Training doctoral students in critical thinking and experimental design using problem-based learning.

BACKGROUND: Traditionally, doctoral student education in the biomedical sciences relies on didactic coursework to build a foundation of scientific knowledge and an apprenticeship model of training in the laboratory of an established investigator. Recent recommendations for revision of graduate training include the utilization of graduate student competencies to assess progress and the introduction of novel curricula focused on development of skills, rather than accumulation of facts. Evidence demonstrates that active learning approaches are effective. Several facets of active learning are components of problem-based learning (PBL), which is a teaching modality where student learning is self-directed toward solving problems in a relevant context. These concepts were combined and incorporated in creating a new introductory graduate course designed to develop scientific skills (student competencies) in matriculating doctoral students using a PBL format. METHODS: Evaluation of course effectiveness was measured using the principals of the Kirkpatrick Four Level Model of Evaluation. At the end of each course offering, students completed evaluation surveys on the course and instructors to assess their perceptions of training effectiveness. Pre- and post-tests assessing students' proficiency in experimental design were used to measure student learning. RESULTS: The analysis of the outcomes of the course suggests the training is effective in improving experimental design. The course was well received by the students as measured by student evaluations (Kirkpatrick Model Level 1). Improved scores on post-tests indicate that the students learned from the experience (Kirkpatrick Model Level 2). A template is provided for the implementation of similar courses at other institutions. CONCLUSIONS: This problem-based learning course appears effective in training newly matriculated graduate students in the required skills for designing experiments to test specific hypotheses, enhancing student preparation prior to initiation of their dissertation research.

Evaluation of the matrix metalloproteinase 9 (MMP9) inhibitor Andecaliximab as an Anti-invasive therapeutic in Head and neck squamous cell carcinoma.

OBJECTIVES: Locoregional and lymphovascular involvement of invasive head and neck squamous cell carcinoma (HNSCC) complicates curative treatment. Matrix metalloproteinase (MMP) 9 is a negative prognostic marker in HNSCC and targets multiple extracellular matrix (ECM) substrates, where it contributes to breaching basement membrane and stromal barriers enabling invasive spread. Andecaliximab (ADX) is a second-generation MMP9 inhibitor well tolerated in clinical trials of gastric and pancreatic adenocarcinoma. The impact of selective MMP9 targeting by ADX in HNSCC has not been evaluated. MATERIALS AND METHODS: Established and patient-derived xenograft (PDX) cell lines were utilized in HNSCC invasion assays to determine the inhibitory ability of MMP9-mediated invasion by ADX. MMP9 expression was confirmed using immunohistochemistry (IHC) and immunoblotting. ECM degradation was evaluated with confocal microscopy. Cell invasion from tumor spheroids was monitored by phase microscopy. Histological evaluation was used to determine ADX efficacy in three-dimensional organotypic cultures containing cancer associated fibroblasts (CAFs). RESULTS: MMP9 was expressed in all established and PDX-derived cell lines. While the broad spectrum clinical MMP inhibitor marimastat (BB2516) blocked HNSCC invadopodia function and tumor spheroid invasion, ADX treatment failed to inhibit invadopodia-based matrix degradation, tumor cell or fibroblast-driven ECM invasion in collagen I-based matrices. CONCLUSION: ADX monotherapy was ineffective at blocking initial MMP-dependent events of HNSCC invasion, likely due to redundant functions of additional non-targeted MMPs produced by tumor cells and microenvironment. Combination of ADX with existing and emerging therapies targeting additional MMP activation pathways may warrant future investigation.

Copy number alterations identify a smoking-associated expression signature predictive of poor outcome in head and neck squamous cell carcinoma.

Cigarette smoking is a risk factor for the development of head and neck squamous cell carcinoma (HNSCC), partially due to tobacco-induced large-scale chromosomal copy-number alterations (CNAs). Identifying CNAs caused by smoking is essential in determining how gene expression from such regions impact tumor progression and patient outcome. We utilized The Cancer Genome Atlas (TCGA) whole genome sequencing data for HNSCC to directly identify amplified or deleted genes correlating with smoking pack-year based on linear modeling. Internal cross-validation identified 35 CNAs that significantly correlated with patient smoking, independent of human papillomavirus (HPV) status. The most abundant CNAs were chromosome 11q13.3-q14.4 amplification and 9p23.1/9p24.1 deletion. Evaluation of patient amplicons reveals four different patterns of 11q13 gene amplification in HNSCC resulting from breakage-fusion-bridge (BFB) events. . Predictive modeling identified 16 genes from these regions that denote poorer overall and disease-free survival with increased pack-year use, constituting a smoking-associated expression signature (SAES). Patients with altered expression of signature genes have increased risk of death and enhanced cervical lymph node involvement. The identified SAES can be utilized as a novel predictor of increased disease aggressiveness and poor outcome in smoking-associated HNSCC.

Disparate survival of late-stage male oropharyngeal cancer in Appalachia.

The United States Appalachian region harbors a higher cancer burden than the rest of the nation, with disparate incidence of head and neck squamous cell carcinomas (HNSCC), including oral cavity and pharynx (OC/P) cancers. Whether elevated HNSCC incidence generates survival disparities within Appalachia is unknown. To address this, HNSCC survival data for 259,737 tumors from the North American Association for Central Cancer Registries 2007-2013 cohort were evaluated, with age-adjusted relative survival (RS) calculated based on staging, race, sex, and Appalachian residence. Tobacco use, a primary HNSCC risk factor, was evaluated through the Behavioral Risk Factor Surveillance System from Appalachian states. Decreased OC/P RS was found in stage IV Appalachian white males within a subset of states. The survival disparity was confined to human papillomavirus (HPV)-associated oropharyngeal cancers, specifically the oropharynx subsite. This correlated with significantly higher smoking and male smokeless tobacco use in most Appalachian disparity states. Lower survival of Appalachian males with advanced-stage HPV-associated oropharyngeal cancers suggests pervasive tobacco consumption likely generates more aggressive tumors at HPV-associated oropharynx subsites than national averages. Comprehensive tobacco and HPV status should therefore be evaluated prior to considering treatment de-intensification regimens for HPV-associated oropharyngeal cancers in populations with high tobacco consumption.

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound-associated extracellular matrix: implications for cancer therapy.

The human-adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen-like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin-C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin-C, are present for several days in the wound extracellular matrix. Scl1-FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin-C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer-associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1-FnIII interaction as a novel method of anti-neoplastic targeting in the tumor microenvironment.

Cortactin Phosphorylation by Casein Kinase 2 Regulates Actin-Related Protein 2/3 Complex Activity, Invadopodia Function, and Tumor Cell Invasion.

Malregulation of the actin cytoskeleton enhances 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 head and neck squamous cell carcinoma (HNSCC) and other cancers, corresponding with elevated tumor progression and poor patient outcome. Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. Although cortactin-mediated activation of Arp2/3 complex and invadopodia regulation has been well established, signaling pathways responsible for governing cortactin binding to Arp2/3 are unknown and potentially present a new avenue for anti-invasive therapeutic targeting. Here we identify casein kinase (CK) 2α phosphorylation of cortactin as a negative regulator of Arp2/3 binding. CK2α directly phosphorylates cortactin at a conserved threonine (T24) adjacent to the canonical Arp2/3 binding motif. Phosphorylation of cortactin T24 by CK2α impairs the ability of cortactin to bind Arp2/3 and activate actin nucleation. Decreased invadopodia activity is observed in HNSCC cells with expression of CK2α phosphorylation-null cortactin mutants, shRNA-mediated CK2α knockdown, and with the CK2α inhibitor Silmitasertib. Silmitasertib inhibits HNSCC collective invasion in tumor spheroids and orthotopic tongue tumors in mice. Collectively these data suggest that CK2α-mediated cortactin phosphorylation at T24 is critical in regulating cortactin binding to Arp2/3 complex and pro-invasive activity, identifying a potential targetable mechanism for impairing HNSCC invasion. IMPLICATIONS: This study identifies a new signaling pathway that contributes to enhancing cancer cell invasion.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/987/F1.large.jpg.

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 µm, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse  model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological  analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

Tumor and stromal-based contributions to head and neck squamous cell carcinoma invasion.

Head and neck squamous cell carcinoma (HNSCC) is typically diagnosed at advanced stages with evident loco-regional and/or distal metastases. The prevalence of metastatic lesions directly correlates with poor patient outcome, resulting in high patient mortality rates following metastatic development. The progression to metastatic disease requires changes not only in the carcinoma cells, but also in the surrounding stromal cells and tumor microenvironment. Within the microenvironment, acellular contributions from the surrounding extracellular matrix, along with contributions from various infiltrating immune cells, tumor associated fibroblasts, and endothelial cells facilitate the spread of tumor cells from the primary site to the rest of the body. Thus far, most attempts to limit metastatic spread through therapeutic intervention have failed to show patient benefit in clinic trails. The goal of this review is highlight the complexity of invasion-promoting interactions in the HNSCC tumor microenvironment, focusing on contributions from tumor and stromal cells in order to assist future therapeutic development and patient treatment.

Use of high frequency ultrasound to monitor cervical lymph node alterations in mice.

Cervical lymph node evaluation by clinical ultrasound is a non-invasive procedure used in diagnosing nodal status, and when combined with fine-needle aspiration cytology (FNAC), provides an effective method to assess nodal pathologies. Development of high-frequency ultrasound (HF US) allows real-time monitoring of lymph node alterations in animal models. While HF US is frequently used in animal models of tumor biology, use of HF US for studying cervical lymph nodes alterations associated with murine models of head and neck cancer, or any other model of lymphadenopathy, is lacking. Here we utilize HF US to monitor cervical lymph nodes changes in mice following exposure to the oral cancer-inducing carcinogen 4-nitroquinoline-1-oxide (4-NQO) and in mice with systemic autoimmunity. 4-NQO induces tumors within the mouse oral cavity as early as 19 wks that recapitulate HNSCC. Monitoring of cervical (mandibular) lymph nodes by gray scale and power Doppler sonography revealed changes in lymph node size eight weeks after 4-NQO treatment, prior to tumor formation. 4-NQO causes changes in cervical node blood flow resulting from oral tumor progression. Histological evaluation indicated that the early 4-NQO induced changes in lymph node volume were due to specific hyperproliferation of T-cell enriched zones in the paracortex. We also show that HF US can be used to perform image-guided fine needle aspirate (FNA) biopsies on mice with enlarged mandibular lymph nodes due to genetic mutation of Fas ligand (Fasl). Collectively these studies indicate that HF US is an effective technique for the non-invasive study of cervical lymph node alterations in live mouse models of oral cancer and other mouse models containing cervical lymphadenopathy.

NEDD9 regulates actin dynamics through cortactin deacetylation in an AURKA/HDAC6-dependent manner.

UNLABELLED: The prometastatic protein NEDD9 (neural precursor cell expressed, developmentally downregulated 9) is highly expressed in many cancers and is required for mesenchymal individual cell migration and progression to the invasive stage. Nevertheless, the molecular mechanisms of NEDD9-driven migration and the downstream targets effecting metastasis are not well defined. In the current study, knockdown of NEDD9 in highly metastatic tumor cells drastically reduces their migratory capacity due to disruption of actin dynamics at the leading edge. Specifically, NEDD9 deficiency leads to a decrease in the persistence and stability of lamellipodial protrusions similar to knockdown of cortactin (CTTN). Mechanistically, it was shown that NEDD9 binds to and regulates acetylation of CTTN in an Aurora A kinase (AURKA)/HDAC6-dependent manner. The knockdown of NEDD9 or AURKA results in an increase in the amount of acetylated CTTN and a decrease in the binding of CTTN to F-actin. Overexpression of the deacetylation mimicking (9KR) mutant of CTTN is sufficient to restore actin dynamics at the leading edge and migration proficiency of the tumor cells. Inhibition of AURKA and HDAC6 activity by alisertib and Tubastatin A in xenograft models of breast cancer leads to a decrease in the number of pulmonary metastases. Collectively, these findings identify CTTN as the key downstream component of NEDD9-driven migration and metastatic phenotypes. IMPLICATIONS: This study provides a mechanistic platform for therapeutic interventions based on AURKA and HDAC6 inhibition for patients with metastatic breast cancer to prevent and/or eradicate metastases.

NEDD9 depletion leads to MMP14 inactivation by TIMP2 and prevents invasion and metastasis.

UNLABELLED: The scaffolding protein NEDD9 is an established prometastatic marker in several cancers. Nevertheless, the molecular mechanisms of NEDD9-driven metastasis in cancers remain ill-defined. Here, using a comprehensive breast cancer tissue microarray, it was shown that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly, it was shown that NEDD9 overexpression is a hallmark of highly invasive breast cancer cells. Moreover, NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo, leading to decreased circulating tumor cells and lung metastases in xenograft models. Mechanistically, NEDD9 localized to invasive pseudopods and was required for local matrix degradation. Depletion of NEDD9 impaired invasion of cancer cells through inactivation of membrane-bound matrix metalloproteinase MMP14 by excess TIMP2 on the cell surface. Inactivation of MMP14 is accompanied by reduced collagenolytic activity of soluble metalloproteinases MMP2 and MMP9. Reexpression of NEDD9 is sufficient to restore the activity of MMP14 and the invasive properties of breast cancer cells in vitro and in vivo. Collectively, these findings uncover critical steps in NEDD9-dependent invasion of breast cancer cells. IMPLICATIONS: This study provides a mechanistic basis for potential therapeutic interventions to prevent metastasis.

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa.

Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis.

Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion.

Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.

Src binds cortactin through an SH2 domain cystine-mediated linkage.

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

Effects of tobacco smoking and nicotine on cancer treatment.

A substantial number of the world's population continues to smoke tobacco, even in the setting of a cancer diagnosis. Studies have shown that patients with cancer who have a history of smoking have a worse prognosis than nonsmokers. Modulation of several physiologic processes involved in drug disposition has been associated with long-term exposure to tobacco smoke. The most common of these processes can be categorized into the effects of smoking on cytochrome P450-mediated metabolism, glucuronidation, and protein binding. Perturbation in the pharmacokinetics of anticancer drugs could result in clinically significant consequences, as these drugs are among the most toxic, but potentially beneficial, pharmaceuticals prescribed. Unfortunately, the effect of tobacco smoking on drug disposition has been explored for only a few marketed anticancer drugs; thus, little prescribing information is available to guide clinicians on the vast majority of these agents. The carcinogenic properties of several compounds found in tobacco smoke have been well studied; however, relatively little attention has been given to the effects of nicotine itself on cancer growth. Data that identify nicotine's effect on cancer cell apoptosis, tumor angiogenesis, invasion, and metastasis are emerging. The implications of these data are still unclear but may lead to important questions regarding approaches to smoking cessation in patients with cancer.

Quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts.

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components. Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Cortactin is a substrate of activated Cdc42-associated kinase 1 (ACK1) during ligand-induced epidermal growth factor receptor downregulation.

BACKGROUND: Epidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization. CONCLUSIONS/SIGNIFICANCE: Cortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.

Further insights into cortactin conformational regulation.

The actin regulatory protein cortactin is involved in multiple signaling pathways impinging on the cortical actin cytoskeleton. Cortactin is phosphorylated by ERK1/2 and Src family tyrosine kinases, resulting in neuronal Wiskott Aldrich Syndrome protein (N-WASp) activation and enhanced actin related protein (Arp)2/3-mediated actin nucleation. Cortactin migrates as an 80/85 kDa doublet when analyzed by SDS-PAGE. Phosphorylation by ERK1/2 is associated with conversion of the 80 kDa to the 85 kDa form, postulated to occur by inducing a conformational alteration that releases the carboxyl-terminal SH3 domain from autoinhibition. Our recent analysis of the 80-85 kDa cortactin "shift" in tumor cells indicates that while ERK1/2 phosphorylation is associated with the 85 kDa shift, this phosphorylation event is not required for the shift to occur, nor does ERK1/2 phosphorylation appreciably alter global cortactin confirmation. These data indicate that additional factors besides ERK1/2 phosphorylation contribute to generating and/or maintaining the activated 85 kDa cortactin form in stimulated cells.

Multi-photon imaging of tumor cell invasion in an orthotopic mouse model of oral squamous cell carcinoma.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1(nu/nu) mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

Revisiting the ERK/Src cortactin switch.

The filamentous (F)-actin regulatory protein cortactin plays an important role in tumor cell movement and invasion by promoting and stabilizing actin related protein (Arp)2/3-mediated actin networks necessary for plasma membrane protrusion. Cortactin is a substrate for ERK1/2 and Src family kinases, with previous in vitro findings demonstrating ERK1/2 phosphorylation of cortactin as a positive and Src phosphorylation as a negative regulatory event in promoting Arp2/3 activation through neuronal Wiskott Aldrich Syndrome protein (N-WASp). Evidence for this regulatory cortactin "switch" in cells has been hampered due to the lack of phosphorylation-specific antibodies that recognize ERK1/2-phosphorylated cortactin. Our findings with phosphorylation-specific antibodies against these ERK1/2 sites (pS405 and pS418) indicate that cortactin can be co-phosphorylated at 405/418 and tyrosine residues targeted by Src family tyrosine kinases. These results indicate that the ERK/Src cortactin switch is not the sole mechanism by which ERK1/2 and tyrosine phosphorylation events regulate cortactin function in cell systems.