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

  • Original Article—Liver, Pancreas, and Biliary Tract
  • Published:
Journal of Gastroenterology Aims and scope Submit manuscript

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.

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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

References

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  4. Bruix J, Llovet JM. Prognostic prediction and treatment strategy in hepatocellular carcinoma. Hepatology. 2002;35:519–24.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  7. Vaupel P, Thews O, Hoechkel M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol. 2001;18:243–59.

    Article  CAS  PubMed  Google Scholar 

  8. Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003;9:685–93.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–21.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  13. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313–26.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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. 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  19. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313–26.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–77.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  34. Kirkegaard K, Taylor MP, Jackson WT. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol. 2004;2:301–14.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed Central  PubMed  Google Scholar 

  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. Weiner LM, Lotze MT. Tumor-cell death, autophagy, and immunity. N Engl J Med. 2012;366:1156–8.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed Central  PubMed  Google Scholar 

  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.

    CAS  PubMed Central  PubMed  Google Scholar 

  46. Boya P, González-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005;25:1025–40.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Mizushima N, et al. Methods for monitoring autophagy. Int J Biochem Cell Biol. 2004;36:2491–502.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  52. Kuma A, Hatano M, Matsui M, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;432:1032–6.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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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.

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Authors and Affiliations

  1. Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Takeo Toshima, Ken Shirabe, Yoshihiro Matsumoto, Shohei Yoshiya, Toru Ikegami, Tomoharu Yoshizumi, Yuji Soejima, Tetsuo Ikeda & Yoshihiko Maehara

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  1. Takeo Toshima
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  2. Ken Shirabe
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Correspondence to Ken Shirabe.

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Toshima, T., Shirabe, K., Matsumoto, Y. et al. Autophagy enhances hepatocellular carcinoma progression by activation of mitochondrial β-oxidation. J Gastroenterol 49, 907–916 (2014). https://doi.org/10.1007/s00535-013-0835-9

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  • DOI: https://doi.org/10.1007/s00535-013-0835-9

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