Loss of E-cadherin-dependent cell-cell adhesion due to mutation of the beta-catenin gene in a human cancer cell line, HSC-39 (original) (raw)

Abstract

Detachment of cell-cell adhesion is indispensable for the first step of invasion and metastasis of cancer. This mechanism is frequently associated with the impairment of either E-cadherin expression or function. However, mechanisms of such abnormalities have not been fully elucidated. In this study, we demonstrated that the function of E-cadherin was completely abolished in the human gastric cancer cell line HSC-39, despite the high expression of E-cadherin, because of mutations in one of the E-cadherin-associated cytoplasmic proteins, beta-catenin. Although immunofluorescence staining of HSC-39 cells by using an anti-E-cadherin antibody (HECD-1) revealed the strong and uniform expression of E-cadherin on the cell surface, cell compaction and cell aggregation were not observed in this cell. Western blotting (immunoblotting) using HECD-1 exhibited a 120-kDa band which is equivalent to normal E-cadherin. Northern (RNA) blotting demonstrated a 4.7-kb band, the same as mature E-cadherin mRNA. Immunoprecipitation of metabolically labeled proteins with HECD-1 revealed three bands corresponding to E-cadherin, alpha-catenin, and gamma-catenin and a 79-kDa band which was apparently smaller than that of normal beta-catenin, indicating truncated beta-catenin. The 79-kDa band was immunologically identified as beta-catenin by using immunoblotting with anti-beta-catenin antibodies. Examination of beta-catenin mRNA by the reverse transcriptase-PCR method revealed a transcript which was shorter than that of normal beta-catenin. The sequencing of PCR product for beta-catenin confirmed deletion in 321 bases from nucleotides +82 to +402. Southern blotting of beta-catenin DNA disclosed mutation at the genomic level. Expression vectors of Beta-catenin were introduced into HSC-39 cells by transfection. In the obtained transfectants, E-cadherin-dependent cell-cell adhesiveness was recovered, as revealed by cell compaction, cell aggregation, and immunoflourescence staining. From these results, it was concluded that in HSC-39 cells, impaired cell-cell adhesion is due to mutations in beta-catenin which results in the dysfunction of E-cadherin.

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Selected References

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  1. Behrens J., Vakaet L., Friis R., Winterhager E., Van Roy F., Mareel M. M., Birchmeier W. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J Cell Biol. 1993 Feb;120(3):757–766. doi: 10.1083/jcb.120.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bringuier P. P., Umbas R., Schaafsma H. E., Karthaus H. F., Debruyne F. M., Schalken J. A. Decreased E-cadherin immunoreactivity correlates with poor survival in patients with bladder tumors. Cancer Res. 1993 Jul 15;53(14):3241–3245. [PubMed] [Google Scholar]
  3. Bussemakers M. J., van Bokhoven A., Mees S. G., Kemler R., Schalken J. A. Molecular cloning and characterization of the human E-cadherin cDNA. Mol Biol Rep. 1993 Feb;17(2):123–128. doi: 10.1007/BF00996219. [DOI] [PubMed] [Google Scholar]
  4. Doki Y., Shiozaki H., Tahara H., Inoue M., Oka H., Iihara K., Kadowaki T., Takeichi M., Mori T. Correlation between E-cadherin expression and invasiveness in vitro in a human esophageal cancer cell line. Cancer Res. 1993 Jul 15;53(14):3421–3426. [PubMed] [Google Scholar]
  5. Dorudi S., Sheffield J. P., Poulsom R., Northover J. M., Hart I. R. E-cadherin expression in colorectal cancer. An immunocytochemical and in situ hybridization study. Am J Pathol. 1993 Apr;142(4):981–986. [PMC free article] [PubMed] [Google Scholar]
  6. Duband J. L., Dufour S., Hatta K., Takeichi M., Edelman G. M., Thiery J. P. Adhesion molecules during somitogenesis in the avian embryo. J Cell Biol. 1987 May;104(5):1361–1374. doi: 10.1083/jcb.104.5.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hamaguchi M., Matsuyoshi N., Ohnishi Y., Gotoh B., Takeichi M., Nagai Y. p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin cell adhesion system. EMBO J. 1993 Jan;12(1):307–314. doi: 10.1002/j.1460-2075.1993.tb05658.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hashimoto M., Niwa O., Nitta Y., Takeichi M., Yokoro K. Unstable expression of E-cadherin adhesion molecules in metastatic ovarian tumor cells. Jpn J Cancer Res. 1989 May;80(5):459–463. doi: 10.1111/j.1349-7006.1989.tb02336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hirano S., Kimoto N., Shimoyama Y., Hirohashi S., Takeichi M. Identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization. Cell. 1992 Jul 24;70(2):293–301. doi: 10.1016/0092-8674(92)90103-j. [DOI] [PubMed] [Google Scholar]
  10. Kadowaki T., Shiozaki H., Inoue M., Tamura S., Oka H., Doki Y., Iihara K., Matsui S., Iwazawa T., Nagafuchi A. E-cadherin and alpha-catenin expression in human esophageal cancer. Cancer Res. 1994 Jan 1;54(1):291–296. [PubMed] [Google Scholar]
  11. Liotta L. A. Tumor invasion and metastases: role of the basement membrane. Warner-Lambert Parke-Davis Award lecture. Am J Pathol. 1984 Dec;117(3):339–348. [PMC free article] [PubMed] [Google Scholar]
  12. Matsuura K., Kawanishi J., Fujii S., Imamura M., Hirano S., Takeichi M., Niitsu Y. Altered expression of E-cadherin in gastric cancer tissues and carcinomatous fluid. Br J Cancer. 1992 Dec;66(6):1122–1130. doi: 10.1038/bjc.1992.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Matsuyoshi N., Hamaguchi M., Taniguchi S., Nagafuchi A., Tsukita S., Takeichi M. Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts. J Cell Biol. 1992 Aug;118(3):703–714. doi: 10.1083/jcb.118.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mayer B., Johnson J. P., Leitl F., Jauch K. W., Heiss M. M., Schildberg F. W., Birchmeier W., Funke I. E-cadherin expression in primary and metastatic gastric cancer: down-regulation correlates with cellular dedifferentiation and glandular disintegration. Cancer Res. 1993 Apr 1;53(7):1690–1695. [PubMed] [Google Scholar]
  15. McCrea P. D., Gumbiner B. M. Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin). Characterization and extractability of the protein complex from the cell cytostructure. J Biol Chem. 1991 Mar 5;266(7):4514–4520. [PubMed] [Google Scholar]
  16. Morton R. A., Ewing C. M., Nagafuchi A., Tsukita S., Isaacs W. B. Reduction of E-cadherin levels and deletion of the alpha-catenin gene in human prostate cancer cells. Cancer Res. 1993 Aug 1;53(15):3585–3590. [PubMed] [Google Scholar]
  17. Nagafuchi A., Takeichi M. Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO J. 1988 Dec 1;7(12):3679–3684. doi: 10.1002/j.1460-2075.1988.tb03249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nagafuchi A., Takeichi M. Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin. Cell Regul. 1989 Nov;1(1):37–44. doi: 10.1091/mbc.1.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nagafuchi A., Takeichi M., Tsukita S. The 102 kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulation of expression. Cell. 1991 May 31;65(5):849–857. doi: 10.1016/0092-8674(91)90392-c. [DOI] [PubMed] [Google Scholar]
  20. Oka H., Shiozaki H., Kobayashi K., Inoue M., Tahara H., Kobayashi T., Takatsuka Y., Matsuyoshi N., Hirano S., Takeichi M. Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship to metastasis. Cancer Res. 1993 Apr 1;53(7):1696–1701. [PubMed] [Google Scholar]
  21. Ozawa M., Baribault H., Kemler R. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 1989 Jun;8(6):1711–1717. doi: 10.1002/j.1460-2075.1989.tb03563.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ozawa M., Kemler R. Molecular organization of the uvomorulin-catenin complex. J Cell Biol. 1992 Feb;116(4):989–996. doi: 10.1083/jcb.116.4.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ozawa M., Ringwald M., Kemler R. Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4246–4250. doi: 10.1073/pnas.87.11.4246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rubinfeld B., Souza B., Albert I., Müller O., Chamberlain S. H., Masiarz F. R., Munemitsu S., Polakis P. Association of the APC gene product with beta-catenin. Science. 1993 Dec 10;262(5140):1731–1734. doi: 10.1126/science.8259518. [DOI] [PubMed] [Google Scholar]
  25. Sasaki K., Zak O., Aisen P. Antisense suppression of transferrin receptor gene expression in a human hepatoma cell (HuH-7) line. Am J Hematol. 1993 Jan;42(1):74–80. doi: 10.1002/ajh.2830420115. [DOI] [PubMed] [Google Scholar]
  26. Shimoyama Y., Hirohashi S. Expression of E- and P-cadherin in gastric carcinomas. Cancer Res. 1991 Apr 15;51(8):2185–2192. [PubMed] [Google Scholar]
  27. Shimoyama Y., Hirohashi S., Hirano S., Noguchi M., Shimosato Y., Takeichi M., Abe O. Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas. Cancer Res. 1989 Apr 15;49(8):2128–2133. [PubMed] [Google Scholar]
  28. Shimoyama Y., Nagafuchi A., Fujita S., Gotoh M., Takeichi M., Tsukita S., Hirohashi S. Cadherin dysfunction in a human cancer cell line: possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesiveness. Cancer Res. 1992 Oct 15;52(20):5770–5774. [PubMed] [Google Scholar]
  29. Su L. K., Vogelstein B., Kinzler K. W. Association of the APC tumor suppressor protein with catenins. Science. 1993 Dec 10;262(5140):1734–1737. doi: 10.1126/science.8259519. [DOI] [PubMed] [Google Scholar]
  30. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991 Mar 22;251(5000):1451–1455. doi: 10.1126/science.2006419. [DOI] [PubMed] [Google Scholar]
  31. Takeichi M. Functional correlation between cell adhesive properties and some cell surface proteins. J Cell Biol. 1977 Nov;75(2 Pt 1):464–474. doi: 10.1083/jcb.75.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wheelock M. J., Knudsen K. A. Cadherins and associated proteins. In Vivo. 1991 Sep-Oct;5(5):505–513. [PubMed] [Google Scholar]
  33. Yanagihara K., Seyama T., Tsumuraya M., Kamada N., Yokoro K. Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene. Cancer Res. 1991 Jan 1;51(1):381–386. [PubMed] [Google Scholar]