Phosphorylation Of Cortactin Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 5067: Casein kinase 2 alpha phosphorylation of cortactin governs actin cytoskeletal regulation of invadopodia function

Abstract Malregulation of signal transduction events controlling cortical actin cytoskeleton dynamics is responsible for to enhancing tumor cell motility and invasion. The actin-binding protein cortactin facilitates branched actin network formation through activation of the actin related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in many cancers, corresponding with increased tumor progression and poor patient outcome. In many cancer types, elevated Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. While cortactin-mediated activation of Arp2/3 complex has been well established, the objective of this study is to identify upstream signaling pathways responsible for modulating the interaction between cortactin and Arp2/3 complex in invasive carcinoma cells. We have determined that casein kinase (CK) 2α is responsible for disrupting the ability of cortactin to bind and/or activate Arp2/3 complex. CK2α directly phosphorylates cortactin at a conserved threonine 24 (T24) adjacent to the canonical Arp2/3 binding motif in the cortactin amino-terminal acidic (NTA) domain. CK2α phosphorylation of cortactin T24 impairs the ability of cortactin to activate Arp2/3 actin nucleation as determined by spectrofluorometric analysis. Decreased invadopodia formation and ECM degradation activity is observed in head and neck squamous cell carcinoma (HNSCC) cells with shRNA-mediated CK2α knockdown, expression of a CK2α phosphorylation-null cortactin mutant (T24A), and with treatment of the Phase I CK2α inhibitor CX-4945 (Silmitasertib). Collectively, these data suggest that CK2α-mediated cortactin phosphorylation at T24 is a critical event in regulating the interaction with Arp2/3 complex, identifying a key anti-invasive mechanism impacted by targeted anti-CK2α therapeutics. Citation Format: Steven M. Markwell, Amanda Gatesman Ammer, Erik T. Interval, Dorothy A. Schafer, River A. Hames, Scott A. Weed. Casein kinase 2 alpha phosphorylation of cortactin governs actin cytoskeletal regulation of invadopodia function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5067.

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.

Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2.

Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.

Oncogenic BRAF-Mediated Melanoma Cell Invasion.

Melanoma patients with oncogenic BRAF(V600E) mutation have poor prognoses. While the role of BRAF(V600E) in tumorigenesis is well established, its involvement in metastasis that is clinically observed in melanoma patients remains a topic of debate. Here, we show that BRAF(V600E) melanoma cells have extensive invasion activity as assayed by the generation of F-actin and cortactin foci that mediate membrane protrusion, and degradation of the extracellular matrix (ECM). Inhibition of BRAF(V600E) blocks melanoma cell invasion. In a BRAF(V600E)-driven murine melanoma model or in patients' tumor biopsies, cortactin foci decrease upon inhibitor treatment. In addition, genome-wide expression analysis shows that a number of invadopodia-related genes are downregulated after BRAF(V600E) inhibition. Mechanistically, BRAF(V600E) induces phosphorylation of cortactin and the exocyst subunit Exo70 through ERK, which regulates actin dynamics and matrix metalloprotease secretion, respectively. Our results provide support for the role of BRAF(V600E) in metastasis and suggest that inhibiting invasion is a potential therapeutic strategy against melanoma.

Tyrosine 397 phosphorylation is critical for FAK-promoted Rac1 activation and invasive properties in oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) is a common cancer worldwide. Despite advances in diagnosis and therapy, treatment options for patients with metastatic OSCC are few, due in part to the limited understanding of the molecular events involved in the invasion and metastasis of OSCC. In this study, we investigated the expression of focal adhesion kinase (FAK) and its tyrosine 397 phosphorylation (pY397) in the tissue specimens of OSCC. The roles of pY397 in regulating the activities of Rac1 and cortactin and the invasive properties of OSCC cells were further determined. Results from immunohistochemical analyses in 9 benign, 19 premalignant, and 19 malignant oral tissues showed that the immunoreactivity of FAK was observed in 5 benign (56%), 19 premalignant (100%), and 18 malignant tissues (95%), whereas the immunoreactivity of pY397 was only found in 1 of 9 (11%) benign lesions but was observed in 9 premalignant (47%) and 12 malignant (63%) lesions. Compared with the low-invading SCC4 cells, the high-invading OECM-1 cells exhibited higher levels of FAK expression and pY397, correlating with higher levels of GTP-bound Rac1 and cortactin phosphorylation. Manipulation of FAK expression or Y397 phosphorylation in SCC4, FaDu, OECM-1, or HSC-3 cells regulated their Rac1 activities and invasive properties. Furthermore, treatment of NSC23766, a Rac1-specific inhibitor, in OECM-1 and HSC-3 cells led to reduced invasive properties. Nevertheless, knockdown of FAK expression or suppression of pY397 had no effect on the cortactin activity in OECM-1 cells. The data collectively suggest that pY397 plays critical roles in the FAK-promoted Rac1 activation and invasive properties in OSCC cells. Thus, the inhibition of FAK phosphorylation at Y397 or Rac1 activity can serve as a therapeutic strategy for treating patients with metastatic OSCC.

Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation.

Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions.

The CD44s splice isoform is a central mediator for invadopodia activity.

The ability for tumor cells to spread and metastasize to distant organs requires proteolytic degradation of extracellular matrix (ECM). This activity is mediated by invadopodia, actin-rich membrane protrusions that are enriched for proteases. However, the mechanisms underlying invadopodia activity are not fully understood. Here, we report that a specific CD44 splice isoform, CD44s, is an integral component in invadopodia. We show that CD44s, but not another splice isoform CD44v, is localized in invadopodia. Small hairpin (sh)RNA-mediated depletion of CD44s abolishes invadopodia activity, prevents matrix degradation and decreases tumor cell invasiveness. Our results suggest that CD44s promotes cortactin phosphorylation and recruits MT1-MMP (also known as MMP14) to sites of matrix degradation, which are important activities for invadopodia function. Importantly, we show that depletion of CD44s inhibits breast cancer cell metastasis to the lung in animals. These findings suggest a crucial mechanism underlying the role of the CD44s splice isoform in breast cancer metastasis.

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.

PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration.

Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein-coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδ activation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin-WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration.

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and fibronectin-enriched matrices.

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche.

Thymosin β4 induces proliferation, invasion, and epithelial-to-mesenchymal transition of oral squamous cell carcinoma.

The epithelial-to-mesenchymal transition (EMT) plays a vital role in carcinogenesis, invasion, and metastasis of many epithelial tumors including oral squamous cell carcinoma (OSCC), a common malignancy of the head and neck. However, the functional role of the actin-sequestering protein thymosin β4 (Tβ4) in the EMT in OSCCs remains unclear. Thus, we investigated whether overexpression of Tβ4 could induce in vitro tumorigenesis such as cell proliferation and anchorage independency and an EMT-like phenotype in OSCCs. Also, we examined whether it affects invasiveness and cell motility-associated signaling molecules. Tβ4-overexpressing OSCCs, SCC-15_Tβ4 and SCC-25_Tβ4, enhanced cell proliferation and colony formation. In addition, we observed that Tβ4 overexpression induced an EMT-like phenotype, accompanied by a decrease in expression of the epithelial cell marker E-cadherin and an increase in expression of mesenchymal cell markers vimentin and N-cadherin. Also, the expression level of Twist1, an EMT-inducing transcription factor, was significantly enhanced in SCC-15_Tβ4 and SCC-25_Tβ4 cells. Tβ4 overexpression augmented in vitro invasion and MMP-2 activity and enhanced the phosphorylation of paxillin and cortactin and expression of LIMK1. Taken together, these results suggest that Tβ4 overexpression could be one of the causes of tumorigenesis and progression in OSCCs. Further investigation on the Tβ4 molecule would encourage the development of specific targets for cancer treatment.

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.

Possible role of cortactin phosphorylation by protein kinase Cα in actin-bundle formation at growth cone.

Cortactin contributes to growth cone morphogenesis by forming with dynamin, ring-shaped complexes that mechanically bundle and stabilise F-actin. However, the regulatory mechanism of cortactin action is poorly understood. Immunofluorescence microscopy revealed that protein kinase C (PKC) α colocalises with cortactin at growth cone filopodia in SH-SY5Y neuroblastoma cells. PKC activation by phorbol 12-myristate 13-acetate causes cortactin phosphorylation, filopodial retraction and F-actin-bundle loss. Moreover, PKCα directly phosphorylates cortactin in vitro at S135/T145/S172, mitigating both cortactin's actin-binding and actin-crosslinking activity, whereas cellular expression of a phosphorylation-mimetic cortactin mutant hinders filopodial formation with a significant decrease of actin bundles. Our results indicate that PKC-mediated cortactin phosphorylation might be implicated in the maintenance of growth cone.

Src tyrosyl phosphorylates cortactin in response to prolactin

The hormone/cytokine prolactin (PRL) is implicated in breast cancer cell invasion and metastasis. PRL-induced pathways are mediated by two non-receptor tyrosine kinases, JAK2 and Src. We previously demonstrated that prolactin stimulates invasion of breast cancer cells TMX2-28 through JAK2 and its target serine/threonine kinase PAK1. We hypothesize herein that the actin-binding protein cortactin, a protein involved in invadopodia formation and cell invasion, is activated by PRL. We demonstrate that TMX2-28 cells are more invasive than T47D breast cancer cells in response to PRL. We determine that cortactin is tyrosyl phosphorylated in response to PRL in a time and dose-dependent manner in TMX2-28 cells, but not in T47D cells. Furthermore, we show that PRL mediates cortactin tyrosyl phosphorylation via Src, but not JAK2. Finally, we demonstrate that maximal PRL-mediated TMX2-28 cell invasion requires both Src and JAK2 kinase activity, while T47D cell invasion is JAK2- but not Src-dependent. Thus PRL may induce cell invasion via two pathways: through a JAK2/PAK1 mediated pathway that we have previously demonstrated, and Src-dependent activation and tyrosyl phosphorylation of cortactin.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A Golgi-based KDELR-dependent signalling pathway controls extracellular matrix degradation.

We recently identified an endomembrane-based signalling cascade that is activated by the KDEL receptor (KDELR) on the Golgi complex. At the Golgi, the KDELR acts as a traffic sensor (presumably via binding to chaperones that leave the ER) and triggers signalling pathways that balance membrane fluxes between ER and Golgi. One such pathway relies on Gq and Src. Here, we examine if KDELR might control other cellular modules through this pathway. Given the central role of Src in extracellular matrix (ECM) degradation, we investigated the impact of the KDELR-Src pathway on the ability of cancer cells to degrade the ECM. We find that activation of the KDELR controls ECM degradation by increasing the number of the degradative structures known as invadopodia. The KDELR induces Src activation at the invadopodia and leads to phosphorylation of the Src substrates cortactin and ASAP1, which are required for basal and KDELR-stimulated ECM degradation. This study furthers our understanding of the regulatory circuitry underlying invadopodia-dependent ECM degradation, a key phase in metastases formation and invasive growth.

α integrin targeting for radiosensitization of three-dimensionally grown human head and neck squamous cell carcinoma cells

Integrin cell adhesion molecules play a crucial role in tumor cell resistance to radio- and chemotherapy and are therefore considered attractive targets for cancer therapy. Here, we assessed the role of β1 integrin-interacting α integrin subunits in more physiological three-dimensional extracellular matrix grown head and neck squamous cell carcinoma (HNSCC) cell cultures for evaluating cytotoxic and radiosensitizing potential. α2, α3, α5 and α6 integrins, which are overexpressed in HNSCC according to Oncomine database analysis, were coprecipitated with β1 integrin. More potently than α2, α5 or α6 integrin inhibition, siRNA-based α3 integrin targeting resulted in reduced clonogenic cell survival, induced apoptosis and enhanced radiosensitivity. These events were associated with diminished phosphorylation of Akt, Cortactin and Paxillin. Cell line-dependently, simultaneous α3 and β1 integrin inhibition led to higher cytotoxicity and radiosensitization than α3 integrin blocking alone. Stable overexpression of wild-type and constitutively active forms of the integrin signaling mediator focal adhesion kinase (FAK) revealed FAK as a key determinant of α3 integrin depletion-mediated radiosensitization. Our findings show that α3 integrin is essentially involved in HNSCC cell radioresistance and critical for a modified cellular radiosensitivity along with β1 integrins.

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