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Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent

Oncogene volume 29, pages 4449–4459 (2010)Cite this article

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Abstract

We previously reported a vascular endothelial growth factor (VEGF) autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach, we screened the HNSCC cell line, squamous cell carcinoma-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation 1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion and matrix metalloproteinase (MMP) expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix-coated coverslips showed enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared with a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also show for the first time that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1's recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.

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References

  • Alexander NR, Branch KM, Parekh A, Clark ES, Iwueke IC, Guelcher SA et al. (2008). Extracellular matrix rigidity promotes invadopodia activity. Curr Biol 18: 1295–1299.

    Article  CAS  Google Scholar 

  • Arold ST, Hoellerer MK, Noble MEM . (2002). The structural basis of localization and signaling by the focal adhesion targeting domain. Structure 10: 319–327.

    Article  CAS  Google Scholar 

  • Ayala I, Baldassarre M, Giacchetti G, Caldieri G, Tete S, Luini A et al. (2008). Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation. J Cell Sci 121: 369–378.

    Article  CAS  Google Scholar 

  • Bachelder RE, Lipscomb EA, Lin X, Wendt MA, Chadborn NH, Eickholt BJ et al. (2003). Competing autocrine pathways involving alternative neuropilin-1 ligands regulate chemotaxis of carcinoma cells. Cancer Res 63: 5230–5233.

    CAS  Google Scholar 

  • Badowski C, Pawlak G, Grichine A, Chabadel A, Oddou C, Jurdic P et al. (2008). Paxillin phosphorylation controls invadopodia/podosomes spatiotemporal organization. Mol Biol Cell 19: 633–645.

    Article  CAS  Google Scholar 

  • Bigbee WL, Grandis JR, Siegfried JM . (2007). Multiple cytokine and growth factor serum biomarkers predict therapeutic response and survival in advanced-stage head and neck cancer patients. Clin Cancer Res 13: 3107–3108.

    Article  CAS  Google Scholar 

  • Bowden ET, Onikoyi E, Slack R, Myoui A, Yoneda T, Yamada KM et al. (2006). Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells. Exp Cell Res 312: 1240–1253.

    Article  CAS  Google Scholar 

  • Brabek J, Constancio SS, Siesser PF, Shin N-Y, Pozzi A, Hanks SK . (2005). Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of Src-transformed cells. Mol Cancer Res 3: 307–315.

    Article  CAS  Google Scholar 

  • Brown MC, Turner CE . (2004). Paxillin: adapting to change. Physiol Rev 84: 1315–1339.

    Article  CAS  Google Scholar 

  • Buday L, Downward J . (2007). Roles of cortactin in tumor pathogenesis. Biochim Biophys Acta 1775: 263–273.

    CAS  Google Scholar 

  • Canel M, Secades P, Rodrigo JP, Cabanillas R, Herrero A, Suarez C et al. (2006). Overexpression of focal adhesion kinase in head and neck squamous cell carcinoma is independent of fak gene copy number. Clin Cancer Res 12: 3272–3279.

    Article  CAS  Google Scholar 

  • Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O et al. (2006). CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7: R100.

    Article  Google Scholar 

  • Clark ES, Whigham AS, Yarbrough WG, Weaver AM . (2007). Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia. Cancer Res 67: 4227–4235.

    Article  CAS  Google Scholar 

  • Cohen LA, Guan JL . (2005). Mechanisms of focal adhesion kinase regulation. Curr Cancer Drug Targets 5: 629–643.

    Article  CAS  Google Scholar 

  • Cooney MM, Tserng KY, Makar V, McPeak RJ, Ingalls ST, Dowlati A et al. (2005). A phase IB clinical and pharmacokinetic study of the angiogenesis inhibitor SU5416 and paclitaxel in recurrent or metastatic carcinoma of the head and neck. Cancer Chemother Pharmacol 55: 295–300.

    Article  CAS  Google Scholar 

  • Descamps FJ, Martens E, Opdenakker G . (2002). Analysis of gelatinases in complex biological fluids and tissue extracts. Lab Invest 82: 1607–1608.

    Article  Google Scholar 

  • Fashena SJ, Einarson MB, O'Neill GM, Patriotis C, Golemis EA . (2002). Dissection of HEF1-dependent functions in motility and transcriptional regulation. J Cell Sci 115: 99–111.

    CAS  PubMed  Google Scholar 

  • Ferrara N . (2004). Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25: 581–611.

    Article  CAS  Google Scholar 

  • Gimona M, Buccione R, Courtneidge SA, Linder S . (2008). Assembly and biological role of podosomes and invadopodia. Curr Opin Cell Biol 20: 235–241.

    Article  CAS  Google Scholar 

  • Guarino M, Rubino B, Ballabio G . (2007). The role of epithelial-mesenchymal transition in cancer pathology. Pathology 39: 305–318.

    Article  CAS  Google Scholar 

  • Izumchenko E, Singh MK, Plotnikova OV, Tikhmyanova N, Little JL, Serebriiskii IG et al. (2009). NEDD9 promotes oncogenic signaling in mammary tumor development. Cancer Res 69: 7198–7206.

    Article  CAS  Google Scholar 

  • Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E et al. (2004). Cancer statistics, 2004. CA Cancer J Clin 54: 8–29.

    Article  Google Scholar 

  • Ji H, Ramsey MR, Hayes DN, Fan C, McNamara K, Kozlowski P et al. (2007). LKB1 modulates lung cancer differentiation and metastasis. Nature 448: 807–810.

    Article  CAS  Google Scholar 

  • Kim M, Gans JD, Nogueira C, Wang A, Paik JH, Feng B et al. (2006). Comparative oncogenomics identifies NEDD9 as a melanoma metastasis gene. Cell 125: 1269–1281.

    Article  CAS  Google Scholar 

  • Kratchmarova I, Kalume DE, Blagoev B, Scherer PE, Podtelejnikov AV, Molina H et al. (2002). A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes. Mol Cell Proteomics 1: 213–222.

    Article  CAS  Google Scholar 

  • Kyzas PA, Stefanou D, Batistatou A, Agnantis NJ . (2005). Potential autocrine function of vascular endothelial growth factor in head and neck cancer via vascular endothelial growth factor receptor-2. Mod Pathol 18: 485–494.

    Article  CAS  Google Scholar 

  • Linder S . (2007). The matrix corroded: podosomes and invadopodia in extracellular matrix degradation. Trends Cell Biol 17: 107–117.

    Article  CAS  Google Scholar 

  • Lua BL, Low BC . (2005). Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control. FEBS Lett 579: 577–585.

    Article  CAS  Google Scholar 

  • Michi Y, Morita I, Amagasa T, Murota S . (2000). Human oral squamous cell carcinoma cell lines promote angiogenesis via expression of vascular endothelial growth factor and upregulation of KDR/flk-1 expression in endothelial cells. Oral Oncol 36: 81–88.

    Article  CAS  Google Scholar 

  • Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD et al. (2005). Genes that mediate breast cancer metastasis to lung. Nature 436: 518–524.

    Article  CAS  Google Scholar 

  • Molinolo AA, Hewitt SM, Amornphimoltham P, Keelawat S, Rangdaeng S, Meneses Garcia A et al. (2007). Dissecting the Akt/mammalian target of rapamycin signaling network: emerging results from the head and neck cancer tissue array initiative. Clin Cancer Res 13: 4964–4973.

    Article  CAS  Google Scholar 

  • Mon NN, Ito S, Senga T, Hamaguchi M . (2006). FAK signaling in neoplastic disorders: a linkage between inflammation and cancer. Ann NY Acad Sci 1086: 199–212.

    Article  CAS  Google Scholar 

  • Nakamoto T, Yamagata T, Sakai R, Ogawa S, Honda H, Ueno H et al. (2000). CIZ, a zinc finger protein that interacts with p130(cas) and activates the expression of matrix metalloproteinases. Mol Cell Biol 20: 1649–1658.

    Article  CAS  Google Scholar 

  • Natarajan M, Stewart JE, Golemis EA, Pugacheva EN, Alexandropoulos K, Cox BD et al. (2006). HEF1 is a necessary and specific downstream effector of FAK that promotes the migration of glioblastoma cells. Oncogene 25: 1721–1732.

    Article  CAS  Google Scholar 

  • O'Neill GM, Seo S, Serebriiskii IG, Lessin SR, Golemis EA . (2007). A new central scaffold for metastasis: parsing HEF1/Cas-L/NEDD9. Cancer Res 67: 8975–8979.

    Article  CAS  Google Scholar 

  • Ormandy CJ, Musgrove EA, Hui R, Daly RJ, Sutherland RL . (2003). Cyclin D1, EMS1 and 11q13 amplification in breast cancer. Breast Cancer Res Treat 78: 323–335.

    Article  CAS  Google Scholar 

  • Patel AM, Incognito LS, Schechter GL, Wasilenko WJ, Somers KD . (1996). Amplification and expression of EMS-1 (cortactin) in head and neck squamous cell carcinoma cell lines. Oncogene 12: 31–35.

    CAS  PubMed  Google Scholar 

  • Rheinwald JG, Beckett MA . (1981). Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res 41: 1657–1663.

    CAS  PubMed  Google Scholar 

  • Ries L, Eisner M, Kosary C, Hankey B, Miller B, Clegg L et al. (2004). SEER Cancer Statistics Review, 1975–2001, National Caner Institute. Bethesda, MD, http://seer.cancer.gov/csr/1975_2001/.

  • Rothschild BL, Shim AH, Ammer AG, Kelley LC, Irby KB, Head JA et al. (2006). Cortactin overexpression regulates actin-related protein 2/3 complex activity, motility, and invasion in carcinomas with chromosome 11q13 amplification. Cancer Res 66: 8017–8025.

    Article  CAS  Google Scholar 

  • Ruest PJ, Shin N-Y, Polte TR, Zhang X, Hanks SK . (2001). Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src. Mol Cell Biol 21: 7641–7652.

    Article  CAS  Google Scholar 

  • Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ et al (2005). Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23: 94–101.

    Article  CAS  Google Scholar 

  • Sabe H, Onodera Y, Mazaki Y, Hashimoto S . (2006). ArfGAP family proteins in cell adhesion, migration and tumor invasion. Curr Opin Cell Biol 18: 558–564.

    Article  CAS  Google Scholar 

  • Sanz-Moreno V, Gadea G, Ahn J, Paterson H, Marra P, Pinner S et al (2008). Rac activation and inactivation control plasticity of tumor cell movement. Cell 135: 510–523.

    Article  CAS  Google Scholar 

  • Simpson KJ, Selfors LM, Bui J, Reynolds A, Leake D, Khvorova A et al (2008). Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat Cell Biol 10: 1027–1038.

    Article  CAS  Google Scholar 

  • Singh MK, Cowell L, Seo S, O'Neill GM, Golemis EA . (2007). Molecular basis for HEF1/NEDD9/Cas-L action as a multifunctional co-ordinator of invasion, apoptosis and cell cycle. Cell Biochem Biophys 48: 54–72.

    Article  CAS  Google Scholar 

  • Singh MK, Dadke D, Nicolas E, Serebriiskii IG, Apostolou S, Canutescu A et al (2008). A novel Cas family member, HEPL, regulates FAK and cell spreading. Mol Biol Cell 19: 1627–1636.

    Article  CAS  Google Scholar 

  • Slack-Davis JK, Martin KH, Tilghman RW, Iwanicki M, Ung EJ, Autry C et al (2007). Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem 282: 14845–14852.

    Article  CAS  Google Scholar 

  • Sloan EK, Anderson RL . (2002). Genes involved in breast cancer metastasis to bone. Cell Mol Life Sci 59: 1491–1502.

    Article  CAS  Google Scholar 

  • Slomiany MG, Black LA, Kibbey MM, Day TA, Rosenzweig SA . (2006). IGF-1 induced vascular endothelial growth factor secretion in head and neck squamous cell carcinoma. Biochem Biophys Res Commun 342: 851–858.

    Article  CAS  Google Scholar 

  • Slomiany MG, Black LA, Kibbey MM, Tingler MA, Day TA, Rosenzweig SA . (2007). Insulin-like growth factor-1 receptor and ligand targeting in head and neck squamous cell carcinoma. Cancer Lett 248: 269–279.

    Article  CAS  Google Scholar 

  • Steen H, Kuster B, Fernandez M, Pandey A, Mann M . (2002). Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. J Biol Chem 277: 1031–1039.

    Article  CAS  Google Scholar 

  • Tachibana K, Urano T, Fujita H, Ohashi Y, Kamiguchi K, Iwata S et al (1997). Tyrosine phosphorylation of Crk-associated substrates by focal adhesion kinase. A putative mechanism for the integrin-mediated tyrosine phosphorylation of Crk-associated substrates. J Biol Chem 272: 29083–29090.

    Article  CAS  Google Scholar 

  • Tehrani S, Tomasevic N, Weed S, Sakowicz R, Cooper JA . (2007). Src phosphorylation of cortactin enhances actin assembly. Proc Natl Acad Sci USA 104: 11933–11938.

    Article  CAS  Google Scholar 

  • Timpson P, Lynch DK, Schramek D, Walker F, Daly RJ . (2005). Cortactin overexpression inhibits ligand-induced down-regulation of the epidermal growth factor receptor. Cancer Res 65: 3273–3280.

    Article  CAS  Google Scholar 

  • van Nimwegen MJ, van de Water B . (2007). Focal adhesion kinase: a potential target in cancer therapy. Biochem Pharmacol 73: 597–609.

    Article  CAS  Google Scholar 

  • Weaver AM . (2008). Cortactin in tumor invasiveness. Cancer Lett 265: 15912–15920.

    Article  Google Scholar 

  • Yu Y-H, Kuo H-K, Chang K-W . (2008). The evolving transcriptome of head and neck squamous cell carcinoma: a systematic review. PLoS ONE 3: e3215.

    Article  Google Scholar 

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Acknowledgements

We thank Amanda M Brock for excellent technical assistance, Jennifer Bethard and Dr Kevin Schey (MUSC Mass Spectrometry Facility), the HCC Cell and Molecular Imaging Shared Resource, the HCC Tissue Biorepository and Dr Elizabeth Garrett-Mayer, Director (HCC Biostatistics Shared Resource) for aid and consultations. We are indebted to Drs Terry Day and M Boyd Gillespie for human specimens and Drs Silvio Gutkind and Alfredo Molinolo (NIDCR) for tissue microarrays. This work was supported by a MUSC Summer Health Research Fellowship, a Paul Calabresi Fellowship (PhRMA Foundation), an Abney Foundation Award, the Southeastern Pre-Doctoral Training in Clinical Research T32 (JTL), NIH grants CA78887 and CA134845 (SAR), DOD award (N6311601MD10004) to HCC and the American Health Assistance Foundation (SAR). Portions of this work were presented at the Annual Meeting of the AACR, 2008, San Diego, CA, USA and the Annual Meeting of the Endocrine Society, 2008, San Francisco, CA, USA.

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  1. Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA

    J T Lucas Jr, B P Salimath & S A Rosenzweig

  2. Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC, USA

    M G Slomiany

  3. Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA

    S A Rosenzweig

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Lucas, J., Salimath, B., Slomiany, M. et al. Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent. Oncogene 29, 4449–4459 (2010). https://doi.org/10.1038/onc.2010.185

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