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Cellular fibronectin 1 promotes VEGF-C expression, lymphangiogenesis and lymph node metastasis associated with human oral squamous cell carcinoma

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Abstract

Lymph node metastasis (LNM) is associated with poor survival in patients with oral squamous cell carcinoma (OSCC). Vascular endothelial growth factor-C (VEGF-C) is thought to be responsible for increased lymphangiogenesis and LNM. Understanding of the mechanism by which VEGF-C expression is regulated in OSCC is thus important to design logic therapeutic interventions. We showed that inoculation of the SAS human OSCC cells expressing the venus GFP (V-SAS cells) into the tongue in nude mice developed LNM. V-SAS cells in LNM were isolated by FACS and re-inoculated into the tongue. This procedure was repeated eight times, establishing V-SAS-LM8 cells. Differential metastasis PCR array between the parental V-SAS and V-SAS-LM8 was performed to identify a molecule responsible for lymphangiogenesis and LNM. Fibronectin 1 (FN1) expression was elevated in V-SAS-LM8 cells compared to V-SAS-cells. V-SAS-LM8 tongue tumor showed increased expression of FN1 and VEGF-C, and promoted lymphangiogenesis and LNM compared with V-SAS tumor. Further, phosphorylation of focal adhesion kinase (FAK), a main downstream signaling molecule of FN1, was up-regulated, and epithelial-mesenchymal transition (EMT) was promoted in V-SAS-LM8 cells. Silencing of FN1 by shRNA in V-SAS-LM8 cells decreased FAK phosphorylation, VEGF-C expression and inhibited lymphangiogenesis and LNM. EMT was also reversed. The FAK phosphorylation inhibitor PF573228 also decreased VEGF-C expression and reversed EMT in V-SAS-LM8 cells. Finally, we detected intense FN1 expression in some clinical specimens obtained from OSCC patients with LNM. These results demonstrate that elevated expression of cellular FN1 and following activation of FAK lead to increased VEGF-C expression, lymphangiogenesis and LNM and promoted EMT in SAS human OSCC cells and suggest that FN1-phosphorylated FAK signaling cascade is a potential therapeutic target in the treatment of LNM in OSCC.

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Abbreviations

LNM:

Lymph node metastasis

LYVE-1:

Lymphatic vessel endothelium-specific marker 1

OSCC:

Oral squamous cell carcinoma

VEGF:

Vascular endothelial growth factor

FN1:

Fibronectin 1

FAK:

Focal adhesion kinase

EMT:

Epithelial-mesenchymal transition

ECM:

Extracellular matrices

References

  1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300

    Article  PubMed  Google Scholar 

  2. Matsuda A, Matsuda T, Shibata A et al (2014) Cancer incidence and incidence rates in Japan in 2008: a study of 25 population-based cancer registries for the Monitoring of Cancer Incidence in Japan (MCIJ) project. Jpn J Clin Oncol 44:388–396

    Article  PubMed  Google Scholar 

  3. Hunter KD, Parkinson EK, Harrison PR (2005) Profiling early head and neck cancer. Nat Rev Cancer 5:127–135

    Article  CAS  PubMed  Google Scholar 

  4. Forastiere A, Koch W, Trotti A, Sidransky D (2001) Head and neck cancer. N Engl J Med 345:1890–1900

    Article  CAS  PubMed  Google Scholar 

  5. Gil Z, Carlson DL, Boyle JO et al (2009) Lymph node density is a significant predictor of outcome in patients with oral cancer. Cancer 115:5700–5710

    Article  PubMed  Google Scholar 

  6. Mamelle G, Pampurik J, Luboinski B et al (1994) Lymph node prognostic factors in head and neck squamous cell carcinomas. Am J Surg 168:494–498

    Article  CAS  PubMed  Google Scholar 

  7. Joukov V, Pajusola K, Kaipainen A et al (1996) A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J 15:1751

    CAS  PubMed Central  PubMed  Google Scholar 

  8. Mandriota SJ, Jussila L, Jeltsch M et al (2001) Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J 20:672–682

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Stacker SA, Caesar C, Baldwin ME et al (2001) VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med 7:186–191

    Article  CAS  PubMed  Google Scholar 

  10. Skobe M, Hawighorst T, Jackson DG et al (2001) Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 7:192–198

    Article  CAS  PubMed  Google Scholar 

  11. Sleeman JP, Thiele W (2009) Tumor metastasis and the lymphatic vasculature. Int J Cancer 125:2747–2756

    Article  CAS  PubMed  Google Scholar 

  12. Makinen T, Veikkola T, Mustjoki S et al (2001) Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J 20:4762–4773

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Veikkola T, Jussila L, Makinen T et al (2001) Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J 20:1223–1231

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Roberts N, Kloos B, Cassella M et al (2006) Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2. Cancer Res 66:2650–2657

    Article  CAS  PubMed  Google Scholar 

  15. Burton JB, Priceman SJ, Sung JL et al (2008) Suppression of prostate cancer nodal and systemic metastasis by blockade of the lymphangiogenic axis. Cancer Res 68:7828–7837

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Alitalo K, Carmeliet P (2002) Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell 1:219–227

    Article  CAS  PubMed  Google Scholar 

  17. Dadras SS, Paul T, Bertoncini J et al (2003) Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival. Am J Pathol 162:1951–1960

    Article  PubMed Central  PubMed  Google Scholar 

  18. Karpanen T, Alitalo K (2001) Lymphatic vessels as targets of tumor therapy? J Exp Med 194:F37–F42

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Morita Y, Morita N, Hata K et al (2014) Cyclooxygenase-2 expression is associated with vascular endothelial growth factor-c and lymph node metastasis in human oral tongue cancer. Oral Surg Oral Med Oral Pathol Oral Radiol 117:502–510

    Article  PubMed  Google Scholar 

  20. Yanase M, Kato K, Yoshizawa K et al (2014) Prognostic value of vascular endothelial growth factors A and C in oral squamous cell carcinoma. J Oral Pathol Med 43:514–520

    Article  CAS  PubMed  Google Scholar 

  21. Morita Y, Hata K, Nakanishi M et al (2012) Cyclooxygenase-2 promotes tumor lymphangiogenesis and lymph node metastasis in oral squamous cell carcinoma. Int J Oncol 41:885–892

    CAS  PubMed  Google Scholar 

  22. Takahashi K, Kanazawa H, Akiyama Y et al (1989) Establishment and characterization of a cell line (SAS) from poorly differentiated human squamous cell carcinoma of the tongue. Jpn Stomatol Soc 38:20–28

    Google Scholar 

  23. Harada M, Miyata K, Wada T et al (1993) Establishment and characterization of a cell line(Sa3) from squamous cell carcinoma of the human gingiva. Japanese J Oral Maxillofac Surg 39:965–971

    Article  Google Scholar 

  24. Horikoshi M, Kimura Y, Nagura H et al (1974) A new human cell line derived from human carcinoma of the gingiva. I. Its establishment and morphological studies. Nihon Koku Geka Gakkai Zasshi 20:100–106

    CAS  PubMed  Google Scholar 

  25. Cailleau R, Young R, Olivé M, Reeves WJ (1974) Breast tumor cell lines from pleural effusions. J Natl Cancer Inst 53:661–674

    CAS  PubMed  Google Scholar 

  26. Yoneda T, Williams PJ, Hiraga T et al (2001) A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res 16:1486–1495

    Article  CAS  PubMed  Google Scholar 

  27. Iwata C, Kano MR, Komuro A et al (2007) Inhibition of cyclooxygenase-2 suppresses lymph node metastasis via reduction of lymphangiogenesis. Cancer Res 67:10181–10189

    Article  CAS  PubMed  Google Scholar 

  28. Hata K, Nishimura R, Ueda M et al (2005) A CCAAT/enhancer binding protein beta isoform, liver-enriched inhibitory protein, regulates commitment of osteoblasts and adipocytes. Mol Cell Biol 25:1971–1979

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Qi W, Chen X, Gilbert RE et al (2007) High glucose-induced thioredoxin-interacting protein in renal proximal tubule cells is independent of transforming growth factor-beta1. Am J Pathol 171:744–754

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Cabrita MA, Jones LM, Quizi JL et al (2011) Focal adhesion kinase inhibitors are potent anti-angiogenic agents. Mol Oncol 5:517–526

    Article  CAS  PubMed  Google Scholar 

  31. Zhao J, Guan J-L (2009) Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Rev 28:35–49

    Article  PubMed  Google Scholar 

  32. Takeshita A, Iwai S, Morita Y et al (2014) Wnt5b promotes the cell motility essential for metastasis of oral squamous cell carcinoma through active Cdc42 and RhoA. Int J Oncol 44:59–68

    CAS  PubMed  Google Scholar 

  33. Berrier AL, Yamada KM (2007) Cell-matrix adhesion. J Cell Physiol 213:565–573

    Article  CAS  PubMed  Google Scholar 

  34. Hu M, Carles-Kinch KL, Zelinski DP, Kinch MS (2004) EphA2 induction of fibronectin creates a permissive microenvironment for malignant cells. Mol Cancer Res 2:533–540

    CAS  PubMed  Google Scholar 

  35. Zheng Y, Ritzenthaler JD, Roman J, Han S (2007) Nicotine stimulates human lung cancer cell growth by inducing fibronectin expression. Am J Respir Cell Mol Biol 37:681–690

    Article  CAS  PubMed  Google Scholar 

  36. Ritzenthaler JD, Han S, Roman J (2008) Stimulation of lung carcinoma cell growth by fibronectin-integrin signalling. Mol BioSyst 4:1160–1169

    Article  CAS  PubMed  Google Scholar 

  37. Pankov R, Yamada KM (2002) Fibronectin at a glance. J Cell Sci 115:3861–3863

    Article  CAS  PubMed  Google Scholar 

  38. Mitra AK, Sawada K, Tiwari P et al (2011) Ligand-independent activation of c-Met by fibronectin and α(5)β(1)-integrin regulates ovarian cancer invasion and metastasis. Oncogene 30:1566–1576

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Scanlon CS, Van Tubergen EA, Inglehart RC, D’Silva NJ (2013) Biomarkers of epithelial-mesenchymal transition in squamous cell carcinoma. J Dent Res 92:114–121

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Tsai JH, Yang J (2013) Epithelial-mesenchymal plasticity in carcinoma metastasis. Genes Dev 27:2192–2206

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  42. Gupta GP, Massagué J (2006) Cancer metastasis: building a framework. Cell 127:679–695

    Article  CAS  PubMed  Google Scholar 

  43. Yoo JS, Kim HB, Won N et al (2011) Evidence for an additional metastatic route: in vivo imaging of cancer cells in the primo-vascular system around tumors and organs. Mol Imaging Biol 13:471–480

    Article  PubMed  Google Scholar 

  44. Margadant C, van den Bout I, van Boxtel AL et al (2012) Epigenetic regulation of galectin-3 expression by β1 integrins promotes cell adhesion and migration. J Biol Chem 287:44684–44693

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Park J, Schwarzbauer JE (2014) Mammary epithelial cell interactions with fibronectin stimulate epithelial-mesenchymal transition. Oncogene 33:1649–1657

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Yang J, Mani SA, Donaher JL et al (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117:927–939

    Article  CAS  PubMed  Google Scholar 

  47. Van Obberghen-Schilling E, Tucker RP, Saupe F et al (2011) Fibronectin and tenascin-C: accomplices in vascular morphogenesis during development and tumor growth. Int J Dev Biol 55:511–525

    Article  PubMed  Google Scholar 

  48. Jerhammar F, Ceder R, Garvin S et al (2010) Fibronectin 1 is a potential biomarker for radioresistance in head and neck squamous cell carcinoma. Cancer Biol Ther 10:1244–1251

    Article  CAS  PubMed  Google Scholar 

  49. Avraamides CJ, Garmy-Susini B, Varner JA (2008) Integrins in angiogenesis and lymphangiogenesis. Nat Rev Cancer 8:604–617

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Garmy-Susini B, Avraamides CJ, Schmid MC et al (2010) Integrin alpha4beta1 signaling is required for lymphangiogenesis and tumor metastasis. Cancer Res 70:3042–3051

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Steffens S, Schrader AJ, Vetter G et al (2012) Fibronectin 1 protein expression in clear cell renal cell carcinoma. Oncol Lett 3:787–790

    PubMed Central  PubMed  Google Scholar 

  52. Takeyama H, Kyoda S, Okamoto T et al (2011) The expression of sialic fibronectin correlates with lymph node metastasis of thyroid malignant neoplasmas. Anticancer Res 31:1395–1398

    PubMed  Google Scholar 

  53. Sudo T, Iwaya T, Nishida N et al (2013) Expression of mesenchymal markers vimentin and fibronectin: the clinical significance in esophageal squamous cell carcinoma. Ann Surg Oncol 20(Suppl 3):S324–S335

    Article  PubMed  Google Scholar 

  54. Kamarajan P, Garcia-Pardo A, D’Silva NJ, Kapila YL (2010) The CS1 segment of fibronectin is involved in human OSCC pathogenesis by mediating OSCC cell spreading, migration, and invasion. BMC Cancer 10:330

    Article  PubMed Central  PubMed  Google Scholar 

  55. Lyons AJ, Bateman AC, Spedding A et al (2001) Oncofetal fibronectin and oral squamous cell carcinoma. Br J Oral Maxillofac Surg 39:471–477

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by Grants-in -Aid, No. 23659870 [TY], 26670806 [TY] and 26293394 [TY] by the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Authors and Affiliations

  1. Departments of Molecular and Cellular Biochemistry, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

    Yoshihiro Morita, Kenji Hata, Masako Nakanishi, Riko Nishimura & Toshiyuki Yoneda

  2. Oral and Maxillofacial Surgery II, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

    Yoshihiro Morita & Yoshiaki Yura

  3. Department of Oral and Maxillofacial Surgery, Seichokai Hannan Municipal Hospital, Hannan, Osaka, Japan

    Yoshihiro Morita & Nobuo Morita

  4. Department of Oral and Maxillofacial Surgery, Kinan Hospital, Tanabe, Wakayama, Japan

    Tetsuji Omata & Nobuo Morita

  5. Department of Oral-Maxillo-facial Surgery, NS Medical & Healthcare Service General Incorporation Foundation, Wakayama, Japan

    Nobuo Morita

  6. Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, Walther Hall, R3-C321D, 980 W Walnut St, Indianapolis, IN, 46202, USA

    Toshiyuki Yoneda

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  1. Yoshihiro Morita
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Correspondence to Toshiyuki Yoneda.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

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Morita, Y., Hata, K., Nakanishi, M. et al. Cellular fibronectin 1 promotes VEGF-C expression, lymphangiogenesis and lymph node metastasis associated with human oral squamous cell carcinoma. Clin Exp Metastasis 32, 739–753 (2015). https://doi.org/10.1007/s10585-015-9741-2

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  • DOI: https://doi.org/10.1007/s10585-015-9741-2

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