Advertisement

Journal of Genetics

, Volume 96, Issue 1, pp 109–118 | Cite as

Cooverexpression of EpCAM and c-myc genes in malignant breast tumours

  • SAMIRA SADEGHI
  • ZOHREH HOJATIEmail author
  • HOSSEIN TABATABAEIAN
RESEARCH ARTICLE

Abstract

The overexpression of epithelial cell adhesion molecule (EpCAM), a proto-oncogene, affects progression, treatment, and diagnosis of many adenocarcinomas. C-myc has been shown to be a downstream target of EpCAM and is also one of the most important proto-oncogenes routinely overexpressed in breast cancer. However, cooverexpression of EpCAM and c-myc genes has not been investigated in breast cancer tissues, particularly in Iranian population. The aim of this study was to assess the expression of EpCAM and c-myc genes in malignant breast cancer tissues using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) followed by analyses of the association between the outcomes. In this study, 122 fresh tissues, including 104 malignant and 18 benign samples, were disrupted by mortar and pestle, and then the RNA was isolated from the samples and converted to cDNA. The relative expression levels of EpCAM and c-myc genes were measured by 2 −ΔΔCt method using RT-qPCR. EpCAM protein level was also assessed in 66 cases using Western blot technique. Using RT-qPCR method, our results showed that EpCAM was overexpressed in 48% of malignant and 11.1% of benign samples. Evaluating EpCAM protein overexpression in a portion of samples depicted the fully concordance rate between Western blot and RT-qPCR techniques. C-myc expression was first evaluated by RT-qPCR method, showing the overexpression rate of 39% and 28% in malignant and benign samples, respectively. These data were also quite concordant with the clinically available immunohistochemistry reports of the same samples studied in this study. Importantly, overexpression of EpCAM and c-myc was significantly associated and showed an agreement of 57.3%. This study demonstrated the cooverexpression of EpCAM and c-myc in breast tumours collected from breast cancer patients of the Iranian population. EpCAM and c-myc positive cases were significantly associated with reduced and enhanced risk of ER/PR positivity respectively. However, both were associated with grade III of breast cancer.

Keywords

breast cancer c-myc gene epithelial cell adhesion molecule real-time reverse transcriptase polymerase chain reaction Western blot 

Notes

Acknowledgements

This study was performed at the University of Isfahan and financially supported by the Graduate Office of the University of Isfahan. We also sincerely appreciate Dr Maryam Tabatabaeian and Dr Mehri Faghighy and Mr Nihar Masurkar for their comprehensive assistance in collecting the samples, follow up information and editions.

References

  1. Ambrogi F., Fornili M., Alberti S., Querzoli P., Piantelli M., Pedriali M. et al. 2012 EpCAM expression is an indicator of increased incidence of relapse in p53-positive breast cancer. Cancer Clin. Oncol. 2, p40.Google Scholar
  2. Baccelli I., Schneeweiss A., Riethdorf S., Stenzinger A., Schillert A., Vogel V. et al. 2013 Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat. Biotechnol. 31, 539–544.CrossRefPubMedGoogle Scholar
  3. Baeuerle P. and Gires O. 2007 EpCAM (CD326) finding its role in cancer. Br. J. Cancer 96, 417–423.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Balzar M., Winter M., De Boer C. and Litvinov S. 1999 The biology of the 17–1A antigen (Ep-CAM). J. Mol. Med. 77, 699–712.CrossRefPubMedGoogle Scholar
  5. Bièche I., Laurendeau I., Tozlu S., Olivi M., Vidaud D., Lidereau R. et al. 1999 Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay. Cancer Res. 59, 2759–2765.PubMedGoogle Scholar
  6. Biroccio A., Benassi B., Amodei S., Gabellini C., Del Bufalo D. and Zupi G. 2001 c-Myc down-regulation increases susceptibility to cisplatin through reactive oxygen species-mediated apoptosis in M14 human melanoma cells. Mol. Pharmacol. 60, 174–182.PubMedGoogle Scholar
  7. Chrzan P., Skokowski J., Karmolinski A. and Pawelczyk T. 2001 Amplification of c- myc gene and overexpression of c-Myc protein in breast cancer and adjacent non-neoplastic tissue. Clin. Biochem. 34, 557–562.CrossRefPubMedGoogle Scholar
  8. de Boer C. J., van Krieken J. H., Janssen-van Rhijn C. M. and Litvinov S. V. 1999 Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J. Pathol. 188, 201–206.Google Scholar
  9. El-Maqsoud N. M. A. and El-Rehim D. M. A. 2014 Clinicopathologic implications of EpCAM and Sox2 expression in breast cancer. Clin. Breast Cancer 14, e1–e9.CrossRefGoogle Scholar
  10. Fagotto F. and Gumbiner B. M. 1996 Cell contact-dependent signaling. Dev. Biol. 180, 445–454.CrossRefPubMedGoogle Scholar
  11. Gastl G., Spizzo G., Obrist P., Dünser M. and Mikuz G. 2000 Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet. 356, 1981–1982.CrossRefPubMedGoogle Scholar
  12. Göitlinger H. G., Funke I., Johnson J. P., Gokel J. M. and Riethmüller G. 1986 The epithelial cell surface antigen 17–1A, a target for antibody-mediated tumor therapy: its biochemical nature, tissue distribution and recognition by different monoclonal antibodies. Int. J. Cancer 38, 47–53.CrossRefGoogle Scholar
  13. Gombert W. M., Farris S. D., Rubio E. D., Morey-Rosler K. M., Schubach W. H. and Krumm A. 2003 The c-myc insulator element and matrix attachment regions define the c-myc chromosomal domain. Mol. Cell. Biol. 23, 9338–9348.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kuhn S., Koch M., Nübel T., Ladwein M., Antolovic D., Klingbeil P. et al. 2007 A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression. Mol. Cancer Res. 5, 553–567.CrossRefPubMedGoogle Scholar
  15. Ladwein M., Pape U. -F., Schmidt D. -S., Schnölzer M., Fiedler S., Langbein L. et al. 2005 The cell–cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7. Exp. Cell. Res. 309, 345–357.CrossRefPubMedGoogle Scholar
  16. Lê M. G., Mathieu M. C., Douc-Rasy S., Le Bihan M. L., Adb El All H., Spielmann M. et al. 1999 c-myc, p53 and bcl-2, apoptosis-related genes in infiltrating breast carcinomas: evidence of a link between bcl-2 protein over-expression and a lower risk of metastasis and death in operable patients. Int. J. Cancer 84, 562–567.CrossRefPubMedGoogle Scholar
  17. Le Naour F., André M., Greco C., Billard M., Sordat B., Emile J.-F. et al. 2006 Profiling of the tetraspanin web of human colon cancer cells. Mol. Cell Proteomics 5, 845–857.CrossRefPubMedGoogle Scholar
  18. Liao D. and Dickson R. 2000 c-Myc in breast cancer. Endocr. Relat. Cancer 7, 143–164.CrossRefPubMedGoogle Scholar
  19. Linke R., Klein A. and Seimetz D. 2010 Catumaxomab: clinical development and future directions. MAbs 2, 129–136.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Linnenbach A., Seng B., Wu S., Robbins S., Scollon M., Pyrc J. et al. 1993 Retroposition in a family of carcinoma-associated antigen genes. Mol. Cell. Biochem. 13, 1507–1515.CrossRefGoogle Scholar
  21. Litvinov S. V., Balzar M., Winter M. J., Bakker H. A., Briaire-de Bruijn I. H., Prins F. et al. 1997 Epithelial cell adhesion molecule (Ep-CAM) modulates cell–cell interactions mediated by classic cadherins. J. Cell. Biol. 139, 1337–1348.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Livak K. J. and Schmittgen T. D. 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2- ΔΔC(T)) method. Methods 25, 402–408.CrossRefPubMedGoogle Scholar
  23. Lutz W., Leon J. and Eilers M. 2002 Contributions of Myc to tumorigenesis. Biochim. et Biophys. Acta 1602, 61–71.Google Scholar
  24. Maetzel D., Denzel S., Mack B., Canis M., Went P., Benk M. et al. 2009 Nuclear signalling by tumour-associated antigen EpCAM. Nat. Cell Biol. 11, 162–171.CrossRefPubMedGoogle Scholar
  25. Magee J. A., Piskounova E. and Morrison S. J. 2012 Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21, 283–296.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Marcu K. B., Bossone S. A. and Patel A. J. 1992 Myc function and regulation. Annu. Rev. Biochem. 61, 809–858.CrossRefPubMedGoogle Scholar
  27. Martowicz A., Rainer J., Lelong J., Spizzo G., Gastl G. and Untergasser G. 2013 EpCAM overexpression prolongs proliferative capacity of primary human breast epithelial cells and supports hyperplastic growth. Mol. Cancer 12, 56.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Meyer N. and Penn L. Z. 2008 Reflecting on 25 years with MYC. Nat. Rev. Cancer 8, 976–990.CrossRefPubMedGoogle Scholar
  29. Mousavi S. M., Gouya M. M., Ramazani R., Davanlou M., Hajsadeghi N. and Seddighi Z. 2009 Cancer incidence and mortality in Iran. Ann. Oncol. 20, 556–563.CrossRefPubMedGoogle Scholar
  30. Münz M., Kieu C., Mack B., Schmitt B., Zeidler R. and Gires O. 2004 The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene 23, 5748–5758.CrossRefPubMedGoogle Scholar
  31. Munz M., Fellinger K., Hofmann T., Schmitt B. and Gires O. 2007 Glycosylation is crucial for stability of tumour and cancer stem cell antigen EpCAM. Front Biosci. 13, 5195–5201.Google Scholar
  32. Naidu R., Wahab N. A., Yadav M. and Kutty M. K. 2002 Protein expression and molecular analysis of c-myc gene in primary breast carcinomas using immunohistochemistry and differential polymerase chain reaction. Int. J. Mol. Med. 9, 189–196.PubMedGoogle Scholar
  33. Niemiec J. A., Adamczyk A., Malecki K., Majchrzyk K. and Rys J. 2012 Relationships between immunophenotype, Ki-67 index, microvascular density, Ep-CAM/P-cadherin, and MMP-2 expression in early-stage invasive ductal breast cancer. Appl. Immunohistochem. Mol. Morphol. 20, 550–560.CrossRefPubMedGoogle Scholar
  34. Nübel T., Preobraschenski J., Tuncay H., Weiss T., Kuhn S., Ladwein M. et al. 2009 Claudin-7 regulates EpCAM-mediated functions in tumor progression. Mol. Cancer Res. 7, 285–299.CrossRefPubMedGoogle Scholar
  35. Pauli C., Münz M., Kieu C., Mack B., Breinl P., Wollenberg B. et al. 2003 Tumor-specific glycosylation of the carcinoma-associated epithelial cell adhesion molecule EpCAM in head and neck carcinomas. Cancer Lett. 193, 25–32.CrossRefPubMedGoogle Scholar
  36. Ponta H., Sherman L. and Herrlich P. A. 2003 CD44: from adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 4, 33–45.CrossRefPubMedGoogle Scholar
  37. Raffel A., Eisenberger C. F., Cupisti K., Schott M., Baldus S. E., Hoffmann I. et al. 2010 Increased EpCAM expression in malignant insulinoma: potential clinical implications. Eur. J. Endocrinol. 162, 391–398.CrossRefPubMedGoogle Scholar
  38. Sánchez-García I., Vicente-Dueñas C. and Cobaleda C. 2007 The theoretical basis of cancer-stem-cell-based therapeutics of cancer: can it be put into practice? BioEssays 29, 1269–1280.CrossRefPubMedGoogle Scholar
  39. Schmidt M., Hasenclever D., Schaeffer M., Boehm D., Cotarelo C., Steiner E. et al. 2008 Prognostic effect of epithelial cell adhesion molecule overexpression in untreated node-negative breast cancer. Clin. Cancer. Res. 14, 5849–5855.CrossRefPubMedGoogle Scholar
  40. Schnell U., Cirulli V. and Giepmans B. N. 2013 EpCAM: structure and function in health and disease. Biochim. Biophys. Acta 1828, 1989–2001.CrossRefPubMedGoogle Scholar
  41. Scorilas A., Trangas T., Yotis J., Pateras C. and Talieri M. 1999 Determination of c-myc amplification and overexpression in breast cancer patients: evaluation of its prognostic value against c-erbB-2, cathepsin-D and clinicopathological characteristics using univariate and multivariate analysis. Br. J. Cancer 81, 1385.CrossRefPubMedPubMedCentralGoogle Scholar
  42. Simon M., Stefan N., Plückthun A. and Zangemeister-Wittke U. 2013 Epithelial cell adhesion molecule-targeted drug delivery for cancer therapy. Expert Opin. Drug Saf. 10, 451–468.CrossRefGoogle Scholar
  43. Sklar M. D. and Prochownik E. V. 1991 Modulation of cis-platinum resistance in friend erythroleukemia cells by c-myc. Cancer Res. 51, 2118–2123.PubMedGoogle Scholar
  44. Skliris G., Parkes A., Limer J., Burdall S., Carder P. and Speirs V. 2002 Evaluation of seven oestrogen receptor β antibodies for immunohistochemistry, western blotting, and flow cytometry in human breast tissue. J. Pathol. 197, 155–162.CrossRefPubMedGoogle Scholar
  45. Spandidos D., Pintzas A., Kakkanas A., Yiagnisis M., Mahera H., Patra E. et al. 1986 Elevated expression of the myc gene in human benign and malignant breast lesions compared to normal tissue. Anticancer Res. 7, 1299–1304.Google Scholar
  46. Spizzo G., Fong D., Wurm M., Ensinger C., Obrist P., Hofer C. et al. 2011 EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis. J. Clin. Pathol. 64, 415–420.CrossRefPubMedPubMedCentralGoogle Scholar
  47. Spizzo G., Gastl G., Obrist P., Went P., Dirnhofer S., Bischoff S. et al. 2004 High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer. Breast Cancer Res. Treat. 86, 207–213.CrossRefPubMedGoogle Scholar
  48. Tabatabaeian H. and Hojati Z. 2013 Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using real time RT-PCR and immunohistochemistry. Gene 531, 39–43.CrossRefPubMedGoogle Scholar
  49. Tanida S., Mizoshita T., Ozeki K., Tsukamoto H., Kamiya T., Kataoka H. et al. 2012 Mechanisms of cisplatin-induced apoptosis and of cisplatin sensitivity: potential of BIN1 to act as a potent predictor of cisplatin sensitivity in gastric cancer treatment. Int. J. Surg. Oncol. 2012, 280–288.Google Scholar
  50. van der Gun B. T., Melchers L. J., Ruiters M. H, de Leij L. F., McLaughlin P. M. and Rots M. G. 2010 EpCAM incarcinogenesis: the good, the bad or the ugly. Carcinogenesis 31, 1913–1921.Google Scholar
  51. Whittaker J. L., Walker R. A. and Varley J. M. 1986 Differential expression of cellular oncogenes in benign and malignant human breast tissue. Int. J. Cancer 38, 651–655.CrossRefPubMedGoogle Scholar
  52. Wierstra I. and Alves J. 2008 The c-myc promoter: still mystery and challenge. Adv. Cancer Res. 99, 113–333.CrossRefPubMedGoogle Scholar
  53. Winter M. J., Nagtegaal I. D., Van Krieken J. and Litvinov S. V. 2003 The epithelial cell adhesion molecule (Ep-CAM) as a morphoregulatory molecule is a tool in surgical pathology. Am. J. Pathol. 163, 2139–2148.Google Scholar
  54. Wong D. J., Liu H., Ridky T. W., Cassarino D., Segal E. and Chang H. Y. 2008 Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2, 333–344.CrossRefPubMedPubMedCentralGoogle Scholar
  55. Xu J., Chen Y. and Olopade O. I. 2010 MYC and breast cancer. Genes Cancer 1, 629–640.CrossRefPubMedPubMedCentralGoogle Scholar
  56. Yilmaz Ö. H., Valdez R., Theisen B. K., Guo W., Ferguson D. O., Wu H. et al. 2006 Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 441, 475–482.CrossRefPubMedGoogle Scholar

Copyright information

© Indian Academy of Sciences 2017

Authors and Affiliations

  • SAMIRA SADEGHI
    • 1
  • ZOHREH HOJATI
    • 1
    Email author
  • HOSSEIN TABATABAEIAN
    • 1
  1. 1.Faculty of Sciences, Division of Genetics, Department of BiologyUniversity of IsfahanIsfahanIran

Personalised recommendations