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. 2005 Aug 19;95(4):321–327. doi: 10.1111/j.1349-7006.2004.tb03210.x

Reduced HGF expression in subcutaneous CT26 tumor genetically modified to secrete NK4 and its possible relation with antitumor effects

Takeshi Kubota 1, Hitoshi Fujiwara 1,✉, Hisashi Amaike 1, Kazuhiro Takashima 1, Satoshi Inada 1, Kiyoto Atsuji 1, Mamoru Yoshimura 1, Kunio Matsumoto 2, Toshikazu Nakamura 2, Hisakazu Yamagishi 1
PMCID: PMC11158525  PMID: 15072590

Abstract

Tumor‐stromal interactions, which are regulated by stromal‐derived HGF and tumor‐derived HGF inducers, are essential for tumor cell acquisition of such malignant properties as invasion and metastasis. NK4, a proteolytic cleavage product of HGF, has anti‐tumor activities as both an HGF antagonist and an angiogenesis inhibitor. In this study, we examined the in vitro and in vivo behaviors of mouse colon adenocarcinoma CT26 cells modified by gene transfer to secrete NK4, and investigated the influence of NK4 on expression of HGF and HGF inducers associated with tumor‐stromal interactions. In vitro cell proliferation rates of NK4 transfectant (CT26‐NK4) and mock transfectant (CT26‐NEO) were essentially the same, and scattering and invasion were stimulated by HGF in CT26‐NEO, but not in CT26‐NK4. In syngeneic BALB/c female mice, subcutaneous tumor growth of CT26‐NK4 was potently suppressed, and the survival was prolonged significantly. Immunohistochemistry showed significantly decreased micro vessels and increased apoptotic cells in CT26‐NK4 tumor compared with control. Interestingly, HGF, strongly expressed in CT26‐NEO tumor stroma, was reduced in CT26‐NK4. In vitro, conditioned medium of CT26‐NK4 inhibited fibroblast‐derived HGF production, which was increased by that of CT26‐NEO. Moreover, although similar constitutive expression levels of PDGF and TGF‐α (both HGF inducers) were detected in CT26‐NK4 and CT26‐NEO in semiquantitative RT‐PCR analyses, the expression was up‐regulated by HGF in CT26‐NEO, but not CT26‐NK4. These results suggest that NK4 may exert antitumor activities not only by antagonizing HGF, but also by inhibiting HGF amplification via tumor‐stromal interactions. Continuous, abundant NK4 production induced at a tumor site by gene transfer should show multiple antitumor activities with potential therapeutic benefit.

Abbreviations:

HGF

hepatocyte growth factor

PDGF

platelet‐derived growth factor

TGF

transforming growth factor

RT‐PCR

reverse transcription‐polymerase chain reaction

References

  • 1. Nakamura T, Matsumoto K, Kiritoshi A, Tano Y, Nakamura T. Induction of hepatocyte growth factor in fibroblasts by tumor‐derived factors affects invasive growth of tumor cells: in vitro analysis of tumor‐stromal interactions. Cancer Res 1997; 57: 3305–13. [PubMed] [Google Scholar]
  • 2. Matsumoto K, Date K, Shimura H, Nakamura T. Acquisition of invasive phenotype in gallbladder cancer cells via mutual interaction of stromal fibro‐blasts and cancer cells mediated by hepatocyte growth factor. Jpn J Cancer Res 1996; 87: 702–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Matsumoto K, Okazaki H, Nakamura T. Up‐regulation of hepatocyte growth factor gene expression by interleukin 1 in human skin fibroblasts. Biochem Biophys Res Commun 1992; 188: 235–43. [DOI] [PubMed] [Google Scholar]
  • 4. Gohda E, Matsunaga T, Kataoka H, Takebe T, Yamamoto I. Induction of hepatocyte growth factor in human skin fibroblasts by epidermal growth factor, platelet‐derived growth factor, and fibroblast growth factor. Cytokine 1994; 6: 633–40. [DOI] [PubMed] [Google Scholar]
  • 5. Seslar SP, Nakamura T, Byers SW. Regulation of fibroblast hepatocyte growth factor/scatter factor expression by human breast carcinoma cell lines and peptide growth factors. Cancer Res 1993; 53: 1233–8. [PubMed] [Google Scholar]
  • 6. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimoniski M, Sugimata A, Tashiro K, Shimizu S. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989; 342: 440–3. [DOI] [PubMed] [Google Scholar]
  • 7. Nakamura T, Nawa K, Ichihara A. Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem Biophys Res Commun 1984; 122: 1450–9. [DOI] [PubMed] [Google Scholar]
  • 8. Hiscox SE, Hallett MB, Puntis MC, Nakamura T, Jiang WG. Expression of the HGF/SF receptor, c‐met, and its ligand in human colorectal cancers. Cancer Invest 1997; 15: 513–21. [DOI] [PubMed] [Google Scholar]
  • 9. Kaji M, Yonemura Y, Harada S, Liu X, Terada I, Yamamoto H. Participation of c‐met in the progression of human gastric cancers: anti‐c‐met oligonucleotides inhibit proliferation or invasiveness of gastric cancer cells. Cancer Gene Ther 1996; 3: 393–404. [PubMed] [Google Scholar]
  • 10. Iwazawa T, Shiozaki H, Doki Y, Inoue M, Tamura S, Matsui S, Monden T, Matsumoto K, Nakamura T, Monden M. Primary human fibroblasts induce diverse tumor invasiveness: involvement of hepatocyte growth factor as an important paracrine factor. Jpn J Cancer Res 1996; 87:1134–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Beviglia L, Matsumoto K, Lin CS, Ziober BL, Kramer RH. Expression of the c‐Met/HGF receptor in human breast carcinoma: correlation with tumor progression. Int J Cancer 1997; 74: 301–9. [DOI] [PubMed] [Google Scholar]
  • 12. Boix L, Rosa JI, Ventura F, Castells A, Bruix J, Rodes J, Bartrons R. c‐met messenger RNA overexpression in human hepatocellular carcinoma. Hepatology 1994; 19: 88–91. [PubMed] [Google Scholar]
  • 13. Hu Z, Evarts RP, Fujio K, Omori N, Omori M, Marsden ER, Thorgeirsson SS. Expression of transforming growth factor alpha/epidermal growth factor receptor, hepatocyte growth factor/c‐met and acidic fibroblast growth factor/fibroblast growth factor receptors during hepatocarcinogenesis. Carcinogenesis 1996; 17: 931–8. [DOI] [PubMed] [Google Scholar]
  • 14. Harvey P, Warn A, Newman P, Perry LJ, Ball RY, Warn RM. Immunoreactivity for hepatocyte growth factor/scatter factor and its receptor, met, in human lung carcinomas and malignant mesotheliomas. J Pathol 1996; 180: 389–94. [DOI] [PubMed] [Google Scholar]
  • 15. Takanami I, Tanana F, Hashizume T, Kikuchi K, Yamamoto Y, Yamamoto T, Kodaira S. Hepatocyte growth factor and c‐Met/hepatocyte growth factor receptor in pulmonary adenocarcinomas: an evaluation of their expression as prognostic markers. Oncology 1996; 53: 392–7. [DOI] [PubMed] [Google Scholar]
  • 16. Bussolino F, DiRenzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, Gaudino G, Tamagnone L, Coffer A, Comoglio PM. Hepatocyte growth fac‐tor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 1992; 119: 629–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Grant DS, Kleinman HK, Goldberg ID, Bhargava MM, Nickoloff BJ, Kinsella JL, Polverini P, Rosen EM. Scatter factor induces blood‐vessel formation in vivo. Proc Natl Acad Sci USA 1993; 90: 1937–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333: 1757–63. [DOI] [PubMed] [Google Scholar]
  • 19. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86: 353–64. [DOI] [PubMed] [Google Scholar]
  • 20. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis‐ correlation in invasive breast carcinoma. N Engl J Med 1991; 324: 1–8. [DOI] [PubMed] [Google Scholar]
  • 21. Macchiarini P, Fontanini G, Hardin MJ, Squartini F, Angeletti CA. Relation of neovascularisation to metastasis of non‐small‐cell lung cancer. Lancet 1992; 340: 145–6. [DOI] [PubMed] [Google Scholar]
  • 22. Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 1993; 143: 401–9. [PMC free article] [PubMed] [Google Scholar]
  • 23. Smith‐MaCune KK, Weidner N. Demonstration and characterization of the angiogenic properties of cervical dysplasia. Cancer Res 1994; 54: 800–4. [PubMed] [Google Scholar]
  • 24. Kitadai Y, Haruma K, Tokutomi T, Tanaka S, Sumii K, Carvalho M, Kuwabara M, Yoshida K, Hirai T, Kajiyama G, Tahara E. Significance of vessel count and vascular endothelial growth factor in human esophageal carcinomas. Clin Cancer Res 1998; 4: 2195–200. [PubMed] [Google Scholar]
  • 25. Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 1995; 55: 3964–8. [PubMed] [Google Scholar]
  • 26. Date K, Matsumoto K, Shimura H, Tanaka M, Nakamura T. HGF/NK4 is a specific antagonist for pleiotrophic actions of hepatocyte growth factor. FEBS Lett 1997; 420: 1–6. [DOI] [PubMed] [Google Scholar]
  • 27. Parr C, Hiscox S, Nakamura T, Matsumoto K, Giang WG. NK4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells. Int J Cancer 2000; 85: 563–70. [PubMed] [Google Scholar]
  • 28. Maehara N, Matsumoto K, Kuba K, Mizumoto K, Tanaka M, Nakamura T. NK4, a four‐kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells. Br J Cancer 2001; 84: 864–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Date K, Matsumoto K, Kuba K, Shimura H, Tanaka M, Nakamura T. Inhibition of tumor growth and invasion by a four‐kringle antagonist (HGF/NK4) for hepatocyte growth factor. Oncogene 1998; 17: 3045–54. [DOI] [PubMed] [Google Scholar]
  • 30. Kuba K, Matsumoto K, Date K, Shimura H, Tanaka M, Nakamura T. HGF/NK4, a four‐kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice. Cancer Res 2000; 60: 6737–43. [PubMed] [Google Scholar]
  • 31. Davies G, Mason MD, Martin TA, Parr C, Watkins G, Lane J, Matsumoto K, Nakamura T, Jiang WG. The HGF/SF antagonist NK4 reverses fibroblast‐ and HGF‐induced prostate tumor growth and angiogenesis in vivo. Int J Cancer 2003; 106: 348–54. [DOI] [PubMed] [Google Scholar]
  • 32. Seki T, Ihara I, Sugimura A, Shimonishi M, Nishizawa T, Asami O, Hagiya M, Nakamura T, Shimizu S. Isolation and expression of cDNA for different forms of hepatocyte growth factor from human leukocytes. Biochem Biophys Res Commun 1990; 172: 321–7. [DOI] [PubMed] [Google Scholar]
  • 33. Corbett TH, Griswold DP, Roberts BJ, Peckham JC, Schabel FJ Jr. Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with note on carcinogen structure. Cancer Res 1975; 35: 2434–9. [PubMed] [Google Scholar]
  • 34. Gherardi E, Gray J, Stoker M, Perryman M, Furlong R. Purification of scatter factor, a fibroblast‐derived basic protein that modulates epithelial interaction and movement. Proc Natl Acad Sci USA 1989; 86: 5844–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Weidner KM, Behrens J, Vanderkerckhove J, Birchmeier W. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J Cell Biol 1990; 111: 2097–108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Tomioka D, Maehara N, Kuba K, Mizumoto K, Tanaka M, Matsumoto K, Nakamura T. Inhibition of growth, invasion, and metastasis if human pancreatic carcinoma cells by NK4 in an orthotopic mouse model. Cancer Res 2001; 61: 7518–24. [PubMed] [Google Scholar]
  • 37. Saimura M, Nagai E, Mizumoto K, Maehara N, Minamishima YA, Katano M, Matsumoto K, Nakamura T, Tanaka M. Tumor suppression through angiogenesis inhibition by SUIT‐2 pancreatic cancer cells genetically engineered to secrete NK4. Clin Cancer Res 2002; 10: 3243–9. [PubMed] [Google Scholar]
  • 38. Giordano S, Zhen Z, Medico E, Gaudino G, Galimi F, Comoglio PM. Transfer of motogenic and invasive response to scatter factor/hepatocyte growth factor by transfection of human MET protooncogene. Proc Natl Acad Sci USA 1993; 90: 649–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Lamszus K, Jin L, Fuchs A, Shi E, Chowdhury S, Yao Y, Polverini PJ, Laterra J, Goldberg ID, Rosen EM. Scatter factor stimulates tumor growth and tumor angiogenesis in human breast cancers in the mammary fat pads of nude mice. Lab Invest 1997; 76: 339–53. [PubMed] [Google Scholar]
  • 40. Saimura M, Nagai E, Mizumoto K, Maehara N, Okino H, Katano M, Matsumoto K, Nakamura T, Narumi K, Nukiwa T, Tanaka M. Intraperitoneal injection of adenovirus‐mediated NK4 gene suppresses peritoneal dissemina‐tion of pancreatic cancer cell line AsPC‐1 in nude mice. Cancer Gene Ther 2002; 9: 799–806. [DOI] [PubMed] [Google Scholar]
  • 41. Maemondo M, Narumi K, Saijo Y, Usui K, Tahara M, Tazawa R, Hagiwara K, Matsumoto K, Nakamura T, Nukiwa T. Targeting angiogenesis and HGF function using an adenoviral vector expressing the HGF antagonist NK4 for cancer therapy. Mol Ther 2002; 5: 177–85. [DOI] [PubMed] [Google Scholar]

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