CDX1/2 and KLF5 Expression and Epigenetic Modulation of Sonic Hedgehog Signaling in Gastric Adenocarcinoma

  • Ali Akbar Samadani
  • Novin Nikbakhsh
  • Hassan Taheri
  • Shahriyar Shafaee
  • Sadegh Fattahi
  • Maryam Pilehchian Langroudi
  • Karimollah Hajian
  • Haleh Akhavan-NiakiEmail author
Original Article


Gastric cancer is among the commonplace causes of cancer death worldwide. Sonic hedgehog (Shh) signaling is an important pathway which may be dysregulated in many cancers.CDX1/2, and KLF5are key transcription factors involved in Shh pathway and cancer stem cells. The aim of this study was to investigate the expression and epigenetic alterations of these genes in gastric cancer patients. DNA methylation’s modifications of CDX1, KLF5 and CDX2 genes alongside with the expressions of these genes in gastric cancer tissues and their non-tumoral counterparts (margin tissues) were analyzed using methylation specific sequencing, and Real time PCR Taq man assays, respectively. The expression of CDX1 (P = 0.002) and KLF5 (P = 0.010) were decreased significantly, but it was considerably increased for CDX2 (P = 0.001). Relatively, the results for the regulatory region methylation status of each CpG site had shown a notable fluctuation in these genes with no significant difference in most places. The creation of metastatic lymph nodes in patients was significantly associated with increased expression of CDX2 gene. The modifications of these genes expression can be considered as a cancer biomarker in future studies. Methylation of the investigated genes is not the main mechanism of gastric cancer development.


Gastric cancer DNA methylation Gene expression Epigenetics 



complementary DNA


caudal type homeobox 1/2


gastric cancer


kruppel like factor 5


polymerase chain reaction



This work was supported by the Research deputy of Babol University of Medical Sciences (Grant No 9237527), and a grant by Iranian National Science Foundation (INSF project code: 92017552). Authors appreciate the staffs of Cellular and Molecular Biology Research Center, Rouhani and Shahid Beheshti hospitals, Razi Medical Laboratory, and all patients who participated in this project.

Compliance with Ethical Standards

Institutional Review Board

This study was approved by the ethics committee of Babol University of Medical Sciences, Iran.

Informed Consent

Written informed consent was obtained for all subjects before their inclusion in the study. All experiments and data comparisons were carried out in compliance with relevant laws and guidelines and in accordance with the ethical standards of the Declaration of Helsinki.

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Akhavan-Niaki H, Samadani AA (2014) Molecular insight in gastric cancer induction: an overview of cancer stemness genes. Cell Biochem Biophys 68(3):463–473CrossRefGoogle Scholar
  2. 2.
    Nikbakhsh N, Shafahi S, Taheri H, Samadani AA, Noroullahi SE, Pilecian M, Saromeh SH, Kolagar A, Ghasemzadeh M, Esmailzadeh Tahmtan S (2016) Histopathological investigation in gastric cancer. J Int Res Med Pharm Sci 8(2):52–56Google Scholar
  3. 3.
    Singh SR (2013) Gastric cancer stem cells: a novel therapeutic target. Cancer Lett 338(1):110–119CrossRefGoogle Scholar
  4. 4.
    Li K, Dan Z, Nie YQ (2014) Gastric cancer stem cells in gastric carcinogenesis, progression, prevention and treatment. World J Gastroenterol 20(18):5420–5426CrossRefGoogle Scholar
  5. 5.
    Xu Z-Y, Tang J-N, Xie H-X, Du Y-A, Huang L, Yu P-F, Cheng X-D (2015) 5-fluorouracil chemotherapy of gastric Cancer generates residual cells with properties of Cancer stem cells. Int J Biol Sci 11(3):284–294CrossRefGoogle Scholar
  6. 6.
    Samadani AA, Akhavan-Niaki H (2015) Interaction of sonic hedgehog (SHH) pathway with cancer stem cell genes in gastric cancer. Med Oncol 32(3):48–55CrossRefGoogle Scholar
  7. 7.
    Fujii Y, Yoshihashi K, Suzuki H, Tsutsumi S, Mutoh H, Maeda S, Yamagata Y, Seto Y, Aburatani H, Hatakeyama M (2012) CDX1 confers intestinal phenotype on gastric epithelial cells via induction of stemness-associated reprogramming factors SALL4 and KLF5. Proc Natl Acad Sci U S A 09(50):20584–20589CrossRefGoogle Scholar
  8. 8.
    Bornschein J, Tothk, Selgrad M, Kuestel D, Wex T, Molnar B, Tulassay Z, Malfertheiner P (2013) Dysregulation of CDX1,CDX2 and SOX2 in patients with gastric cancer also affects the non-malignant mucosa. J Clin Pathol 66(9):819–822CrossRefGoogle Scholar
  9. 9.
    Hryniuk A, Grainger S, Savory JG, Lohnes D (2014) CDX1 and CDX2 function as tumor suppressors. J Biol Chem 289(48):33343–33354CrossRefGoogle Scholar
  10. 10.
    Samadani AA, Nikbakhsh N, Pilehchian M, Fattahi S, Akhavan-Niaki H (2016) Epigenetic changes of CDX2 in gastric adenocarcinoma. J Cell Commun Signal 10(4):267–272CrossRefGoogle Scholar
  11. 11.
    Chia NY, Deng N, Das K, Huang D, Hu L, Zhu Y, Lim KH, Lee MH, Wu J, Sam XX, Tan GS, Wan WK, Yu W, Gan A, Tan AL, Tay ST, Soo KC, Wong WK, Dominguez LT, Ng HH, Rozen S, Goh LK, Teh BT, Tan P (2015) Regulatory crosstalk between lineage-survival oncogenes KLF5, GATA4 and GATA6 cooperatively promotes gastric cancer development. Gut 64(5):707–719CrossRefGoogle Scholar
  12. 12.
    Kwak MK, Lee HJ, Hur K, Park DJ, Lee HS, Kim WH, Lee KU, Choe KJ, Guilford P (2008) Expression of Krüppel-like factor 5 in human gastric carcinomas. J Cancer Res Clin Oncol 134(2):163–167CrossRefGoogle Scholar
  13. 13.
    Kang JM, Lee BH, Kim N, Lee HS, Lee HE, Park JH, Kim JS, Jung HC, Song IS (2011) CDX1 and CDX2 expression in intestinal metaplasia, dysplasia and gastric cancer. J Korean Med Sci 26(5):647–653CrossRefGoogle Scholar
  14. 14.
    Mutoh H, Sakurai S, Satoh K, Osawa H, Hakamata Y, Takeuchi T, Sugano K (2004) Cdx1 induced intestinal metaplasia in the transgenic mouse stomach: comparative study with Cdx2 transgenic mice. Gut 53(10):1416–1423CrossRefGoogle Scholar
  15. 15.
    Visvader JE, Linderman GJ (2008) Cancer stem cells in solid tumors: accumulating evidence and unresolved questions. Nat Rev Cancer 8(10):755–768CrossRefGoogle Scholar
  16. 16.
    Akhavan-Niaki H, Samadani AA (2013) DNA methylation and cancer development: molecular mechanism. Cell Biochem Biophys 67(2):501–513CrossRefGoogle Scholar
  17. 17.
    Hur K, Niwa T, Toyoda T, Tsukamoto T, Tatematsu M, Yang HK, Ushijima T (2011) Insufficient role of cell proliferation in aberrant DNA methylation induction and involvement of specific types of inflammation. Carcinogenesis 32(1):35–41CrossRefGoogle Scholar
  18. 18.
    Fattahi S, Amirbozorgi G, Lotfi M, Navaei BA, Kavoosian S, Asouri M, Akhavan-Niaki H (2018) Development of a universal Taqman probe for mRNA gene expression analysis. Iran J Sci Technol Trans A Sci 42(2):363–370CrossRefGoogle Scholar
  19. 19.
    Fattahi S, Pilehchian Langroudi M, Samadani AA, Nikbakhsh N, Asouri M, Akhavan-Niaki H (2017) Application of unique sequence index (USI) barcode to gene expression profiling in gastric adenocarcinoma. J Cell Commun Signal 11(1):97–104CrossRefGoogle Scholar
  20. 20.
    Chia N-Y, Deng N, Das K, Huang D, Hu L, Zhu Y, Lim KH, Lee MH, Wu J, Sam XX, Tan GS, Wan WK, Yu W, Gan A, Tan ALK, Tay ST, Soo KC, Wong WK, Dominguez LTM, Ng HH, Rozen S, Goh LK, Teh BT, Tan P (2015) Regulatory crosstalk between lineage-survival oncogenes KLF5, GATA4 and GATA6 cooperatively promotes gastric cancer development. Gut 64(5):707–719CrossRefGoogle Scholar
  21. 21.
    Eda A, Osawa H, Yanaka I, Satoh K, Mutoh H, Kihira K, Sugano K (2002) Expression of homeobox gene CDX2 precedes that of CDX1 during the progression of intestinal metaplasia. J Gastroenterol 37:94–100CrossRefGoogle Scholar
  22. 22.
    Baba Y, Nosho K et al (2009) Ralationship of CDX2 loss with molecular features and prognosis in colorectal cancer. Clin Cancer Res 15:4665–4673CrossRefGoogle Scholar
  23. 23.
    Mizoshita T et al (2001) Expression of CDX1 and CDX2 mRNAs and relevance of this expression to differentiation in human gastrointestinal mucosa with special emphasis on participation in intestinal metaplasia of the human stomach. Gastric Cancer 4(4):185–191CrossRefGoogle Scholar
  24. 24.
    Chan CW et al (2009) Gastrointestial differentiation marker cytokeratin 20 is regulated by homeobox CDX1. Proc Natl Acad Sci 16:1936–1941CrossRefGoogle Scholar
  25. 25.
    Silberg DG, Sullivan J, Kang E, Swain GP, Moffett J, Sund NJ, Sackett SD, Kaestner KH (2002) CDX2 ectopic expression induces gastric intestinal metaplasia in transgenic mice. Gastroenterology 122(3):689–696CrossRefGoogle Scholar
  26. 26.
    Liu Y, Chidgey M, Yang VW1, Bialkowska AB (2017) Krüppel-like factor 5 is essential for maintenance of barrier function in mouse colon. Am J Physiol Gastrointest Liver Physiol 313(5):G478–G491CrossRefGoogle Scholar
  27. 27.
    Hryniuk A, Grainger S, Savory JG, Lohnes D (2014) Cdx1 and Cdx2 function as tumor suppressors. J Biol Chem 289(48):33343–33354CrossRefGoogle Scholar
  28. 28.
    Pilozzi E, Onelli MR, Ziparo V, Mercantini P, Ruco L (2004) CDX1 expression is reduced in colorectal carcinoma and is associated with promoter hypermethylation. J Pathol 204:289–295CrossRefGoogle Scholar
  29. 29.
    Zeraati H, Amiri Z (2016) Estimating postoperative survival of gastric cancer patients and factors affecting it in Iran: based on a TNM-7 staging system. Acta Med Iran 54(2):114–8.5Google Scholar
  30. 30.
    Binto L, Yong J, Antebi YE, McCue K, Kazuki Y, Uno N, Oshimura M, Elowitz MB (2016) Dynamics of epigenetic regulation at the single-cell level. Acta Med Iran 351(6274):720–724Google Scholar
  31. 31.
    Cropley JE, Dang TH, Martin DI, Suter CM (2012) The penetrance of an epigenetic trait in mice is progressively yet reversibly increased by selection and environment. Proc Boil Sci 279(1737):2347–2353CrossRefGoogle Scholar
  32. 32.
    Lee JY, Kong G (2016) Roles and epigenetic regulation of epithelial-mesenchymal transition and its transcription factors in cancer initiation and progression. Proc Biol Sci 73(24):4643–4660Google Scholar
  33. 33.
    Chen Z1, Wu Q, Ding Y, Zhou W, Liu R, Chen H, Zhou J, Feng J (2018) Discovery of novel mifepristone derivatives via suppressing KLF5 expression for the treatment of triple-negative breast cancer. Eur J Med Chem 146:354–367CrossRefGoogle Scholar
  34. 34.
    Bialkowska A, Crisp M, Madoux F, Spicer T, Knapinska A, Mercer B, Bannister TD, He Y, Chowdhury S, Cameron M, Yang VW, Hodder P. ML264: an antitumor agent that potently and selectively inhibits Krüppel-like factor five (KLF5) expression: a probe for studying Colon Cancer development and progression. Probe reports from the NIH molecular libraries program [internet]. Bethesda (MD): National Center for biotechnology information (US); 2010–2011 Oct 31 [updated 2013 Mar 7]Google Scholar
  35. 35.
    Fattahi S, Pilehchian Langroudi M, Akhavan-Niaki H (2018) Hedgehog signaling pathway: epigenetic regulation and role in disease and cancer development. J Cell Physiol 233(8):5726–5735CrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2019

Authors and Affiliations

  • Ali Akbar Samadani
    • 1
    • 2
  • Novin Nikbakhsh
    • 3
  • Hassan Taheri
    • 4
  • Shahriyar Shafaee
    • 5
  • Sadegh Fattahi
    • 1
  • Maryam Pilehchian Langroudi
    • 1
  • Karimollah Hajian
    • 6
  • Haleh Akhavan-Niaki
    • 1
    • 7
    Email author
  1. 1.Cellular and Molecular Biology Research Center, Health Research InstituteBabol University of Medical SciencesBabolIran
  2. 2.Gastrointestinal and liver diseases research center (GLDRC)Guilan University of Medical SciencesRashtIran
  3. 3.Department of Surgery, Cancer Research CenterBabol University of Medical SciencesBabolIran
  4. 4.Department of GastroenterologyBabol University of Medical SciencesBabolIran
  5. 5.Department of PathologyBabol University of Medical SciencesBabolIran
  6. 6.Department of Social medicine and statistics, Faculty of MedicineBabol University of Medical SciencesBabolIran
  7. 7.Department of Genetics, Faculty of MedicineBabol University of Medical SciencesBabolIran

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