Clinical & Experimental Metastasis

, Volume 27, Issue 7, pp 539–547 | Cite as

Cold shock domain protein A (CSDA) overexpression inhibits tumor growth and lymph node metastasis in a mouse model of squamous cell carcinoma

  • Goichi Matsumoto
  • Nobuyuki Yajima
  • Hiroyuki Saito
  • Hironori Nakagami
  • Yasushi Omi
  • Ushaku Lee
  • Yasufumi Kaneda
Research Paper


Cancer cells metastasize by entering the lymphatic system. Regional lymph-node dissemination is the first detectable step in the metastasis of oral squamous cell carcinoma (SCC) and is highly correlated to the prognosis of the disease. Cold shock domain protein A (CSDA) is a DNA-binding protein that represses angiogenesis and lymphangiogenesis by directly binding to hypoxia response element (HRE) and serum response element (SRE). In our study we used the cell line NR-S1M, a mouse SCC model with a high rate of lymph-node metastasis. Into these cells we transfected the expression-plasmid coding for full-length mouse CSDA. Of importance, we showed that overexpression of CSDA significantly inhibits the production of VEGF-A and VEGF-C in NR-S1M cells. The overexpression of CSDA in NR-S1M cells inhibited tumor growth, inhibited regional lymph-node metastasis, and reduced the density of blood vessels and lymphatic vessels in the primary tumors in vivo. Our results support the hypothesis that VEGF-A and VEGF-C are crucial regulators of angiogenesis and lymphangiogenesis in NR-S1M cells. Therefore, they are promising targets for CSDA overexpression gene therapy to inhibit tumor growth and lymph-node metastasis in SCC.


Cold shock domain protein A Angiogenesis Lymphangiogenesis VEGF-A VEGF-C Squamous cell carcinoma 



The Ministry of Education, Culture, Sports, Science and Technology of Japan supported this work with a Grant-in-Aid for the High-Tech Research Center Project.


  1. 1.
    Rennie J, Rusting R (1996) Making headway against cancer. Sci Am 275:56–59CrossRefPubMedGoogle Scholar
  2. 2.
    Fidler IJ (2001) Molecular biology of cancer: invasion and metastasis. In: DeVita VT, Hellman S Jr, Rosenburg SA (eds) Cancer Principles and Practice of Oncology. Lippincott Raven, Philadelphia, pp 135–153Google Scholar
  3. 3.
    Karpanen T, Alitalo K (2001) Lymphatic vessels as targets of tumor therapy? J Exp Med 194:37–42CrossRefGoogle Scholar
  4. 4.
    Fisher B, Bauer M, Wickerham DL et al (1983) Relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer. An NSABP update. Cancer 52:1551–1557CrossRefPubMedGoogle Scholar
  5. 5.
    Pepper MS (2001) Lymphangiogenesis and tumor metastasis: myth or reality? Clin Cancer Res 7:462–468PubMedGoogle Scholar
  6. 6.
    Wells KE, Rapaport DP, Cruse CW et al (1997) Sentinel lymph node biopsy in melanoma of the head and neck. Plast Reconstr Surg 100:591–594CrossRefPubMedGoogle Scholar
  7. 7.
    Albertini JJ, Lyman GH, Cox C et al (1996) Lymphatic mapping and sentinel node biopsy in the patient with breast cancer. J Am Med Assoc 276:1818–1822CrossRefGoogle Scholar
  8. 8.
    Albertini JJ, Cruse CW, Rapaport D et al (1996) Intraoperative radio-lympho-scintigraphy improves sentinel lymph node identification for patients with melanoma. Ann Surg 223:217–224CrossRefPubMedGoogle Scholar
  9. 9.
    Mandriota SJ, Jussila L, Jeltsch M et al (2001) Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J 20:672–682CrossRefPubMedGoogle Scholar
  10. 10.
    Skobe M, Hawighorst T, Jackson DG et al (2001) Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 7:192–198CrossRefPubMedGoogle Scholar
  11. 11.
    Stacker SA, Caesar C, Baldwin ME et al (2001) VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat Med 7:186–191CrossRefPubMedGoogle Scholar
  12. 12.
    Karpanen T, Egeblad M, Karkkainen MJ et al (2001) Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res 61:1786–1790PubMedGoogle Scholar
  13. 13.
    Shannon MF, Coles LS, Vadas MA et al (1997) Signals for activation of the GM-CSF promoter and enhancer in T cells. Crit Rev Immunol 17:301–323PubMedGoogle Scholar
  14. 14.
    Shannon MF, Coles LS, Attema J et al (2001) The role of architectural transcription factors in cytokine gene transcription. J Leukoc Biol 69:21–32PubMedGoogle Scholar
  15. 15.
    Coles LS, Diamond P, Lambrusco L et al (2002) A novel mechanism of repression of the vascular endothelial growth factor promoter, by single strand DNA binding cold shock domain (Y-box) proteins in normoxic fibroblasts. Nucleic Acids Res 30:4845–4854CrossRefPubMedGoogle Scholar
  16. 16.
    Saito Y, Nakagami H, Kurooka M et al (2008) Cold shock domain protein A represses angiogenesis and lymphangiogenesis via inhibition of serum response element. Oncogene 27:1821–1833CrossRefPubMedGoogle Scholar
  17. 17.
    Usui S, Urano M, Koike S et al (1976) Effect of PS-K, a protein polysaccharide, on pulmonary metastases of a C3H mouse squamous cell carcinoma. J Natl Cancer Inst 56:185–187PubMedGoogle Scholar
  18. 18.
    Clauss M, Gerlach M, Gerlach H et al (1990) Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med 172:1535–1545CrossRefPubMedGoogle Scholar
  19. 19.
    Krishnan J, Kirkin V, Steffen A et al (2003) Differential in vivo and in vitro expression of vascular endothelial growth factor (VEGF)-C and VEGF-D in tumors and its relationship to lymphatic metastasis in immunocompetent rats. Cancer Res 63:713–722PubMedGoogle Scholar
  20. 20.
    Gleadle JM, Ratcliffe PJ (1997) Induction of hypoxia-inducible factor-1, erythropoietin, vascular endothelial growth factor, and glucose transporter-1 by hypoxia: evidence against a regulatory role for Src kinase. Blood 89:503–509PubMedGoogle Scholar
  21. 21.
    Forsythe JA, Jiang BH, Iyer NV et al (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16:4604–4613PubMedGoogle Scholar
  22. 22.
    Hong YK, Lange-Asschenfeldt B, Velasco P et al (2004) VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins. FASEB J 18:1111–1113PubMedGoogle Scholar
  23. 23.
    Saaristo A, Veikkola T, Enholm B et al (2002) Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J 16:1041–1049CrossRefPubMedGoogle Scholar
  24. 24.
    Kimura H, Konishi K, Nukui T et al (2001) Prognostic significance of expression of thymidine phosphorylase and vascular endothelial growth factor in human gastric carcinoma. J Surg Oncol 7:31–36CrossRefGoogle Scholar
  25. 25.
    Hirakawa S, Kodama S, Kunstfeld R et al (2005) VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. J Exp Med 201:1089–1099CrossRefPubMedGoogle Scholar
  26. 26.
    Skobe M, Detmar M (2000) Structure, function, and molecular control of the skin lymphatic system. J Investig Dermatol Symp Proc 5:14–19CrossRefPubMedGoogle Scholar
  27. 27.
    Yonemura Y, Endo Y, Fujita H et al (1999) Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer. Clin Cancer Res 5:1823–1829PubMedGoogle Scholar
  28. 28.
    Akagi K, Ikeda Y, Miyazaki M et al (2000) Vascular endothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues. Br J Cancer 83:887–891CrossRefPubMedGoogle Scholar
  29. 29.
    Niki T, Iba S, Tokunou M et al (2000) Expression of vascular endothelial growth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma. Clin Cancer Res 6:2431–2439PubMedGoogle Scholar
  30. 30.
    Sugiura T, Inoue Y, Matsuki R et al (2009) VEGF-C and VEGF-D expression is correlated with lymphatic vessel density and lymph node metastasis in oral squamous cell carcinoma: implications for use as a prognostic marker. Int J Oncol 34:673–680CrossRefPubMedGoogle Scholar
  31. 31.
    Isaka N, Padera TP, Hagendoorn J et al (2004) Peritumor lymphatics induced by vascular endothelial growth factor-C exhibit abnormal function. Cancer Res 64:4400–4404CrossRefPubMedGoogle Scholar
  32. 32.
    Chilov D, Kukk E, Taira S et al (1997) Genomic organization of human and mouse genes for vascular endothelial growth factor C. J Biol Chem 272:25176–25183CrossRefPubMedGoogle Scholar
  33. 33.
    Okada K, Osaki M, Araki K et al (2005) Expression of hypoxia-inducible factor (HIF-1alpha), VEGF-C and VEGF-D in non-invasive and invasive breast ductal carcinomas. Anticancer Res 25:3003–3009PubMedGoogle Scholar
  34. 34.
    Nilsson I, Shibuya M, Wennström S (2004) Differential activation of vascular genes by hypoxia in primary endothelial cells. Exp Cell Res 299:476–485CrossRefPubMedGoogle Scholar
  35. 35.
    Joukov V, Sorsa T, Kumar V et al (1997) Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J 16:3898–3911CrossRefPubMedGoogle Scholar
  36. 36.
    Fujii T, Otsuki T, Moriya T et al (2002) Effect of hypoxia on human seminoma cells. Int J Oncol 20:955–962PubMedGoogle Scholar
  37. 37.
    Katsuta M, Miyashita M, Makino H et al (2005) Correlation of hypoxia inducible factor-1alpha with lymphatic metastasis via vascular endothelial growth factor-C in human esophageal cancer. Exp Mol Pathol 78:123–130CrossRefPubMedGoogle Scholar
  38. 38.
    Beasley NJ, Prevo R, Banerji S et al (2002) Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res 62:1315–1326PubMedGoogle Scholar
  39. 39.
    Sanna-Mari M, Marjaana L, Reidar G et al (2003) Intratumoral lymphatics are essential for the metastatic spread and prognosis in squamous sell carcinomas of the head and neck region. Cancer Res 63:1920Google Scholar
  40. 40.
    Kishimoto K, Sasaki A, Yoshihama Y et al (2003) Expression of vascular endothelial growth factor-C predicts regional lymph node metastasis in early oral squamous cell carcinoma. Oral Oncol 39:391–396CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Goichi Matsumoto
    • 1
  • Nobuyuki Yajima
    • 2
  • Hiroyuki Saito
    • 3
  • Hironori Nakagami
    • 4
  • Yasushi Omi
    • 1
  • Ushaku Lee
    • 1
  • Yasufumi Kaneda
    • 4
  1. 1.Department of Oral and Maxillofacial SurgeryKanagawa Dental CollegeYokosukaJapan
  2. 2.Department of Biochemistry and Molecular BiologyKanagawa Dental CollegeYokosukaJapan
  3. 3.Department of SurgeryAsahikawa Medical UniversityAsahikawaJapan
  4. 4.Division of Gene Therapy ScienceGraduate School of Medicine, Osaka UniversityOsakaJapan

Personalised recommendations