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C-terminal Src kinase (Csk) targets Src family kinases (SFKs) and thereby inactivates them. We have previously shown that Csk binds to phosphorylated tyrosine 685 of VE-cadherin, an adhesion molecule of major importance for the regulation of endothelial junctions. This tyrosine residue is an SFK target, and its mutation (VE-cadherin-Y685F) inhibits the induction of vascular permeability in various inflammation models. Nevertheless, surprisingly, it increases leukocyte extravasation. Here, we investigated whether endothelial Csk is involved in these effects. We found that the deficiency of Csk in endothelial cells augments SFK activation and the phosphorylation of VE-cadherin-Y685 but had no net effect on vascular leak formation. In contrast, the lack of endothelial Csk enhanced leukocyte adhesion and transmigration in vitro and in vivo. Furthermore, the silencing of Csk increased tyrosine phosphorylation of the SFK substrate cortactin. Importantly, the effects of Csk silencing on the increase in SFK activation, cortactin phosphorylation, and neutrophil diapedesis were all dependent on Y685 of VE-cadherin. Deletion of cortactin, in turn, erased the supporting effect of Csk silencing on leukocyte transmigration. We have previously shown that leukocyte transmigration is regulated by endothelial cortactin in an ICAM-1-dependent manner. In line with this, blocking of ICAM-1 erased the supporting effect of Csk silencing on leukocyte transmigration. Collectively, our results establish a negative feedback loop that depends on the phosphorylation of VE-cadherin-Y685, which recruits Csk, which in turn dampens the activation of SFK and cortactin and thereby the clustering of ICAM-1 and the extravasation of neutrophils. [ABSTRACT FROM AUTHOR]

Many viral, protozoal, and fungal pathogens represent major human and animal health problems due to their great potential of causing infectious diseases. Research on these pathogens has contributed substantially to our current understanding of both microbial virulence determinants and host key factors during infection. Countless studies have also shed light on the molecular mechanisms of host–pathogen interactions that are employed by these microbes. For example, actin cytoskeletal dynamics play critical roles in effective adhesion, host cell entry, and intracellular movements of intruding pathogens. Cortactin is an eminent host cell protein that stimulates actin polymerization and signal transduction, and recently emerged as fundamental player during host–pathogen crosstalk. Here we review the important role of cortactin as major target for various prominent viral, protozoal and fungal pathogens in humans, and its role in human disease development and cancer progression. Most if not all of these important classes of pathogens have been reported to hijack cortactin during infection through mediating up‐ or downregulation of cortactin mRNA and protein expression as well as signaling. In particular, pathogen‐induced changes in tyrosine and serine phosphorylation status of cortactin at its major phospho‐sites (Y‐421, Y‐470, Y‐486, S‐113, S‐298, S‐405, and S‐418) are addressed. As has been reported for various Gram‐negative and Gram‐positive bacteria, many pathogenic viruses, protozoa, and fungi also control these regulatory phospho‐sites, for example, by activating kinases such as Src, PAK, ERK1/2, and PKD, which are known to phosphorylate cortactin. In addition, the recruitment of cortactin and its interaction partners, like the Arp2/3 complex and F‐actin, to the contact sites between pathogens and host cells is highlighted, as this plays an important role in the infection process and internalization of several pathogens. However, there are also other ways in which the pathogens can exploit the function of cortactin for their needs, as the cortactin‐mediated regulation of cellular processes is complex and involves numerous different interaction partners. Here, the current state of knowledge is summarized. [ABSTRACT FROM AUTHOR]

Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN , which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors. • ICMT overexpression promotes metastasis in vivo. • ICMT enhances migration, invasion and invadopodia formation in cancer cells. • The ICMT gene is overexpressed in several human cancers. • High expression of ICMT and CTTN correlate with por clinical outcome in lung cancer. [ABSTRACT FROM AUTHOR]

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis. [ABSTRACT FROM AUTHOR]

C-terminal Src kinase (Csk) targets Src family kinases (SFKs) and thereby inactivates them. We have previously shown that Csk binds to phosphorylated tyrosine 685 of VE-cadherin, an adhesion molecule of major importance for the regulation of endothelial junctions. This tyrosine residue is an SFK target, and its mutation (VE-cadherin-Y685F) inhibits the induction of vascular permeability in various inflammation models. Nevertheless, surprisingly, it increases leukocyte extravasation. Here, we investigated whether endothelial Csk is involved in these effects. We found that the deficiency of Csk in endothelial cells augments SFK activation and the phosphorylation of VE-cadherin-Y685 but had no net effect on vascular leak formation. In contrast, the lack of endothelial Csk enhanced leukocyte adhesion and transmigration in vitro and in vivo. Furthermore, the silencing of Csk increased tyrosine phosphorylation of the SFK substrate cortactin. Importantly, the effects of Csk silencing on the increase in SFK activation, cortactin phosphorylation, and neutrophil diapedesis were all dependent on Y685 of VE-cadherin. Deletion of cortactin, in turn, erased the supporting effect of Csk silencing on leukocyte transmigration. We have previously shown that leukocyte transmigration is regulated by endothelial cortactin in an ICAM-1-dependent manner. In line with this, blocking of ICAM-1 erased the supporting effect of Csk silencing on leukocyte transmigration. Collectively, our results establish a negative feedback loop that depends on the phosphorylation of VE-cadherin-Y685, which recruits Csk, which in turn dampens the activation of SFK and cortactin and thereby the clustering of ICAM-1 and the extravasation of neutrophils.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actindecorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14. [ABSTRACT FROM AUTHOR]

Background: One prevalent malignant tumor in the digestive system is gastric cancer (GC). Cortactin is an intracellular cytoskeleton protein and exerts the crucial function in GC development. However, the roles and mechanisms of cortactin in the invasion and metastasis of GC need further exploration. Materials and Methods: Cortactin expression in GC tissues and cells via western blot and quantitative reverse transcription PCR. Cell migration and invasion were detected by the Transwell assays. Immunofluorescence staining and extracellular matrix (ECM) degradation assays verified the ability to invadopodium formation and ECM degradation. We then used gelatin zymography to identify the relationship between cortactin and matrix metalloproteinases (MMPs). The xenograft tumor model proved that cortactin can accelerate tumor growth and intraperitoneal metastasis in mice. Results: We found that cortactin is overexpressed in GC. cortactin overexpression facilitated cell migration and invasion, whereas cortactin silencing exerted the opposite function. cortactin can facilitate invadopodium formation and ECM degradation in GC cells. Cortactin can positively regulate matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) levels. Furthermore, Cortactin accelerate GC progression in vivo. Conclusion: In short, this study confirmed that cortactin enhanced invadopodium formation to accelerate GC development through upregulating MMP2 and MMP9. [ABSTRACT FROM AUTHOR]

BackgroundColorectal carcinoma (CRC) has emerged as one of the most widespread cancers and was the third leading cause of cancer-related mortality in 2020. The role of the podosomal protein Tks4 in tumor formation and progression is well established, including its involvement in gastric carcinoma and hepatocellular carcinoma; however, exploration of Tks4 and its associated EMT-regulating interactome in the context of colon cancer remains largely unexplored.MethodsWe conducted a comprehensive bioinformatic analysis to investigate the mRNA and protein expression levels of Tks4 and its associated partner molecules (CD2AP, GRB2, WASL, SRC, CTTN, and CAPZA1) across different tumor types. We quantified the expression levels of Tks4 and its partner molecules using qPCR, utilizing a TissueScan colon cancer array. We then validated the usefulness of Tks4 and its associated molecules as biomarkers via careful statistical analyses, including Pearson’s correlation analysis, principal component analysis (PCA), multiple logistic regression, confusion matrix analysis, and ROC analysis.ResultsOur findings indicate that the co-expression patterns of the seven examined biomarker candidates better differentiate between tumor and normal samples compared with the expression levels of the individual genes. Moreover, variable importance analysis of these seven genes revealed four core genes that yield consistent results similar to the seven genes. Thus, these four core genes from the Tks4 interactome hold promise as potential combined biomarkers for colon adenocarcinoma diagnosis and prognosis.ConclusionOur proposed biomarker set from the Tks4 interactome shows promising sensitivity and specificity, aiding in colon cancer prevention and diagnosis.

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals.

Objective(s): The prognosis of endometrial cancer (EC) is significantly affected by tumor infiltration and metastasis. Cortactin (CTTN) regulates infiltration and metastasis in other tumors. Studies on the role and mechanism of CTTN in EC are limited and further studies are needed.Materials and Methods: Quantitative PCR and immunohistochemistry were used to detect Ras-associated C3 botulinum toxin substrate 1 (Rac1) and CTTN in EC and normal tissues. The relationship between the expression of these two genes and their prognostic factors was analyzed. A CTTN-RNAi lentiviral system was constructed and transfected into EC cells. Migration and invasion were evaluated by scratch assay, transwell migration, and invasion assays. Pseudopodia formation was observed by immunofluorescence staining. Western blotting was performed to detect the expression of Rac1.Results: The expression levels of Rac1 and CTTN in EC tissues were significantly higher than those in normal tissues. In the EC group, Rac1 and CTTN levels were correlated. The protein expression levels of Rac1 and CTTN were related to myometrial invasion and stage. After CTTN knockdown, the migration rate, invasiveness, and migratory ability of EC cells decreased significantly. Lamellipodia was observed to disappear with the appearance of blebs. Rac1 protein expression was decreased after CTTN knockdown.Conclusion: CTTN may promote the invasion and migration of EC by lamellipodia. This effect may be related to the regulation of Rac1 by CTTN.

Background: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. Methods: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. Results: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. Conclusion: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy. [ABSTRACT FROM AUTHOR]

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF’s branch destabilizing activities are potently blocked by cortactin (IC50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin’s on-rate (3 ×107 s−1 M−1) and off-rate (0.03 s−1) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (Kd = 0.9 nM) and filament sides (Kd = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.

Successful treatment for any type of carcinoma largely depends on understanding the patterns of invasion and migration. For oral squamous cell carcinoma (OSCC), these processes are not entirely understood as of now. Invadopodia and podosomes, called invadosomes, play an important role in cancer cell invasion and migration. Previous research has established that cortactin (CTTN) is a major inducer of invadosome formation. However, less is known about the expression patterns of CTTN and other genes related to it or invadopodia formation in OSCC during tumor progression in particular. In this study, gene expression patterns of CTTN and various genes (n = 36) associated with invadopodia formation were analyzed to reveal relevant expression patterns and give a comprehensive overview of them. The genes were analyzed from a whole genome dataset of 83 OSCC samples relating to tumor size, grading, lymph node status, and UICC (Union for Internatioanl Cancer Control). The data revealed significant overexpression of 18 genes, most notably CTTN, SRC (SRC proto-onocogene, non-receptor tyrosine kinase), EGFR (epidermal growth factor receptor), SYK (spleen associated tyrosine kinase), WASL (WASP like actin nucleation promotion factor), and ARPC2 (arrestin beta 1) due to their significant correlation with further tumor parameters. This study is one of the first to summarize the expression patterns of CTTN and related genes in a complex group of OSCC samples.

Background: Cortactin is overexpressed in several types of invasive cancers. However, the role of cortactin expression in breast cancer prognosis has not been sufficiently elucidated. Therefore, we investigated the clinicopathological significance of cortactin in breast cancer. Methods: Tissue microarrays were prepared from a cohort of 506 patients with breast cancer, and cortactin expression was evaluated using immunohistochemistry. The cortactin immunoreactivity score (IRS) was quantified as the product of the intensity score and the percentage of immunoreactive cells. Cortactin expression was classified as low or high using the IRS (IRS ≤ 4 as a cortactin-low value and IRS > 4 as a cortactin-high value). We compared cortactin expression and clinicopathological factors according to the molecular subtypes of breast cancer. Results: Of 506 breast cancer cases, 333 and 173 showed high and low cortactin expression, respectively. Of the 333 patients with high cortactin expression, 204, 58, and 71 had luminal, HER2, and triple-negative breast cancer (TNBC), respectively. In the univariate and multivariate analyses of patients with TNBC, cortactin expression was found to be a significant prognostic factor for overall survival (OS). However, in all patients with non-TNBC, cortactin expression had no significant association with prognosis or overall survival. Survival curves revealed that among patients with TNBC, the high-cortactin group had a better prognosis in disease-free survival and OS. Conclusions: Cortactin expression may be a good biomarker for predicting the prognosis of patients with TNBC. [ABSTRACT FROM AUTHOR]

Ubiquitin‐specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating β‐catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on β‐catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin‐binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of β‐catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer. [ABSTRACT FROM AUTHOR]

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that is still fatal in many cases. T cell blasts are characterized by hyperactivation and strong proliferative and migratory capacities. The chemokine receptor CXCR4 is involved in mediating malignant T cell properties, and cortactin has been shown to control CXCR4 surface localization in T-ALL cells. We have previously shown that cortactin overexpression is correlated with organ infiltration and relapse in B-ALL. However, the role of cortactin in T cell biology and T-ALL remains elusive. Here, we analyzed the functional relevance of cortactin for T cell activation and migration and the implications for T-ALL development. We found that cortactin is upregulated in response to T cell receptor engagement and recruited to the immune synapse in normal T cells. Loss of cortactin caused reduced IL-2 production and proliferation. Cortactin-depleted T cells showed defects in immune synapse formation and migrated less due to impaired actin polymerization in response to T cell receptor and CXCR4 stimulation. Leukemic T cells expressed much higher levels of cortactin compared to normal T cells that correlated with greater migratory capacity. Xenotransplantation assays in NSG mice revealed that cortactin-depleted human leukemic T cells colonized the bone marrow significantly less and failed to infiltrate the central nervous system, suggesting that cortactin overexpression drives organ infiltration, which is a major complication of T-ALL relapse. Thus, cortactin could serve as a potential therapeutic target for T-ALL and other pathologies involving aberrant T cell responses. [ABSTRACT FROM AUTHOR]

Gastric cancer is a malignant tumor with high mortality and high incidence. This study aims to explore the function and molecular mechanism of Cortactin on gastric cancer progression in vitro and in vivo. A bioinformatics analysis from TCGA displayed that Cortactin was highly expressed in gastric cancer samples, and patients with a high Cortactin level had a worse survival rate. Subsequently, we investigated the specific mechanism of action of A in gastric cancer by collecting patient samples for immunohistochemistry, WB, qRT-PCR, cell transfection, cell invasion and metastasis, and constructing tumor xenografts in nude mice. Overexpression of Cortactin inhibited apoptosis and enhanced cellular proliferation and mobility in AGS cells, while those activities were reversed by the knockdown of MMP2 or MMP9. Conversely, the deletion of Cortactin induced apoptosis and suppressed cell growth and metastasis in SGC7901 cells, whereas those behaviors were inhibited by overexpression of MMP2 or MMP9. Additionally, the ERK pathway was activated by Cortactin upregulation. In vivo studies presented that overexpression of Cortactin promoted tumor growth, increased Ki67 expression, and reduced caspase 3 expression, which was reversed by ERK inhibitor treatment. In conclusion, Cortactin acted as an oncogene in gastric cancer and exerted its function by ERK/MMP2/MMP9 signaling pathway.

Chronic lymphocytic leukemia cells (CLL) migrate between the blood and lymphoid tissues in response to chemokines. Such migration requires structured cytoskeletal-actin polymerization, which may involve the protein cortactin. We discovered that treatment of CLL cells with Wnt5a causes Receptor tyosin kinase-like orphan receptor 1 (ROR1) to bind cortactin, which undergoes tyrosine phosphorylation at Y421, recruits ARHGEF1, and activates RhoA, thereby enhancing leukemia-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. We transfected the CLL-cell-line MEC1 with either full-length ROR1 or various mutant forms of ROR1 to examine the structural features required for binding cortactin. We found that the proline-rich domain (PRD) was necessary for ROR1 to recruit cortactin. We generated MEC1 cells that each expressed a mutant form of ROR1 with a single amino-acid substitution of alanine (A) for proline (P) in potential SH3-binding sites in the ROR1-PRD at positions 784, 808, 826, 841, or 850. In contrast to wild-type ROR1, or other ROR1P=>A mutants, ROR1P(841)A failed to complex with cortactin or ARHGEF1 in response to Wnt5a. Moreover, Wnt5a could not induce MEC1-ROR1P(841)A to phosphorylate cortactin or enhance CLL-cell F-actin polymerization. Taken together, these studies show that cortactin plays an important role in ROR1-dependent Wnt5a-enhanced CLL-cell migration.

Pathogenic bacteria possess a great potential of causing infectious diseases and represent a serious threat to human and animal health. Understanding the molecular basis of infection development can provide new valuable strategies for disease prevention and better control. In host‐pathogen interactions, actin‐cytoskeletal dynamics play a crucial role in the successful adherence, invasion, and intracellular motility of many intruding microbial pathogens. Cortactin, a major cellular factor that promotes actin polymerization and other functions, appears as a central regulator of host‐pathogen interactions and different human diseases including cancer development. Various important microbes have been reported to hijack cortactin signaling during infection. The primary regulation of cortactin appears to proceed via serine and/or tyrosine phosphorylation events by upstream kinases, acetylation, and interaction with various other host proteins, including the Arp2/3 complex, filamentous actin, the actin nucleation promoting factor N‐WASP, focal adhesion kinase FAK, the large GTPase dynamin‐2, the guanine nucleotide exchange factor Vav2, and the actin‐stabilizing protein CD2AP. Given that many signaling factors can affect cortactin activities, several microbes target certain unique pathways, while also sharing some common features. Here we review our current knowledge of the hallmarks of cortactin as a major target for eminent Gram‐negative and Gram‐positive bacterial pathogens in humans. [ABSTRACT FROM AUTHOR]

Metastasis is the primary stumbling block to the treatment of bladder cancer (BC). In order to spread, tumor cells must acquire increased migratory and invasive capacity, which is tightly linked with pseudopodia formation. Here, we unravel the effects of sulforaphane (SFN), an isothiocyanate in cruciferous vegetables, on the assembly of pseudopodia and BC metastasis, and its molecular mechanism in the process. Our database analysis revealed that in bladder tumor, pseudopodia-associated genes, CTTN , WASL and ACTR2/ARP2 are upregulated. SFN caused lamellipodia to collapse in BC cells by blocking the CTTN-ARP2 axis. SFN inhibited invadopodia formation and cell invasion by reducing WASL in different invasive BC cell lines. The production of ATP, essential for the assembly of pseudopodia, was significantly increased in bladder tumors and strongly inhibited by SFN. Overexpressing AKT1 reversed the downregulation of ATP in SFN-treated bladder cancer cells and restored filopodia and lamellipodia morphology and function. Bioluminescent imaging showed that SFN suppressed BC metastases to the lung of nude mice while downregulating Cttn and Arp2 expression. Our study thus reveals mechanisms of SFN action in inhibiting pseudopodia formation and highlights potential targeting options for the therapy of metastatic bladder cancer. Pseudopodia formation in bladder cancer (BC) cells is driven by the upregulation of multiple pseudopodia-associated genes, including CDC42, ACTR2/ARP2, WAS and CTTN. Dataset analysis shows that high expression of the gene set CDC42 , WAS and ACTR2/ARP2 , accompanied by low expression of ARPC3 in bladder tumors represents the high-risk patient group with poorer overall survival. Sulforaphane (SFN), a plant-derived isothiocyanate from cruciferous vegetables, causes pseudopodia to collapse in BC cells by downregulating the CTTN (cortactin)-ARP2 axis. SFN also suppresses WASL, thus blocking invadopodia formation and BC cell invasion. Notably, SFN inhibits AKT1-mediated ATP production, which results in decreased ARP2 expression and impaired lamellipodia and filopodia, thereby reducing BC cell migration. [Display omitted] • High expression of CDC42 , WAS and ACTR2 with low expression of ARPC3 in bladder tumor represents the high-risk patient group. • SFN causes pseudopodia to collapse in bladder cancer cells by inhibiting the cortactin (CTTN)-ARP2 axis. • SFN suppresses WASL, thereby blocking invadopodia formation and bladder cancer cell invasion. • SFN inhibits AKT1-mediated ATP production, impairing filopodia and lamellipodia and bladder cancer cell migration. • SFN suppresses bladder cancer metastases to the lung of nude mice while downregulating Cttn and Actr 2 expression. [ABSTRACT FROM AUTHOR]

Background: Cortactin is overexpressed in several types of invasive cancers. However, the role of cortactin expression in breast cancer prognosis has not been sufficiently elucidated. Therefore, we investigated the clinicopathological significance of cortactin in breast cancer. Methods: Tissue microarrays were prepared from a cohort of 506 patients with breast cancer, and cortactin expression was evaluated using immunohistochemistry. The cortactin immunoreactivity score (IRS) was quantified as the product of the intensity score and the percentage of immunoreactive cells. Cortactin expression was classified as low or high using the IRS (IRS ≤ 4 as a cortactin-low value and IRS > 4 as a cortactin-high value). We compared cortactin expression and clinicopathological factors according to the molecular subtypes of breast cancer. Results: Of 506 breast cancer cases, 333 and 173 showed high and low cortactin expression, respectively. Of the 333 patients with high cortactin expression, 204, 58, and 71 had luminal, HER2, and triple-negative breast cancer (TNBC), respectively. In the univariate and multivariate analyses of patients with TNBC, cortactin expression was found to be a significant prognostic factor for overall survival (OS). However, in all patients with non-TNBC, cortactin expression had no significant association with prognosis or overall survival. Survival curves revealed that among patients with TNBC, the high-cortactin group had a better prognosis in disease-free survival and OS. Conclusions: Cortactin expression may be a good biomarker for predicting the prognosis of patients with TNBC.

Objective To explore the role of ezrin in the formation of invadopodia in hepatocellular carcinoma (HCC) cells. Methods GEPIA database was used to analyze the expression correlation between ezrin and the invadopodia marker cortactin in HCC tissues at mRNA level. Tissue specimens obtained from 105 HCC patients were made into tissue microarrays, which were subsequently detected by immunohistochemistry to analyze the expression correlation of ezrin with cortactin at protein level. Moreover, the relationship between ezrin level and the clinicopathological features as well as the prognosis of HCC patients were analyzed. Finally, ezrin expression was knocked down or up-regulated in HCC cells, respectively, and Transwell assay was performed to observe the effects of ezrin expression changes on the invasion and migration of HCC cells; Western blotting and immunofluorescence assay were also adopted to detect the role of ezrin in the formation of invadopodia in HCC cells. Results Bioinformatics analysis revealed that the mRNA level of ezrin was positively correlated with that of cortactin in HCC tissues (r=0.420, P < 0.01), and immunohistochemistry assay also confirmed the positive correlation between ezrin and cortactin expression at protein level (r= 0.491, P < 0.001). Analysis of clinical data showed that high expression of ezrin protein was correlated with vascular invasion rate (P=0.016) and tumor-related mortality rate (P=0.018) in HCC patients, and patients with higher ezrin level had both lower 5-year postoperative survival rate and 5-year tumor-free survival rate (P < 0.05). In cell experiments, up-regulation of ezrin promoted the invasion and metastasis (P < 0.05), and facilitated the invadopodia formation of HCC cells (P < 0.05). In contrast, knockdown of ezrin suppressed the invadopodia formation and inhibited the invasion and metastasis of HCC cells. Conclusion Ezrin enhances the invasive and migratory abilities of HCC cells by regulating invadopodia formation and thus promotes the progression of HCC.

Intracerebral hemorrhage (ICH) represents the deadliest subtype of all strokes. The development of brain edema, a consequence of blood-brain barrier (BBB) disruption, is the most life-threatening event after ICH. Pathophysiological conditions activate the endothelium, one of the components of BBB, inducing rearrangement of the actin cytoskeleton. Upon activation, globular actin assembles into a filamentous actin resulting in the formation of contractile actin bundles, stress fibers. The contraction of stress fibers leads to the formation of intercellular gaps between endothelial cells increasing the permeability of BBB. In the present study, we investigated the effect of ICH on stress fiber formation in CD1 mice. We hypothesized that ICH-induced formation of stress fiber is triggered by the activation of PDGFR-β and mediated by the cortactin/RhoA/LIMK pathway. We demonstrated that ICH induces formation of stress fibers. Furthermore, we demonstrated that the inhibition of PDGFR-β and its downstream reduced the number of stress fibers, preserving BBB and resulting in the amelioration of brain edema and improvement of neurological functions in mice after ICH.

Background Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by leukocytosis and an accumulation of granulocytes and their precursors. Cortactin is an actin-binding protein substrate of Src kinase. High cortactin expression in many hematological malignancies has been correlated with adverse prognostic factors. Aim The aim of our study was to measure cortactin levels in patients with CML at diagnosis and correlate such levels with other prognostic factors. Patients and methods This is a case–control study that was executed at hematology unit, Ain-Shams University Hospital during the period between January 2021 and October 2021. The study included 25 newly diagnosed patients with chronic phase CML and 25 healthy controls. Accelerated phase and blast crisis were excluded from the study. Results Cortactin level at diagnosis was higher in the patients group compared with the control group (71.04 ± 20.04 vs. 36.8 ± 11.6%, P<0.001). Cortactin level was significantly higher in patients who did not achieve complete hematological remission (CHR) at 3 months in comparison with those who achieved CHR (88.49 ± 8.02 vs. 61.23 ± 17.98, P<0.001). Patients who failed to attain CHR at 3 months had a significantly worse prognostic score at diagnosis using Sokal, Hasford, and ELTS scores (P=0.016, 0.035, and 0.009, respectively), but this did not apply to EUTOS score (P=0.089). Conclusion Higher cortactin levels are associated with delayed CHR in newly diagnosed patients with chronic phase CML, and it can be used as a prognostic marker for patients with CML at diagnosis. [ABSTRACT FROM AUTHOR]

Purpose: Metastasis is a major leading cause of mortality in female breast cancer (BrCa). Cellular motility is a pathological process of metastasis remarked by the overexpression of cortactin (CTTN), Ras homolog family member-A (RhoA), and Rho-associated kinase (ROCK) genes. Their balance is responsible for upholding the integrity of healthy epithelial cell junctions. This study aimed to explore the associations between a posteriori dietary patterns and the expression levels of pro-metastatic genes in primary BrCa. Methods: In this consecutive case series, 215 eligible women, newly diagnosed with histologically confirmed non-metastatic BrCa (stage I-IIIA), were recruited from Hospitals in Tabriz, Northwestern Iran (2015–2017). The tumoral expression levels of genes were quantified using real-time reverse transcription-polymerase chain reaction. Dietary data assessment was carried out using a validated food frequency questionnaire. Results: Three dietary patterns were identified using principal component analysis (KMO = 0.699). Adherence to the "vegan" pattern (vegetables, fruits, legumes, nuts, seeds, and whole grains) was inversely associated with the expression levels of RhoA (ORAdj.T3vs.T1 = 0.24, 95%CI 0.07–0.79) and ROCK (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08–0.87). In addition, the highest adherence to the "prudent" pattern (spices, seafood, dairy, and vegetable oils) decreased the odds of overexpressions at RhoA (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08–0.84) and ROCK genes (ORAdj.T3vs.T1 = 0.29, 95%CI 0.09–0.95). The highest adherence to "Western" pattern (meat, processed meat, hydrogenated fat, fast food, refined cereals, sweets, and soft drinks) was a risk factor associated with the overexpression of RhoA (ORAdj.T3vs.T1 = 3.15, 95%CI 1.12–8.85). Conclusion: Adherence to healthy dietary patterns was significantly associated with the downregulation of pro-metastatic genes. Findings provided new implications to advance the nutrigenomic knowledge to prevent the odds of over-regulations in pro-metastatic genes of the primary BrCa. [ABSTRACT FROM AUTHOR]

Rationale: Cortactin, an actin-binding cytoskeletal protein, plays a crucial role in maintaining endothelial cell (EC) barrier integrity and regulating vascular permeability. The gene encoding cortactin, CTTN, is implicated in various lung inflammatory disorders. Despite this, the transcriptional regulation of CTTN by inflammatory stimuli and promoter SNPs remains unexplored., Methods: We transfected human lung ECs with a full-length CTTN promoters linked to a luciferase reporter to measure promoter activity. SNP-containing CTTN promoter was created via site-directed mutagenesis. Transfected ECs were exposed to LPS (PAMP), TNF-α (cytokine), cyclic stretch (CS), FG-4592 (HIF-inducer), NRF2 (anti-oxidant modulator), FTY-(S)-phosphate (endothelial barrier enhancer), and 5'-Aza (demethylation inducer). Immunohistochemistry was used to assess cortactin expression in mouse lungs exposed to LPS., Results: LPS, TNF-α, and 18%CS significantly increased CTTN promoter activities in a time-dependent manner (P<0.05). The variant rs34612166 (-212T/C) markedly enhanced LPS- and 18%CS- induced CTTN promoter activities (P<0.05). FG-4592 significantly boosted CTTN promoter activities (P<0.01), which were partially inhibited by HIF1α (KC7F2) and HIF2α (PT2385) inhibitors (P<0.05). NRF2 activator Bixin increased CTTN promoter activities, whereas NRF2 inhibitor Brusatol reduced them (P<0.05). 5'-Aza increased CTTN promoter activities by 2.9-fold (P<0.05). NF-κB response element mutations significantly reduced CTTN promoter activities response to LPS and TNFα. FTY-(S)-phosphate significantly increased CTTN promoter activities in 24 h. In vivo, cortactin levels were significantly elevated in inflammatory mouse lungs exposed to LPS for 18 h., Conclusion: CTTN transcriptional is significantly influenced by inflammatory factors and promoter variants. Cortactin, essential in mitigating inflammatory edema, presents a promising therapeutic target to alleviate severe inflammatory disorders., (© 2024 The Author(s).)

Respiratory syncytial virus (RSV) infection is the leading cause of acute lower respiratory tract infection in young children worldwide. Our group recently revealed that RSV infection disrupts the airway epithelial barrier in vitro and in vivo. However, the underlying molecular pathways were still elusive. Here, we report the critical roles of the filamentous actin (F-actin) network and actin-binding protein cortactin in RSV infection. We found that RSV infection causes F-actin depolymerization in 16HBE cells, and that stabilizing the F-actin network in infected cells reverses the epithelial barrier disruption. RSV infection also leads to significantly decreased cortactin in vitro and in vivo. Cortactin-knockout 16HBE cells presented barrier dysfunction, whereas overexpression of cortactin protected the epithelial barrier against RSV. The activity of Rap1 (which has Rap1A and Rap1B forms), one downstream target of cortactin, declined after RSV infection as well as in cortactin-knockout cells. Moreover, activating Rap1 attenuated RSV-induced epithelial barrier disruption. Our study proposes a key mechanismin which RSV disrupts the airway epithelial barrier via attenuating cortactin expression and destabilizing the F-actin network. The identified pathways will provide new targets for therapeutic intervention toward RSV-related disease. [ABSTRACT FROM AUTHOR]

To study the role of HER2/cortactin co-overexpression in advanced gastric cancer (GC). This study retrospectively enrolled 246 patients with stage III GC from January 2015 to December 2016 at our hospital. We explored, using immunostaining techniques, the role of the expression of cortactin and HER2 in the progression of advanced GC. The patient data, including age, sex, cortactin and HER2 expression, pathological parameters and survival, were collected. Univariate and multivariate analyses were used to analyze the characteristics, survival, and prognostic factors of the patients. The results showed that the expression of cortactin was significantly associated with vascular-lymphatic invasion (P < 0.001), N stage (P = 0.001), and TNM stage (P = 0.046). HER2 overexpression correlated with tumor size (P = 0.002), neural invasion (P = 0.002), Lauren classification (P = 0.005) and N stage (P = 0.034). Through univariate analysis using the Kaplan–Meier method, vascular-lymphatic invasion (P = 0.015), neural invasion (P = 0.021), N stage (P < 0.003), and HER2/cortactin co-overexpression (P < 0.028) were shown to be significantly associated with overall survival. Multivariate analysis demonstrated that vascular lymphatic invasion (hazard ratio = 1.481, 95% CI, 1.064 to 2.061, P = 0.020), neural invasion (hazard ratio = 1.505, 95% CI, 1.084 to 2.089, P = 0.015), N stage (N2/N1: hazard ratio = 1.655, 95% CI, 1.048 to 2.641, P < 0.031, N3/N1: hazard ratio = 2.089, 95% CI, 1.325 to 3.295, P < 0.002), and HER2/cortactin co-overexpression (hazard ratio = 1.427, 95% CI, 1.007 to 2.024, P = 0.046) were independent prognostic factors for poor overall survival. The results suggested that HER2/cortactin co-overexpression is an important predictive biomarker for GC patients. GC patients with HER2/cortactin co-overexpression may receive dual-targeted therapy to improve survival prognosis in the future. [ABSTRACT FROM AUTHOR]

Plexin-domain containing 2 (PLXDC2) has been reported as an oncoprotein in several human malignancies. However, its expression and roles in gastric cancer remain largely unclear. In this study, we found that PLXDC2 was highly expressed in gastric cancer tissues, and the expression levels were positively correlated with clinicopathological features, but negatively with the patients' outcome. Cox regression analysis identified PLXDC2 as an independent prognostic indicator for the patients. Knockdown of PLXDC2 markedly suppressed the in vitro invasion and in vivo metastasis of gastric cancer cells, while overexpression of PLXDC2 resulted in opposite effects. Mechanistically, PLXDC2 enhanced the level of phosphorylated Cortactin (p-Cortactin) by physically interacting with protein tyrosine phosphatase 1B (PTP1B), an important dephosphorylase, to prevent its dephosphorylating of p-Cortactin, thereby promoting the formation of invadopodia. Collectively, our results indicate that PLXDC2 contributes to the invasion and metastasis of gastric cancer by inhibiting PTP1B to facilitate the invadopodium formation, and may serve as a potential prognostic biomarker and a therapeutic target for this disease. [ABSTRACT FROM AUTHOR]

Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodiaassociated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. [ABSTRACT FROM AUTHOR]

In patients with sickle cell disease (SCD), acute chest syndrome (ACS) is a common form of acute lung injury and a major cause of morbidity and mortality. The pathophysiology of ACS is complex, and hemin, the prosthetic moiety of hemoglobin, has been implicated in endothelial cell (EC) activation and subsequent acute lung injury (ALI) and ACS in vitro and in animal studies. Here, we examined the role of cortactin (CTTN), a cytoskeletal protein that regulates EC function, in response to hemin-induced ALI and ACS. Cortactin heterozygous (Cttn+/-) mice (n = 8) and their wild-type siblings (n = 8) were irradiated and subsequently received bone marrow cells (BMCs) extruded from the femurs of SCD mice (SS) to generate SS Cttn+/- and SS CttnWT chimeras. Following hemoglobin electrophoretic proof of BMC transplantation, the mice received 35 mmol/kg of hemin. Within 24 h, surviving mice were euthanized, and bronchoalveolar fluid (BAL) and lung samples were analyzed. For in vitro studies, human lung microvascular endothelial cells (HLMVECs) were used to determine hemin-induced changes in gene expression and reactive oxygen species (ROS) generation in cortactin deficiency and control conditions. When compared with wild-type littermates, the mortality for SS Cttn+/- mice trended to be lower after hemin infusion and these mice exhibited less severe lung injury and less necroptotic cell death. In vitro studies confirmed that cortactin deficiency is protective against hemin-induced injury in HMLVECs, by decreasing protein expression of p38/HSP27, improving cell barrier function, and decreasing the production of ROS. Further studies examining the role of CTTN in ACS are warranted and may open a new avenue of potential treatment for this devastating disease. [ABSTRACT FROM AUTHOR]

Abstract Background Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development. Methods Co-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis. Results The data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas. Conclusion The main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs. Video abstract

ObjectiveCortactin translocates to the cell periphery in vascular endothelial cells (ECs) on cortical-actin assembly in response to pulsatile shear stress. Because cortactin has putative sites for AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 1 (SIRT1) deacetylation, we examined the hypothesis that AMPK and SIRT1 coregulate cortactin dynamics in response to shear stress.Approach and resultsAnalysis of the ability of AMPK to phosphorylate recombinant cortactin and oligopeptides whose sequences matched AMPK consensus sequences in cortactin pointed to Thr-401 as the site of AMPK phosphorylation. Mass spectrometry confirmed Thr-401 as the site of AMPK phosphorylation. Immunoblot analysis with AMPK siRNA and SIRT1 siRNA in human umbilical vein ECs and EC-specific AMPKα2 knockout mice showed that AMPK phosphorylation of cortactin primes SIRT1 deacetylation in response to shear stress. Immunoblot analyses with cortactin siRNA in human umbilical vein ECs, phospho-deficient T401A and phospho-mimetic T401D mutant, or aceto-deficient (9K/R) and aceto-mimetic (9K/Q) showed that cortactin regulates endothelial nitric oxide synthase activity. Confocal imaging and sucrose-density gradient analyses revealed that the phosphorylated/deacetylated cortactin translocates to the EC periphery facilitating endothelial nitric oxide synthase translocation from lipid to nonlipid raft domains. Knockdown of cortactin in vitro or genetic reduction of cortactin expression in vivo in mice substantially decreased the endothelial nitric oxide synthase-derived NO bioavailability. In vivo, atherosclerotic lesions increase in ApoE-/-/cortactin+/- mice, when compared with ApoE-/-/cortactin+/+ littermates.ConclusionsAMPK phosphorylation of cortactin followed by SIRT1 deacetylation modulates the interaction of cortactin and cortical-actin in response to shear stress. Functionally, this AMPK/SIRT1 coregulated cortactin-F-actin dynamics is required for endothelial nitric oxide synthase subcellular translocation/activation and is atheroprotective.

Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals. [Display omitted] • Cortactin is present in the postsynaptic machinery of the neuromuscular junction. • Cortactin interacts with αDsytrobrevin1 at the neuromuscular junction. • Cortactin knockout mice have impaired organization of the neuromuscular junction. [ABSTRACT FROM AUTHOR]

Cortactin (CTTN) is an actin-binding and cytoskeletal protein that is found in abundance in the cell cortex and other peripheral structures of most cell types. It was initially described as a target for Src-mediated phosphorylation at several tyrosine sites within CTTN, and post-translational modifications at these tyrosine sites are a primary regulator of its function. CTTN participates in multiple cellular functions that require cytoskeletal rearrangement, including lamellipodia formation, cell migration, invasion, and various other processes dependent upon the cell type involved. The role of CTTN in vascular endothelial cells is particularly important for promoting barrier integrity and inhibiting vascular permeability and tissue edema. To mediate its functional effects, CTTN undergoes multiple post-translational modifications and interacts with numerous other proteins to alter cytoskeletal structures and signaling mechanisms. In the present review, we briefly describe CTTN structure, post-translational modifications, and protein binding partners and then focus on its role in regulating cellular processes and well-established functional mechanisms, primarily in vascular endothelial cells and disease models. We then provide insights into how CTTN function affects the pathophysiology of multiple lung disorders, including acute lung injury syndromes, COPD, and asthma. [ABSTRACT FROM AUTHOR]

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity. [ABSTRACT FROM AUTHOR]

Background: A glandular odontogenic cyst (GOC) has an intriguing, aggressive behaviour whose mechanisms have not yet been clarified. Objective: To conduct a collaborative cross‐sectional study on the clinical, demographic, microscopic and immunohistochemical characteristics of GOCs, emphasizing the histopathological characteristics and expression of proteins related to invasiveness. Methods: Twenty‐two cases of GOC from three oral and maxillofacial pathology services in Brazil were selected from 1988 to 2018. Clinical and demographic data were collected. Histopathological features were evaluated in detail. Sixteen cases of GOC were also submitted to immunohistochemistry to detect MT1‐MMP, TKS4, TKS5 and cortactin, the key regulators of invadopodia formation. Results: Glandular odontogenic cysts were primarily seen in men over 40 years of age, in the posterior mandible and the anterior maxilla as a unilocular, radiolucent lesion. All cases presented hobnail cells, clear cells and variable thickness of the lining epithelium, 3 of the 10 key histopathological parameters to be evaluated in GOCs. Immunohistochemistry revealed a greater expression of the studied proteins in the GOCs than in the controls (p < 0.0001). Conclusion: Overexpression of proteins that regulate cell invasiveness was identified, and the present study's findings suggest that invadopodia activity is a possible mechanism used by GOCs to promote local invasion, which could partly explain its intriguing biological behaviour. [ABSTRACT FROM AUTHOR]

Objective To investigate the expression of cortactin in colorectal cancer and its correlation with clinicopathological parameters and prognosis. Methods The expressions of cortactin in normal colorectal mucosal tissue and colorectal cancer tissue in paraffin-embedded tissue microarray from 319 patients who were diagnosed as colorectal cancer and treated in Cancer Hospital of Chinese Academy of Medical Sciences from 2006 to 2009 was detected by immunohistochemistry. Kaplan-Meier method and Log rank test were used for survival analysis, and Cox proportional risk regression model was used for multivariate analysis. Results The positive expression rates of cortactin in colorectal cancer tissue and normal colorectal mucosal tissue were 61.1% (195/319) and 5.6% (18/319, P<0.001), respectively. T-stage, N-stage, American Joint Committee on Cancer (AJCC) stage, degree of tumor differentiation, neural invasion and preoperative carcinoembryonic antigen (CEA) levels were associated with the expression of cortactin (P< 0.05). The positive expression of cortactin was associated with poorer disease-free survival (P=0.036) and overall survival (P=0.043), and the effect was more significant in patients with stage I to Ill. For patients with stage - colorectal cancer, postoperative adjuvant therapy was associated with disease-free survival (P=0.007) and overall survival (P=0.015). The vascular tumor embolus, pathological type, preoperative CEA level and cortactin expression were independent influencing factors for disease-free survival (P<0.05). The age. AJCC stage, preoperative CEA level and cortactin expression were independent influencing factors for overall survival (P<0.05). Preoperative CEA level and cortactin expression were independent influencing factors for disease-free survival and overall survival (P<0.05). Conclusion Cortactin is expressed in colorectal cancer and in stage II-III patients, it is a potential predictor of colorectal cancer prognosis. [ABSTRACT FROM AUTHOR]

Abstract Background Metastasis accounts for ninety percent of breast cancer (BrCa) mortality. Cortactin, Ras homologous gene family member A (RhoA), and Rho-associated kinase (ROCK) raise cellular motility in favor of metastasis. Claudins (CLDN) belong to tight junction integrity and are dysregulated in BrCa. Thus far, epidemiologic evidence regarding the association of different pro-metastatic genes with pathological phenotypes of BrCa is largely inconsistent. This study aimed to determine the possible transcriptional models of pro-metastatic genes incorporate in holding the integrity of epithelial cell–cell junctions (CTTN, RhoA, ROCK, CLDN-1, CLDN-2, and CLDN-4), for the first time, in association with clinicopathological features of primary BrCa. Methods In a consecutive case-series design, 206 newly diagnosed non-metastatic eligible BrCa patients with histopathological confirmation (30–65 years) were recruited in Tabriz, Iran (2015–2017). Real-time RT-PCR was used. Then fold changes in the expression of target genes were measured. Results ROCK amplification was associated with the involvement of axillary lymph node metastasis (ALNM; ORadj. = 3.05, 95%CI 1.01–9.18). Consistently, inter-correlations of CTTN-ROCK (β = 0.226, P

Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.

Axon branching is a fundamental aspect of neuronal morphogenesis, neuronal circuit formation, and response of the nervous system to injury. Sterile alpha and TIR motif containing 1 (SARM1) was initially identified as promoting Wallerian degeneration of axons. We now report a novel function of SARM1 in postnatal sensory neurons; the suppression of axon branching. Axon collateral branches develop from axonal filopodia precursors through the coordination of the actin and microtubule cytoskeleton. In vitro analysis revealed that cultured P0-2 dorsal root ganglion sensory neurons from a SARM1 knockout (KO) mouse exhibit increased numbers of collateral branches and axonal filopodia relative to wild-type neurons. In SARM1 KO mice, cutaneous sensory endings exhibit increased branching in the skin in vivo with normal density of innervation. Transient axonal actin patches serve as cytoskeletal platforms from which axonal filopodia emerge. Live imaging analysis of axonal actin dynamics showed that SARM1 KO neurons exhibit increased rates of axonal actin patch formation and increased probability that individual patches will give rise to a filopodium before dissipating. SARM1 KO axons contain elevated levels of drebrin and cortactin, two actin regulatory proteins that are positive regulators of actin patches, filopodia formation, and branching. Live imaging of microtubule plus tip dynamics revealed an increase in the rate of formation and velocity of polymerizing tips along the axons of SARM1 KO neurons. Stationary mitochondria define sites along the axon where branches may arise, and the axons of SARM1 KO sensory neurons exhibit an increase in stationary mitochondria. These data reveal SARM1 to be a negative regulator of axonal cytoskeletal dynamics and collateral branching. [ABSTRACT FROM AUTHOR]

To evaluate and compare the immunohistochemical expression of cortactin in the epithelial lining of orthokeratinized odontogenic cyst (OOC), sporadic odontogenic keratocyst (OKC), and syndromic OKC. Formalin-fixed paraffin-embedded tissue blocks of histopathologically diagnosed cases of OOC, OKC, syndromic OKC, normal buccal mucosa (NBM), and oral squamous cell carcinoma (OSCC) were examined for immunohistochemical expression of cortactin. Clear brown cytoplasmic and membranous staining was considered positive. A statistically significant difference was observed between OOC and syndromic OKC (p < 0.001), as well as between sporadic OKC and syndromic OKC (p < 0.001). Although not statistically significant, the expression of cortactin was slightly higher in the basal layer of NBM (mean = 0.47), OOC (mean = 0.27), sporadic OKC (mean = 0.47) syndromic OKC (mean = 1.53), and OSCC (mean = 0.67) than in the parabasal layers of NBM (mean = 0.27), OOC (mean = 0.20), sporadic OKC (mean = 0.47), syndromic OKC (mean = 1.27), and OSCC (mean = 0.60). The expression of cortactin in the basal layer may suggest the formation of invadopodia in the basal layer where the invasion mechanism occurs. This finding is further supported by the higher localization of cortactin in areas of epithelial budding and daughter cysts in syndromic OKC, thereby reaffirming its possible association with recurrence. • Local invasion is the direct extension of lesional cells via proteolytic membranous protrusions/invadopodia for migration. • Cortactin is a biomarker for invadopodia having a significant role in invasion and cell migration. • We suggest that presence of invadopodia in syndromic OKC plays a significant role in its biological behavior. [ABSTRACT FROM AUTHOR]

Transcription factors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) family control important signaling pathways in the regulation of the host innate immune system. Various bacterial pathogens in the human gastrointestinal tract induce NF-kB activity and provoke proinflammatory signaling events in infected epithelial cells. NF-kB activation requires the phosphorylation-dependent proteolysis of inhibitor of kB (IkB) molecules including the NF-kB precursors through ubiqitin-mediated proteolysis. The canonical NF-kB pathway merges on IkB kinases (IKKs), which are required for signal transduction. Using CRISPR-Cas9 technology, secreted embryonic alkaline phosphatase (SEAP) reporter assays and cytokine enzyme-linked immunosorbent assay (ELISA), we demonstrate that the actin-binding protein cortactin is involved in NF-kB activation and subsequent interleukin-8 (IL-8) production upon infection by Helicobacter pylori, Salmonella enterica and Pseudomonas aeruginosa. Our data indicate that cortactin is needed to efficiently activate the c-Sarcoma (Src) kinase, which can positively stimulate NF-kB during infection. In contrast, cortactin is not involved in activation of NF-kB and IL-8 expression upon infection with Campylobacter species C. jejuni, C. coli or C. consisus, suggesting that Campylobacter species pluralis (spp.) induce a different signaling pathway upstream of cortactin to trigger the innate immune response. [ABSTRACT FROM AUTHOR]

Abstract Abnormal synaptic formation and signaling is one of the key molecular features of autism spectrum disorders (ASD). Cortactin binding protein 2 (CTTNBP2), an ASD-linked gene, is known to regulate the subcellular distribution of synaptic proteins, such as cortactin, thereby controlling dendritic spine formation and maintenance. However, it remains unclear how ASD-linked mutations of CTTNBP2 influence its function. Here, using cultured hippocampal neurons and knockin mouse models, we screen seven ASD-linked mutations in the short form of the Cttnbp2 gene and identify that M120I, R533* and D570Y mutations impair CTTNBP2 protein–protein interactions via divergent mechanisms to reduce dendritic spine density in neurons. R533* mutation impairs CTTNBP2 interaction with cortactin due to lack of the C-terminal proline-rich domain. Through an N–C terminal interaction, M120I mutation at the N-terminal region of CTTNBP2 also negatively influences cortactin interaction. D570Y mutation increases the association of CTTNBP2 with microtubule, resulting in a dendritic localization of CTTNBP2, consequently reducing the distribution of CTTNBP2 in dendritic spines and impairing the synaptic function of CTTNBP2. Finally, we generated heterozygous M120I knockin mice to mimic the genetic variation of patients and found they exhibit reduced social interaction. Our study elucidates that different ASD-linked mutations of CTTNBP2 result in diverse molecular deficits, but all have the similar consequence of synaptic impairment.

Axon branching is a fundamental aspect of neuronal morphogenesis, neuronal circuit formation, and response of the nervous system to injury. Sterile alpha and TIR motif containing 1 (SARM1) was initially identified as promoting Wallerian degeneration of axons. We now report a novel function of SARM1 in postnatal sensory neurons; the suppression of axon branching. Axon collateral branches develop from axonal filopodia precursors through the coordination of the actin and microtubule cytoskeleton. In vitro analysis revealed that cultured P0-2 dorsal root ganglion sensory neurons from a SARM1 knockout (KO) mouse exhibit increased numbers of collateral branches and axonal filopodia relative to wild-type neurons. In SARM1 KO mice, cutaneous sensory endings exhibit increased branching in the skin in vivo with normal density of innervation. Transient axonal actin patches serve as cytoskeletal platforms from which axonal filopodia emerge. Live imaging analysis of axonal actin dynamics showed that SARM1 KO neurons exhibit increased rates of axonal actin patch formation and increased probability that individual patches will give rise to a filopodium before dissipating. SARM1 KO axons contain elevated levels of drebrin and cortactin, two actin regulatory proteins that are positive regulators of actin patches, filopodia formation, and branching. Live imaging of microtubule plus tip dynamics revealed an increase in the rate of formation and velocity of polymerizing tips along the axons of SARM1 KO neurons. Stationary mitochondria define sites along the axon where branches may arise, and the axons of SARM1 KO sensory neurons exhibit an increase in stationary mitochondria. These data reveal SARM1 to be a negative regulator of axonal cytoskeletal dynamics and collateral branching.