Clinical and Experimental Medicine

, Volume 19, Issue 1, pp 121–132 | Cite as

mTOR and ERK regulate VKORC1 expression in both hepatoma cells and hepatocytes which influence blood coagulation

  • Yaofu Liu
  • Huiling Li
  • Jianyi Dong
  • Lei Ma
  • Aijun Liao
  • Zhuona Rong
  • Zhi Zhou
  • Liang Cao
  • Fujin Wang
  • Jingyu WangEmail author
  • Aiguo WangEmail author
Original Article


Deficiency of γ-glutamyl carboxylation of coagulation factors, as evidenced by the elevated level of Des-γ-carboxyl prothrombin (DCP), is a common feature in hepatocellular carcinoma patients. Additionally, treatment of cancer patients with mTOR inhibitors significantly increases hemorrhagic events. However, the underlying mechanisms remain unknown. In the present study, Vitamin K epoxide reductase complex subunit 1 (VKORC1) was found to be significantly down-regulated in clinical hepatoma tissues and most tested hepatoma cell lines. In vitro investigations showed that VKORC1 expression was promoted by p-mTOR at the translational level and repressed by p-ERK at the transcriptional level. By exploring Hras12V transgenic mice, a hepatic tumor model, VKROC1 was significantly down-regulated in hepatic tumors and showed prolonged activated partial prothrombin time (APTT). In vivo investigations further showed that VKORC1 expression was promoted by p-mTOR and repressed by p-ERK in both hepatoma and hepatocytes. Consistently, APTT and prothrombin time were significantly prolonged under the mTOR inhibitor treatment and significantly shortened under the ERK inhibitor treatment. Conclusively, these findings indicate that mTOR and ERK play crucial roles in controlling VKORC1 expression in both hepatoma and hepatocytes, which provides a valuable molecular basis for preventing hemorrhage in clinical therapies.


Hepatocellular carcinoma Coagulopathy VKORC1 mTOR ERK 



Activated partial prothrombin time


Des-γ-carboxyl prothrombin


Extracellular signal-regulated kinase


Gamma-glutamyl carboxylase


Hepatocellular carcinoma


Mammalian target of rapamycin


Next-generation sequencing


Hepatic tumor-adjacent normal liver tissues


Prothrombin time

Ras-Tg mice

Hras12V transgenic mice


Hepatic tumor tissues


Tissue factor


Vitamin K


Vitamin K deficiency-related bleeding


Vitamin K epoxide reductase complex subunit 1


Wild-type mice or normal liver tissues of wild-type mice



This work was supported by the National Natural Science Foundation of China (30872950). A. G. Wang and J. Y. Wang conceived of the research ideas, supervised the project and revised the manuscript. Y. F. Liu and H. L. Li performed experiments, analyzed data and wrote the manuscript. J. Y. Dong, L. Ma, A. J. Liao, Z. N. Rong, L. Cao, Z, Zhou. F. J. Wang conducted animal and molecular experiments. L. Ma, A. J. Liao recruited clinical data and human samples in the clinical study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10238_2018_528_MOESM1_ESM.docx (2.5 mb)
Supplementary material 1 (DOCX 2540 kb)
10238_2018_528_MOESM2_ESM.doc (40 kb)
Supplementary material 2 (DOC 39 kb)
10238_2018_528_MOESM3_ESM.docx (36 kb)
Supplementary material 3 (DOCX 36 kb)
10238_2018_528_MOESM4_ESM.docx (17 kb)
Supplementary material 4 (DOCX 16 kb)
10238_2018_528_MOESM5_ESM.docx (31 kb)
Supplementary material 5 (DOCX 31 kb)
10238_2018_528_MOESM6_ESM.docx (23 kb)
Supplementary material 6 (DOCX 22 kb)
10238_2018_528_MOESM7_ESM.docx (3.2 mb)
Supplementary material 7 (DOCX 3271 kb)


  1. 1.
    Yang JD, Roberts LR. Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol. 2010;7(8):448–58. Scholar
  2. 2.
    Okonkwo UC, Nwosu MN, Ukah C, Okpala OC, Ahaneku JI. The clinical and pathological features of hepatocellular carcinoma in Nnewi, Nigeria. Niger J Med. 2011;20(3):366–71.Google Scholar
  3. 3.
    Jaka H, Mshana SE, Rambau PF, Masalu N, Chalya PL, Kalluvya SE. Hepatocellular carcinoma: clinicopathological profile and challenges of management in a resource-limited setting. World J Surg Oncol. 2014;12:246. Scholar
  4. 4.
    Friedman LS. The risk of surgery in patients with liver disease. Hepatology. 1999;29(6):1617–23. Scholar
  5. 5.
    Mucino-Bermejo J, Carrillo-Esper R, Uribe M, Mendez-Sanchez N. Coagulation abnormalities in the cirrhotic patient. Ann Hepatol. 2013;12(5):713–24.Google Scholar
  6. 6.
    Zhang DY, Friedman SL. Fibrosis-dependent mechanisms of hepatocarcinogenesis. Hepatology. 2012;56(2):769–75. Scholar
  7. 7.
    Ferron M, Lacombe J, Germain A, Oury F, Karsenty G. GGCX and VKORC1 inhibit osteocalcin endocrine functions. J Cell Biol. 2015;208(6):761–76. Scholar
  8. 8.
    Oldenburg J, Marinova M, Muller-Reible C, Watzka M. The vitamin K cycle. Vitam Horm. 2008;78:35–62. Scholar
  9. 9.
    Spohn G, Kleinridders A, Wunderlich FT, Watzka M, Zaucke F, Blumbach K, et al. VKORC1 deficiency in mice causes early postnatal lethality due to severe bleeding. Thromb Haemost. 2009;101(6):1044–50.CrossRefGoogle Scholar
  10. 10.
    Zhu A, Sun H, Raymond RM Jr, Furie BC, Furie B, Bronstein M, et al. Fatal hemorrhage in mice lacking gamma-glutamyl carboxylase. Blood. 2007;109(12):5270–5. Scholar
  11. 11.
    Cui SX, Yu XF, Qu XJ. Roles and signaling pathways of Des-gamma-carboxyprothrombin in the progression of hepatocellular carcinoma. Cancer Invest. 2016;34(9):459–64. Scholar
  12. 12.
    Younossi ZM, Otgonsuren M, Henry L, Venkatesan C, Mishra A, Erario M, et al. Association of nonalcoholic fatty liver disease (NAFLD) with hepatocellular carcinoma (HCC) in the United States from 2004 to 2009. Hepatology. 2015;62(6):1723–30. Scholar
  13. 13.
    Newell P, Toffanin S, Villanueva A, Chiang DY, Minguez B, Cabellos L, et al. Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. J Hepatol. 2009;51(4):725–33. Scholar
  14. 14.
    Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, et al. Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma. Gastroenterology. 2011;140(3):1071–83. Scholar
  15. 15.
    Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology. 2008;135(6):1972–83, 83 e1–11.
  16. 16.
    Wang C, Cigliano A, Delogu S, Armbruster J, Dombrowski F, Evert M, et al. Functional crosstalk between AKT/mTOR and Ras/MAPK pathways in hepatocarcinogenesis: implications for the treatment of human liver cancer. Cell Cycle. 2013;12(13):1999–2010. Scholar
  17. 17.
    Whittaker S, Marais R, Zhu AX. The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene. 2010;29(36):4989–5005. Scholar
  18. 18.
    Duffy A, Wilkerson J, Greten TF. Hemorrhagic events in hepatocellular carcinoma patients treated with antiangiogenic therapies. Hepatology. 2013;57(3):1068–77. Scholar
  19. 19.
    Di Costanzo GG, Tortora R, Iodice L, Lanza AG, Lampasi F, Tartaglione MT, et al. Safety and effectiveness of sorafenib in patients with hepatocellular carcinoma in clinical practice. Dig Liver Dis. 2012;44(9):788–92. Scholar
  20. 20.
    Zavaglia C, Airoldi A, Mancuso A, Vangeli M, Vigano R, Cordone G, et al. Adverse events affect sorafenib efficacy in patients with recurrent hepatocellular carcinoma after liver transplantation: experience at a single center and review of the literature. Eur J Gastroenterol Hepatol. 2013;25(2):180–6. Scholar
  21. 21.
    Bhoori S, Toffanin S, Sposito C, Germini A, Pellegrinelli A, Lampis A, et al. Personalized molecular targeted therapy in advanced, recurrent hepatocellular carcinoma after liver transplantation: a proof of principle. J Hepatol. 2010;52(5):771–5. Scholar
  22. 22.
    O’Neil BH, Goff LW, Kauh JS, Strosberg JR, Bekaii-Saab TS, Lee RM, et al. Phase II study of the mitogen-activated protein kinase 1/2 inhibitor selumetinib in patients with advanced hepatocellular carcinoma. J Clin Oncol. 2011;29(17):2350–6. Scholar
  23. 23.
    Armstrong AJ, Shen T, Halabi S, Kemeny G, Bitting RL, Kartcheske P, et al. A phase II trial of temsirolimus in men with castration-resistant metastatic prostate cancer. Clin Genitourin Cancer. 2013;11(4):397–406. Scholar
  24. 24.
    Altomare I, Bendell JC, Bullock KE, Uronis HE, Morse MA, Hsu SD, et al. A phase II trial of bevacizumab plus everolimus for patients with refractory metastatic colorectal cancer. Oncologist. 2011;16(8):1131–7. Scholar
  25. 25.
    Assi H, Abdel-Samad N. Severe gastrointestinal hemorrhage during targeted therapy for advanced breast carcinoma. Curr Oncol. 2014;21(5):e732–5. Scholar
  26. 26.
    Junpaparp P, Sharma B, Samiappan A, Rhee JH, Young KR. Everolimus-induced severe pulmonary toxicity with diffuse alveolar hemorrhage. Ann Am Thorac Soc. 2013;10(6):727–9. Scholar
  27. 27.
    Kakimoto M, Nakata T, Imaizumi K, Hirano T, Murata T, Okuno K, et al. Subclavian artery hemorrhage related to everolimus in a patient with recurrent breast cancer—a case report. Gan To Kagaku Ryoho. 2015;42(12):1806–8.Google Scholar
  28. 28.
    Coleman RL, Sill MW, Thaker PH, Bender DP, Street D, McGuire WP, et al. A phase II evaluation of selumetinib (AZD6244, ARRY-142886), a selective MEK-1/2 inhibitor in the treatment of recurrent or persistent endometrial cancer: an NRG Oncology/Gynecologic Oncology Group study. Gynecol Oncol. 2015;138(1):30–5. Scholar
  29. 29.
    Wang AG, Moon HB, Lee MR, Hwang CY, Kwon KS, Yu SL, et al. Gender-dependent hepatic alterations in H-ras12V transgenic mice. J Hepatol. 2005;43(5):836–44. Scholar
  30. 30.
    Wang AG, Song YN, Chen J, Li HL, Dong JY, Cui HP, et al. Activation of RAS/ERK alone is insufficient to inhibit RXRalpha function and deplete retinoic acid in hepatocytes. Biochem Biophys Res Commun. 2014;452(3):801–7. Scholar
  31. 31.
    Wada H, Usui M, Sakuragawa N. Hemostatic abnormalities and liver diseases. Semin Thromb Hemost. 2008;34(8):772–8. Scholar
  32. 32.
    Lisman T, Caldwell SH, Burroughs AK, Northup PG, Senzolo M, Stravitz RT, et al. Hemostasis and thrombosis in patients with liver disease: the ups and downs. J Hepatol. 2010;53(2):362–71. Scholar
  33. 33.
    Amitrano L, Guardascione MA, Brancaccio V, Balzano A. Coagulation disorders in liver disease. Semin Liver Dis. 2002;22(1):83–96. Scholar
  34. 34.
    Slichter SJ. Evidence-based platelet transfusion guidelines. Hematology. 2007. Scholar
  35. 35.
    Falanga A, Marchetti M, Vignoli A. Coagulation and cancer: biological and clinical aspects. J Thromb Haemost (JTH). 2013;11(2):223–33. Scholar
  36. 36.
    Falanga A, Panova-Noeva M, Russo L. Procoagulant mechanisms in tumour cells. Best Pract Res Clin Haematol. 2009;22(1):49–60. Scholar
  37. 37.
    Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hortnagel K, Pelz HJ, et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature. 2004;427(6974):537–41. Scholar
  38. 38.
    Ladu S, Calvisi DF, Conner EA, Farina M, Factor VM, Thorgeirsson SS. E2F1 inhibits c-Myc-driven apoptosis via PIK3CA/Akt/mTOR and COX-2 in a mouse model of human liver cancer. Gastroenterology. 2008;135(4):1322–32. Scholar
  39. 39.
    Dai J, Bercury KK, Macklin WB. Interaction of mTOR and Erk1/2 signaling to regulate oligodendrocyte differentiation. Glia. 2014;62(12):2096–109. Scholar
  40. 40.
    Woo HY, Heo J. Sorafenib in liver cancer. Expert Opin Pharmacother. 2012;13(7):1059–67. Scholar
  41. 41.
    Cui SX, Shi WN, Song ZY, Wang SQ, Yu XF, Gao ZH, et al. Des-gamma-carboxy prothrombin antagonizes the effects of Sorafenib on human hepatocellular carcinoma through activation of the Raf/MEK/ERK and PI3 K/Akt/mTOR signaling pathways. Oncotarget. 2016. Scholar
  42. 42.
    Inagaki Y, Tang W, Makuuchi M, Hasegawa K, Sugawara Y, Kokudo N. Clinical and molecular insights into the hepatocellular carcinoma tumour marker des-gamma-carboxyprothrombin. Liver Int. 2011;31(1):22–35. Scholar
  43. 43.
    Tamano M, Sugaya H, Oguma M, Iijima M, Yoneda M, Murohisa T, et al. Serum and tissue PIVKA-II expression reflect the biological malignant potential of small hepatocellular carcinoma. Hepatol Res. 2002;22(4):261–9.CrossRefGoogle Scholar
  44. 44.
    Marrero JA, Feng Z, Wang Y, Nguyen MH, Befeler AS, Roberts LR, et al. Alpha-fetoprotein, des-gamma carboxyprothrombin, and lectin-bound alpha-fetoprotein in early hepatocellular carcinoma. Gastroenterology. 2009;137(1):110–8. Scholar
  45. 45.
    Zhang YS, Chu JH, Cui SX, Song ZY, Qu XJ. Des-gamma-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma. Cell Physiol Biochem. 2014;34(3):903–15. Scholar
  46. 46.
    Matsubara M, Shiraha H, Kataoka J, Iwamuro M, Horiguchi S, Nishina S, et al. Des-gamma-carboxyl prothrombin is associated with tumor angiogenesis in hepatocellular carcinoma. J Gastroenterol Hepatol. 2012;27(10):1602–8. Scholar
  47. 47.
    Fujikawa T, Shiraha H, Ueda N, Takaoka N, Nakanishi Y, Matsuo N, et al. Des-gamma-carboxyl prothrombin-promoted vascular endothelial cell proliferation and migration. J Biol Chem. 2007;282(12):8741–8. Scholar
  48. 48.
    Furie B, Furie BC. Molecular basis of vitamin K-dependent gamma-carboxylation. Blood. 1990;75(9):1753–62.Google Scholar
  49. 49.
    Ueda N, Shiraha H, Fujikawa T, Takaoka N, Nakanishi Y, Suzuki M, et al. Exon 2 deletion splice variant of gamma-glutamyl carboxylase causes des-gamma-carboxy prothrombin production in hepatocellular carcinoma cell lines. Mol Oncol. 2008;2(3):241–9. Scholar
  50. 50.
    Bertino G, Ardiri AM, Boemi PM, Ierna D, Interlandi D, Caruso L, et al. A study about mechanisms of des-gamma-carboxy prothrombin’s production in hepatocellular carcinoma. Panminerva Med. 2008;50(3):221–6.Google Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Yaofu Liu
    • 1
    • 2
  • Huiling Li
    • 1
  • Jianyi Dong
    • 1
  • Lei Ma
    • 1
  • Aijun Liao
    • 1
  • Zhuona Rong
    • 1
  • Zhi Zhou
    • 1
  • Liang Cao
    • 3
  • Fujin Wang
    • 1
  • Jingyu Wang
    • 1
    Email author
  • Aiguo Wang
    • 1
    Email author
  1. 1.Department of Comparative Medicine, Laboratory Animal CenterDalian Medical UniversityDalianPeople’s Republic of China
  2. 2.Suzhou Institute of Systems Medicine, Center for Systems MedicineChinese Academy of Medical SciencesSuzhouPeople’s Republic of China
  3. 3.Department of General Surgery, The First Affiliated HospitalDalian Medical UniversityDalianPeople’s Republic of China

Personalised recommendations