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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-actin-decorated 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., 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-actin-decorated 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.

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-actin-decorated 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., 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-actin-decorated 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.

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.

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.

Growth factors play an essential role in abnormalities in both intracellular trafficking and signal transduction pathways responsible in normal and cancer cells. Growth factors, such as epidermal growth factor, represent a main course on the activation of mitogenic signal that contribute to the mitogen activated protein kinase (MAPK) pathway affecting cell proliferation, which is driven by Ras GTPases. However, it also induces a profound morphological change by reorganization actin and other cytoskeleton proteins, which are driven by other GTPases (i.e., Rho and Rac). Ras interference 1 (Rin1) is a key cytosolic protein, that regulates both membrane trafficking and signaling pathways through the Rin1:Vps9, which activates Rab5, and Rin1:RA, which interacts with Ras, respectively. In addition, Rin1 proline rich domain (Rin1: PR) may also help to orchestrate this complex regulation by interacting with Cortactin, which is a key molecule in actin organization at the plasma membrane via Cortactin:SH3 domain. Thus, it is possible connection between Rin1, an effector of the active form of Ras as well as an activator of Rab5, and subsequent interaction with Cortactin is of particular interest as Cortactin is frequently overexpressed in cancers. In this study, Rin1 works with a dual action: First, it sequesters Cortactin and thus, it blocks the cellular migration and invasion driven by Cortactin. Second, interaction of RIN1 with Ras in the GTP-bound form decreased serine phosphorylation of Cortactin. This inhibitory effect of RIN1 on the phosphorylation of Cortactin may be due since the interaction of Ras, which blocks both Erk and Akt activities upon EGF stimulation. In addition, Rin1 inhibits cell proliferation in cancer cells through Ras-Raf-Erk and PI3K-AKT signaling cascades by selectively affecting FOXO1 and c-Myc. This selective inhibitory effect seems to be observed on breast cancer cells but not in other cancer cell lines (human melanoma and human glioblastoma cells

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies against the acetylcholine receptor (AChR), muscle-specific kinase (MuSK) or other AChR-related proteins in the postsynaptic muscle membrane. Localized or general muscle weakness is the predominant symptom and is induced by the antibodies. Patients are grouped according to the presence of antibodies, symptoms, age at onset and thymus pathology. Diagnosis is straightforward in most patients with typical symptoms and a positive antibody test, although a detailed clinical and neurophysiological examination is important in antibody-negative patients. MG therapy should be ambitious and aim for clinical remission or only mild symptoms with near-normal function and quality of life. Treatment should be based on MG subgroup and includes symptomatic treatment using acetylcholinesterase inhibitors, thymectomy and immunotherapy. Intravenous immunoglobulin and plasma exchange are fast-acting treatments used for disease exacerbations, and intensive care is necessary during exacerbations with respiratory failure. Comorbidity is frequent, particularly in elderly patients. Active physical training should be encouraged.

Compartmentalization of calcium-dependent plasticity allows for rapid actin remodeling in dendritic spines. However, molecular mechanisms for the spatio-temporal regulation of filamentous actin (F-actin) dynamics by spinous Ca2+-transients are still poorly defined. We show that the postsynaptic Ca2+ sensor caldendrin orchestrates nano-domain actin dynamics that are essential for actin remodeling in the early phase of long-term potentiation (LTP). Steep elevation in spinous [Ca2+]i disrupts an intramolecular interaction of caldendrin and allows cortactin binding. The fast on and slow off rate of this interaction keeps cortactin in an active conformation, and protects F-actin at the spine base against cofilin-induced severing. Caldendrin gene knockout results in higher synaptic actin turnover, altered nanoscale organization of spinous F-actin, defects in structural spine plasticity, LTP, and hippocampus-dependent learning. Collectively, the data indicate that caldendrin-cortactin directly couple [Ca2+]i to preserve a minimal F-actin pool that is required for actin remodeling in the early phase of LTP.

Normal cerebral cortical function requires a highly ordered balance between projection neurons and interneurons. During development these two neuronal populations migrate from distinct progenitor zones to form the cerebral cortex, with interneurons originating in the more distant ganglionic eminences. Moreover, deficits in interneurons have been linked to a variety of neurodevelopmental disorders underscoring the importance of understanding interneuron development and function. We, and others, have identified SDF1 signaling as one important modulator of interneuron migration speed and leading process branching behavior in mice, although how SDF1 signaling impacts these behaviors remains unknown. We previously found SDF1 inhibited leading process branching while increasing the rate of migration. We have now mechanistically linked SDF1 modulation of leading process branching behavior to a dual regulation of both actin and microtubule organization. We find SDF1 consolidates actin at the leading process tip by de-repressing calpain protease and increasing proteolysis of branched-actin-supporting cortactin. Additionally, SDF1 stabilizes the microtubule array in the leading process through activation of the microtubule-associated protein doublecortin (DCX). DCX stabilizes the microtubule array by bundling microtubules within the leading process, reducing branching. These data provide mechanistic insight into the regulation of interneuron leading process dynamics during neuronal migration in mice and provides insight into how cortactin and DCX, a known human neuronal migration disorder gene, participate in this process.

Cortactin stimulates cell migration, invasion, and experimental metastasis. Overexpression of cortactin has been reported in several human cancers. CRK was originally identified as an oncogene product of v-CRK in a CT10 chicken retrovirus system. Overexpression of CRKII has been reported in several human cancers. CRKII regulates cell migration, morphogenesis, invasion, phagocytosis, and survival; however, the underlying mechanisms are not well understood. We evaluated the possibility of the combination of cortactin and CRKII as an appropriate molecular target for cancer gene therapy. The expression of cortactin and CRKII in 70 primary oral squamous cell carcinomas and 10 normal oral mucosal specimens was determined immunohistochemically, and the correlation of cortactin and CRKII co-overexpression with clinicopathological factors was evaluated. Co-overexpression of cortactin and CRKII was detected in 31 of 70 oral squamous cell carcinomas, the frequency being significantly greater than in normal oral mucosa. In addition, cortactin and CRKII co-overexpression was more frequent in higher-grade cancers according to the T classification, N classification, and invasive pattern. RNAi-mediated co-suppression of cortactin and CRKII expression reduced the migration and invasion potential of an oral squamous cell carcinoma cell line, OSC20. Downregulation of cortactin and CRKII expression also reduced the expression of vimentin, fibronectin, and N-cadherin. These results indicate that the co-overexpression of cortactin and CRKII may be tightly associated with an aggressive phenotype of oral squamous cell carcinoma. Therefore, we propose that the combination of cortactin and CRKII could be a potential molecular target of gene therapy by RNAi-targeting in oral squamous cell carcinoma., Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology, 26(1), pp.14-21; 2014

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactin's movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin's interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein's use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactin's movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin's interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein's use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.

BackgroundCell migration plays an important role in many physiological and pathological processes, including immune cell chemotaxis and cancer metastasis. It is a coordinated process that involves dynamic changes in the actin cytoskeleton and its interplay with focal adhesions. At the leading edge of a migrating cell, it is the re-arrangement of actin and its attachment to focal adhesions that generates the driving force necessary for movement. However, the mechanisms involved in the attachment of actin filaments to focal adhesions are still not fully understood.ResultsSignaling by the FAK-Src complex plays a crucial role in regulating the formation of protein complexes at focal adhesions to which the actin filaments are attached. Cortactin, an F-actin associated protein and a substrate of Src kinase, was found to interact with FAK through its SH3 domain and the C-terminal proline-rich regions of FAK. We found that the autophosphorylation of Tyr(397) in FAK, which is necessary for FAK activation, was not required for the interaction with cortactin, but was essential for the tyrosine phosphorylation of the associated cortactin. At focal adhesions, cortactin was phosphorylated at tyrosine residues known to be phosphorylated by Src. The tyrosine phosphorylation of cortactin and its ability to associate with the actin cytoskeleton were required in tandem for the regulation of cell motility. Cell motility could be inhibited by truncating the N-terminal F-actin binding domains of cortactin or by blocking tyrosine phosphorylation (Y421/466/475/482F mutation). In addition, the mutant cortactin phosphorylation mimic (Y421/466/475/482E) had a reduced ability to interact with FAK and promoted cell motility. The promotion of cell motility by the cortactin phosphorylation mimic could also be inhibited by truncating its N-terminal F-actin binding domains.ConclusionsOur results suggest that cortactin acts as a bridging molecule between actin filaments and focal adhesions. The cort

BackgroundCell migration plays an important role in many physiological and pathological processes, including immune cell chemotaxis and cancer metastasis. It is a coordinated process that involves dynamic changes in the actin cytoskeleton and its interplay with focal adhesions. At the leading edge of a migrating cell, it is the re-arrangement of actin and its attachment to focal adhesions that generates the driving force necessary for movement. However, the mechanisms involved in the attachment of actin filaments to focal adhesions are still not fully understood.ResultsSignaling by the FAK-Src complex plays a crucial role in regulating the formation of protein complexes at focal adhesions to which the actin filaments are attached. Cortactin, an F-actin associated protein and a substrate of Src kinase, was found to interact with FAK through its SH3 domain and the C-terminal proline-rich regions of FAK. We found that the autophosphorylation of Tyr(397) in FAK, which is necessary for FAK activation, was not required for the interaction with cortactin, but was essential for the tyrosine phosphorylation of the associated cortactin. At focal adhesions, cortactin was phosphorylated at tyrosine residues known to be phosphorylated by Src. The tyrosine phosphorylation of cortactin and its ability to associate with the actin cytoskeleton were required in tandem for the regulation of cell motility. Cell motility could be inhibited by truncating the N-terminal F-actin binding domains of cortactin or by blocking tyrosine phosphorylation (Y421/466/475/482F mutation). In addition, the mutant cortactin phosphorylation mimic (Y421/466/475/482E) had a reduced ability to interact with FAK and promoted cell motility. The promotion of cell motility by the cortactin phosphorylation mimic could also be inhibited by truncating its N-terminal F-actin binding domains.ConclusionsOur results suggest that cortactin acts as a bridging molecule between actin filaments and focal adhesions. The cort

We find that the integrity of the cortical actin cytoskeleton is an important regulator of microtentacle formation, which can be mediated by genetic alteration of the cortactin protein that is commonly over-expressed in invasive breast cancers. Studies are underway to determine how these cortactin mutants influence the plasticity of detached tumor cells, using optical stretcher technology, and how these mechanical phenotypes translate to survival of circulating tumor cells in live animals. This genetic approach additionally led to the discovery of some interesting effects of cytoskeletally-targeted anti-mitotic chemotherapeutics, such as paclitaxel (Taxol). Studies of how these compounds affect cortical integrity and microtentacle formation revealed that although commonly used in clinical settings, very little was previously known about how acute exposures to these drugs impacts the behavior of suspended tumor cells. A manuscript was published on these studies by the PI, detailing how common anti-mitotic chemotherapeutics may inadvertently promote the survival of disseminated, chemotherapy-resistant tumor cells by impairing cortical integrity and enhancing microtentacle formation. Additionally, in vivo data now supports the initial hypothesis that MT-stabilizing chemotherapies can enhance circulating tumor cell binding and organ retention.

Cortactin, an F-actin binding protein, stabilizes F-actin networks and promotes actin polymerization by activating the Arp2/3 complex. Overexpression of cortactin has been reported in several human cancers. Cortactin stimulates cell migration, invasion, and experimental metastasis. However, the underlying mechanism is not still understood. In the present study, we therefore evaluated the possibility that cortactin could be appropriate as a molecular target for cancer gene therapy. In 70 primary oral squamous cell carcinomas and 10 normal oral mucosal specimens, cortactin expression was evaluated by immunological analyses, and the correlations of the overexpression of cortactin with clinicopathologic factors were evaluated. Overexpression of cortactin was detected in 32 of 70 oral squamous cell carcinomas; significantly more frequently than in normal oral mucosa. Cortactin overexpression was more frequent in higher grade cancers according to T classification, N classifications, and invasive pattern. Moreover, RNAi-mediated decrease in cortactin expression reduced invasion. Downregulation of cortactin expression increased the expression levels of E-cadherin, beta-catenin, and EpCAM. The siRNA of cortactin also reduced PTHrP expression via EGF signaling. These results consistently indicate that the overexpression of cortactin is strongly associated with an aggressive phenotype of oral squamous cell carcinoma. In conclusion, we propose that cortactin could be a potential molecular target of gene therapy by RNAi targeting in oral squamous cell carcinoma., Pathology & Oncology Research, 16(4), pp.523-531; 2010

We find that the integrity of the cortical actin cytoskeleton is an important regulator of microtentacle formation, which can be mediated by genetic alteration of the cortactin protein that is commonly over-expressed in invasive breast cancers. Studies are underway to determine how these cortactin mutants influence the plasticity of detached tumor cells, using optical stretcher technology, and how these mechanical phenotypes translate to survival of circulating tumor cells in live animals. This genetic approach additionally led to the discovery of some interesting effects of cytoskeletally targeted anti-mitotic chemotherapeutics, such as paclitaxel (Taxol). Studies of how these compounds affect cortical integrity and microtentacle formation revealed that although commonly used in clinical settings, very little was previously known about how acute exposures to these drugs impacts the behavior of suspended tumor cells. A manuscript was published on these studies by the PI, detailing how common anti-mitotic chemotherapeutics may inadvertently promote the survival of disseminated, chemotherapy-resistant tumor cells by impairing cortical integrity and enhancing microtentacle formation., The original document contains color images.

Cortactin is a ubiquitously expressed actin filament (F-actin)-binding protein that stabilizes F-actin networks and promotes actin polymerization by activating the actin-related protein 2/3 (Arp2/3) complex. Overexpression of cortactin in cancer cells stimulate cell migration, invasion, and experimental metastasis; however, the underlying mechanism in cortactin involvement in tumor progression is not fully understood. Recently, a direct interaction between zonula occludens-1 (ZO-1) and cortactin in epithelial cells was reported. The present study aimed to further clarify the significance of the interaction between cortactin and ZO-1 in cancer progression. Cortactin expression and localization in colorectal human cancer tissues were evaluated by immunohistochemistry and immunofluorescence. Co-immunoprecipitation and immunofluorescence analysis revealed cortactin and ZO-1 interaction and localization in cancer cells. In our study, the localization of cortactin is a crucial marker for lymph node metastasis in colorectal cancer. We show how the localization of cortactin effects cancer development. A molecular interaction between cortactin and ZO-1 in migrating or polarized cancer cells was revealed. This is the first report to show the interaction of cortactin and ZO-1 in colorectal cancer progression. We conclude that localization of cortactin in cancer cells and interaction between ZO-1 and cortactin are crucial for cancer progression., International journal of oncology, 35(6), pp.1271-1276; 2009

Chromosome 11ql3 is amplified in 13% of primary breast cancers and is indicative of a poor prognosis (1). The region amplified includes the cyclin Dl gene, hst-l, int-2, and emsl (cortactin); however, only cyclin Dl, a cell cycle regulator, and cortactin, a cytoskeletal protein, are overexpressed. Several studies have shown increased levels of these proteins in different breast cancer cell lines and their amplification is associated with a more invasive phenotype; however whether or not these proteins are directly contributing to the enhanced tumorigenic potential has not been clearly addressed (2). While the function of cyclin Dl is fairly well understood very little is known about the function of cortactin overexpression in mammary tumorigenesis. The goals of this proposal are to 1) understand the function of cortactin in normal cell growth and differentiation; 2) determine the consequences of cortactin overexpression in mammary epithelial cells; and 3) identify% cellular targets of cortactin. These goals will be achieved using genetic, biochemical, and cell biological approaches including generation of mice carrying a targeted disruption in the cortactin gene, derivation of mammary epithelial cells overexpressing wild type and mutant cortactin, and analysis of a two-hybrid screen using full length cortactin.

Chromosome 11q13 is amplified in 13% of primary breast cancers and is indicative of a poor prognosis (1). The region amplified includes the cyclin Dl gene, hst-1, int-2, and ems1 (cortactin); however, only cyclin D1, a cell cycle regulator, and cortactin, a cytoskeletal protein, are overexpressed. Several studies have shown increased levels of these proteins in different breast cancer cell lines and their amplification is associated with a more invasive phenotype; however whether or not these proteins are directly contributing to the enhanced tumorigenic potential has not been clearly addressed (2). While the function of cyclin Dl is fairly well understood very little is known about the function of cortactin overexpression in mammary tumorigenesis. The goals of this proposal are to (1) understand the function of cortactin in normal cell growth and differentiation; (2) determine the consequences of cortactin overexpression in mammary epithelial cells; and (3) identify cellular targets of cortactin. These goals will be achieved using genetic, biochemical, and cell biological approaches including generation of mice carrying a targeted disruption in the cortactin gene, derivation of mammary epithelial cells overexpressing wild type and mutant cortactin, and analysis of a two-hybrid screen using full length cortactin.

Cortactin is an actin cytoskeleton associated protein, frequently amplified and overexpressed along with the chromosome 11q13 in breast cancer, and acts as a prominent substrate of protein tyrosine kinase Src. We have hypothesized that cortactin plays a role in tumor progression by promoting metastasis. To test this hypothesis, we conducted a series of experiments aimed at characterizing the effects of cortactin and its mutant with a defect in tyrosine phosphorylation on cell motility in vitro and metastasis in vivo. These studies have eventually led to following conclusions: (1) cortactin is primarily implicated in actin cytoskeleton reorganization rather than DNA synthesis; (2) overexpression of wild type cortactin can increase cell motility and cell shape changes in response to growth factors and reactive oxygen species in a tyrosine phosphorylation dependent manner; (3) introduction of a cortactin mutant deficient in tyrosine phosphorylation can effectively inhibit cell motility and cell shape changes and depress tumor metastasis in vivo; and (4), the primary biochemical function of cortactin is to modulate actin polymerization by regulating the activity of Arp2/3 complex, a key machinery of actin polymerization. Our studies provide first evidence for the role of actin polymerization in tumor invasion and indicate a novel approach to suppress metastasis by targeting at actin polymerization.

Cortactin is a prominent substrate of protein tyrosine kinase Src and is frequently amplified and overexpressed along with the chromosome 11q13 in breast cancer. In vitro and in vivo, cortactin binds to and cross-links filamentous actin (F-actin). The F-actin cross-linking activity of cortactin is down-regulated by Src, resulting in increase in dynamics of actin cytoskeleton. We hypothesize that cortactin is implicated in tumorigenesis by promoting metastasis. To test this hypothesis, we developed several retroviruses encoding cortactin variants and infected the viruses into MDA-MB-231 cells. The cells overexpressing wild type cortactin exhibited increase in the motility as analyzed by a Trans-well apparatus, whereas cells overexpressing a cortactin mutant deficient in tyrosine phosphorylation reduced cell motility. We also examined these cells with an in vivo metastasis model in which cortactin overexpressors were injected into the left cardiac ventricle of female nude mice to develop lesions in bone. The preliminary data showed that overexpression of wild-type cortactin increases in metastases of MDA-MB-231 cells in bone and cachexia, whereas cells overexpressing a cortactin mutant deficient in tyrosine phosphorylation showed an impaired capacity to induce osteolytic metastases and cachexia. Furthermore, mice injected with the cells expressing the cortactin mutant tend to have longer life span compared to mice injected with wild-type cortactin transfected cells. These results suggest that tyrosine phosphorylation of cortactin plays an important role in the osteolytic metastases of human breast cancer., Original contains color plates: All DTIC reproductions will be in black and white.

Breast cancer is frequently associated with gene amplification at the chromosome 11 q 13. Studies have demonstrated that cortactin (also EMS1), a filamentous actin (F-actin) associated protein and a substrate of protein tyrosine kinase Src, plays an important role in the amplification. However, the pathological role of cortactin in breast cancer is unknown. We have developed a form of cortactin mutant that is deficient in tyrosine phosphorylation and is no longer to be regulated by Src. The mutant and wild-type forms of cortactin and oncogenic Src were introduced into MDA-MB-231 cells, a highly invasive breast cancer cell line. Overexpression of wild-type cortactin or oncogenic Src potentiate the motility of the cells, while overexpression of the mutant form impairs the cell migration as well as cell invasion into extracellular matrix. This study indicates that cortactin may play an important role in metastasis associated with breast cancer.