ALEX1, a novel tumor suppressor gene, inhibits gastric cancer metastasis via the PAR-1/Rho GTPase signaling pathway

  • Li Pang
  • Jian-fang Li
  • Liping Su
  • Mingde Zang
  • Zhiyuan Fan
  • Beiqin Yu
  • Xiongyan Wu
  • Chen Li
  • Min Yan
  • Zheng-gang Zhu
  • Bingya Liu
Original Article—Alimentary Tract



The ALEX is a novel member of the armadillo family and ALEX1 was reported to be reduced or even lost in multiple solid tumors. However, its expression profile and oncogenic role in gastric cancer (GC) remains largely unknown.


ALEX1 expression was detected in 161 GC samples by immunohistochemistry staining. NCI-N87 cells transfected by ALEX1 lentivirus vectors and MKN28 cells transfected by ALEX1 shRNA were used for biological function investigation. Western blot was applied to explore the molecular mechanism and pull-down assays were applied to measure the activity of Rho GTPases. In vivo tumorigenicity, peritoneal and lung metastasis experiments were performed by tumor cell engraftment into nude mice. Bisulfite genomic sequencing and methylation-specific PCR were applied to check the methylation status of the ALEX1 gene.


The expression rate of ALEX1 was significantly reduced in gastric tumor samples compared to non-tumor samples (43.5 vs. 90.2%), and its expression was closely related to the tumor differentiation, TNM staging, and lymph nodes metastasis. ALEX1 overexpression in NCI-N87 cells significantly inhibited cell proliferation, migration, and invasion in vitro, and disrupted the structure of the cytoskeleton. ALEX1 overexpression attenuated xenografts growth, peritoneal, and lung metastasis in nude mice. Mechanistically, the overexpression of ALEX1 inhibits thrombin-induced metastasis and Rho GTPases activation. Bisulfite genomic sequencing and methylation-specific PCR revealed that the promoter of ALEX1 is highly methylated in GC cells and tissues.


ALEX1 expression is reduced in GC and is involved in diverse cellular functions. ALEX1 inhibits metastasis through the PAR-1/Rho GTPase signaling pathway.


Gastric cancer ALEX1 Cell metastasis PAR-1 Rho GTPase 



Gastric cancer


Arm protein lost in epithelial cancers, on chromosome X


Protease-activated receptor 1

Rho GTPase

Rho family of GTPase


Epithelial-to-mesenchymal transition


Cell Counting Kit-8


Methylation-specific PCR




Quantitative reverse transcription-polymerase chain reaction

Supplementary material

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Supplementary material 1 (DOCX 7387 kb)
535_2017_1329_MOESM2_ESM.docx (16 kb)
Supplementary material 2 (DOCX 16 kb)
535_2017_1329_MOESM3_ESM.docx (16 kb)
Supplementary material 3 (DOCX 16 kb)


  1. 1.
    Fitzmaurice C, Dicker D, Pain A, et al. The global burden of cancer 2013. JAMA Oncol. 2015;1(4):505–27.CrossRefPubMedGoogle Scholar
  2. 2.
    Van Cutsem E, Sagaert X, Topal B, et al. Gastric cancer. Lancet. 2016;388(10060):2654–64.CrossRefPubMedGoogle Scholar
  3. 3.
    Hall A. The cytoskeleton and cancer. Cancer Metastasis Rev. 2009;28(1–2):5–14.CrossRefPubMedGoogle Scholar
  4. 4.
    Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer. 2002;2(2):133–42.CrossRefPubMedGoogle Scholar
  5. 5.
    Fukata M, Nakagawa M, Kaibuchi K. Roles of Rho-family GTPases in cell polarisation and directional migration. Curr Opin Cell Biol. 2003;15(5):590–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Shi XL, Gangadharan B, Brass LF, et al. Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res. 2004;2(7):395–402.PubMedGoogle Scholar
  7. 7.
    Spiegelberg BD, Hamm HE. Roles of G-protein-coupled receptor signaling in cancer biology and gene transcription. Curr Opin Genet Dev. 2007;17(1):40–4.CrossRefPubMedGoogle Scholar
  8. 8.
    Fujimoto D, Hirono Y, Goi T, et al. The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion. BMC Cancer. 2010;10:443.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pan Y, Bi F, Liu N, et al. Expression of seven main Rho family members in gastric carcinoma. Biochem Biophys Res Commun. 2004;315(3):686–91.CrossRefPubMedGoogle Scholar
  10. 10.
    Du T, Qu Y, Li J, et al. Maternal embryonic leucine zipper kinase enhances gastric cancer progression via the FAK/Paxillin pathway. Mol Cancer. 2014;13(1):100.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kurochkin IV, Yonemitsu N, Funahashi SI, et al. ALEX1, a novel human armadillo repeat protein that is expressed differentially in normal tissues and carcinomas. Biochem Biophys Res Commun. 2001;280(1):340–7.CrossRefPubMedGoogle Scholar
  12. 12.
    Hatzfeld M. The armadillo family of structural proteins. Int Rev Cytol. 1999;186:179–224.CrossRefPubMedGoogle Scholar
  13. 13.
    Gao Y, Wu JY, Zeng F, et al. ALEX1 regulates proliferation and apoptosis in breast cancer cells. Asian Pac J Cancer Prev. 2015;16(8):3293–9.CrossRefPubMedGoogle Scholar
  14. 14.
    Iseki H, Takeda A, Andoh T, et al. ALEX1 suppresses colony formation ability of human colorectal carcinoma cell lines. Cancer Sci. 2012;103(7):1267–71.CrossRefPubMedGoogle Scholar
  15. 15.
    Zender L, Xue W, Zuber J, et al. An oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer. Cell. 2008;135(5):852–64.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Liu X, Tang X, Zhang S, et al. Methylation and expression of retinoblastoma and transforming growth factor-beta1 genes in Epstein-Barr virus-associated and -negative gastric carcinomas. Gastroenterol Res Pract. 2012;2012:906017.PubMedPubMedCentralGoogle Scholar
  17. 17.
    Otsuki T, Fujimoto D, Hirono Y, et al. Thrombin conducts epithelial–mesenchymal transition via proteasea-ctivated receptor1 in human gastric cancer. Int J Oncol. 2014;45(6):2287–94.PubMedGoogle Scholar
  18. 18.
    Fujimoto D, Hirono Y, Goi T, et al. The activation of proteinase-activated receptor-1 (PAR1) promotes gastric cancer cell alteration of cellular morphology related to cell motility and invasion. Int J Oncol. 2013;42(2):565–73.PubMedGoogle Scholar
  19. 19.
    Calcagno DQ, Gigek CO, Chen ES, et al. DNA and histone methylation in gastric carcinogenesis. World J Gastroenterol. 2013;19(8):1182–92.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    McCarthy N. Epigenetics: worth another look? Nat Rev Cancer. 2012;12(1):2.Google Scholar
  21. 21.
    Mou Z, Tapper AR, Gardner PD. The armadillo repeat-containing protein, ARMCX3, physically and functionally interacts with the developmental regulatory factor Sox10. J Biol Chem. 2009;284(20):13629–40.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Smith CA, McClive PJ, Sinclair AH. Temporal and spatial expression profile of the novel armadillo-related gene, Alex2, during testicular differentiation in the mouse embryo. Dev Dyn. 2005;233(1):188–93.CrossRefPubMedGoogle Scholar
  23. 23.
    Weber GF, Bronson RT, Ilagan J, et al. Absence of the CD44 gene prevents sarcoma metastasis. Cancer Res. 2002;62(8):2281–6.PubMedGoogle Scholar
  24. 24.
    Lefebvre O, Chenard MP, Masson R, et al. Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science. 1996;274(5285):259–62.CrossRefPubMedGoogle Scholar
  25. 25.
    Talmadge JE, Fidler IJ. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res. 2010;70(14):5649–69.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kim SJ, Shin JY, Lee KD, et al. Galectin-3 facilitates cell motility in gastric cancer by up-regulating protease-activated receptor-1 (PAR-1) and matrix metalloproteinase-1 (MMP-1). PLoS One. 2011;6(9):e25103.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Boire A, Covic L, Agarwal A, et al. PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell. 2005;120(3):303–13.CrossRefPubMedGoogle Scholar
  28. 28.
    Martin CB, Mahon GM, Klinger MB, et al. The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways. Oncogene. 2001;20(16):1953–63.CrossRefPubMedGoogle Scholar
  29. 29.
    Greenberg DL, Mize GJ, Takayama TK. Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells. Biochemistry. 2003;42(3):702–9.CrossRefPubMedGoogle Scholar
  30. 30.
    Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 2005;6(2):167–80.CrossRefPubMedGoogle Scholar
  31. 31.
    Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128(4):683–92.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Li Y, Liang J, Hou P. Hypermethylation in gastric cancer. Clin Chim Acta. 2015;448:124–32.CrossRefPubMedGoogle Scholar
  33. 33.
    Tahara T, Arisawa T. DNA methylation as a molecular biomarker in gastric cancer. Epigenomics. 2015;7(3):475–86.CrossRefPubMedGoogle Scholar
  34. 34.
    Niwa T, Tsukamoto T, Toyoda T, et al. Inflammatory processes triggered by Helicobacter pylori infection cause aberrant DNA methylation in gastric epithelial cells. Cancer Res. 2010;70(4):1430–40.CrossRefPubMedGoogle Scholar

Copyright information

© Japanese Society of Gastroenterology 2017

Authors and Affiliations

  • Li Pang
    • 1
  • Jian-fang Li
    • 1
  • Liping Su
    • 1
  • Mingde Zang
    • 1
  • Zhiyuan Fan
    • 1
  • Beiqin Yu
    • 1
  • Xiongyan Wu
    • 1
  • Chen Li
    • 1
  • Min Yan
    • 1
  • Zheng-gang Zhu
    • 1
  • Bingya Liu
    • 1
  1. 1.Shanghai Key Laboratory of Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin HospitalShanghai Jiao Tong University School of MedicineShanghaiPeople’s Republic of China

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