Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Hypoxia-induced TUFT1 promotes the growth and metastasis of hepatocellular carcinoma by activating the Ca2+/PI3K/AKT pathway


Tuftelin1 (TUFT1), an acidic protein constituent of developing and mineralizing tooth tissues, is regulated by hypoxia and the Hedgehog signaling pathway. We investigated the role of TUFT1 in hepatocellular carcinoma (HCC). qRT-PCR, immunohistochemistry and western blot were employed to evaluate TUFT1 level in HCC. MTT, BrdU, 3D culture and Transwell assays were used to assess cell viability, proliferation, in vitro growth, migration, and invasion. Subcutaneous and tail vein injection models were established to investigate in vivo growth and metastasis. Chromatin immunoprecipitation was performed to assess binding of hypoxia-inducible factor 1α (HIF-1α) to TUFT1 promoter. A microRNA array was used to identify hypoxia-related microRNAs. TUFT1 was elevated in HCC, and correlated with unfavorable clinicopathologic characteristics and poor survival. TUFT1 promoted HCC cell growth, metastasis and epithelial-mesenchymal transition in vitro and in vivo via activation of Ca2+/PI3K/AKT pathway. Hypoxia induced TUFT1 expression in an HIF-1α dependent manner, and TUFT1 expression was positively correlated with HIF-1α level in HCC tissues. Hypoxiaenhanced TUFT1 expression by downregulating miR-671-5p rather than by directly promoting the binding of HIF-1α to TUFT1 promoter. MiR-671-5p interacted with the 3′-UTR of TUFT1 mRNA and subsequently inhibited TUFT1 expression. Consequently, knockdown of TUFT1 blocked the effects of hypoxia in promoting HCC progression. TUFT1 promoted the growth, metastasis and EMT of HCC cells through activating Ca2+/PI3K/AKT pathway. The hypoxic microenvironment increased the expression of TUFT1 via downregulation of miR-671-5p. TUFT1 may function as a potential therapeutic target for the intervention and treatment of HCC.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


  1. 1.

    El–Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132:2557–76.

    Article  Google Scholar 

  2. 2.

    Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology. 2011;53:1020–2.

    Article  Google Scholar 

  3. 3.

    Maluccio M, Covey A. Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma. CA Cancer J Clin. 2012;62:394–9.

    Article  Google Scholar 

  4. 4.

    Pouysségur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006;441:437–43.

    Article  Google Scholar 

  5. 5.

    Harris AL. Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer. 2002;2:38–47.

    CAS  Article  Google Scholar 

  6. 6.

    Rankin EB, Giaccia AJ. Hypoxic control of metastasis. Science. 2016;352:175–80.

    CAS  Article  Google Scholar 

  7. 7.

    Soni S, Padwad YS. HIF-1 in cancer therapy: two decade long story of a transcription factor. Acta Oncol (Madr). 2017;56:503–15.

    CAS  Article  Google Scholar 

  8. 8.

    Carnero A, Lleonart M. The hypoxic microenvironment: a determinant of cancer stem cell evolution. Bioessays. 2016;38:S65–S74.

    Article  Google Scholar 

  9. 9.

    Bristow RG, Hill RP. Hypoxia and metabolism: hypoxia, DNA repair and genetic instability. Nat Rev Cancer. 2008;8:180–92.

    CAS  Article  Google Scholar 

  10. 10.

    Giatromanolaki A, Harris AL. Tumour hypoxia, hypoxia signaling pathways and hypoxia inducible factor expression in human cancer. Anticancer Res. 2000;21:4317–24.

    Google Scholar 

  11. 11.

    Jubb A, Hillan K. Expression of HIF-1α in human tumours. J Clin Pathol. 2005;58:1344–1344.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Wu XZ, Xie GR, Chen D. Hypoxia and hepatocellular carcinoma: the therapeutic target for hepatocellular carcinoma. J Gastroenterol Hepatol. 2007;22:1178–82.

    CAS  Article  Google Scholar 

  13. 13.

    Gwak G-Y, Yoon J-H, Kim KM, Lee H-S, Chung JW, Gores GJ. Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression. J Hepatol. 2005;42:358–64.

    CAS  Article  Google Scholar 

  14. 14.

    Mao Z, Shay B, Hekmati M, Fermon E, Taylor A, Dafni L, et al. The human tuftelin gene: cloning and characterization. Gene. 2001;279:181–96.

    CAS  Article  Google Scholar 

  15. 15.

    Deutsch D, Palmon A, Dafni L, Mao Z, Leytin V, Young M, et al. Tuftelin–aspects of protein and gene structure. Eur J Oral Sci. 1998;106:315–23.

    CAS  Article  Google Scholar 

  16. 16.

    Deutsch D, Palmon A, Dafni L, Catalano-Sherman J, Young M, Fisher L. The enamelin (tuftelin) gene. Int J Dev Biol. 2003;39:135–43.

    Google Scholar 

  17. 17.

    Leiser Y, Blumenfeld A, Haze A, Dafni L, Taylor AL, Rosenfeld E, et al. Localization, quantification, and characterization of tuftelin in soft tissues. Anat Rec. 2007;290:449–54.

    Article  Google Scholar 

  18. 18.

    Sliz E, Taipale M, Welling M, Skarp S, Alaraudanjoki V, Ignatius J, et al. TUFT1, a novel candidate gene for metatarsophalangeal osteoarthritis, plays a role in chondrogenesis on a calcium-related pathway. PLoS ONE. 2017;12:e0175474.

    Article  Google Scholar 

  19. 19.

    Leiser Y, Silverstein N, Blumenfeld A, Shilo D, Haze A, Rosenfeld E, et al. The induction of tuftelin expression in PC12 cell line during hypoxia and NGF‐induced differentiation. J Cell Physiol. 2011;226:165–72.

    CAS  Article  Google Scholar 

  20. 20.

    Oliveira F, Bellesini L, Defino H, da Silva Herrero C, Beloti M, Rosa A. Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells. J Cell Biochem. 2012;113:204–8.

    CAS  Article  Google Scholar 

  21. 21.

    Zheng X, Zeng W, Gai X, Xu Q, Li C, Liang Z, et al. Role of the Hedgehog pathway in hepatocellular carcinoma. Oncol Rep. 2013;30:2020–6.

    CAS  Article  Google Scholar 

  22. 22.

    Zheng X, Vittar NBR, Gai X, Fernandez-Barrena MG, Moser CD, Hu C, et al. The transcription factor GLI1 mediates TGFβ1 driven EMT in hepatocellular carcinoma via a SNAI1-dependent mechanism. PLoS ONE. 2012;7:e49581.

    CAS  Article  Google Scholar 

  23. 23.

    Thiery JP. Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002;2:442–54.

    CAS  Article  Google Scholar 

  24. 24.

    Altomare DA, Testa JR. Perturbations of the AKT signaling pathway in human cancer. Oncogene. 2005;24:7455–64.

    CAS  Article  Google Scholar 

  25. 25.

    Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 2007;26:3291–310.

    CAS  Article  Google Scholar 

  26. 26.

    Wagner EF, Nebreda ÁR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer. 2009;9:537–49.

    CAS  Article  Google Scholar 

  27. 27.

    Sliz E, Taipale M, Welling M, Skarp S, Alaraudanjoki V, Ignatius J. TUFT1, a novel candidate gene for metatarsophalangeal osteoarthritis, plays a role in chondrogenesis on a calcium-related pathway. 2017;12: e0175474.

  28. 28.

    Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4:517–29.

    CAS  Article  Google Scholar 

  29. 29.

    Zhang Y, Zhang T, Wu C, Xia Q, Xu D. ASIC1a mediates the drug resistance of human hepatocellular carcinoma via the Ca2+/PI3-kinase/AKT signaling pathway. Lab Investig; a J Tech Methods Pathol. 2017;97:53–69.

    CAS  Article  Google Scholar 

  30. 30.

    Davis FM, Azimi I, Faville RA, Peters AA, Jalink K, Putney JW Jr., et al. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent. Oncogene. 2014;33:2307–16.

    CAS  Article  Google Scholar 

  31. 31.

    Wen L, Liang C, Chen E, Chen W, Liang F, Zhi X, et al. Regulation of multi-drug resistance in hepatocellular carcinoma cells is TRPC6/calcium dependent. Sci Rep. 2016;6:23269.

    CAS  Article  Google Scholar 

  32. 32.

    Poon E, Harris AL, Ashcroft M. Targeting the hypoxia-inducible factor (HIF) pathway in cancer. Expert Rev Mol Med. 2009;11:e26.

    Article  Google Scholar 

  33. 33.

    Lee J-W, Bae S-H, Jeong J-W, Kim S-H, Kim K-W. Hypoxia-inducible factor (HIF-1)[alpha]: its protein stability and biological functions. Exp Mol Med. 2004;36:1.

    Article  Google Scholar 

  34. 34.

    Choudhry H, Harris AL, McIntyre A. The tumour hypoxia induced non-coding transcriptome. Mol Asp Med. 2016;47:35–53.

    Article  Google Scholar 

  35. 35.

    Kulshreshtha R, Ferracin M, Wojcik SE, Garzon R, Alder H, Agosto-Perez FJ, et al. A microRNA signature of hypoxia. Mol Cell Biol. 2007;27:1859–67.

    CAS  Article  Google Scholar 

  36. 36.

    Chang Y-N, Zhang K, Hu Z-M, Qi H-X, Shi Z-M, Han X-H, et al. Hypoxia-regulated lncRNAs in cancer. Gene. 2016;575:1–8.

    CAS  Article  Google Scholar 

  37. 37.

    Dou C, Liu Z, Xu M, Jia Y, Wang Y, Li Q, et al. miR-187-3p inhibits the metastasis and epithelial–mesenchymal transition of hepatocellular carcinoma by targeting S100A4. Cancer Lett. 2016;381:380–90.

    CAS  Article  Google Scholar 

  38. 38.

    Xu S, Huang H, Chen Y-N, Deng Y-T, Zhang B, Xiong X-D, et al. DNA damage responsive miR-33b-3p promoted lung cancer cells survival and cisplatin resistance by targeting p21WAF1/CIP1. Cell Cycle. 2016;15:2920–30.

    CAS  Article  Google Scholar 

  39. 39.

    Meng H, Wang K, Chen X, Guan X, Hu L, Xiong G, et al. MicroRNA-330-3p functions as an oncogene in human esophageal cancer by targeting programmed cell death 4. Am J Cancer Res. 2015;5:1062.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Tan X, Fu Y, Chen L, Lee W, Lai Y, Rezaei K, et al. miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer. Oncotarget. 2016;7:293.

    PubMed  Google Scholar 

  41. 41.

    Li X, Zhang G, Luo F, Ruan J, Huang D, Feng D, et al. Identification of aberrantly expressed miRNAs in rectal cancer. Oncol Rep. 2012;28:77–84.

    PubMed  Google Scholar 

  42. 42.

    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

    CAS  Article  Google Scholar 

  43. 43.

    Kawasaki N, Isogaya K, Dan S, Yamori T, Takano H, Yao R, et al. TUFT1 interacts with RABGAP1 and regulates mTORC1 signaling. Cell Discov. 2018;4:1.

    CAS  Article  Google Scholar 

  44. 44.

    Ruan K, Song G, Ouyang G. Role of hypoxia in the hallmarks of human cancer. J Cell Biochem. 2009;107:1053–62.

    CAS  Article  Google Scholar 

  45. 45.

    Zhou B, Zhan H, Tin L, Liu S, Xu J, Dong Y, et al. TUFT1 regulates metastasis of pancreatic cancer through HIF1-Snail pathway induced epithelial–mesenchymal transition. Cancer Lett. 2016;382:11–20.

    CAS  Article  Google Scholar 

Download references


This study was supported by grants from the National Natural Science Foundation of China (81874049, 81773123, 81572847, 81502092); Innovation Capacity Support Plan in Shaanxi Province of China (2018KJXX-045); the Fundamental Research Funds for the Central Universities (7N010011015).


This study was supported by grants from the National Natural Science Foundation of China (81874049, 81773123, 81572847, 81502092); Innovation Capacity Support Plan in Shaanxi Province of China (2018KJXX-045); the Fundamental Research Funds for the Central Universities (7N010011015).

Author information



Corresponding authors

Correspondence to Qingguang Liu or Kangsheng Tu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

These authors contributed equally: Changwei Dou, Zhenyu Zhou, Qiuran Xu, Zhikui Liu

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dou, C., Zhou, Z., Xu, Q. et al. Hypoxia-induced TUFT1 promotes the growth and metastasis of hepatocellular carcinoma by activating the Ca2+/PI3K/AKT pathway. Oncogene 38, 1239–1255 (2019).

Download citation

Further reading


Quick links