking chronic stress to depression- and anxiety-like behavior. These SFK/JAK/FAK nRTKs are abundantly expressed in the prefrontal cortex and hippocampus, two core limbic regions implicated in depression, and are enriched at synaptic sites. In various acute or chronic animal models of depression, the nRTKs were significantly altered (up- or downregulated) in their phosphorylation, expression, subcellular/subsynaptic distribution, and/or function. Stress that precipitates depressive behavior also influenced the interaction of nRTKs with other signaling molecules and downstream substrates, including ionotropic and metabotropic glutamate receptors. The commonly-used antidepressants showed the ability to alter nRTK activity. In sum, the limbic SFK/JAK/FAK nRTKs are sensitive to stress and undergo drastic adaptations in response to chronic depression. These long-lasting adaptations contribute to the remodeling of signaling network or synaptic plasticity critical for the vulnerability to depression and the therapeutic efficacy of antidepressants.

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2.

Background: Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood. Methods: In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression. Results: COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor. Conclusion: COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC. [ABSTRACT FROM AUTHOR]

BackgroundHere, we explore the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, including members of the Src kinase family, EphA2, and focal adhesion kinase for the treatment of ovarian cancer.MethodsWe examined the effects of dasatinib on proliferation, invasion, apoptosis, cell-cycle arrest, and kinase activity using a panel of 34 established human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions with chemotherapeutic drugs.ResultsConcentration-dependent anti-proliferative effects of dasatinib were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC(50) values (IC(50) range: 0.001-11.3 micromol l(-1)). Dasatinib significantly inhibited invasion, and induced cell apoptosis, but less cell-cycle arrest. At a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for dasatinib plus carboplatin (mean CI values, range: 0.73-1.11) or paclitaxel (mean CI values, range: 0.76-1.05). In this study, 24 out of 34 (71%) representative ovarian cancer cell lines were highly sensitive to dasatinib, compared with only 8 out of 39 (21%) representative breast cancer cell lines previously reported. Cell lines with high expression of Yes, Lyn, Eph2A, caveolin-1 and 2, moesin, annexin-1, and uPA were particularly sensitive to dasatinib.ConclusionsThese data provide a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in patients with ovarian cancer.

Oxidative stress is associated with functional disorder of trophoblast cells. Our previous studies have demonstrated that cyclosporin A (CsA) promotes the activity of normal human trophoblast cells. We further investigated the role and mechanism of CsA on oxidative stress in trophoblast cells. JEG-3 cells were co-cultured with H 2 O 2 and CsA. Cell viability and morphology were measured by MTT assay and inverted microscope. Reactive oxygen species (ROS) was analyzed by fluorescence microscopy. Cell mitochondrial membrane potential (MMP) was determined by flow cytometric analysis. Malondialdehyde (MDA) production, superoxide dismutase (SOD) and catalase (CAT) activities were examined using colorimetric assays. The expression and phosphorylation of FAK and Src kinase proteins were examined by western blotting. CsA increased JEG-3 cell viability and reduced the morphologic injury induced by H 2 O 2 treatment. CsA decreased ROS and MDA production, increased SOD and CAT activities, and restored the MMP of H 2 O 2 treated JEG-3 cells. CsA administration suppressed H 2 O 2 -induced reduction of FAK and Src phosphorylation. Blocking the activation of FAK or Src attenuated the protective effect of CsA on JEG-3 cells in H 2 O 2 -induced oxidative injury. CsA protects JEG-3 cells from H 2 O 2 -induced oxidative injury, and the FAK/Src signaling pathway plays an important role in this process. • H 2 O 2 -induced oxidative stress in trophoblast cells. • CsA recovered JEG-3 cell viability and morphologic injury induced by H 2 O 2. • CsA decreased oxidative injury by increasing anti-oxidation system in JEG-3 cells. • CsA restored the mitochondrial membrane potential of JEG-3 cells damaged by H 2 O 2. • FAK/Src pathway is necessary for CsA anti-oxidative stress activity in JEG-3 cells. [ABSTRACT FROM AUTHOR]

Study Question: 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?Summary Answer: 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.What Is Known Already: 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.Study Design, Size, Duration: 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.Participants/materials, Setting, Methods: 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.Main Results and the Role Of Chance: 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%).Limitations, Reasons For Caution: Human decidual stromal cells were obtained from women having terminations after 6-12 weeks of pregnancy and differences in timing could affect their properties.Wider Implications Of the Findings: Kisspeptin acting within the endometrium has a potential modulatory role on embryo implantation and decidual programming of human pregnancy.Study Funding/competing Interest(s): 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.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

The biophysical microenvironment of the tumor site has significant impact on breast cancer progression and metastasis. The importance of altered mechanotransduction in cancerous tissue has been documented, yet its role in the regulation of cellular metabolism and the potential link between cellular energy and cell migration remain poorly understood. In this study, we investigated the role of mechanotransduction in AMP-activated protein kinase (AMPK) activation in breast cancer cells in response to interstitial fluid flow (IFF). Additionally, we explored the involvement of AMPK in breast cancer cell migration. IFF was applied to the 3D cell-matrix construct. The subcellular signaling activity of Src, FAK, and AMPK was visualized in real-time using fluorescent resonance energy transfer (FRET). We observed that breast cancer cells (MDA-MB-231) are more sensitive to IFF than normal epithelial cells (MCF-10A). AMPK was activated at the mitochondria of MDA-MB-231 cells by IFF, but not in other subcellular compartments (i.e., cytosol, plasma membrane, and nucleus). The inhibition of FAK or Src abolished flow-induced AMPK activation in the mitochondria of MDA-MB-231 cells. We also observed that global AMPK activation reduced MDA-MB-231 cell migration. Interestingly, specific AMPK inhibition in the mitochondria reduced cell migration and blocked flow-induced cell migration. Our results suggest the linkage of FAK/Src and mitochondria-specific AMPK in mechanotransduction and the differential role of AMPK in breast cancer cell migration depending on its subcellular compartment-specific activation. • In response to interstitial fluid flow, AMPK was activated at the mitochondria of MDA-MB-231 cells. • The inhibition of FAK or Src abolished flow-induced AMPK activation at the mitochondria. • AMPK inhibition at the mitochondria reduced cell migration and blocked flow-induced cell migration. • AMPK inhibition at the mitochondria triggered cytosolic AMPK increase. [ABSTRACT FROM AUTHOR]

Aim: To study the carcinogenetic mechanism of HOXB7 in gastric cancer (GC) remains. Methods: Two human GC cell lines — SGC7901 and SNU1 — were used for this study. SGC7901 cells were transfected with siRNA-HOXB7 (siHOXB7) to knock down HOXB7 expression, whereas, SNU1 cells were transduced with pCDNA3.1-HOXB7 to overexpress HOXB7. After transfection, cancer progression was assessed by determining cell proliferation, wound-healing process, cell cycle, apoptosis, invasion, and migration. The effect of HOXB7 on epithelial–mesenchymal transition (EMT) was measured by observing changes in F-actin cytoskeleton and evaluating the expression of EMT markers. p-Scr and p-FAK were evaluated to assess the mechanism. Results: Knockdown of HOXB7 suppressed cell proliferation, alleviated the wound-healing process, inhibited cell migration and invasion, and arrested the cell cycle while promoting cell apoptosis, suggesting the tumor-suppressive effect of siHOXB7 in human GC cells. On the contrary, HOXB7 overexpression showed a tumor-promoting effect on human GC cells. Moreover, we confirmed an inhibitory effect of siHOXB7 on the EMT process by preventing epithelial cells from acquiring a mesenchymal phenotype and downregulating mesenchymal markers (vimentin, β-catenin, N-cadherin, Twist) while upregulating epithelial markers (E-cadherin). Our data revealed that HOXB7 was associated with Src/FAK and favored the activation of the Src–FAK pathway in human GC cells. Conclusion: HOXB7 accelerated the malignancy of GC, by facilitating EMT and regulating the Scr–FAK pathway. [ABSTRACT FROM AUTHOR]

Abstract Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the β 1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether β 1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1β (IL-1β)-induced inflammatory in vitro BBB model. We demonstrate that IL-1β increases cell-matrix adhesion and induces a redistribution of active β 1 integrins to the basal surface. In particular, binding of α 5 β 1 integrin to its ligand fibronectin is enhanced and α 5 β 1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α 5 β 1 integrin reduces the IL-1β-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1β-induced inflammation not only destabilizes tight junctions but also increases α 5 β 1 integrin-dependent cell-matrix adhesion to fibronectin. Graphical abstract fx1 Highlights • IL-1β-induced inflammation of THBMECs increases cell-matrix adhesion. • Active β 1 integrins are redistributed under inflammatory conditions. • IL-1β activates the FN/α 5 β 1 integrin/FAK/Src-signalling pathway. • Blocking the α 5 integrin subunit reduces IL-1β-induced transendothelial migration. [ABSTRACT FROM AUTHOR]

Abstract Purpose Statins extended their hypocholestremic effect to show a promising anticancer activity. Hepatocellular carcinoma (HCC), the third common cause of cancer-related death, responded positively to statins. Some in-vitro studies reveal the rosuvastatin antitumor effect, but barely in-vivo studies. Hence, we evaluated the antitumor potential of rosuvastatin in a HCC model, the possible signaling cues involved, and whether it augments the dasatinib anticancer effect. Method For the in-vitro study, the IC 50 and the combination (CI)/dose reduction (DRI) indices were determined for HCC cell line (HepG2) treated with dasatinib and/or rosuvastatin. For the in-vivo study, mice with diethylnitrosamine-induced HCC were treated for 21 days with dasatinib and/or rosuvastatin (10 and 20 mg/kg, respectively). The p -focal adhesion kinase/ p- rous sarcoma oncogene cellular homolog (p -FAK/ p -Src) cascade and its downstream molecules were assessed. Results The in-vitro study confirmed the synergistic effect of rosuvastatin with dasatinib, which entailed the in-vivo results. The two drugs decreased the p -FAK/ p -Src cue along with p -Ras/c-Raf, p -STAT-3, and p -Akt levels to enhance apoptosis by an increase in caspase-3 level and a decline in survivin level. Additionally, they inhibited HGF, VEGF, and the MMP-9. Moreover, the different treatments downregulated the expression of proliferative cell nuclear antigen (PCNA) and Ki-67. The best effect was mediated by the combination regimen that surpassed the effect of either drug alone. Conclusion Our results highlighted some of the signals involved in rosuvastatin antitumor effect and nominate it as an adds-on therapy with dasatinib to yield a better effect in HCC through inhibiting the FAK/Src cascade. [ABSTRACT FROM AUTHOR]

EPLIN is frequently downregulated or lost in various cancers. The purpose of this study was to evaluate the importance of EPLIN in prostate cancer progression, with particular focus on the mechanistic implications to elucidate EPLIN's tumor suppressive function in cancer. EPLIN expression was evaluated in prostate cancer cell lines and tissues. PC‐3 and LNCaP EPLINα overexpression models were generated through transfection with EPLINα sequence and EPLIN knockdown was achieved using shRNA in CA‐HPV‐10 cells. Functional assays were performed to evaluate cellular characteristics and potential mechanisms were evaluated using a protein microarray, and validated using western blot analysis. EPLIN expression was reduced in clinical prostate cancer sections, including hyperplasia (p ≤ 0.001) and adenocarcinoma (p = 0.005), when compared to normal prostate tissue. EPLINα overexpression reduced cell growth, migration and invasion, and influenced transcript, protein and phosphoprotein expression of paxillin, FAK and Src. EPLIN knockdown increased the invasive and migratory nature of CA‐HPV‐10 cells and also induced changes to FAK and Src total and/or phospho expression. Functional characterization of cellular migration and invasion in addition to FAK and Src inhibition demonstrated differential effects between control and EPLINα overexpression and EPLIN knockdown cell lines. This study highlights that EPLIN expression in prostate cancer is able to influence several aspects of cancer cell characteristics, including cell growth, migration and invasion. The mechanism of the tumor suppressive action of EPLIN remains to be fully elucidated; and this study proposes a role for EPLIN's ability to regulate the aggressive characteristics of prostate cancer cells partially through regulating FAK/Src signaling. What's new? There is growing evidence that EPLIN is a tumor suppressor molecule in several human cancers. The underlying mechanisms, however, remain unclear. Here, the authors provide functional evidence that EPLIN is a negative regulator of prostate cancer and acts as a tumor suppressor to impede metastatic traits. They document downregulation of EPLIN in clinical prostate cancer and a potential mechanistic link between EPLIN and proto‐oncogene tyrosine‐protein kinase (Src)/Focal adhesion kinase (FAK) signaling. This study provides insights into molecular associations which drive the regulatory actions of EPLIN, and establishes a broader picture of the signaling cascades coordinating cellular functions in prostate cancer. [ABSTRACT FROM AUTHOR]

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

Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.

During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2. [ABSTRACT FROM AUTHOR]

Methylmercury (MeHg) is an environmental neurotoxicant that inhibits neuronal migration. This process requires several cyclic steps involving the formation of membrane protrusions (lamellipodia and filopodia) and focal adhesion turnover. FAK and Src are critical proteins that regulate both processes. The FAK-Src complex promotes the activation of Rac1 and Cdc42, two GTPases involved in the remodeling of the actin cytoskeletal network. Here, we studied the effect of MeHg (1, 10, 100, 500 and 1000 nM) on cell migration, the formation of cell protrusions, focal adhesion location and the activation of FAK, Src, Rac1 and Cdc42 using the SH-SY5Y neuroblastoma cell line stimulated with PDGF-BB (PDGF). The data show that MeHg (1–500 nM) inhibited PDGF-stimulated cell migration. In PDGF-stimulated cells, MeHg (100–1000 nM) decreased protrusions and increased the size of the p-FAK Y397 clusters. MeHg also inhibited PDGF-induced FAK and Src activation and, at 100 nM, MeHg inhibited the activation of Rac1 and Cdc42. Altogether, the findings show that low concentrations of MeHg inhibit SH-SY5Y cell migration by disrupting the activation and disassembly of FAK. This negatively affects the activation of Src, Rac1 and Cdc42, all of which are critical proteins for the regulation of cell movement. These effects could be related to the MeHg-mediated inhibition of PDGF-induced formation of lamellipodia and filopodia, focal adhesion disassembly and PDGF-induced movement. [ABSTRACT FROM AUTHOR]

Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described ‘spatial rheostat’ controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin one and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs.

Summary Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT) by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity.

Leptin is one of the main adipokines secreted in breast tissue. Leptin promotes epithelial−mesenchymal transition (EMT), cell migration and invasion in epithelial breast cells, leading to tumor progression. Although, the molecular mechanisms that underlie these events are not fully understood, the activation of different signaling pathways appears to be essential. In this sense, the effects of leptin on the activation of kinases like Src and FAK, which regulate signaling pathways that activate the EMT program, are not completely described. Therefore, we investigated the involvement of these kinases using an in vitro model for leptin-induced EMT process in the non-tumorigenic MCF10A cell line. To this end, MCF10A cells were stimulated with leptin, and Src and FAK activation was assessed. Specific events occurring during EMT were also evaluated in the presence or absence of the kinases’ chemical inhibitors PP2 and PF-573228. For instance, we tested the expression and subcellular localization of the EMT-related transcription factors Twist and β-catenin, by western blot and immunofluorescence. We also evaluated the secretion and activation of matrix metalloproteases (MMP-2 and MMP-9) by gelatin zymography. Invasiveness properties of leptin-stimulated cells were determined by invadopodia formation assays, and by the Transwell chamber method. Our results showed that leptin promotes EMT through Src and FAK activation, which leads to the secretion and activation of MMP-2 and MMP-9, invadopodia formation and cell invasion in MCF10A cells. In conclusion, our data suggest that leptin promotes an increase in the expression levels of Twist and β-catenin, the secretion of MMP-2, MMP-9, the invadopodia formation and invasion in MCF10A cells in a Src and FAK-dependent manner.

Arecoline is the primary alkaloid in betel nuts, which are known as a risk factor for oral submucosal fibrosis and oral cancer. Lung cancer is a severe type of carcinoma with high cell motility that is difficult to treat. However, the detailed mechanisms of the correlation between Arecoline and lung cancer are not fully understood. Here, we investigated the effect of Arecoline on migration in lung cancer cell lines and its potential mechanism through the muscarinic acetylcholine receptor 3 (mAChR3)-triggered EGFR/Src/FAK pathway. Our results indicate that different concentrations of Arecoline treatment (10 µM, 20 µM, and 40 µM) significantly increased the cell migration ability in A549 and CL1-0 cells and promoted the formation of the filamentous actin (F-actin) cytoskeleton, which is a crucial element for cell migration. However, migration of H460, CL1-5, and H520 cell lines, which have a higher migration ability, was not affected by Arecoline treatment. The EGFR/c-Src/Fak pathway, which is responsible for cell migration, was activated by Arecoline treatment, and a decreased expression level of E-cadherin, which is an epithelial marker, was observed in Arecoline-treated cell lines. Blockade of the EGFR/c-Src/Fak pathway with the inhibitors of EGFR (Gefitinib) or c-Src (Dasatinib) significantly prevented Arecoline-promoted migration in A549 cells. Gefitinib or Dasatinib treatment significantly disrupted the Arecoline-induced localization of phospho-Y576-Fak during focal adhesion in A549 cells. Interestingly, Arecoline-promoted migration in A549 cells was blocked by a specific mAChR3 inhibitor (4-DAMP) or a neutralizing antibody of matrix metalloproteinase (MMP7 or Matrilysin). Taken together, our findings suggest that mAChR3 might play an essential role in Arecoline-promoted EGFR/c-Src/Fak activation and migration in an A549 lung cancer cell line.

Tumor angiogenesis is one of the major hallmarks of tumor progression. Nobiletin is a natural flavonoid isolated from citrus peel that has anti-angiogenic activity. Steroid receptor coactivator (Src) is an intracellular tyrosine kinase so that focal adhesion kinase (FAK) binds to Src to play a role in tumor angiogenesis. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis which interacts with Src. Paxillin (PXN) acts as a downstream target for both FAK and STAT3. The main goal of this study was to assess inhibition of tumor angiogenesis by nobiletin in estrogen receptor positive (ER+) breast cancer cells via Src, FAK, and STAT3-mediated signaling through PXN. Treatment with nobiletin in MCF-7 and T47D breast cancer cells inhibited angiogenesis markers, based on western blotting and RT-PCR. Validation of in vitro angiogenesis in the human umbilical vein endothelial cells (HUVEC) endothelial cell line proved the anti-angiogenic activity of nobiletin. Electrophoretic mobility shift assay and the ChIP assay showed that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the PXN gene promoter. We also investigated the migration and invasive ability of nobiletin in ER+ cells. Nobiletin inhibited tumor angiogenesis by regulating Src, FAK, and STAT3 signaling through PXN in ER+ breast cancer cells. [ABSTRACT FROM AUTHOR]

Background:Neuroblastoma (NB) often presents with metastatic disease and poor survival. The need for new prognostic markers remains invaluable. The FAK-Src-Paxillin protein system is associated with aggressive phenotype in adult malignancies but is largely unexplored in pediatric NB.Objective:To assess FAK-Src-Paxillin protein expression in human NB cell lines and clinical cytology material and to delineate its association with survival.Design/Methods:Western blot and immunohistochemistry were applied for FAK-Src-Paxillin expression in NB cell lines and 23 human cytology specimens, respectively. Protein expression in human clinical samples was correlated with clinicopathological parameters,MYCNamplification and survival.Results:FAK, Src and Paxillin proteins are expressed in human NB cells lines, and can be detected in clinical cytology specimens from NB patients, (59%, 32% and 33% respectively). Simultaneous FAK-Src-Paxillin expression was noted in 30% of NB patients. Children with concomitant positivity FAK, Src, and Paxillin tumors, as well asMYCNamplification, had increased mortality compared to those without.Conclusions:FAK-Src-Paxillin system is a marker of unfavorable prognosis for human NB patients but also a promising therapeutic target. [ABSTRACT FROM AUTHOR]

Interleukin-1β (IL-1b) is a pleiotropic cytokine that is important in tumor progression and invasion. Matrix metalloproteinase-9 (MMP-9), which is a secreted matrix-degrading enzyme, is one of the key regulators of tumor invasion and metastasis. The current report indicated that IL-1b promotes MMP-9 production and cell invasion in non-metastatic MCF-7 breast cancer cells. IL-1b activated focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src). Moreover, inhibiting the Src/FAK pathway reduced the IL-1b-induced production of MMP-9 and cell invasion. To investigate the functional role of FAK in MMP-9 production cell lines expressing mutant FAK in FAK knock-out mouse fibroblasts were generated. In wild-type FAK-expressing cells, MMP-9 production was induced by IL-1b stimulation. By contrast, IL-1b-induced MMP-9 production was abrogated in FAK knock-out, FAK Y397F, FAK Y925F, and kinase dead mutant-expressing cells. Therefore the results of the current study indicate that FAK and Src kinases are activated by IL-1b and play a critical role in MMP-9 production and tumor cell invasion. [ABSTRACT FROM AUTHOR]