Publications by authors named "Scott A Weed"

39 Publications

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

Cancer Genet 2021 Aug 28;256-257:136-148. Epub 2021 May 28.

Department of Biochemistry, Program in Cancer Cell Biology USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cancergen.2021.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8273756PMC
August 2021

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

Sci Rep 2020 07 15;10(1):11612. Epub 2020 Jul 15.

Department of Biochemistry, Program in Cancer Cell Biology, West Virginia University Cancer Institute, West Virginia University, P.O. Box 9300, Morgantown, WV, 26506, USA.

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.
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http://dx.doi.org/10.1038/s41598-020-68380-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363863PMC
July 2020

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

Mol Microbiol 2019 09 12;112(3):800-819. Epub 2019 Jun 12.

Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, WV, USA.

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.
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http://dx.doi.org/10.1111/mmi.14317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736723PMC
September 2019

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

Mol Cancer Res 2019 04 4;17(4):987-1001. Epub 2019 Jan 4.

Program in Cancer Cell Biology, Department of Biochemistry, West Virginia University, Morgantown, West Virginia.

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. http://mcr.aacrjournals.org/content/molcanres/17/4/987/F1.large.jpg.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445698PMC
April 2019

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes.

J Vis Exp 2015 Jul 25(101):e52718. Epub 2015 Jul 25.

Department of Neurobiology and Anatomy, West Virginia University; Mary Babb Randolph Cancer Center, West Virginia University;

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.
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http://dx.doi.org/10.3791/52718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545045PMC
July 2015

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

Cancers (Basel) 2015 Feb 27;7(1):382-406. Epub 2015 Feb 27.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA.

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.
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http://dx.doi.org/10.3390/cancers7010382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381264PMC
February 2015

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

PLoS One 2014 23;9(6):e100185. Epub 2014 Jun 23.

Department of Neurobiology and Anatomy, West Virginia University, Morgantown, West Virginia, United States of America; Program in Cancer Cell Biology, West Virginia University, Morgantown, West Virginia, United States of America; Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100185PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067293PMC
October 2015

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

Mol Cancer Res 2014 May 26;12(5):681-93. Epub 2014 Feb 26.

Authors' Affiliations: Mary Babb Randolph Cancer Center; Departments of 2Biochemistry, 3Pathology, and 4Neurobiology and Anatomy, West Virginia University School of Medicine, Morgantown, West Virginia.

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.
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http://dx.doi.org/10.1158/1541-7786.MCR-13-0654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020952PMC
May 2014

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

Mol Cancer Res 2014 Jan 7;12(1):69-81. Epub 2013 Nov 7.

Department of Biochemistry and Mary Babb Randolph Cancer Center, PO Box 9142, 1 Medical Center Drive, West Virginia University School of Medicine, Morgantown, WV 26506.

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.
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http://dx.doi.org/10.1158/1541-7786.MCR-13-0300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946989PMC
January 2014

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

Biol Open 2013 3;2(9):891-900. Epub 2013 Jul 3.

Department of Microbiology, Immunology and Cell Biology, West Virginia University Health Sciences Center , Morgantown, WV 26506-9177 , USA.

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.
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http://dx.doi.org/10.1242/bio.20133632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3773335PMC
October 2013

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

Nucleic Acids Res 2013 May 21;41(9):4949-62. Epub 2013 Mar 21.

Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425, USA.

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.
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http://dx.doi.org/10.1093/nar/gkt170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3643583PMC
May 2013

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

J Cell Sci 2012 Dec 24;125(Pt 24):6185-97. Epub 2012 Oct 24.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA.

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.
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http://dx.doi.org/10.1242/jcs.121046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585525PMC
December 2012

Effects of tobacco smoking and nicotine on cancer treatment.

Pharmacotherapy 2012 Oct;32(10):920-31

School of Pharmacy, West Virginia University, Morgantown, West Virginia 26506, USA.

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.
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http://dx.doi.org/10.1002/j.1875-9114.2012.01117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499669PMC
October 2012

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

PLoS One 2012 30;7(8):e44363. Epub 2012 Aug 30.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, USA.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044363PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431376PMC
February 2013

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

J Vis Exp 2012 Aug 27(66):e4119. Epub 2012 Aug 27.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, USA.

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.
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http://dx.doi.org/10.3791/4119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3606055PMC
August 2012

Further insights into cortactin conformational regulation.

Bioarchitecture 2011 Jan;1(1):21-23

Department of Neurobiology and Anatomy; Program in Cancer Cell Biology; Mary Babb Randolph Cancer Center; West Virginia University; Morgantown, WV USA.

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.
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http://dx.doi.org/10.4161/bioa.1.1.14631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158636PMC
January 2011

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

J Vis Exp 2011 Jul 25(53). Epub 2011 Jul 25.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, USA.

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.
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http://dx.doi.org/10.3791/2941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196194PMC
July 2011

Revisiting the ERK/Src cortactin switch.

Commun Integr Biol 2011 Mar;4(2):205-7

Department of Neurobiology and Anatomy; Program in Cancer Cell Biology; Mary Babb Randolph Cancer Center; West Virginia University, Morgantown, WV USA.

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.
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http://dx.doi.org/10.4161/cib.4.2.14420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104580PMC
March 2011

Recently identified biomarkers that promote lymph node metastasis in head and neck squamous cell carcinoma.

Cancers (Basel) 2011 Feb 22;3(1):747-72. Epub 2011 Feb 22.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous cancer that arises in the upper aerodigestive tract. Despite advances in knowledge and treatment of this disease, the five-year survival rate after diagnosis of advanced (stage 3 and 4) HNSCC remains approximately 50%. One reason for the large degree of mortality associated with late stage HNSCC is the intrinsic ability of tumor cells to undergo locoregional invasion. Lymph nodes in the cervical region are the primary sites of metastasis for HNSCC, occurring before the formation of distant metastases. The presence of lymph node metastases is strongly associated with poor patient outcome, resulting in increased consideration being given to the development and implementation of anti-invasive strategies. In this review, we focus on select proteins that have been recently identified as promoters of lymph node metastasis in HNSCC. The discussed proteins are involved in a wide range of critical cellular functions, and offer a more comprehensive understanding of the factors involved in HNSCC metastasis while additionally providing increased options for consideration in the design of future therapeutic intervention strategies.
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http://dx.doi.org/10.3390/cancers3010747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756388PMC
February 2011

Cortactin phosphorylated by ERK1/2 localizes to sites of dynamic actin regulation and is required for carcinoma lamellipodia persistence.

PLoS One 2010 Nov 4;5(11):e13847. Epub 2010 Nov 4.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, United States of America.

Background: Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement.

Methodology/principal Findings: In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging.

Conclusions/significance: Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013847PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2973953PMC
November 2010

Oncogenic Src requires a wild-type counterpart to regulate invadopodia maturation.

J Cell Sci 2010 Nov 27;123(Pt 22):3923-32. Epub 2010 Oct 27.

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia 26506-9300, USA.

The proto-oncogene Src tyrosine kinase (Src) is overexpressed in human cancers and is currently a target of anti-invasive therapies. Activation of Src is an essential catalyst of invadopodia production. Invadopodia are cellular structures that mediate extracellular matrix (ECM) proteolysis, allowing invasive cell types to breach confining tissue barriers. Invadopodia assembly and maturation is a multistep process, first requiring the targeting of actin-associated proteins to form pre-invadopodia, which subsequently mature by recruitment and activation of matrix metalloproteases (MMPs) that facilitate ECM degradation. We demonstrate that active, oncogenic Src alleles require the presence of a wild-type counterpart to induce ECM degradation at invadopodia sites. In addition, we identify the phosphorylation of the invadopodia regulatory protein cortactin as an important mediator of invadopodia maturation downstream of wild-type Src. Distinct phosphotyrosine-based protein-binding profiles in cells forming pre-invadopodia and mature invadopodia were identified by SH2-domain array analysis. These results indicate that although elevated Src kinase activity is required to target actin-associated proteins to pre-invadopodia, regulated Src activity is required for invadopodia maturation and matrix degradation activity. Our findings describe a previously unappreciated role for proto-oncogenic Src in enabling the invasive activity of constitutively active Src alleles.
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http://dx.doi.org/10.1242/jcs.075200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2972274PMC
November 2010

Saracatinib Impairs Head and Neck Squamous Cell Carcinoma Invasion by Disrupting Invadopodia Function.

J Cancer Sci Ther 2009 Nov;1(2):52-61

Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, 26506-9300.

Elevated Src kinase activity is linked to the progression of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Src regulates HNSCC proliferation and tumor invasion, with the Src-targeted small molecule inhibitor saracatinib displaying potent anti-invasive effects in preclinical studies. However, the pro-invasive cellular mechanism(s) perturbed by saracatinib are unclear. The anti-proliferative and anti-invasive effects of saracatinib on HNSCC cell lines were therefore investigated in pre-clinical cell and mouse model systems. Saracatinib treatment inhibited growth, cell cycle progression and transwell Matrigel invasion in HNSCC cell lines. Dose-dependent decreases in Src activation and phosphorylation of the invasion-associated substrates focal adhesion kinase, p130 CAS and cortactin were also observed. While saracatinib did not significantly impact HNSCC tumor growth in a mouse orthotopic model of tongue squamous cell carcinoma, impaired perineural invasion and cervical lymph node metastasis was observed. Accordingly, saracatinib treatment displayed a dose-dependent inhibitory effect on invadopodia formation, extracellular matrix degradation and matrix metalloprotease 9 activation. These results suggest that inhibition of Src kinase by saracatinib impairs the pro-invasive activity of HNSCC by inhibiting Src substrate phosphorylation important for invadopodia formation and associated matrix metalloprotease activity.
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http://dx.doi.org/10.4172/1948-5956.1000009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875552PMC
November 2009

Cortactin is a functional target of E-cadherin-activated Src family kinases in MCF7 epithelial monolayers.

J Biol Chem 2009 Jul 19;284(28):18913-22. Epub 2009 May 19.

Division of Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane 4072, Australia.

Src family kinases (SFKs) signal in response to E-cadherin to support cadherin adhesion and the integrity of cell-cell contacts (McLachlan, R. W., Kraemer, A., Helwani, F. M., Kovacs, E. M., and Yap, A. S. (2007) Mol. Biol. Cell 18, 3214-3223). We now identify the actin-regulatory protein, cortactin, as a target of E-cadherin-activated SFK signaling. Tyr-phosphorylated cortactin was found at cell-cell contacts in established epithelial monolayers, and cortactin became acutely tyrosine-phosphorylated when E-cadherin adhesion was engaged. In all circumstances, cortactin tyrosine phosphorylation was blocked by inhibiting SFK signaling. Importantly, Tyr-phosphorylated cortactin was necessary to preserve the integrity of cadherin contacts and the perijunctional actin cytoskeleton. Moreover, expression of a phosphomimetic cortactin mutant could prevent SFK blockade from disrupting cadherin organization, thereby placing cortactin functionally downstream of SFK signaling at cadherin adhesions. We conclude that SFK and cortactin constitute an important signaling pathway that functionally links E-cadherin adhesion and the actin cytoskeleton.
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http://dx.doi.org/10.1074/jbc.M109.000307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2707247PMC
July 2009

Cortactin branches out: roles in regulating protrusive actin dynamics.

Cell Motil Cytoskeleton 2008 Sep;65(9):687-707

Department of Neuroscience and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia 26506-9300, USA.

Since its discovery in the early 1990's, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures.
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http://dx.doi.org/10.1002/cm.20296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2561250PMC
September 2008

Phosphorylation of AFAP-110 affects podosome lifespan in A7r5 cells.

J Cell Sci 2008 Jul 24;121(Pt 14):2394-405. Epub 2008 Jun 24.

The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA.

AFAP-110 is an actin-binding and -crosslinking protein that is enriched in Src and phorbol ester (PE)-induced podosomes. In vascular smooth muscle cells endogenous AFAP-110 localized to actin stress fibers and, in response to treatment with phorbol-12,13-dibutyrate (PDBu), to actin-rich podosomes. Since PEs can activate PKCalpha, AFAP-110 is a substrate of PKCalpha and PKCalpha-AFAP-110 interactions direct podosome formation, we sought to identify a PE-induced phosphorylation site in AFAP-110 and determine whether phosphorylation is linked to the formation of podosomes. Mutational analysis revealed Ser277 of AFAP-110 to be phosphorylated in PE-treated cells. The use of a newly generated, phospho-specific antibody directed against phosphorylated Ser277 revealed that PKCalpha activation is associated with PE-induced AFAP-110 phosphorylation. In PDBu-treated A7r5 rat vascular smooth muscle cells, immunolabeling using the phospho-specific antibody showed that phospho-AFAP-110 is primarily associated with actin in podosomes. Although mutation of Ser at position 277 to Ala (AFAP-110(S277A)) did not alter the ability of AFAP-110 to localize to podosomes, overexpression of AFAP-110(S277A) in treated and untreated A7r5 cells resulted in an increased number of cells that display podosomes. Video microscopy demonstrated that AFAP-110(S277A) expression correlates with an increased number of long-lived podosomes. Therefore, we hypothesize that AFAP-110 phosphorylation and/or dephosphorylation is involved in the regulation of podosome stability and lifespan.
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http://dx.doi.org/10.1242/jcs.026187DOI Listing
July 2008

Actin cytoskeletal mediators of motility and invasion amplified and overexpressed in head and neck cancer.

Clin Exp Metastasis 2008 7;25(4):289-304. Epub 2008 Mar 7.

Department of Neuroscience and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506-9300, USA.

Coordinated regulation of the actin cytoskeleton is central to cell motility, invasion and metastasis. Head and neck squamous cell carcinoma (HNSCC) is a highly invasive disease displaying frequent lymph node metastasis, compounding patient management. HNSCC progression is characterized by frequent amplification of chromosome segments 3q26-29, 8q23-24 and 11q13, events that are associated with poor patient outcome. The relative frequency of these amplification events and correlation with invasive disease raises the potential that these regions harbor actin regulatory genes important in facilitating reorganization of the actin cytoskeleton to promote tumor invasion. Identification of the actin cytoskeletal regulatory genes located within the 3q26-29, 8q23-24 and 11q13 amplicons will provide an important first step towards the comprehensive understanding of the molecular events that govern invasion and metastasis in HNSCC and other tumors containing these amplifications. We utilized Ensembl MartView to conduct a gene mining analysis within chromosome segments 3q26-29, 8q23-24 and 11q13 to identify known and predicted regulators of actin-based cell movement, tumor invasion and metastasis. All examined chromosomal regions contain genes known that regulate the actin cytoskeleton, with several (PI3-kinase alpha, focal adhesion kinase (FAK) and cortactin) known to promote invasion in HNSCC and other carcinomas. Additional genes known to regulate motility and invasion were also identified. Amplification of chromosome 3q26-29, 8q23-24 and 11q13 therefore results in known or predicted overexpression of several key mediators that can act alone or potentially act in concert to promote actin-based cell invasion in HNSCC and other cancer types.
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http://dx.doi.org/10.1007/s10585-008-9154-6DOI Listing
August 2008

PI3K activation is required for PMA-directed activation of cSrc by AFAP-110.

Am J Physiol Cell Physiol 2007 Jul 14;293(1):C119-32. Epub 2007 Mar 14.

The Mary Babb Randolph Cancer Center, Dept. of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA.

Activation of PKCalpha will induce the cSrc binding partner AFAP-110 to colocalize with and activate cSrc. The ability of AFAP-110 to colocalize with cSrc is contingent on the integrity of the amino-terminal pleckstrin homology (PH1) domain, while the ability to activate cSrc is dependent on the integrity of its SH3 binding motif, which engages the cSrc SH3 domain. The outcome of AFAP-110-directed cSrc activation is a change in actin filament integrity and the formation of podosomes. Here, we address what cellular signals promote AFAP-110 to colocalize with and activate cSrc, in response to PKCalpha activation or PMA treatment. Because PH domain integrity in AFAP-110 is required for colocalization, and PH domains are known to interact with both protein and lipid binding partners, we sought to determine whether phosphatidylinositol 3-kinase (PI3K) activation played a role in PMA-induced colocalization between AFAP-110 and cSrc. We show that PMA treatment is able to direct activation of PI3K. Treatment of mouse embryo fibroblast with PI3K inhibitors blocked PMA-directed colocalization between AFAP-110 and cSrc and subsequent cSrc activation. PMA also was unable to induce colocalization or cSrc activation in cells that lacked the p85alpha and -beta regulatory subunits of PI3K. This signaling pathway was required for migration in a wound healing assay. Cells that were null for cSrc or the p85 regulatory subunits or expressed a dominant-negative AFAP-110 also displayed a reduction in migration. Thus PI3K activity is required for PMA-induced colocalization between AFAP-110 and cSrc and subsequent cSrc activation, and this signaling pathway promotes cell migration.
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http://dx.doi.org/10.1152/ajpcell.00525.2006DOI Listing
July 2007

Overexpression of cyclin D1 and cortactin is primarily independent of gene amplification in salivary gland adenoid cystic carcinoma.

Oral Oncol 2007 Sep 17;43(8):735-41. Epub 2006 Nov 17.

Department of Diagnostic and Biological Sciences, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80010, USA.

Adenoid cystic carcinoma (ACC) of the salivary glands exhibits persistent growth, invasion and metastasis. Chromosome 11q13 amplification is a frequent event associated with tumor progression in a number of carcinomas and is associated with poor prognosis. Two genes within the 11q13 amplicon that are overexpressed as a result of 11q13 amplification are the cell cycle regulatory protein cyclin D1 (CCND1) and cortactin (CTTN), a protein involved cell motility and invasion. To determine the expression and gene status of cyclin D1 and cortactin in ACC, we evaluated 39 ACC cases by immunohistochemistry (IHC) for cyclin D1 and cortactin expression. Amplification of CCND1 and CTTN was determined by fluorescent in situ hybridization (FISH). Cyclin D1 overexpression was present in 90% (35/39) and cortactin expression in 62% (24/39) of evaluated cases, although CCND1 and CTTN levels were elevated in only two cases (5%) as determined by FISH. Our results indicate that chromosome 11q13 amplification is uncommon in ACC, but that cyclin D1 and cortactin are frequently overexpressed and may therefore contribute to the growth and invasive potential of ACC.
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http://dx.doi.org/10.1016/j.oraloncology.2006.09.007DOI Listing
September 2007

Cortactin overexpression regulates actin-related protein 2/3 complex activity, motility, and invasion in carcinomas with chromosome 11q13 amplification.

Cancer Res 2006 Aug;66(16):8017-25

Department of Craniofacial Biology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado, USA.

Carcinoma cell motility and invasion are prerequisites for tumor cell metastasis, which requires regulation of the actin cytoskeleton. Cortactin is an actin-related protein 2/3 (Arp2/3) complex-activating and filamentous (F)-actin-binding protein that is implicated in tumor cell motility and metastasis, partially by its ability to become tyrosine phosphorylated. Cortactin is encoded by the CTTN gene and maps to chromosome 11q13, a region amplified in many carcinomas, including head and neck squamous cell carcinoma (HNSCC). CTTN gene amplification is associated with lymph node metastasis and poor patient outcome, and cortactin overexpression enhances motility in tumor cells lacking 11q13 amplification. However, a direct link between increased motility and invasion has not been reported in tumor cells with chromosome 11q13 amplification and cortactin overexpression. In this study, we have examined the relationship between CTTN amplification and tumor cell motility in HNSCC. In 11 of 39 (28%) HNSCC cases, cortactin overexpression determined by immunohistochemistry correlates with lymph node metastasis and CTTN gene amplification. HNSCC cells containing cortactin gene amplification and protein overexpression display increased binding and activation of Arp2/3 complex, and were more motile and invasive than HNSCC cells lacking CTTN amplification. Down-regulation of cortactin expression in CTTN-amplified HNSCC cells by small interfering RNA impairs HNSCC motility and invasion. Treatment of HNSCC cells with the epidermal growth factor receptor inhibitor gefitinib inhibits HNSCC motility and down-regulates cortactin tyrosine phosphorylation. These data suggest that cortactin may be a valid prognostic and therapeutic marker for invasive and metastatic HNSCC and other carcinomas with 11q13 amplification.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-4490DOI Listing
August 2006

Protein kinase Calpha activates c-Src and induces podosome formation via AFAP-110.

Mol Cell Biol 2004 Sep;24(17):7578-97

The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA.

We report that the actin filament-associated protein AFAP-110 is required to mediate protein kinase Calpha (PKCalpha) activation of the nonreceptor tyrosine kinase c-Src and the subsequent formation of podosomes. Immunofluorescence analysis demonstrated that activation of PKCalpha by phorbol 12-myristate 13-acetate (PMA), or ectopic expression of constitutively activated PKCalpha, directs AFAP-110 to colocalize with and bind to the c-Src SH3 domain, resulting in activation of the tyrosine kinase. Activation of c-Src then directs the formation of podosomes, which contain cortactin, AFAP-110, actin, and c-Src. In a cell line (CaOV3) that has very little or no detectable AFAP-110, PMA treatment was unable to activate c-Src or effect podosome formation. Ectopic expression of AFAP-110 in CaOV3 cells rescued PKCalpha-mediated activation of c-Src and elevated tyrosine phosphorylation levels and subsequent formation of podosomes. Neither expression of activated PKCalpha nor treatment with PMA was able to induce these changes in CAOV3 cells expressing mutant forms of AFAP-110 that are unable to bind to, or colocalize with, c-Src. We hypothesize that one major function of AFAP-110 is to relay signals from PKCalpha that direct the activation of c-Src and the formation of podosomes.
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http://dx.doi.org/10.1128/MCB.24.17.7578-7597.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC506973PMC
September 2004
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