Tumor Biology

, Volume 37, Issue 4, pp 4295–4304 | Cite as

Glucose-regulated protein 94 mediates cancer progression via AKT and eNOS in hepatocellular carcinoma

  • Chien-Yu Huang
  • Uyanga Batzorig
  • Wan-Li Cheng
  • Ming-Te Huang
  • Wei- Yu Chen
  • Po-Li Wei
  • Yu-Jia Chang
Original Article


Hepatocellular carcinoma (HCC) is a crucial health issue worldwide. High glucose-regulated protein 94 (GRP94) expression has been observed in different types of cancer, suggesting a link between tumor progression and GRP94 expression. However, the mechanisms underlying the role of GRP94 in HCC progression remain unclear. We used specific small hairpin RNA (shRNA) to manipulate GRP94 expression in HCC cells. Tissue arrays, MTT assays, xCELLigence assays, and in vivo xenograft model were performed to identify clinicopathological correlations and to analyze cell growth. We found that high GRP94 expression reflected a poor response and a lower survival rate. In vitro and in vivo studies showed that silencing GRP94 suppressed cancer progression. Mechanistically, GRP94 knockdown reduced AKT, phospho-AKT, and eNOS levels but did not influence the AMPK pathway. Our results demonstrated that GRP94 is a key molecule in HCC progression that modulates the AKT pathway and eNOS levels. Our findings suggest that GRP94 may be a new prognostic and therapeutic target for HCC.


GRP94 HCC eNOS AKT Proliferation 



Glucose-regulated protein 94


Hepatocellular carcinoma



This work was supported by grants from the Ministry of Science and Technology, ROC (NSC101-2314-B-038-030-MY2 and MOST103-2314-B-038-039), and Taipei Medical University–Shuang Ho Hospital (104TMU-SHH-12).


  1. 1.
    Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59:225–49.CrossRefPubMedGoogle Scholar
  2. 2.
    El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007;132:2557–76.CrossRefPubMedGoogle Scholar
  3. 3.
    Hung H. Treatment modalities for hepatocellular carcinoma. Curr Cancer Drug Targets. 2005;5:131–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet. 2003;362:1907–17.CrossRefPubMedGoogle Scholar
  5. 5.
    Llovet JM, Bru C, Bruix J. Prognosis of hepatocellular carcinoma: the BCLC staging classification. Semin Liver Dis. 1999;19:329–38.CrossRefPubMedGoogle Scholar
  6. 6.
    Bartolozzi C, Lencioni R. Ethanol injection for the treatment of hepatic tumours. Eur Radiol. 1996;6:682–96.PubMedGoogle Scholar
  7. 7.
    McGhana JP, Dodd 3rd GD. Radiofrequency ablation of the liver: current status. AJR Am J Roentgenol. 2001;176:3–16.CrossRefPubMedGoogle Scholar
  8. 8.
    Okada S. Local ablation therapy for hepatocellular carcinoma. Semin Liver Dis. 1999;19:323–8.CrossRefPubMedGoogle Scholar
  9. 9.
    Bismuth H, Majno PE. Hepatobiliary surgery. J Hepatol. 2000;32:208–24.CrossRefPubMedGoogle Scholar
  10. 10.
    Rossi S, Di Stasi M, Buscarini E, Quaretti P, Garbagnati F, Squassante L, et al. Percutaneous RF interstitial thermal ablation in the treatment of hepatic cancer. AJR Am J Roentgenol. 1996;167:759–68.CrossRefPubMedGoogle Scholar
  11. 11.
    Choi D, Lim HK, Kim MJ, Lee SH, Kim SH, Lee WJ, et al. Recurrent hepatocellular carcinoma: percutaneous radiofrequency ablation after hepatectomy. Radiology. 2004;230:135–41.CrossRefPubMedGoogle Scholar
  12. 12.
    Minami Y, Kudo M. Radiofrequency ablation of hepatocellular carcinoma: a literature review. Int J Hepatol. 2011;2011:104685.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Solmi L, Nigro G, Roda E. Therapeutic effectiveness of echo-guided percutaneous radiofrequency ablation therapy with a LeVeen needle electrode in hepatocellular carcinoma. World J Gastroenterol. 2006;12:1098–104.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Samuel M, Chow PK, Chan Shih-Yen E, Machin D, Soo KC. Neoadjuvant and adjuvant therapy for surgical resection of hepatocellular carcinoma. Cochrane Database Syst Rev. 2009:CD001199.Google Scholar
  15. 15.
    Lamb JR, Bal V, Mendez-Samperio P, Mehlert A, So A, Rothbard J, et al. Stress proteins may provide a link between the immune response to infection and autoimmunity. Int Immunol. 1989;1:191–6.CrossRefPubMedGoogle Scholar
  16. 16.
    Pockley AG, Muthana M, Calderwood SK. The dual immunoregulatory roles of stress proteins. Trends Biochem Sci. 2008;33:71–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Wang XP, Liu GZ, Song AL, Chen RF, Li HY, Liu Y. Expression and significance of heat shock protein 70 and glucose-regulated protein 94 in human esophageal carcinoma. World J Gastroenterol. 2005;11:429–32.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Heike M, Frenzel C, Meier D, Galle PR. Expression of stress protein gp96, a tumor rejection antigen, in human colorectal cancer. Int J Cancer. 2000;86:489–93.CrossRefPubMedGoogle Scholar
  19. 19.
    Chang JT, Chan SH, Lin CY, Lin TY, Wang HM, Liao CT, et al. Differentially expressed genes in radioresistant nasopharyngeal cancer cells: Gp96 and gdf15. Mol Cancer Ther. 2007;6:2271–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Gazit G, Lu J, Lee AS. De-regulation of GRP stress protein expression in human breast cancer cell lines. Breast Cancer Res Treat. 1999;54:135–46.CrossRefPubMedGoogle Scholar
  21. 21.
    Zheng HC, Takahashi H, Li XH, Hara T, Masuda S, Guan YF, et al. Overexpression of GRP78 and GRP94 are markers for aggressive behavior and poor prognosis in gastric carcinomas. Hum Pathol. 2008;39:1042–9.CrossRefPubMedGoogle Scholar
  22. 22.
    Chen WT, Tseng CC, Pfaffenbach K, Kanel G, Luo B, Stiles BL, et al. Liver-specific knockout of GRP94 in mice disrupts cell adhesion, activates liver progenitor cells, and accelerates liver tumorigenesis. Hepatology. 2014;59:947–57.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Rachidi S, Sun S, Wu BX, Jones E, Drake RR, Ogretmen B, et al. Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis. J Hepatol. 2015;62:879–88.CrossRefPubMedGoogle Scholar
  24. 24.
    Chiou JF, Tai CJ, Huang MT, Wei PL, Wang YH, An J, et al. Glucose-regulated protein 78 is a novel contributor to acquisition of resistance to sorafenib in hepatocellular carcinoma. Ann Surg Oncol. 2010;17:603–12.CrossRefPubMedGoogle Scholar
  25. 25.
    Chang YJ, Chiu CC, Wu CH, An J, Wu CC, Liu TZ, et al. Glucose-regulated protein 78 (GRP78) silencing enhances cell migration but does not influence cell proliferation in hepatocellular carcinoma. Ann Surg Oncol. 2010;17:1703–9.CrossRefPubMedGoogle Scholar
  26. 26.
    Wang SK, Liang PH, Astronomo RD, Hsu TL, Hsieh SL, Burton DR, et al. Targeting the carbohydrates on hiv-1: interaction of oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN. Proc Natl Acad Sci U S A. 2008;105:3690–5.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, Kohler K, et al. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol. 2008;10:211–9.CrossRefPubMedGoogle Scholar
  28. 28.
    Wei PL, Chang YJ, Ho YS, Lee CH, Yang YY, An J, et al. Tobacco-specific carcinogen enhances colon cancer cell migration through alpha7-nicotinic acetylcholine receptor. Ann Surg. 2009;249:978–85.CrossRefPubMedGoogle Scholar
  29. 29.
    Tai CJ, Wang JW, Su HY, Wang CK, Wu CT, Lien YC, et al. Glucose-regulated protein 94 modulates the therapeutic efficacy to taxane in cervical cancer cells. Tumour Biol: J Int Soc Oncodev Biol Med. 2014;35:403–10.CrossRefGoogle Scholar
  30. 30.
    Chang YJ, Li LT, Chen HA, Hung CS, Wei PL. Silencing survivin activates autophagy as an alternative survival pathway in HCC cells. Tumour Biol: J Int Soc Oncodev Biol Med. 2014;35:9957–66.CrossRefGoogle Scholar
  31. 31.
    Chang YJ, Huang CY, Hung CS, Chen WY, Wei PL. Grp78 mediates the therapeutic efficacy of curcumin on colon cancer. Tumour Biol: J Int Soc Oncodevel Biol Med. 2014.Google Scholar
  32. 32.
    Kuo LJ, Hung CS, Chen WY, Chang YJ, Wei PL. Glucose-regulated protein 78 silencing down-regulates vascular endothelial growth factor/vascular endothelial growth factor receptor 2 pathway to suppress human colon cancer tumor growth. J Surg Res. 2013;185:264–72.CrossRefPubMedGoogle Scholar
  33. 33.
    Lim KH, Ancrile BB, Kashatus DF, Counter CM. Tumour maintenance is mediated by eNOS. Nature. 2008;452:646–9.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Behne T, Copur MS. Biomarkers for hepatocellular carcinoma. Int J Hepatol. 2012;2012:859076.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Lin CY, Lin TY, Wang HM, Huang SF, Fan KH, Liao CT, et al. Gp96 is over-expressed in oral cavity cancer and is a poor prognostic indicator for patients receiving radiotherapy. Radiat Oncol (Lond, Engl). 2011;6:136.CrossRefGoogle Scholar
  36. 36.
    Wang HX, Liu YF, Yang SJ, Duan CG, Wang YX, Zhao J, et al. Expression of Hsp70 GRP94 and igg in human lung carcinoma. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi = Chin J Cell Mol Immunol. 2008;24:447–9.Google Scholar
  37. 37.
    Li J, Lee AS. Stress induction of GRP78/BiP and its role in cancer. Curr Mol Med. 2006;6:45–54.CrossRefPubMedGoogle Scholar
  38. 38.
    Srivastava PK. Therapeutic cancer vaccines. Curr Opin Immunol. 2006;18:201–5.CrossRefPubMedGoogle Scholar
  39. 39.
    Pan Z, Erkan M, Streit S, Friess H, Kleeff J. Silencing of GRP94 expression promotes apoptosis in pancreatic cancer cells. Int J Oncol. 2009;35:823–8.PubMedGoogle Scholar
  40. 40.
    Fu Z, Zhen H, Zou F, Wang X, Chen Y, Liu L. Involvement of the AKT signaling pathway in ER-alpha36/GRP94-mediated signaling in gastric cancer. Oncol Lett. 2014;8:2077–80.PubMedPubMedCentralGoogle Scholar
  41. 41.
    Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer. 2002;2:489–501.CrossRefPubMedGoogle Scholar
  42. 42.
    Janku F, Kaseb AO, Tsimberidou AM, Wolff RA, Kurzrock R. Identification of novel therapeutic targets in the PI3K/AKT/mTOR pathway in hepatocellular carcinoma using targeted next generation sequencing. Oncotarget. 2014;5:3012–22.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Ying L, Hofseth LJ. An emerging role for endothelial nitric oxide synthase in chronic inflammation and cancer. Cancer Res. 2007;67:1407–10.CrossRefPubMedGoogle Scholar
  44. 44.
    Patel PD, Yan P, Seidler PM, Patel HJ, Sun W, Yang C, et al. Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2. Nat Chem Biol. 2013;9:677–84.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Duerfeldt AS, Peterson LB, Maynard JC, Ng CL, Eletto D, Ostrovsky O, et al. Development of a GRP94 inhibitor. J Am Chem Soc. 2012;134:9796–804.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Hua Y, White-Gilbertson S, Kellner J, Rachidi S, Usmani SZ, Chiosis G, et al. Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma. Clin Cancer Res. 2013;19:6242–51.CrossRefPubMedGoogle Scholar
  47. 47.
    Muth A, Crowley V, Khandelwal A, Mishra S, Zhao J, Hall J, et al. Development of radamide analogs as GRP94 inhibitors. Bioorg Med Chem. 2014;22:4083–98.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Polivka Jr J, Janku F. Molecular targets for cancer therapy in the pI3K/AKT/mTOR pathway. Pharmacol Ther. 2014;142:164–75.CrossRefPubMedGoogle Scholar
  49. 49.
    Lampson BL, Kendall SD, Ancrile BB, Morrison MM, Shealy MJ, Barrientos KS, et al. Targeting eNOS in pancreatic cancer. Cancer Res. 2012;72:4472–82.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Chien-Yu Huang
    • 1
  • Uyanga Batzorig
    • 2
    • 3
    • 11
  • Wan-Li Cheng
    • 4
  • Ming-Te Huang
    • 1
    • 3
    • 5
    • 6
  • Wei- Yu Chen
    • 3
    • 9
    • 10
  • Po-Li Wei
    • 5
    • 6
    • 7
    • 8
  • Yu-Jia Chang
    • 3
    • 5
    • 6
    • 7
  1. 1.Division of General Surgery, Department of Surgery, Shuang Ho HospitalTaipei Medical UniversityTaipeiTaiwan
  2. 2.Mongolian National University of Medical SciencesUlaanbaatarMongolia
  3. 3.Graduate Institute of Clinical Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  4. 4.Department of Internal Medicine, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  5. 5.Department of Surgery, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  6. 6.Division of General SurgeryTaipei Medical University and HospitalTaipei CityTaiwan
  7. 7.Cancer Research CenterTaipei Medical University and HospitalTaipei CityTaiwan
  8. 8.Graduate Institute of Cancer Biology and Drug DiscoveryTaipei Medical UniversityTaipeiTaiwan
  9. 9.Department of Pathology, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  10. 10.Department of Pathology, Wan Fang HospitalTaipei Medical UniversityTaipeiTaiwan
  11. 11.National Dermatology CenterUlaanbaatarMongolia

Personalised recommendations