Tyrosine Phosphorylation Of Cortactin Research Articles

Requirement of Site-Specific Tyrosine Phosphorylation of Cortactin in Retinal Neovascularization and Vascular Leakage.

Retinal neovascularization is a major cause of vision impairment. Therefore, the purpose of this study is to investigate the mechanisms by which hypoxia triggers the development of abnormal and leaky blood vessels. A variety of cellular and molecular approaches as well as tissue-specific knockout mice were used to investigate the role of Cttn (cortactin) in retinal neovascularization and vascular leakage. We found that VEGFA (vascular endothelial growth factor A) stimulates Cttn phosphorylation at Y421, Y453, and Y470 residues in human retinal microvascular endothelial cells. In addition, we observed that while blockade of Cttn phosphorylation at Y470 inhibited VEGFA-induced human retinal microvascular endothelial cell angiogenic events, suppression of Y421 phosphorylation protected endothelial barrier integrity from disruption by VEGFA. In line with these observations, while blockade of Cttn phosphorylation at Y470 negated oxygen-induced retinopathy-induced retinal neovascularization, interference with Y421 phosphorylation prevented VEGFA/oxygen-induced retinopathy-induced vascular leakage. Mechanistically, while phosphorylation at Y470 was required for its interaction with Arp2/3 and CDC6 facilitating actin polymerization and DNA synthesis, respectively, Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, resulting in adherens junction disruption. Furthermore, whereas Cttn phosphorylation at Y470 residue was dependent on Lyn, its phosphorylation at Y421 residue required Syk activation. Accordingly, lentivirus-mediated expression of shRNA targeting Lyn or Syk levels inhibited oxygen-induced retinopathy-induced retinal neovascularization and vascular leakage, respectively. The above observations show for the first time that phosphorylation of Cttn is involved in a site-specific manner in the regulation of retinal neovascularization and vascular leakage. In view of these findings, Cttn could be a novel target for the development of therapeutics against vascular diseases such as retinal neovascularization and vascular leakage.

DAB2IP suppresses invadopodia formation through destabilizing ALK by interacting with USP10 in breast cancer

Invadopodia, being actin-rich membrane protrusions, play a vital role in tumor cell invasion and metastasis. Our previous studies have revealed some functions of the DOC-2/DAB2 interacting protein (DAB2IP) as a tumor suppressor. Nevertheless, the specific role and mechanism of DAB2IP in invadopodia formation remain unclear. Here, we find that DAB2IP effectively suppresses invadopodia formation and metastasis in breast cancer, both invitro and invivo. Additionally, DAB2IP could downregulate anaplastic lymphoma kinase (ALK), resulting in the inhibition of tyrosine phosphorylation of Cortactin and the prevention of invadopodia formation. DAB2IP competitively antagonizes the interaction between the deubiquitinating enzyme Ubiquitin-specific peptidase 10 (USP10) and ALK, leading to a decrease in the abundance of ALK protein. In summary, DAB2IP impairs the stability of ALK through USP10-dependent deubiquitination, suppressing Cortactin phosphorylation, thereby inhibiting invadopodia formation and metastasis of breast cancer cells. Furthermore, this study suggests a potential therapeutic strategy for breast cancer treatment.

A novel Pyk2-derived peptide inhibits invadopodia-mediated breast cancer metastasis.

Dissemination of cancer cells from the primary tumor into distant body tissues and organs is the leading cause of death in cancer patients. While most clinical strategies aim to reduce or impede the growth of the primary tumor, no treatment to eradicate metastatic cancer exists at present. Metastasis is mediated by feet-like cytoskeletal structures called invadopodia which allow cells to penetrate through the basement membrane and intravasate into blood vessels during their spread to distant tissues and organs. The non-receptor tyrosine kinase Pyk2 is highly expressed in breast cancer, where it mediates invadopodia formation and function via interaction with the actin-nucleation-promoting factor cortactin. Here, we designed a cell-permeable peptide inhibitor that contains the second proline-rich region (PRR2) sequence of Pyk2, which binds to the SH3 domain of cortactin and inhibits the interaction between Pyk2 and cortactin in invadopodia. The Pyk2-PRR2 peptide blocks spontaneous lung metastasis in immune-competent mice by inhibiting cortactin tyrosine phosphorylation and actin polymerization-mediated maturation and activation of invadopodia, leading to reduced MMP-dependent tumor cell invasiveness. The native structure of the Pyk2-PRR2:cortactin-SH3 complex was determined using nuclear magnetic resonance (NMR), revealing an extended class II interaction surface spanning the canonical binding groove and a second hydrophobic surface which significantly contributes to ligand affinity. Using structure-guided design, we created a mutant peptide lacking critical residues involved in binding that failed to inhibit invadopodia maturation and function and consequent metastatic dissemination in mice. Our findings shed light on the specific molecular interactions between Pyk2 and cortactin and may lead to the development of novel strategies for preventing dissemination of primary breast tumors predicted at the time of diagnosis to be highly metastatic, and of secondary tumors that have already spread to other parts of the body.

Helicobacter pylori CagA Induces Cortactin Y-470 Phosphorylation-Dependent Gastric Epithelial Cell Scattering via Abl, Vav2 and Rac1 Activation.

Simple SummaryVarious microbial pathogens target the actin-binding protein cortactin to promote their own uptake, proliferation and spread, and exhibit proposed roles in human cancerogenesis. We aimed to study the molecular mechanisms of how the gastric pathogen Helicobacter pylori hijacks cortactin phosphorylation via tyrosine kinase Abl to trigger cancer-related signal transduction events. We discovered that cortactin phosphorylated at Y-470 recruits the signaling factor Vav2 to activate the small Rho GTPase Rac1, and finally, a cancer cell motility phenotype. We also demonstrate that phosphorylation of cortactin at Y-470 can be completely inhibited by the well-known Abl inhibitor imatinib. Imatinib is an established oral chemotherapy medication, employed for efficient systemic treatment of various cancers. These results reveal a comprehensive novel pathway for how precisely H. pylori manipulates host signaling in gastric disease development, and may pave the way for new opportunities of treatment of the outcome of infections with this pathogen, i.e., through using imatinib.The pathogen Helicobacter pylori is the first reported bacterial type-1 carcinogen playing a role in the development of human malignancies, including gastric adenocarcinoma. Cancer cell motility is an important process in this scenario, however, the molecular mechanisms are still not fully understood. Here, we demonstrate that H. pylori subverts the actin-binding protein cortactin through its type-IV secretion system and injected oncoprotein CagA, e.g., by inducing tyrosine phosphorylation of cortactin at Y-470, which triggers gastric epithelial cell scattering and motility. During infection of AGS cells, cortactin was discovered to undergo tyrosine dephosphorylation at residues Y-421 and Y-486, which is mediated through inactivation of Src kinase. However, H. pylori also profoundly activates tyrosine kinase Abl, which simultaneously phosphorylates cortactin at Y-470. Phosphorylated cortactin interacts with the SH2-domain of Vav2, a guanine nucleotide exchange factor for the Rho-family of GTPases. The cortactin/Vav2 complex then stimulates a previously unrecognized activation cascade including the small GTPase Rac1, to effect actin rearrangements and cell scattering. We hypothesize that injected CagA targets cortactin to locally open the gastric epithelium in order to get access to certain nutrients. This may disturb the cellular barrier functions, likely contributing to the induction of cell motility, which is important in gastric cancer development.

CX3 CL1 promotes tumour cell by inducing tyrosine phosphorylation of cortactin in lung cancer.

It has been reported that chemokine CX3CL1 can regulate various tumours by binding to its unique receptor CX3CR1. However, the effect of CX3CL1‐CX3CR1 on the lung adenocarcinoma and lung squamous cell carcinoma is still unclear. Here, we showed that CX3CL1 can further invasion and migration of lung adenocarcinoma A549 and lung squamous cell carcinoma H520. In addition, Western blot and immunofluorescence test indicated CX3CL1 up‐regulated the phosphorylation level of cortactin, which is a marker of cell pseudopodium. Meanwhile, the phosphorylation levels of c‐Src and c‐Abl, which are closely related to the regulation of cortactin phosphorylation, are elevated. Nevertheless, the src/abl inhibitor bosutinib and mutations of cortactin phosphorylation site could inhibit the promotion effect of CX3CL1 on invasion and migration of A549 and H520. Moreover, these results of MTT, Hoechst staining and Western blot suggested that CX3CL1 had no effect on the proliferation and apoptosis of A549 and H520 in vitro. The effects of CX3CL1 were also verified by the subcutaneous tumour formation in nude mice, which showed that it could promote proliferation and invasion of A549 in vivo. In summary, our results indicated that CX3CL1 furthered invasion and migration in lung cancer cells partly via activating cortactin, and CX3CL1 may be a potential molecule in regulating the migration and invasion of lung cancer.

Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis

ROR1 is a conserved oncoembryonic surface protein expressed in breast cancer. Here we report that ROR1 associates with cortactin in primary breast-cancer cells or in MCF7 transfected to express ROR1. Wnt5a also induced ROR1-dependent tyrosine phosphorylation of cortactin (Y421), which recruited ARHGEF1 to activate RhoA and promote breast-cancer-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. Furthermore, treatment of mice bearing breast-cancer xenograft with cirmtuzumab inhibited cortactin phosphorylation in vivo and impaired metastatic development. We established that the proline at 841 of ROR1 was required for it to recruit cortactin and ARHGEF1, activate RhoA, and enhance breast-cancer-cell migration in vitro or development of metastases in vivo. Collectively, these studies demonstrate that the interaction of ROR1 with cortactin plays an important role in breast-cancer-cell migration and metastasis.

A single tyrosine phosphorylation site in cortactin is important for filopodia formation in neuronal growth cones.

Cortactin is a Src tyrosine phosphorylation substrate that regulates multiple actin-related cellular processes. While frequently studied in nonneuronal cells, the functions of cortactin in neuronal growth cones are not well understood. We recently reported that cortactin mediates the effects of Src tyrosine kinase in regulating actin organization and dynamics in both lamellipodia and filopodia of Aplysia growth cones. Here, we identified a single cortactin tyrosine phosphorylation site (Y499) to be important for the formation of filopodia. Overexpression of a 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of a 499E phospho-mimetic mutant increased filopodia length. Using an antibody against cortactin pY499, we showed that tyrosine-phosphorylated cortactin is enriched along the leading edge. The leading edge localization of phosphorylated cortactin is Src2-dependent, F-actin–independent, and important for filopodia formation. In vitro kinase assays revealed that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro. Finally, we provide evidence that Arp2/3 complex acts downstream of phosphorylated cortactin to regulate density but not length of filopodia. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation.

The Pathogenic Effect of Cortactin Tyrosine Phosphorylation in Cutaneous Squamous Cell Carcinoma.

Cortactin (CTTN) has been considered a promising molecular prognostic factor in various types of cancers. In this study, we aimed to investigate the role of CTTN in the pathogenesis of cutaneous squamous cell carcinoma (CSCC). CTTN and phospho-CTTN (p-CTTN) expression was determined in 10 healthy controls and 38 CSCC tissue samples by immunohistochemistry. The influence of CTTN on the biological behavior of CSCC cells was also investigated. p-CTTN expression was significantly increased in CSCC than control samples. In contrast, no significant difference in CTTN expression was found between control and CSCC tissues. Moreover, a significant association was found between recurrence-free survival with p-CTTN expression, but not with CTTN expression. Furthermore, the proliferative, migratory, and invasive abilities of CSCC cells were significantly decreased by CTTN-siRNA transfection. CTTN phosphorylation is strongly associated with CSCC pathogenesis and may serve as a molecular biomarker of CSCC.

Cortactin regulates endo-lysosomal sorting of AMPARs via direct interaction with GluA2 subunit

AMPA receptor (AMPAR) trafficking is a key determinant of synaptic strength and synaptic plasticity. Under basal conditions, constitutive trafficking maintains surface AMPARs by internalization into the endosomal system, where the majority are sorted and targeted for recycling back to the plasma membrane. NMDA receptor (NMDAR)-dependent Long-Term Depression (LTD) is characterised by a reduction in synaptic strength, and involves endosomal sorting of AMPARs away from recycling pathways to lysosomes. The mechanisms that determine whether AMPARs are trafficked to lysosomes or to recycling endosomes, especially in response to NMDAR stimulation, are unclear. Here, we define a role for the actin-regulatory protein cortactin as a mediator of AMPAR endosomal sorting by direct interaction with the GluA2 subunit. Disrupting GluA2-cortactin binding in neurons causes the targeting of GluA2/A3-containing receptors to lysosomes and their consequent degradation, resulting in a loss of surface and synaptic GluA2 under basal conditions and an occlusion of subsequent LTD expression. Furthermore, we show that NMDAR stimulation causes a dissociation of endogenous cortactin from GluA2 via tyrosine phosphorylation of cortactin. These results demonstrate that cortactin maintains GluA2/A3 levels by directing receptors away from lysosomes, and that disrupting GluA2-cortactin interactions to target GluA2/A3 to lysosomes is an essential component of LTD expression.

Abstract P1-01-05: Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells

Abstract Background: ROR1is an evolutionarily conserved, oncoembryonic surface-antigen expressed in breast cancer. Previously, we found that ROR1 can serve as a receptor for Wnt5a, which can induce non-canonical Wnt signaling that enhances cancer-cell migration. Recently we found that Wnt5a could induce ROR1 to complex with a cytoskeletal protein designated HS1, which recruited ARHGEF1, enhanced activation of RhoA, and promoted leukemia-cell migration. These effects of Wnt5a on CLL cells could be inhibited by cirmtuzumab, a humanized, high-affinity monoclonal antibody (mAb) specific for ROR1. As expression of HS1 is limited to hematopoietic cells we examined breast cancer cell lines and primary breast cancer cells for expression of cortactin. Cortactin (also called EMS1), is a cytoskeletal protein, which is homologous in structure and function to HS1 and is broadly expressed in human cancers, including breast cancer. Methods: We performed immunoprecipitation studies using high-affinity mAb to ROR1 or cortactin and immunoblot analysis to examine for the association of ROR1 with cortactin and tyrosine phosphorylation of cortactin at Y421. We also assessed the expression levels of cortactin, ROR1, and ARHGEF1, in the MCF7 breast cancer cell line and in breast-cancer patient-derived xenografts (PDX). We also generated MCF7-ROR1 cells, which were stably transfected to express ROR1 or various mutant forms of ROR1 generated to study the structure-function requirements for effective ROR1-cortactin interactions. Results: We found that ROR1 associates with cortactin in freshly-isolated breast cancer PDX tumors or in PDX cells cultured with exogenous Wnt5a. We corroborated these results in breast cancer cell line MCF7-ROR1 cells, which were transfected to express ROR1 and found to migrate more effectively than the parental MCF7 lacking expression of ROR1. Wnt5a also induced cortactin tyrosine phosphorylation at Y421, recruitment of ARHGEF1, and activation of RhoA, which we found associated with enhanced breast-cancer cell migation. The capacity of Wnt5a to induce such changes could be blocked by treatment of the cells with cirmtuzumab. We generated truncated forms of ROR1 without a proline-rich domain (PRD) and found PRD was necessary for this association or Wnt5a-induced cortactin phosphorylation and enhanced cancer-cell migration. Accordingly, we introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→A mutants, ROR1P(841)A had impaired capacity to recruit cortactin and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin or activate ARHGEF1, and was unable to enhance the motility of the MCF7 cells transfected with this mutant form of ROR1. Conclusions: Collectively, these studies indicate the capacity of cortactin to complex with ROR1 plays an important role in ROR1-dependent Wnt5a-enhanced breast cancer cell migration. These studies also demonstrate that cirmtuzumab can inhibit the formation of cortactin-ROR1 complexes, cortactin phosphorylation, and cancer-cell migration, and metastasis. Citation Format: Hasan MK, Tripple V, Widhopf II GF, Yu J, Zhang S, Parker BA, Kipps TJ. Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-05.

Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells.

The nonreceptor tyrosine kinase Pyk2 is highly expressed in invasive breast cancer, but the mechanism by which it potentiates tumor cell invasiveness is unclear at present. Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion. Both Pyk2 and the closely related focal adhesion kinase (FAK) regulate tumor cell invasion, albeit via distinct mechanisms. Although Pyk2 regulates tumor cell invasion by controlling invadopodium-mediated functions, FAK controls invasiveness of tumor cells by regulating focal adhesion-mediated motility. Collectively, our findings identify Pyk2 as a unique mediator of invadopodium formation and function and also provide a novel insight into the mechanisms by which Pyk2 mediates tumor cell invasion.

The Microtubule Plus End Tracking Protein TIP150 Interacts with Cortactin to Steer Directional Cell Migration

Cell migration is orchestrated by dynamic interactions of microtubules with the plasma membrane cortex. How these interactions facilitate these dynamic processes is still being actively investigated. TIP150 is a newly characterized microtubule plus end tracking protein essential for mitosis and entosis (Ward, T., Wang, M., Liu, X., Wang, Z., Xia, P., Chu, Y., Wang, X., Liu, L., Jiang, K., Yu, H., Yan, M., Wang, J., Hill, D. L., Huang, Y., Zhu, T., and Yao, X. (2013) Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis. J. Biol. Chem. 288, 15771-15785; Xia, P., Zhou, J., Song, X., Wu, B., Liu, X., Li, D., Zhang, S., Wang, Z., Yu, H., Ward, T., Zhang, J., Li, Y., Wang, X., Chen, Y., Guo, Z., and Yao, X. (2014) Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction. J. Mol. Cell Biol. 6, 240-254). Here we show that TIP150 links dynamic microtubules to steer cell migration by interacting with cortactin. Mechanistically, TIP150 binds to cortactin via its C-terminal tail. Interestingly, the C-terminal TIP150 proline-rich region (CT150) binds to the Src homology 3 domain of cortactin specifically, and such an interaction is negatively regulated by EGF-elicited tyrosine phosphorylation of cortactin. Importantly, suppression of TIP150 or overexpression of phospho-mimicking cortactin inhibits polarized cell migration. In addition, CT150 disrupts the biochemical interaction between TIP150 and cortactin in vitro, and perturbation of the TIP150-cortactin interaction in vivo using a membrane-permeable TAT-CT150 peptide results in an inhibition of directional cell migration. We reason that a dynamic TIP150-cortactin interaction orchestrates directional cell migration via coupling dynamic microtubule plus ends to the cortical cytoskeleton.

Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1.

Monocyte chemotactic protein 1 (MCP1) stimulates phosphorylation of cortactin on Y421 and Y446 residues in a time-dependent manner and phosphorylation at Y446 but not Y421 residue is required for MCP1-induced CDK-interacting protein 1 (p21Cip1) nuclear export and degradation in facilitating human aortic smooth muscle cell (HASMC) proliferation. In addition, MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation are dependent on Fyn activation. Upstream to Fyn, MCP1 stimulated C-C chemokine receptor type 2 (CCR2) and Gi/o and inhibition of either one of these molecules using their specific antagonists or inhibitors attenuated MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Cortactin phosphorylation at Y446 residue is also required for another G protein-coupled receptor (GPCR) agonist, thrombin-induced p21Cip1 nuclear export and its degradation in promoting HASMC proliferation. Quite interestingly, the receptor tyrosine kinase (RTK) agonist, platelet-derived growth factor-BB (PDGF-BB)-induced p21Cip1 degradation and HASMC proliferation do not require cortactin tyrosine phosphorylation. Together, these findings demonstrate that tyrosine phosphorylation of cortactin at Y446 residue is selective for only GPCR but not RTK agonist-induced nuclear export and proteolytic degradation of p21Cip1 in HASMC proliferation.

The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9β1 Affects Cancer Cell Behaviour

Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9β1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9β1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9β1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9β1 to focal adhesions where active β1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions.

CBIO-25NEW HYPOXIA-INDUCED INVASION FACTOR IN HUMAN GLIOBLASTOMA INVASION

Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.

MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated degradation of the extracellular matrix. To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. For evaluation of the matrix degradation properties of the HNSCC lines, an invadopodial matrix degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases. Increased miR-375 expression in HNSCC cells suppresses extracellular matrix degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases. MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.

Abstract 1452: MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity

Abstract Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our goal is to improve patient outcome through understanding the mechanisms determining HNSCC invasiveness. Our research group previously reported that miR-375 expression levels correlate with patient survival, recurrence and distant metastasis. We have previously observed diminished HNSCC invasion in vitro correlates with increased miR-375 expression. To assess whether the miR-375 over-expressing transductant lines have reduced matrix degradation properties, a fluorescent matrix degradation assay was used. We have observed that miR-375 over-expression in HNSCC cells suppresses extracellular matrix degradation. We further determined whether miR-375 over-expressing cells show reduced invadopodium formation and maturation. We observed miR-375 expression in HNSCC cell lines reduces the number of mature invadopodia, but does not affect levels of invadopodium precursors. We examined the phosphorylation and total levels of key invadopodia components (Tks5 and cortactin) to determine whether changes in regulation or expression of these components may lead impaired matrix degradation properties. We determined that miR-375 expression in HNSCC cell lines does not reduce cellular levels of cortactin and Tks5, nor is tyrosine phosphorylation of cortactin altered. To identify candidate proteins regulated by miR-375, we are utilizing stable isotope labeling of cells in cell culture (SILAC). Candidate target proteins are being validated as targets of miR-375 and tested for involvement in the diminished invasive properties of HNSCC as a result of miR-375 expression. In conclusion, increased miR-375 expression may suppress the invasive properties of HNSCC through diminished invadopodia activity. Citation Format: Lizandra Jimenez, Ved P. Sharma, Jihyeon Lim, Ruth Angeletti, John Condeelis, Thomas Harris, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall. MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1452. doi:10.1158/1538-7445.AM2014-1452

Receptor protein tyrosine phosphatase RPTPα controls epithelial adherens junctions, linking E-cadherin engagement to c-Src signaling to cortactin

Epithelial junctions are fundamental determinants of tissue organization, subject to regulation by tyrosine phosphorylation. Homophilic binding of E-cadherin activates tyrosine kinases, such as Src, that control junctional integrity. Protein tyrosine phosphatases (PTPs) also contribute to cadherin-based adhesion and signaling, but little is known about their specific identity or functions at epithelial junctions. Here, we report that the receptor PTP RPTPα (human gene name PTPRA) is recruited to epithelial adherens junctions at the time of cell-cell contact, where it is in molecular proximity to E-cadherin. RPTPα is required for appropriate cadherin-dependent adhesion and for cyst architecture in three-dimensional culture. Loss of RPTPα impairs adherens junction integrity, as manifested by defective E-cadherin accumulation and peri-junctional F-actin density. These effects correlate with a role for RPTPα in cellular (c)-Src activation at sites of E-cadherin engagement. Mechanistically, RPTPα is required for appropriate tyrosine phosphorylation of cortactin, a major Src substrate and a cytoskeletal actin organizer. Expression of a phosphomimetic cortactin mutant in RPTPα-depleted cells partially rescues F-actin and E-cadherin accumulation at intercellular contacts. These findings indicate that RPTPα controls cadherin-mediated signaling by linking homophilic E-cadherin engagement to cortactin tyrosine phosphorylation through c-Src.

Role of Cortactin in Dynamic Actin Remodeling Events in Gonadotrope Cells

GnRH induces marked activation of the actin cytoskeleton in gonadotropes; however, the physiological consequences and cellular mechanisms responsible have yet to be fully elucidated. The current studies focus on the actin scaffolding protein cortactin. Using the gonadotrope-derived αT3-1 cell line, we found that cortactin is phosphorylated at Y(421), S(405), and S(418) in a time-dependent manner in response to the GnRH agonist buserelin (GnRHa). GnRHa induced translocation of cortactin to the leading edge of the plasma membrane where it colocalizes with actin and actin-related protein 3 (Arp3). Incubation of αT3-1 cells with the c-src inhibitor phosphoprotein phosphatase 1, blocked tyrosine phosphorylation of cortactin, reduced cortactin association with Arp3, and blunted actin reorganization in response to GnRHa. Additionally, we used RNA silencing strategies to knock down cortactin in αT3-1 cells. Knockdown of cortactin blocked the ability of αT3-1 cells to generate filopodia, lamellipodia, and membrane ruffles in response to GnRHa. We show that lamellipodia and filopodia are capable of LHβ mobilization in primary pituitary culture after GnRHa treatment, and disruption of these structures using jasplakinolide reduces LH secretion. Collectively, our findings suggest that after GnRHa activation, src activity leads to tyrosine phosphorylation of cortactin, which facilitates its association with Arp3 to engage the actin cytoskeleton. The reorganization of actin by cortactin potentially underlies GnRHa-induced secretory events within αT3-1 cells.

Cyclin G2 Promotes Hypoxia- Driven Local Invasion of Glioblastoma by Orchestrating Cytoskeletal Dynamics

Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.