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Background: Recycling of integrin via endosomal vesicles is critical for the migration of cancer cells, which leads to the metastasis of pancreatic cancer and devastating cancer-related death. So, new diagnostic and therapeutic molecules which target the recycling of endosomal vesicles need to be developed. Methods: Public databases including TCGA, ICGC, GSE21501, GSE28735, and GENT are analyzed to derive diagnostic and therapeutic targets. To reveal biological roles and underlying mechanisms of molecular targets, various molecular biological experiments were conducted. Results: First, we identified UNC13D's overexpression in patients with pancreatic cancer (n = 824) and its prognostic significance and high hazard ratio (HR) in four independent pancreatic cancer cohorts (TCGA, n = 178, p = 0.014, HR = 3.629; ICGC, n = 91, p = 0.000, HR = 4.362; GSE21501, n = 102, p = 0.002, HR = 2.339; GSE28735, n = 45, p = 0.022, HR = 2.681). Additionally, its expression is associated with the clinicopathological progression of pancreatic cancer. Further biological studies have shown that UNC13D regulates the migration of pancreatic cancer cells by coupling the exocytosis of recycling endosomes with focal adhesion turnover via the regulation of FAK phosphorylation. Immunoprecipitation and immunocytochemistry showed the formation of the RAB11-UNC13D-FAK axis in endosomes during integrin recycling. We observed that UNC13D directly interacted with the FERM domain of FAK and regulated FAK phosphorylation in a calcium-dependent manner. Finally, we found co-expression of UNC13D and FAK showed the poorest survival (TCGA, p = 0.000; ICGC, p = 0.036; GSE28735, p = 0.006). Conclusions: We highlight that UNC13D, a novel prognostic factor, promotes pancreatic cancer progression by coupling integrin recycling with focal adhesion turnover via the RAB11-UNC13D-FAK axis for the migration of pancreatic cancer cells. [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]

Cancer is one of the deadliest human diseases, causing high rates of illness and death. Lung cancer has the highest mortality rate among all malignancies worldwide. Effusanin B, a diterpenoid derived from Isodon serra, showed therapeutic potential in treating non-small-cell lung cancer (NSCLC). Further research on the mechanism indicated that effusanin B inhibited the proliferation and migration of A549 cells both in vivo and in vitro. The in vitro activity assay demonstrated that effusanin B exhibited significant anticancer activity. Effusanin B induced apoptosis, promoted cell cycle arrest, increased the production of reactive oxygen species (ROS), and altered the mitochondrial membrane potential (MMP). Based on mechanistic studies, effusanin B was found to inhibit the proliferation and migration of A549 cells by affecting the signal transducer and activator of transcription 3 (STAT3) and focal adhesion kinase (FAK) pathways. Moreover, effusanin B inhibited tumor growth and spread in a zebrafish xenograft model and demonstrated anti-angiogenic effects in a transgenic zebrafish model. [ABSTRACT FROM AUTHOR]

Hepatoblastoma is the most frequent liver malignancy in children. HepG2 has been discovered as a hepatoblastoma-derived cell line and tends to form clumps in culture. Intriguingly, we observed that the addition of calcium ions reduced cell clumping and disassociated HepG2 cells. The calcium signal is in connection with a series of processes critical in the tumorigenesis. Here, we demonstrated that extracellular calcium ions induced morphological changes and enhanced the epithelial-mesenchymal transition in HepG2 cells. Mechanistically, calcium ions promoted HepG2 proliferation and migration by up-regulating the phosphorylation levels of focal adhesion kinase (FAK), protein kinase B, and p38 mitogen-activated protein kinase. The inhibitor of FAK or Ca2+/calmodulin-dependent kinase II (CaMKII) reversed the Ca2+-induced effects on HepG2 cells, including cell proliferation and migration, epithelial-mesenchymal transition protein expression levels, and phosphorylation levels of FAK and protein kinase B. Moreover, calcium ions decreased HepG2 cells' sensitivity to cisplatin. Furthermore, we found that the expression levels of FAK and CaMK II were increased in hepatoblastoma. The group with high expression levels of FAK and CaMK II exhibited significantly lower ImmunoScore as well as CD8+ T and NK cells. The expression of CaMK II was positively correlated with that of PDCD1 and LAG3. Correspondingly, the expression of FAK was negatively correlated with that of TNFSF9, TNFRSF4, and TNFRSF18. Collectively, extracellular calcium accelerates HepG2 cell proliferation and migration via FAK and CaMKII and enhances cisplatin resistance. FAK and CaMKII shape immune cell infiltration and responses in tumor microenvironments, thereby serving as potential targets for hepatoblastoma. [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. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs) play important roles in the progression of glioma. Here, we examined the potential functions of a lncRNA, LINC01003, in glioma and characterized the underlying molecular mechanisms. Methods: The GEIPA2 and Chinese Glioma Genome Atlas (CCGA) databases were employed to analyze gene expression and the overall survival curve in patients with glioma. The functions of LINC01003 in glioma growth and migration were assessed by loss-of-function experiments in vitro and in vivo. RNA sequencing was used to determine the signaling pathways effected by LINC01003. Bioinformatics analysis and RNA immunoprecipitation (RIP) assays were used to explore the mechanism underlying the N6-methyladenine (m6A) modification-dependent upregulation of LINC01003 in glioma. Results: LINC01003 expression was upregulated in glioma cell lines and tissues. Higher LINC01003 expression predicted shorter overall survival time in glioma patients. Functionally, LINC01003 knockdown inhibited the cell cycle and cell proliferation and migration in glioma cells. Mechanistically, RNA sequencing revealed that LINC01003 mediated the focal adhesion signaling pathway. Furthermore, LINC01003 upregulation is induced by m6A modification regulated by METTL3. Conclusion: This study characterized LINC01003 as a lncRNA that contributes to tumorigenesis in glioma and demonstrated that the LINC01003-CAV1-FAK axis serves as a potential therapeutic target for glioma. [ABSTRACT FROM AUTHOR]

FAK mediated tumour cell migration, invasion, survival, proliferation and regulation of tumour stem cells through its kinase-dependent enzymatic functions and kinase-independent scaffolding functions. At present, the development of FAK PROTACs has become one of the hotspots in current pharmaceutical research to solve above problems. Herein, we designed and synthesised a series of FAK-targeting PROTACs consisted of PF-562271 derivative 1 and Pomalidomide. All compounds showed significant in vitro FAK kinase inhibitory activity, the IC50 value of the optimised PROTAC A13 was 26.4 nM. Further, A13 exhibited optimal protein degradation (85% degradation at 10 nM). Meantime, compared with PF-562271, PROTAC A13 exhibited better antiproliferative activity and anti-invasion ability in A549 cells. More, A13 had excellent plasma stability with T1/2 >194.8 min. There are various signs that PROTAC A13 could be useful as expand tool for studying functions of FAK in biological system and as potential therapeutic agents. [ABSTRACT FROM AUTHOR]

ZINC40099027 (ZN27) is a specific focal adhesion kinase (FAK) activator that promotes murine mucosal wound closure after ischemic or NSAID-induced injury. Diverse motogenic pathways involve FAK, but the direct consequences of pure FAK activation have not been studied, and how ZN27-induced FAK activation stimulates wound closure remained unclear. We investigated signaling and focal adhesion (FA) turnover after FAK activation by ZN27 in Caco-2 cells, confirming key results in CCD841 cells. ZN27 increased Caco-2 FAK-Y-397, FAK-Y-576/7, paxillin-Y-118, and ERK 1/2 phosphorylation and decreased FAK-Y-925 phosphorylation, without altering FAK-Y-861, p38, Jnk, or Akt phosphorylation. ZN27 increased FAK-paxillin interaction while decreasing FAK-Grb2 association. ZN27 increased membrane-associated FAK-Y-397 and FAK-Y-576/7 phosphorylation and paxillin-Y-118 and ERK 1/2 phosphorylation but decreased FAK-Y-925 phosphorylation without altering Src or Grb2. Moreover, ZN27 increased the fluorescence intensity of GFP-FAK and pFAK-Y397 in FAs and increased the total number of FAs but reduced their size in GFP-FAK-transfected Caco-2 cells, consistent with increased FA turnover. In contrast, FAK-Y397F transfection prevented ZN27 effects on FAK size and number and FAK and pFAK fluorescent intensity in FAs. We confirmed the proposed FAK/paxillin/ERK pathway using PP2 and U0126 to block Src and MEK1/2 in Caco-2 and CCD841 cells. These results suggest that ZN27 promotes intestinal epithelial monolayer defect closure by stimulating autophosphorylation of FAK in the cytosol, distinct from classical models of FAK activation in the FA. Phosphorylated FAK translocates to the membrane, where its downstream substrates paxillin and ERK are phosphorylated, leading to FA turnover and human intestinal epithelial cell migration. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a significant global health concern, with high rates of morbidity and mortality. Bucidarasin A, a natural diterpenoid, has been shown to exert notable cytotoxic effects across a range of tumor cell lines. However, the underlying mechanisms responsible for this cytotoxicity remain unclear. In this study, we sought to elucidate the antitumor mechanisms of bucidarasin A, a natural diterpenoid derived from Casearia graveolens , with a particular focus on its effects on HCC. Furthermore, we employed surface plasmon resonance (SPR), molecular docking, and cellular thermal shift assay (CETSA) to gain further insight into the target protein of bucidarasin A. Our findings revealed that bucidarasin A exhibited pronounced cytotoxicity towards HepG2 cells. In vitro analysis indicated that bucidarasin A interrupted the cell cycle at the S phase and inhibited the proliferation and metastasis of HepG2 cells by modulating the FAK and STAT3 signaling pathways. Moreover, in vivo studies demonstrated that bucidarasin A not only exhibited antitumor effects but also impeded neovascularization, a finding that was corroborated by SPR interactions between vascular endothelial growth factor (VEGF) and bucidarasin A. This research substantiated that bucidarasin A, a clerodane diterpenoid, held promise as a therapeutic candidate against HCC, showcasing substantial antitumor efficacy both in vitro and in vivo through direct targeting of the STAT3 and FAK signaling pathways. [Display omitted] • The mechanism of bucidarasin A in the treatment of HCC was elucidated. • Bucidarasin A could inhibit the proliferation and migration of HepG2 cells in vitro and in vivo. • The target protein of bucidarasin A was confirmed. • Bucidarasin A could inhibit the formation of new vessels in vivo. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK; also known as PTK2) was discovered three decades ago and is now recognised as a key player in the regulation of cell-matrix adhesion and mesenchymal cell migration. Although it is essential during development, FAK also drives invasive cancer progression and metastasis. On a structural level, the basic building blocks of FAK have been described for some time. However, a picture of how FAK integrates into larger assemblies in various cellular environments, including one of its main cellular locations, the focal adhesion (FA) complex, is only beginning to emerge. Nanoresolution data from cellular studies, as well as atomic structures from reconstituted systems, have provided first insights, but also point to challenges that remain for obtaining a full structural understanding of how FAK is integrated in the FA complex and the structural changes occurring at different stages of FA maturation. In this Review, we discuss the known structural features of FAK, the interactions with its partners within the FA environment on the cell membrane and propose how its initial assembly in nascent FAs might change during FA maturation under force. [ABSTRACT FROM AUTHOR]

Background: Metastasis is still the major cause of endometrial cancer (EC)-related death. Because of their biological function and regenerative properties, exosomes have been applied to therapeutic regimens. SERPINA5 expression is downregulated in several tumors and linked to tumor cell migration and invasion. However, the expression and biological functions of SERPINA5 in EC remain unclear. Methods: The levels of SERPINA5 in plasma exosomes were determined with ELISAs. SERPINA5 expression in EC and its relationship with survival outcomes were analyzed using the TCGA database and clinical EC tissue samples. The effect of SERPINA5 overexpression or exosomal SERPINA5 on EC metastasis was examined by cell migration and invasion assays in vitro. Mechanistically, overexpression of SERPINA5 or high exosomal SERPINA5 levels mediated the regulation of the integrin β1/FAK signaling pathway in EC cell lines. The therapeutic effect of exosomal SERPINA5 was determined with xenograft models. Results: This study revealed that the level of exosomal SERPINA5 was increased in the circulating plasma of EC patients. In addition, the expression of SERPINA5 was decreased in EC patients with distant metastasis, and low expression of SERPINA5 indicated worse survival. In addition, SERPINA5 was elevated in normal tissues adjacent to EC tumors. Moreover, overexpression of SERPINA5 inhibited metastatic potential of EC cell lines in vitro. Furthermore, SERPINA5 loaded on secreted exosomes reduced the metastatic ability of EC cells. Notably, overexpression of SERPINA5 or high exosomal SERPINA5 levels suppressed EC metastatic potential by suppressing integrin β1/FAK signaling pathway activation. Finally, exosomal SERPINA5 impeded tumor growth and metastasis in xenograft models. Conclusions: Our findings revealed that a low level of SERPINA5 expression indicated poor survival outcomes in EC and that exogenous SERPINA5 loading of exosomes may be a novel therapeutic strategy for metastatic EC. [ABSTRACT FROM AUTHOR]

Aggressive invasiveness is a common feature of malignant gliomas, despite their high level of tumor heterogeneity and possible diverse cell origins. Therefore, it is important to explore new therapeutic methods. In this study, we evaluated and compared the effects of graphene (GN) and reduced graphene oxides (rGOs) on a highly invasive and neoplastic cell line, U87. The surface functional groups of the GN and rGO flakes were characterized by X-ray photoelectron spectroscopy. The antitumor activity of these flakes was obtained by using the neutral red assay and their anti-migratory activity was determined using the wound healing assay. Further, we investigated the mRNA and protein expression levels of important cell adhesion molecules involved in migration and invasiveness. The rGO flakes, particularly rGO/ATS and rGO/TUD, were found highly toxic. The migration potential of both U87 and Hs5 cells decreased, especially after rGO/TUD treatment. A post-treatment decrease in mobility and FAK expression was observed in U87 cells treated with rGO/ATS and rGO/TUD flakes. The rGO/TUD treatment also reduced β-catenin expression in U87 cells. Our results suggest that rGO flakes reduce the migration and invasiveness of U87 tumor cells and can, thus, be used as potential antitumor agents. [ABSTRACT FROM AUTHOR]

Background: High glucose conditions cause some changes in the vessels of diabetes through the signal transduction pathways. Dexamethasone and other corticosteroids have a wide range of biological effects in immunological events. In the present study, the effects of dexamethasone were investigated on the VSMC (vascular smooth muscle cell) proliferation, and migration based on the FAK gene and protein changes in high glucose conditions. Methods and materials: The vascular smooth muscle cells were cultured in DMEM and were treated with dexamethasone (10–7 M, 10–6 M, and 10–5 M) for 24, and 48 h in high glucose conditions. The cell viability was estimated by MTT method. The FAK gene expression levels and pFAK protein values were determined by RT-qPCR and western blotting techniques, respectively. A scratch assay was used to evaluate cellular migration. Results: The FAK gene expression levels decreased significantly dependent on dexamethasone doses at 24 and 48 h. The pFAK protein values decreased significantly with a time lag at 24- and 48-h periods as compared with gene expression levels. Conclusion: The results showed that the inhibition of VSMC proliferation and migration by dexamethasone in the high glucose conditions may be related to the changes of FAK. [ABSTRACT FROM AUTHOR]

Intercellular adhesion molecule-1 (ICAM-1) in endothelial cells is critical for neutrophil adhesion and transmigration across the endothelium. Focal adhesion kinase (FAK), which controls the turnover of focal adhesion to regulate cell adhesion and migration, plays a role in the resolution of inflammation. However, the coordinated involvement of ICAM-1 and FAK during endothelial inflammation has yet to be elucidated. This study reports that, as part of an inflammatory response, ICAM-1 controls FAK expression in endothelial cells via the microRNA miR-15b-5p. Induction of lung injury by lipopolysaccharide (LPS) resulted in higher levels of FAK expression in inflammatory tissues, while in ICAM-1 knockout mice, FAK expression was reduced in the lungs. FAK expression was also reduced in endothelial cells following ICAM-1 siRNA downregulation. Furthermore, ICAM-1 inhibited miR-15b-5p expression while increasing FAK mRNA and protein expression via binding of miR-15b-5p to the 3′ untranslated region (UTR) of FAK. ICAM-1 inhibited miR-15b-5p promoter activity and hence reduced miR-15b-5p expression. FAK increased endothelial cell proliferation and migration, whereas miR-15b-5p inhibited cell proliferation and migration. These findings indicate that the inflammatory molecule ICAM-1 regulates FAK expression via miR-15b-5p levels, which in turn controls endothelial cell proliferation and migration. [ABSTRACT FROM AUTHOR]

Elevated levels of Mucin-16 (MUC16) in conjunction with a high expression of truncated O-glycans is implicated in playing crucial roles in the malignancy of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms by which such aberrant glycoforms present on MUC16 itself promote an increased disease burden in PDAC are yet to be elucidated. This study demonstrates that the CRISPR/Cas9-mediated genetic deletion of MUC16 in PDAC cells decreases tumor cell migration. We found that MUC16 enhances tumor malignancy by activating the integrin-linked kinase and focal adhesion kinase (ILK/FAK)-signaling axis. These findings are especially noteworthy in truncated O-glycan (Tn and STn antigen)-expressing PDAC cells. Activation of these oncogenic-signaling pathways resulted in part from interactions between MUC16 and integrin complexes (α4β1), which showed a stronger association with aberrant glycoforms of MUC16. Using a monoclonal antibody to functionally hinder MUC16 significantly reduced the migratory cascades in our model. Together, these findings suggest that truncated O-glycan containing MUC16 exacerbates malignancy in PDAC by activating FAK signaling through specific interactions with α4 and β1 integrin complexes on cancer cell membranes. Targeting these aberrant glycoforms of MUC16 can aid in the development of a novel platform to study and treat metastatic pancreatic cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Sphingosine 1-phosphate (S1P) is a lipid metabolite involved in cell proliferation, survival or migration. S1P is a ligand for five high-affinity G protein-coupled receptors (S1P1-5), which differ in their tissue distribution, and the specific effects of S1P depend on the suite of S1P receptor subtypes expressed. To date, information regarding the role of S1P5 in cell proliferation is limited and ambiguous. Our results suggest that, unlike other S1P receptors, the S1P5 receptor has an anti-proliferative function. We found that S1P5 deficiency promotes cell immortalization and proliferation by controlling the spatial activation of ERK. Sphingosine 1-phosphate (S1P), a bioactive lipid, interacts with five widely expressed G protein-coupled receptors (S1P1-5), regulating a variety of downstream signaling pathways with overlapping but also opposing functions. To date, data regarding the role of S1P5 in cell proliferation are ambiguous, and its role in controlling the growth of untransformed cells remains to be fully elucidated. In this study, we examined the effects of S1P5 deficiency on mouse embryonic fibroblasts (MEFs). Our results indicate that lack of S1P5 expression profoundly affects cell morphology and proliferation. First, S1P5 deficiency reduces cellular senescence and promotes MEF immortalization. Second, it decreases cell size and leads to cell elongation, which is accompanied by decreased cell spreading and migration. Third, it increases proliferation rate, a phenotype rescued by the reintroduction of exogenous S1P5. Mechanistically, S1P5 promotes the activation of FAK, controlling cell spreading and adhesion while the anti-proliferative function of the S1P/S1P5 signaling is associated with reduced nuclear accumulation of activated ERK. Our results suggest that S1P5 opposes the growth-promoting function of S1P1-3 through spatial control of ERK activation and provides new insights into the anti-proliferative function of S1P5. [ABSTRACT FROM AUTHOR]

Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin β1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin β1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin β1 and promotes neuroblastoma cell migration.

Background: We aimed to analyze the regulatory effects of SIPA1 (signal-induced proliferation-associated protein 1) on glioma progression and the dominant signaling pathway. Methods: Differential level of SIPA1 in glioma and normal tissues and cells was determined. Migratory, proliferative, apoptotic and cell cycle progression changes in A172 cells with overexpression or knockdown of SIPA1 were examined. Finally, protein levels of phosphorylated FAKs in A172 cells intervened by SIPA1, and the fAk inhibitor PF-562271 were detected. Results: SIPA1 was upregulated in glioma cases. Knockdown of SIPA1 reduced migratory and proliferative rates of glioma cells, increased apoptotic cell rate, and declined cell ratio in the S phase. The knockdown of SIPA1 also down-regulated cell cycle proteins. In addition, SIPA1 upregulated phosphorylated FAKs in A172 cells and thus boosted malignant phenotypes of glioma. Conclusions: SIPA1 is upregulated in glioma that boosts migratory and proliferative potentials of glioma cells by activating the phosphorylation of the FAK signaling pathway. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cell (VSMC) migration plays a critical role in the pathogenesis of many cardiovascular diseases. We recently showed that TMEM16A is involved in hypertension-induced cerebrovascular remodeling. However, it is unclear whether this effect is related to the regulation of VSMC migration. Here, we investigated whether and how TMEM16A contributes to migration in basilar artery smooth muscle cells (BASMCs). We observed that AngII increased the migration of cultured BASMCs, which was markedly inhibited by overexpression of TMEM16A. TMEM16A overexpression inhibited AngII-induced RhoA/ROCK2 activation, and myosin light chain phosphatase (MLCP) and myosin light chain (MLC20) phosphorylation. But AngII-induced myosin light chain kinase (MLCK) activation was not affected by TMEM16A. Furthermore, a suppressed activation of integrin β 3/FAK pathway, determined by reduced integrin β 3 expression, FAK phosphorylation and F-actin rearrangement, was observed in TMEM16A-overexpressing BASMCs upon AngII stimulation. Contrary to the results of TMEM16A overexpression, silencing of TMEM16A showed the opposite effects. These in vitro results were further demonstrated in vivo in basilar arteries from VSMC-specific TMEM16A transgenic mice during AngII-induced hypertension. Moreover, we observed that the inhibitory effect of TMEM16A on BASMC migration was mediated by decreasing the activation of WNK1, a Cl−-sensitive serine/threonine kinase. In conclusion, this study demonstrated that TMEM16A suppressed AngII-induced BASMC migration, thus contributing to the protection against cerebrovascular remodeling during AngII-infused hypertension. TMEM16A may exert this effect by suppressing the RhoA/ROCK2/MLCP/MLC20 and integrin β 3/FAK signaling pathways via inhibiting WNK1. Our results suggest that TMEM16A may serve as a novel therapeutic target for VSMC migration-related diseases, such as vascular remodeling. This study suggests that modulation of TMEM16A expression and/or activity might be a novel strategy to prevent hypertension-induced vascular remodeling. [Display omitted] [ABSTRACT FROM AUTHOR]

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.

DUSP3 is a phosphatase expressed and active in several tissues that dephosphorylates tyrosine residues in many regulatory proteins of cellular activities such as proliferation, survival, and cell death. Recently, two new independent functions were assigned to this enzyme: dephosphorylation of focal adhesion kinase (FAK) and regulation of nucleotide-excision repair (NER) pathway. Genotoxic stress by UV radiation is known to affect cell morphology, adhesion, and migration for affecting, for example, the Rho GTPases that regulate actin cytoskeleton. This work investigated the intersection of DUSP3 function, XPA protein activity, and UV toxicity by examining cell migration, FAK, and SRC kinase phosphorylation status, in addition to cell morphology, in fibroblast cells proficient (MRC-5) or deficient (XPA) of the NER pathway. DUSP3 loss reduced cell migration of normal cells, which was stimulated by the genotoxic stress, effects evidenced in presence of serum mitogenic stimulus. However, NER-deficient cells migration response was the opposite since DUSP3 loss increased migration, especially after cells being exposed to UV stress. The levels of pFAK(Y397) peaked 15 min and 1 h after UV radiation in normal cells, but only slightly increased in repair-deficient cells. However, the DUSP3 knockdown strongly raised pFAK(Y397) levels in both cells, but especially in XPA cells as supported by the higher SRC activity. These effects impacted on the dynamics of actin-based structures formation, such as stress fibres, apparently dependent on DUSP3 and DNA-repair (NER) proficiency of the cells. Altogether our findings suggest this dual-phosphatase is bridging gaps between the complex regulation of cell morphology, motility, and genomic stability. [ABSTRACT FROM AUTHOR]

Prostate cancer is a life‐threatening condition worldwide. As the tumor progresses, smooth muscle cells (SMCs) become atrophic/dedifferentiated, within a series of stromal changes named stromal reaction. Here, we tested whether a laminin 111‐rich extracellular matrix (Lr‐ECM) could affect SMCs phenotype and differentiation status. Using time‐lapse microscopy, image analyses, quantitative real‐time reverse transcription polymerase chain reaction, immunohistochemistry and immunoblotting, and transmission electron microscopy, we showed that SMCs acquires a migratory behavior with a decreased expression of differentiation markers and relocation of focal adhesion kinase. SMCs set homotypic cell junctions and were active in autophagy/phagocytosis. Analysis of the migratory behavior showed that SMCs polarized and migrated toward each other, recognizing long‐distance signals such as matrix tensioning. However, half of the cell population were immotile, irrespective of the nearest neighbor distance, suggesting they do not engage in productive interactions, possibly as a result of back‐to‐back positioning. In conclusion, the Lr‐ECM, mimics the effects of the proliferating and infiltrating tumor epithelium, causing SMCs phenotypical change similar to that observed in the stromal reaction, in addition to a hitherto undescribed, stereotyped pattern of cell motility resulting from cell polarization. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.

This study aims to investigate how Maspin affects the EMT and angiogenesis of gastric cancer (GC) cells via ITGB1/FAK pathway. Immunohistochemistry was used to evaluate the expressions of Maspin, ITGB1, FAK, E-cadherin, Vimentin, D2-40, and CD34 in GC and adjacent normal tissues from 160 patients. Then, the human GC cells with different degree of differentiation were transfected with Maspin CRISPR activation plasmid, ITGB1 siRNA and/or Maspin siRNA, followed by the following experiments, including qRT-PCR, western blotting, tube formation assay, Transwell assay and wound healing. GC tumor tissues manifested decreased Maspin with the activated ITGB1/FAK pathway. In tumor tissues, Maspin was negatively correlated with the expressions of ITGB1 and FAK, as well as Lauren's classification, differentiation degree, and TNM stage. Besides, Maspin was negatively related with lymphatic vessel density (LVD) and microvessel density (MVD), Vimentin and VEGF, but was positive correlated with E-cadherin. Maspin expression decreased, but ITGB1 and p-FAK expressions increased gradually in MKN-28 (well differentiated), SGC-7901 (moderate differentiated), and MKN-45 (poorly differentiated). Maspin CRISPR and ITGB1 siRNA increased E-cadherin with the decreased Vimentin, VEGF and bFGF, and the reductions of tube length. In comparison with the ITGB1 siRNA group, cells in the Maspin siRNA + ITGB1 siRNA group presented the more evident EMT and angiogenesis. Furthermore, ITGB1 siRNA reduced the malignancies of GC cells, which could be restored by Maspin siRNA. Maspin was downregulated in GC tissues, which could inhibit the EMT and angiogenesis by blocking the ITGB1/FAK pathway, thereby decreasing cell invasion and migration of GC. [ABSTRACT FROM AUTHOR]

Recent studies revealed that the histone demethylase KDM2B regulates the epithelial markers E-Cadherin and ZO-1, the RhoA/B/C-small-GTPases and actin cytoskeleton organization, in DU-145 prostate- and HCT-116 colon-tumor cells. Here we addressed the role of KDM2B in the activation of Focal Adhesion Kinase (FAK)-signaling and its involvement in regulating tumor cell motility. We used RT-PCR for gene transcriptional analysis, Western blotting for the assessment of protein expression and activity and wound-healing assay for the study of cell migration. KDM2B overexpression or silencing controls the activity of FAK in DU-145 prostate- and HCT-116 colon-tumor cells without affecting gene transcription and protein expression of this kinase. Upon KDM2B overexpression in DU-145 cells, significantly enhanced migration was observed, which was abolished in cells pretreated by the specific phosphoinositide-3 kinase (PI3 K) inhibitor LY294002, implying involvement of FAK/PI3 K signaling in the migration process. In line with this, the p85-PI3 K-subunit was downregulated upon knockdown of KDM2B in DU-145 cells, while the opposite effect became evident in KDM2B-overexpressing cells. These results revealed a novel functional role of KDM2B in regulating the activation of the FAK/PI3 K signaling in prostate cancer cells that participates in the control of cell motility. [ABSTRACT FROM AUTHOR]

Background: Breast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer.Methods: To understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested.Results: We found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival.Conclusions: In summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Introduction: Odd-skipped related transcription factor 1 (OSR1) is a newly identified tumor suppressor in many tumor types. However, the role and mechanism of OSR1 in colon adenocarcinoma (COAD) remain unknown. Methods: OSR1 expression was detected in COAD tissues and cells. COAD cells with OSR1 overexpression or knockdown were analyzed by in vitro CCK-8, transwell and flow cytometry assays, and by in vivo xenograft model. Results: OSR1 expression was downregulated in COAD and low expression level of OSR1 was positively correlated with tumor stage and lymph node metastasis. Furthermore, low OSR1 expression was significantly associated with poor overall survival (OS) and distant metastasis-free survival (DMFS). Lentivirus-mediated restoration of OSR1 expression-inhibited proliferation, invasion and migration while induced cell cycle arrest and apoptosis in COAD cells in vitro, and inhibited tumor growth in vivo. In contrast, OSR1 knockdown promoted proliferation, invasion and migration in COAD cells in vitro. Mechanistically, OSR1 exerted anticancer effects by inhibiting FAK-mediated activation of Akt and MAPK pathways. Conclusion: Our findings suggest that OSR1 functions as a tumor suppressor in COAD by suppressing FAK-mediated activation of Akt and MAPK pathways. [ABSTRACT FROM AUTHOR]

Background: Fibroblasts executing directional migration position their centrosome, and their Golgi apparatus, in front of the nucleus towards the cell leading edge. Centrosome positioning relative to the nucleus has been associated to mechanical forces exerted on the centrosome by the microtubule‐dependent molecular motor cytoplasmic dynein 1, and to nuclear movements such as rearward displacement and rotation events. Dynein has been proposed to regulate the position of the centrosome by exerting pulling forces on microtubules from the cell leading edge, where the motor is enriched during migration. However, the mechanism explaining how dynein acts at the front of the cells has not been elucidated. Results: We present here results showing that the protein Focal Adhesion Kinase (FAK) interacts with dynein and regulates the enrichment of the dynein/dynactin complex at focal adhesions at the cell the leading edge of migrating fibroblasts. This suggests that focal adhesions provide anchoring sites for dynein during the polarisation process. In support of this, we present evidence indicating that the interaction between FAK and dynein, which is regulated by the phosphorylation of FAK on its Ser732 residue, is required for proper centrosome positioning. Our results further show that the polarisation of the centrosome can occur independently of nuclear movements. Although FAK regulates both nuclear and centrosome motilities, downregulating the interaction between FAK and dynein affects only the nuclear independent polarisation of the centrosome. Conclusions: Our work highlights the role of FAK as a key player in the regulation of several aspects of cell polarity. We thus propose a model in which the transient localisation of dynein with focal adhesions provides a tuneable mechanism to bias dynein traction forces on microtubules allowing proper centrosome positioning in front of the nucleus. Significance: We unravel here a new role for the cancer therapeutic target FAK in the regulation of cell morphogenesis. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.

Background and purpose:  Verticillin A is a fungal epipolythiodioxopiperazine (ETP) metabolite that was isolated from Amanita flavorubescens Alk infected by Verticillium sp. It was previously proven to possess potent anti-tumor cell growth activity, and we have recently determined that verticillin A is a selective inhibitor of H3K9me3-specific histone methyltransferase. The objective of this study was to find out whether verticillin A is an effective agent for suppression of gastric and cervical tumor progression. Materials and methods: Wound healing and transwell assays was performed to evaluate the effect of verticillin A on hepatocyte growth factor (HGF)-induced AGS and HeLa cells migration and invasion in vitro. Western blot was used to detect signaling proteins verticillin A affected. Results: We determined that verticillin A effectively suppressed hepatocyte growth factor (HGF)-induced AGS and HeLa cells migration and invasion in vitro. At the molecular level, we demonstrated that verticillin A inhibited HGF-induced c-Met phosphorylation and repressed the expression of total c-Met protein in AGS and HeLa cells, resulting from reduced expression of fatty acid synthase. In addition, verticillin A could suppress c-Met downstream FAK/Src signaling pathways by impairing c-Met phosphorylation induced by HGF. Conclusion:  Our study demonstrated verticillin A inhibits the migration ability of human gastric cancer (AGS) cells and cervical cancer (HeLa) cells by targeting c-Met and its downstream FAK/Src signaling pathways, and suggested that verticillin A acts as a novel HGF/c-Met inhibitor by reducing expression of this receptor. [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]

Cells express a family of three inositol hexakisphosphate kinases (IP6Ks). Although sharing the same enzymatic activity, individual IP6Ks mediate different cellular processes. Here we report that IP6K3 is enriched at the leading edge of migrating cells where it associates with dynein intermediate chain 2 (DIC2). Using immunofluorescence microscopy and total internal reflection fluorescence microscopy, we found that DIC2 and IP6K3 are recruited interdependently to the leading edge of migrating cells, where they function coordinately to enhance the turnover of focal adhesions. Deletion of IP6K3 causes defects in cell motility and neuronal dendritic growth, eventually leading to brain malformations. Our results reveal a mechanism whereby IP6K3 functions in coordination with DIC2 in a confined intracellular microenvironment to promote focal adhesion turnover. [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 LNCaP cells are derived from a metastatic lesion of human prostate adenocarcinoma. They express the classical androgen receptor (AR) and ZIP9, a Zn2+ transporter that also binds testosterone and mediates signaling by interacting with G-proteins. Our results show that LNCaP cells respond to testosterone by mobilizing their migratory machinery. Their exposure to testosterone triggers the formation of lamellipodia, reorganization of the actin cytoskeleton, phosphorylation of focal adhesion kinase (FAK) at Tyr925 and of paxillin at Tyr118, expression of matrix metalloproteinase 2 (MMP-2), and cell migration. Silencing ZIP9 expression by means of siRNA does not affect the responsiveness of the classical AR to testosterone; however, it prevents all of the testosterone effects described above: formation of lamellipodia cannot be induced, stimulation of FAK or paxillin phosphorylation or MMP-2 expression is prevented, and cell migration does not take place in the absence of ZIP9. The data presented show that testosterone/ZIP9 interactions might have not only physiological but also pathophysiological relevance. The fact that the migratory machinery of a metastatic prostate cancer cell line is activated exclusively through testosterone/ZIP9 and not through testosterone/AR interactions suggests that targeting specific inhibition of testosterone/ZIP9-mediated events might help in developing new therapeutic strategies against androgen-induced progression of prostate cancer. Graphical abstract Unlabelled Image Highlights • LNCaP cells are derived from a metastatic lesion of human prostate adenocarcinoma. • LNCaP cells respond to testosterone by mobilizing their migratory machinery. • This includes actin re-arrangement, activation of FAK and paxillin and expression of MMP-2. • Testosterone effects are mediated though ZIP9 and not through the classical AR. • The results indicate a pathophysiological relevance of ZIP9/testosterone interactions. [ABSTRACT FROM AUTHOR]

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children and adolescents. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 is at high risk. The fusion negative subgroup, FN-RMS, has a good prognosis when non-metastatic. Despite a multimodal therapeutic approach, FP-RMS and metastatic FN-RMS often show a dismal prognosis with 5-year survival of less than 30%. Therefore, novel targets need to be discovered to develop therapies that halt tumor progression, reducing long-term side effects in young patients. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates focal contacts at the cellular edges. It plays a role in cell motility, survival, and proliferation in response to integrin and growth factor receptors’ activation. FAK is often dysregulated in cancer, being upregulated and/or overactivated in several adult and pediatric tumor types. In RMS, both in vitro and preclinical studies point to a role of FAK in tumor cell motility/invasion and proliferation, which is inhibited by FAK inhibitors. In this review, we summarize the data on FAK expression and modulation in RMS. Moreover, we give an overview of the approaches to inhibit FAK in both preclinical and clinical cancer settings.

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]

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP 7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP 7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP 7 , which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP 7 , thus augments 5-InsP 7 -mediated inhibition of Arp2/3 complex and reduces 5-InsP 7 -mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP 7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility. • IP6K1 binds Arp2 and generates 5-InsP 7 to recruit coronin to Arp2/3 complex. • IP6K1 generates 5-InsP 7 in focal adhesion to promote FAK dimerization. • Itraconazole inhibits Arp2/3 complex by enhancing the binding of IP6K1 to Arp2. • Itraconazole lowers FAK phosphorylation by removing IP6K1 from focal adhesion. • Itraconazole disrupts 5-InsP 7 -mediated focal adhesion and cytoskeletal dynamics. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK) is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP)-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

Background/Aims: Erinacine A, isolated from the ethanol extract of the Hericium erinaceus mycelium, has been demonstrated as a new alternative anticancer medicine. Drawing upon current research, this study presents an investigation of the molecular mechanism of erinacine A inhibition associated with gastric cancer cell growth. Methods: Cell viability was determined by Annexin V-FITC/ propidium iodide staining and migration using a Boyden chamber assay to determine the effects of erinacine A treatment on the proliferation capacity and invasiveness of gastric cancer cells. A proteomic assay provided information that was used to identify the differentially-expressed proteins following erinacine A treatment, as well as the mechanism of its targets in the apoptotic induction of erinacine A. Results: Our results demonstrate that erinacine A treatment of TSGH 9201 cells increased cytotoxicity and the generation of reactive oxygen species (ROS), as well as decreased the invasiveness. Treatment of TSGH 9201 cells with erinacine A resulted in the activation of caspases and the expression of TRAIL. Erinacine A induction of apoptosis was accompanied by sustained phosphorylation of FAK/AKT/p70S6K and the PAK1 pathways, as well as the generation of ROS. Furthermore, the induction of apoptosis and anti-invasion properties by erinacine A could involve the differential expression of the 14-3-3 sigma protein (1433S) and microtubule-associated tumor suppressor candidate 2 (MTUS2), with the activation of the FAK/AKT/p70S6K and PAK1 signaling pathways. Conclusions: These results lead us to speculate that erinacine A may generate an apoptotic cascade in TSGH 9201 cells by activating the FAK/AKT/p70S6K/PAK1 pathway and upregulating proteins 1433S and MTUS2, providing a new mechanism underlying the anti-cancer effects of erinacine A in human gastric cancer cells. [ABSTRACT FROM AUTHOR]

Focal adhesions (FAs) are specialized sites within the cell where clustered integrin receptors interact with the extracellular matrix on the outside of cells and with the actin cytoskeleton on the inside. They provide strong adhesion to the matrix and transmit mechanical tension generated within cells across the plasma membrane to the external environment. Additionally, they act as scaffolds for many signaling pathways triggered by integrin engagement or mechanical force exerted on cells. Here I describe my personal perspective on FA research which I have witnessed since the initial discovery and description of FAs as electron dense regions of the ventral plasma nearly half a century ago. [ABSTRACT FROM AUTHOR]

Background/Aims: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. Methods: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. Results: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF- 00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. Conclusion: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development. [ABSTRACT FROM AUTHOR]

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y RGE mutant receptors. Consistent with the role of P2Y receptors in vasodilation, receptor knockdown and overexpression of P2Y RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y receptor in shear stress-induced cytoskeletal alterations in HUVECs. [ABSTRACT FROM AUTHOR]

During heat stress (HS), the intestinal epithelium suffers damage due to imbalance of tissue homeostasis. However, the specific mechanism by which intestinal stem cells (ISCs) migrate and differentiate along the crypt–villus axis to heal lesions upon insult is unclear. In our study, C57BL/6 mice and IPEC-J2 cells were subjected to normal ambient conditions (25 °C for 7 days in vivo and 37 °C for 18 h in vitro) or 41 °C. The results showed that HS impaired intestinal morphology and barrier function. The numbers of ISCs (SOX9+ cells), mitotic cells (PCNA+ cells), and differentiated cells (Paneth cells marked by lysozyme, absorptive cells marked by Villin, goblet cells marked by Mucin2, enteroendocrine cells marked by Chromogranin A, and tuft cells marked by DCAMKL1) were reduced under high temperature. Importantly, BrdU incorporation confirmed the decreased migration ability of jejunal epithelial cells exposed to 41 °C. Furthermore, intestinal organoids (IOs) expanded from jejunal crypt cells in the HS group exhibited greater growth disadvantages. Mechanistically, the occurrence of these phenotypes was accompanied by FAK/paxillin/F-actin signaling disruption in the jejunum. The fact that the FAK agonist ZINC40099027 reversed the HS-triggered inhibition of IPEC-J2 cell differentiation and migration further confirmed the dominant role of FAK in response to high-temperature conditions. Overall, the present investigation is the first to reveal a major role of FAK/paxillin/F-actin signaling in HS-induced ISC migration and differentiation along the crypt–villus axis, which indicates a new therapeutic target for intestinal epithelial regeneration after heat injuries. • Heat stress disrupts the differentiation of intestinal stem cells into functional cells. • Heat stress suppresses intestinal stem cell migration along the crypt-villus axis. • FAK/paxillin/F-actin signaling mediates the intestinal stem cell transdifferentiation under high-temperature conditions. [ABSTRACT FROM AUTHOR]