Journal of Gastroenterology

, Volume 49, Issue 5, pp 907–916 | Cite as

Autophagy enhances hepatocellular carcinoma progression by activation of mitochondrial β-oxidation

  • Takeo Toshima
  • Ken Shirabe
  • Yoshihiro Matsumoto
  • Shohei Yoshiya
  • Toru Ikegami
  • Tomoharu Yoshizumi
  • Yuji Soejima
  • Tetsuo Ikeda
  • Yoshihiko Maehara
Original Article—Liver, Pancreas, and Biliary Tract

Abstract

Background

Several types of cancers, including hepatocellular carcinoma (HCC), show resistance to hypoxia and nutrient starvation. Autophagy is a means of providing macromolecules for energy generation under such stressed-conditions. The aim of this study was to clarify the role of autophagy in HCC development under hypoxic conditions.

Methods

The expression of microtubule-associated protein 1 light chain 3 (LC3), which is a key gene involved in autophagosome formation, was evaluated in human HCC using immunohistochemistry and western blot. The relationship between LC3 and hypoxia-induced factor 1α (HIF1α) expression was examined using real-time PCR. In addition, human HCC cell line Huh7 was treated with pharmacological autophagy-inhibitor and inactive mutant of Atg4B (Atg4BC74A) under hypoxic condition to evaluate the effects of hypoxia-induced autophagy on cell survival, intracellular ATP, and mitochondrial β-oxidation.

Results

LC3 was significantly highly expressed in HCC as compared with noncancerous tissues. LC3 expression, correlated with HIF1α expression, was also significantly correlated with tumor size, and only in the context of large tumors, was an independent predictor of HCC recurrence after surgery. In addition, Huh7 treated with autophagy-inhibitor under hypoxia had lower viability, with low levels of intracellular ATP due to impaired mitochondrial β-oxidation.

Conclusions

Autophagy in HCC works to promote HIF1α-mediated proliferation through the maintenance of intracellular ATP, depending on the activation of mitochondrial β-oxidation. These findings demonstrated the feasibility of anti-autophagic treatment as a potential curative therapy for HCC, and improved understanding of the factors determining adaptive metabolic responses to hypoxic conditions.

Keywords

Autophagy Cancer progression Hepatocellular carcinoma 

Abbreviations

AFP

Alpha-fetoprotein

Atg

Autophagy-related genes

ATP

Adenosine 5′-triphosphate

DCP

Des-gamma-carboxy prothrombin

HCC

Hepatocellular carcinoma

HIF1α

Hypoxia-induced factor 1α

ICG R15

Indocyanine green retention test at 15 min

LC3

Microtubule-associated protein 1 light chain 3

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

PI3K

Phosphatidylinositol 3-kinase

ROS

Reactive oxygen species

SD

Standard deviation

3MA

3-Methyladenine

Notes

Acknowledgments

We are grateful to T. Yoshimori (Osaka University) for kindly providing the inactive mutant of Atg4B (Atg4BC74A). We also thank N. Yamashita (Kyushu University) for her expert advice related to statistical analysis.

Conflict of interest

The authors have no conflicts of interest to declare and have no financial interests linked to this work.

Supplementary material

535_2013_835_MOESM1_ESM.tif (1.5 mb)
Supplementary material 1 (TIFF 1520 kb)
535_2013_835_MOESM2_ESM.doc (88 kb)
Supplementary material 2 (DOC 88 kb)

References

  1. 1.
    Shirabe K, Toshima T, Taketomi A, et al. Hepatic aflatoxin B1-DNA adducts and TP53 mutations in patients with hepatocellular carcinoma despite low exposure to aflatoxin B1 in southern Japan. Liver Int. 2011;31:1366–72.PubMedCrossRefGoogle Scholar
  2. 2.
    Shirabe K, Itoh S, Yoshizumi T, et al. The predictors of microvascular invasion in candidates for liver transplantation with hepatocellular carcinoma-with special reference to the serum levels of des-gamma-carboxy prothrombin. J Surg Oncol. 2007;95:235–40.PubMedCrossRefGoogle Scholar
  3. 3.
    Shirabe K, Kajiyama K, Harimoto N, Tsujita E, Wakiyama S, Maehara Y. Risk factors for massive bleeding during major hepatectomy. World J Surg. 2010;34:1555–62.PubMedCrossRefGoogle Scholar
  4. 4.
    Bruix J, Llovet JM. Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepatology. 2002;35:519–24.PubMedCrossRefGoogle Scholar
  5. 5.
    Yamashita Y, Taketomi A, Shirabe K, et al. Outcomes of hepatic resection for huge hepatocellular carcinoma (≥10 cm in diameter). J Surg Oncol. 2011;104:292–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Taketomi A, Sanefuji K, Soejima Y, et al. Impact of des-gamma-carboxy prothrombin and tumor size on the recurrence of hepatocellular carcinoma after living donor liver transplantation. Transplantation. 2009;87:531–7.PubMedCrossRefGoogle Scholar
  7. 7.
    Vaupel P, Thews O, Hoechkel M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol. 2001;18:243–59.PubMedCrossRefGoogle Scholar
  8. 8.
    Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003;9:685–93.PubMedCrossRefGoogle Scholar
  9. 9.
    Harris AL. Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer. 2002;2:38–47.PubMedCrossRefGoogle Scholar
  10. 10.
    Kitano M, Kudo M, Maekawa K, et al. Dynamic imaging of pancreatic diseases by contrast enhanced coded phase inversion harmonic ultrasonography. Gut. 2004;53:854–9.PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–21.PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Tanida I, Tanida-Miyake E, Ueno T, Kominami E. The human homolog of Saccharomyces cerevisiae Apg7p is protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3. J Biol Chem. 2001;276:1701–6.PubMedGoogle Scholar
  13. 13.
    Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313–26.PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Fleming A, Noda T, Yoshimori T, Rubinsztein DC. Chemical modulators of autophagy as biological probes and potential therapeutics. Nat Chem Biol. 2011;7:9–17.PubMedCrossRefGoogle Scholar
  15. 15.
    Liver Cancer Study Group. The general rules for the clinical and pathological study of primary liver cancer. 7th ed. Tokyo: Kanehara Publications; 2006.Google Scholar
  16. 16.
    Fujita N, Hayashi-Nishino M, Fukumoto H, et al. An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol Biol Cell. 2008;19:4651–9.PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Aishima S, Fujita N, Mano Y, et al. Different roles of S100P overexpression in intrahepatic cholangiocarcinoma: carcinogenesis of perihilar type and aggressive behavior of peripheral type. Am J Surg Pathol. 2011;35:590–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci. 2004;117:2805–12.PubMedCrossRefGoogle Scholar
  19. 19.
    Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313–26.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 2012;8:445–544.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Anegawa G, Kawanaka H, Yoshida D, et al. Defective endothelial nitric oxide synthase signaling is mediated by rho-kinase activation in rats with secondary biliary cirrhosis. Hepatology. 2008;47:966–77.PubMedCrossRefGoogle Scholar
  22. 22.
    Sadagurski M, Cheng Z, Rozzo A, et al. IRS2 increases mitochondrial dysfunction and oxidative stress in a mouse model of Huntington disease. J Clin Invest. 2011;121:4070–81.PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Nanjundan M, Nakayama Y, Cheng KW, et al. Amplification of MDS1/EVI1 and EVI1, located in the 3q26.2 amplicon, is associated with favorable patient prognosis in ovarian cancer. Cancer Res. 2007;67:3074–84.PubMedCrossRefGoogle Scholar
  24. 24.
    Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008;4:151–75.PubMedCentralPubMedGoogle Scholar
  25. 25.
    Kasahara A, Ishikawa K, Yamaoka M, et al. Generation of trans-mitochondrial mice carrying homoplasmic mtDNAs with a missense mutation in a structural gene using ES cells. Hum Mol Genet. 2006;15:871–81.PubMedCrossRefGoogle Scholar
  26. 26.
    Du H, Yang W, Chen L, Shen B, Peng C, Li H, et al. Emerging role of autophagy during ischemia-hypoxia and reperfusion in hepatocellular carcinoma. Int J Oncol. 2012;40:2049–57.PubMedGoogle Scholar
  27. 27.
    Chang Y, Yan W, He X, Zhang L, Li C, Huang H, et al. miR-375 inhibits autophagy and reduces viability of hepatocellular carcinoma cells under hypoxic conditions. Gastroenterology. 2012;143:177–87.PubMedCrossRefGoogle Scholar
  28. 28.
    Petit PX, et al. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J Cell Biol. 1995;130:157–67.PubMedCrossRefGoogle Scholar
  29. 29.
    Hanson GT, Aggeler R, Oglesbee D, et al. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem. 2004;279:13044–53.PubMedCrossRefGoogle Scholar
  30. 30.
    Aleksunes LM, Reisman SA, Yeager RL, Goedken MJ, Klaassen CD. Nuclear factor erythroid 2-related factor 2 deletion impairs glucose tolerance and exacerbates hyperglycemia in type 1 diabetic mice. J Pharmacol Exp Ther. 2010;333:140–51.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–77.PubMedCrossRefGoogle Scholar
  32. 32.
    Kuwahara Y, Oikawa T, Ochiai Y, et al. Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy. Cell Death Dis. 2011;2:e177.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Colell A, Ricci JE, Tait S, et al. GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation. Cell. 2007;129:983–7.PubMedCrossRefGoogle Scholar
  34. 34.
    Kirkegaard K, Taylor MP, Jackson WT. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol. 2004;2:301–14.PubMedCrossRefGoogle Scholar
  35. 35.
    Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5:726–34.PubMedCrossRefGoogle Scholar
  36. 36.
    Thomas HE, Mercer CA, Carnevalli LS, Park J, Andersen JB, Conner EA, et al. mTOR inhibitors synergize on regression, reversal of gene expression, and autophagy in hepatocellular carcinoma. Sci Transl Med. 2012;4:139ra84.PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Altmeyer A, Josset E, Denis JM, Gueulette J, Slabbert J, Mutter D, Noël G, Bischoff P. The mTOR inhibitor RAD001 augments radiation-induced growth inhibition in a hepatocellular carcinoma cell line by increasing autophagy. Int J Oncol. 2012. doi:10.3892/ijo.2012.1583.
  38. 38.
    Weiner LM, Lotze MT. Tumor-cell death, autophagy, and immunity. N Engl J Med. 2012;366:1156–8.PubMedCrossRefGoogle Scholar
  39. 39.
    Lu Z, Dono K, Gotoh K, et al. Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation. Arch Histol Cytol. 2005;68:71–80.PubMedCrossRefGoogle Scholar
  40. 40.
    Degenhardt K, Mathew R, Beaudoin B, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006;10:51–64.PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Pouyssegur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006;441:437–43.PubMedCrossRefGoogle Scholar
  42. 42.
    Ding ZB, Shi YH, Zhou J, et al. Association of autophagy defect with a malignant phenotype and poor prognosis of hepatocellular carcinoma. Cancer Res. 2008;68:9167–75.PubMedCrossRefGoogle Scholar
  43. 43.
    Scarlatti F, Maffei R, Beau I, Codogno P, Ghidoni R. Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells. Cell Death Differ. 2008;15:1318–29.PubMedCrossRefGoogle Scholar
  44. 44.
    Chu CT, Zhu J, Dagda R. Beclin 1-independent pathway of damage-induced mitophagy and autophagic stress: implications for neurodegeneration and cell death. Autophagy. 2007;3:663–6.PubMedCentralPubMedGoogle Scholar
  45. 45.
    Pickford F, Masliah E, Britschgi M, et al. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. J Clin Invest. 2008;118:2190–9.PubMedCentralPubMedGoogle Scholar
  46. 46.
    Boya P, González-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005;25:1025–40.PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Ait-Mohamed O, Battisti V, Joliot V, et al. Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells. PLoS One. 2011;6:e24537.PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Mizushima N, et al. Methods for monitoring autophagy. Int J Biochem Cell Biol. 2004;36:2491–502.PubMedCrossRefGoogle Scholar
  49. 49.
    Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19:5720–8.PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Sato K, Tsuchihara K, Fujii S, et al. Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation. Cancer Res. 2007;67:9677–84.PubMedCrossRefGoogle Scholar
  51. 51.
    Esumi H, Izuishi K, Kato K, et al. Hypoxia and nitric oxide treatment confer tolerance to glucose starvation in a 5′-AMP-activated protein kinase-dependent manner. J Biol Chem. 2002;277:32791–8.PubMedCrossRefGoogle Scholar
  52. 52.
    Kuma A, Hatano M, Matsui M, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;432:1032–6.PubMedCrossRefGoogle Scholar
  53. 53.
    Rouschop KM, van den Beucken T, Dubois L, et al. The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5. J Clin Invest. 2010;120:127–41.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2013

Authors and Affiliations

  • Takeo Toshima
    • 1
  • Ken Shirabe
    • 1
  • Yoshihiro Matsumoto
    • 1
  • Shohei Yoshiya
    • 1
  • Toru Ikegami
    • 1
  • Tomoharu Yoshizumi
    • 1
  • Yuji Soejima
    • 1
  • Tetsuo Ikeda
    • 1
  • Yoshihiko Maehara
    • 1
  1. 1.Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan

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