Digestive Diseases and Sciences

, Volume 60, Issue 2, pp 420–426 | Cite as

Evidence for Heightened Hexokinase II Immunoexpression in Hepatocyte Dysplasia and Hepatocellular Carcinoma

  • Grace GuzmanEmail author
  • Rohini Chennuri
  • Alexander Chan
  • Bryan Rea
  • Ada Quintana
  • Roshan Patel
  • Pei-Zhang Xu
  • Hui Xie
  • Nissim Hay
Original Article



Normal hepatocytes exhibit low-affinity hexokinase (glucokinase [HKIV]), but during oncogenesis, there is a switch from HKIV to HKII expression. The aims of this study were to compare the immunoexpression of HKII in non-dysplastic cirrhosis (NDC), liver cell change/dysplasia in cirrhosis (LCD), HCC, and normal liver control tissues, and to correlate HKII expression with clinical and histopathological parameters.


Immunohistochemistry was performed on a liver cancer progression tissue array consisting of specimens from explants with cirrhosis, including 45 tissue samples with HCC, 108 without HCC, 143 with LCD, and 8 normal liver control tissues. HKII expression was quantified as positive pixel counts/square millimeter (ppc/mm2) by image analysis.


There was a stepwise increase in HKII level from normal liver tissue to NDC, to LCD, and to HCC (p = 0.001). HKII levels were significantly higher in areas of LCD versus NDC (p ≤ 0.001), and in LCD and HCC versus NDC (p = 0.007). HKII levels were similar in LCD and HCC (p = 0.124). HKII levels were higher in grade 2–4 versus grade 1 HCCs (p = 0.044), and in pleomorphic versus non-pleomorphic HCC variants (p = 0.041). Higher levels of HKII expression in LCD and HCC versus NDC and in higher tumor grade remained significant in multivariate analysis.


Higher levels of HKII immunoexpression in LDC and HCC compared with NDC suggest that upregulation of HKII occurs during the process of hepatocarcinogenesis in humans. In HCC, higher levels of HKII are associated with more aggressive histological features.


Hepatocellular carcinoma Liver cell change/dysplasia in cirrhosis Non-dysplastic cirrhosis Hexokinase II immunohistochemistry Tissue microarray Image analysis 



This Project was supported by the University of Illinois at Chicago (UIC) Center for Clinical and Translational Sciences (CCTS) to G.G., N.H., and H.X. Award number ULRR029879 from the National Center for Research Resources, by Grants CA090764, AG025953 from the National Institutes of Health to N.H., and VA merit award BX000733 to N.H. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. The authors recognize the contribution of the Research Histology and Tissue Imaging Core at the University of Illinois at Chicago to the completion of this Project (GG/gbg08232014).

Conflict of interest



  1. 1.
    Altekruse SF, McGlynn KA, Reichman ME. Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. J Clin Oncol. 2009;27:1485–1491.PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet. 2002;31:339–346.PubMedCrossRefGoogle Scholar
  3. 3.
    El-Serag HB. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 2002;36:S74–S83.Google Scholar
  4. 4.
    Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet. 2003;362:1907–1917.PubMedCrossRefGoogle Scholar
  5. 5.
    Starley BQ, Calcagno CJ, Harrison SA. Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection. Hepatology. 2010;51:1820–1832.PubMedCrossRefGoogle Scholar
  6. 6.
    Bugianesi E. Non-alcoholic steatohepatitis and cancer. Clin Liver Dis. 2007;11:191–207, x–xi.Google Scholar
  7. 7.
    Takamatsu S, Noguchi N, Kudoh A, et al. Influence of risk factors for metabolic syndrome and non-alcoholic fatty liver disease on the progression and prognosis of hepatocellular carcinoma. Hepatogastroenterology. 2008;55:609–614.PubMedGoogle Scholar
  8. 8.
    Guzman G, Brunt EM, Petrovic LM, Chejfec G, Layden TJ, Cotler SJ. Does nonalcoholic fatty liver disease predispose patients to hepatocellular carcinoma in the absence of cirrhosis? Arch Pathol Lab Med. 2008;132:1761–1766.PubMedGoogle Scholar
  9. 9.
    Torres DM, Harrison SA. Nonalcoholic steatohepatitis and noncirrhotic hepatocellular carcinoma: fertile soil. Semin Liver Dis. 2012;32:30–38.PubMedCrossRefGoogle Scholar
  10. 10.
    Rosmorduc O, Fartoux L. HCC and NASH: how strong is the clinical demonstration? Clin Res Hepatol Gastroenterol. 2012;36:202–208.PubMedCrossRefGoogle Scholar
  11. 11.
    Chu CA, Fujimoto Y, Igawa K, et al. Rapid translocation of hepatic glucokinase in response to intraduodenal glucose infusion and changes in plasma glucose and insulin in conscious rats. Am J Physiol Gastrointest Liver Physiol. 2004;286:G627–G634.PubMedCrossRefGoogle Scholar
  12. 12.
    Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 2004;4:891–899.PubMedCrossRefGoogle Scholar
  13. 13.
    Bustamante E, Pedersen PL. High aerobic glycolysis of rat hepatoma cells in culture: role of mitochondrial hexokinase. Proc Natl Acad Sci U S A. 1977;74:3735–3739.PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Robey RB, Hay N. Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt. Oncogene. 2006;25:4683–4696.PubMedCrossRefGoogle Scholar
  15. 15.
    Robey RB, Hay N. Mitochondrial hexokinases: guardians of the mitochondria. Cell Cycle. 2005;4:654–658.PubMedCrossRefGoogle Scholar
  16. 16.
    Dombrowski F, Filsinger E, Bannasch P, Pfeifer U. Altered liver acini induced in diabetic rats by portal vein islet isografts resemble preneoplastic hepatic foci in their enzymic pattern. Am J Pathol. 1996;148:1249–1256.PubMedCentralPubMedGoogle Scholar
  17. 17.
    Goel A, Mathupala SP, Pedersen PL. Glucose metabolism in cancer. Evidence that demethylation events play a role in activating type II hexokinase gene expression. J Biol Chem. 2003;278:15333–15340.PubMedCrossRefGoogle Scholar
  18. 18.
    Mathupala SP, Rempel A, Pedersen PL. Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase. J Bioenerg Biomembr. 1997;29:339–343.PubMedCrossRefGoogle Scholar
  19. 19.
    Brown RS, Goodman TM, Zasadny KR, Greenson JK, Wahl RL. Expression of hexokinase II and Glut-1 in untreated human breast cancer. Nucl Med Biol. 2002;29:443–453.PubMedCrossRefGoogle Scholar
  20. 20.
    Engles JM, Quarless SA, Mambo E, Ishimori T, Cho SY, Wahl RL. Stunning and its effect on 3H-FDG uptake and key gene expression in breast cancer cells undergoing chemotherapy. J Nucl Med. 2006;47:603–608.PubMedGoogle Scholar
  21. 21.
    Kim JE, Ahn BC, Hwang MH, et al. Combined RNA interference of hexokinase II and (131)I-sodium iodide symporter gene therapy for anaplastic thyroid carcinoma. J Nucl Med. 2011;52:1756–1763.PubMedCrossRefGoogle Scholar
  22. 22.
    Peng SY, Lai PL, Pan HW, Hsiao LP, Hsu HC. Aberrant expression of the glycolytic enzymes aldolase B and type II hexokinase in hepatocellular carcinoma are predictive markers for advanced stage, early recurrence and poor prognosis. Oncol Rep. 2008;19:1045–1053.PubMedGoogle Scholar
  23. 23.
    Guzman G, Chan A, Chennuri R, Patel R, Hay N, Cotler S. High level of hexokinase 2 (HK2) is observed in diabetes (DM), biologically aggressive hepatocellular carcinomas (HCCs), and in the progression of HCCs. e-poster European Association for the Study of the Liver, Nice, France, Jun 28–29, 2011.
  24. 24.
    Kwee SA, Hernandez B, Chan O, Wong L. Choline kinase alpha and hexokinase-2 protein expression in hepatocellular carcinoma: association with survival. PLoS ONE. 2012;7:e46591.PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Gong L, Cui Z, Chen P, Han H, Peng J, Leng X. Reduced survival of patients with hepatocellular carcinoma expressing hexokinase II. Med Oncol. 2012;29:909–914.PubMedCrossRefGoogle Scholar
  26. 26.
    Ruby SG. Protocol for the examination of specimens from patients with hepatocellular carcinoma and cholangiocarcinoma, including intrahepatic bile ducts. Cancer Committee of the College of American Pathologists. Arch Pathol Lab Med. 2000;124:41–45.PubMedGoogle Scholar
  27. 27.
    Park YN, Roncalli M. Large liver cell dysplasia: a controversial entity. J Hepatol. 2006;45:734–743.PubMedCrossRefGoogle Scholar
  28. 28.
    Roncalli M, Borzio M, Tombesi MV, Ferrari A, Servida E. A morphometric study of liver cell dysplasia. Hum Pathol. 1988;19:471–474.PubMedCrossRefGoogle Scholar
  29. 29.
    Tiniakos DG, Brunt EM. Proliferating cell nuclear antigen and Ki-67 labeling in hepatocellular nodules: a comparative study. Liver. 1999;19:58–68.PubMedCrossRefGoogle Scholar
  30. 30.
    Gong L, Li YH, Su Q, Chu X, Zhang W. Clonality of nodular lesions in liver cirrhosis and chromosomal abnormalities in monoclonal nodules of altered hepatocytes. Histopathology. 2010;56:589–599.PubMedCrossRefGoogle Scholar
  31. 31.
    Anthony PP, Vogel CL, Barker LF. Liver cell dysplasia: a premalignant condition. J Clin Pathol. 1973;26:217–223.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Anthony PP. Precursor lesions for liver cancer in humans. Cancer Res. 1976;36:2579–2583.Google Scholar
  33. 33.
    Rebello Pinto M, Koelma IA, Kumar D. Fine needle aspiration of focal liver lesions. Cytopathology 1994;5:359–368.Google Scholar
  34. 34.
    Sobin LH, Compton CC. TNM seventh edition: what’s new, what’s changed: communication from the International Union Against Cancer and the American Joint Committee on Cancer. Cancer. 2010;116:5336–5339.PubMedCrossRefGoogle Scholar
  35. 35.
    Ferrell LD. Benign and Malignant Tumors of the Liver. In: Odze RD, Goldblum JR, Crawford R, eds. Surgical pathology of the GI tract, liver, biliary tract, and pancreas. Philadelphia: Saunders; 2004:999–1014.Google Scholar
  36. 36.
    Ferrell L. Malignant liver tumors that mimic benign lesions: analysis of five distinct lesions. Semin Diagn Pathol. 1995;12:64–76.PubMedGoogle Scholar
  37. 37.
    Vogel UF, Bueltmann BD. Simple, inexpensive, and precise paraffin tissue microarrays constructed with a conventional microcompound table and a drill grinder. Am J Clin Pathol. 2006;126:342–348.PubMedCrossRefGoogle Scholar
  38. 38.
    Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol. 1981;75:734–738.PubMedGoogle Scholar
  39. 39.
    Uhlen M, Oksvold P, Fagerberg L, et al. Towards a knowledge-based Human Protein Atlas. Nat Biotechnol. 2010;28:1248–1250.PubMedCrossRefGoogle Scholar
  40. 40.
    Theodorsson-Norheim E. Friedman and Quade tests: BASIC computer program to perform nonparametric two-way analysis of variance and multiple comparisons on ranks of several related samples. Comput Biol Med. 1987;17:85–99.PubMedCrossRefGoogle Scholar
  41. 41.
    Yasuda S, Arii S, Mori A, et al. Hexokinase II and VEGF expression in liver tumors: correlation with hypoxia-inducible factor 1 alpha and its significance. J Hepatol. 2004;40:117–123.PubMedCrossRefGoogle Scholar
  42. 42.
    Majors BS, Betenbaugh MJ, Chiang GG. Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering. Metab Eng. 2007;9:317–326.PubMedCrossRefGoogle Scholar
  43. 43.
    Kim W, Yoon JH, Jeong JM, et al. Apoptosis-inducing antitumor efficacy of hexokinase II inhibitor in hepatocellular carcinoma. Mol Cancer Ther. 2007;6:2554–2562.PubMedCrossRefGoogle Scholar
  44. 44.
    Shergill IS, Shergill NK, Arya M, Patel HR. Tissue microarrays: a current medical research tool. Curr Med Res Opin. 2004;20:707–712.PubMedCrossRefGoogle Scholar
  45. 45.
    Jawhar NM. Tissue Microarray: a rapidly evolving diagnostic and research tool. Ann Saudi Med. 2009;29:123–127.PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Hewitt SM. Tissue microarrays as a tool in the discovery and validation of predictive biomarkers. Methods Mol Biol. 2012;823:201–214.PubMedCrossRefGoogle Scholar
  47. 47.
    Zhang D, Salto-Tellez M, Putti TC, Do E, Koay ES. Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer. Mod Pathol. 2003;16:79–84.PubMedCrossRefGoogle Scholar
  48. 48.
    Riddle SR, Ahmad A, Ahmad S, et al. Hypoxia induces hexokinase II gene expression in human lung cell line A549. Am J Physiol Lung Cell Mol Physiol. 2000;278:L407–L416.PubMedGoogle Scholar
  49. 49.
    Patra KC, Wang Q, Bhaskar PT, et al. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer. Cancer Cell. 2013;24:213–228.PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Gwak GY, Yoon JH, Kim KM, Lee HS, Chung JW, Gores GJ. Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression. J Hepatol. 2005;42:358–364.PubMedCrossRefGoogle Scholar
  51. 51.
    Gosmain Y, Lefai E, Ryser S, Roques M, Vidal H. Sterol regulatory element-binding protein-1 mediates the effect of insulin on hexokinase II gene expression in human muscle cells. Diabetes. 2004;53:321–329.PubMedCrossRefGoogle Scholar
  52. 52.
    Kemmer N, Neff G, Secic M, Zacharias V, Kaiser T, Buell J. Ethnic differences in hepatocellular carcinoma: implications for liver transplantation. Dig Dis Sci. 2008;53:551–555.PubMedCrossRefGoogle Scholar
  53. 53.
    Sloane D, Chen H, Howell C. Racial disparity in primary hepatocellular carcinoma: tumor stage at presentation, surgical treatment and survival. J Natl Med Assoc. 2006;98:1934–1939.PubMedCentralPubMedGoogle Scholar
  54. 54.
    Artinyan A, Mailey B, Sanchez-Luege N, et al. Race, ethnicity, and socioeconomic status influence the survival of patients with hepatocellular carcinoma in the United States. Cancer. 2010;116:1367–1377.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Grace Guzman
    • 1
    Email author
  • Rohini Chennuri
    • 1
  • Alexander Chan
    • 1
  • Bryan Rea
    • 1
  • Ada Quintana
    • 1
  • Roshan Patel
    • 1
  • Pei-Zhang Xu
    • 2
  • Hui Xie
    • 3
  • Nissim Hay
    • 2
  1. 1.Pathology, College of Medicine, Cancer CenterUniversity of Illinois Hospital and Health Science SystemChicagoUSA
  2. 2.Biochemistry and Molecular Genetics, College of MedicineUniversity of Illinois Hospital and Health Science SystemChicagoUSA
  3. 3.Epidemiology and Biostatistics, School of Public HealthUniversity of Illinois Hospital and Health Science SystemChicagoUSA

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