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Original article
Expression of the type 3 InsP3 receptor is a final common event in the development of hepatocellular carcinoma
  1. Mateus T Guerra1,
  2. Rodrigo M Florentino2,
  3. Andressa Franca2,
  4. Antonio C Lima Filho2,
  5. Marcone L dos Santos2,
  6. Roberta C Fonseca2,
  7. Fernanda O Lemos2,
  8. Matheus C Fonseca3,
  9. Emma Kruglov1,
  10. Albert Mennone1,
  11. Basile Njei1,
  12. Joanna Gibson4,
  13. Fulan Guan5,
  14. Yung-Chi Cheng5,
  15. Meenakshisundaram Ananthanarayanam1,
  16. Jianlei Gu6,7,
  17. Jianping Jiang6,7,
  18. Hongyu Zhao6,
  19. Cristiano X Lima8,
  20. Paula T Vidigal9,
  21. Andre G Oliveira2,
  22. Michael H Nathanson1,
  23. Maria Fatima Leite2
  1. 1 Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, Connecticut, USA
  2. 2 Department of Physiology and Biophysics, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
  3. 3 Centro Nacional de Pesquisa em Energia e Materiais, Campinas, Brazil
  4. 4 Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA
  5. 5 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA
  6. 6 Department of Biostatistics, Yale University School of Medicine, New Haven, Connecticut, USA
  7. 7 Department of Bioinformatics and Biostatistics, SJTU-Yale Joint Center for Biostatistics, School of Life Science and Biotechnology, Shanghai Jiao Tong University, China
  8. 8 Department of Surgery, School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
  9. 9 Department of Pathological Anatomy and Forensic Medicine, School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
  1. Correspondence to Dr Michael H Nathanson, Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT 06519, USA; michael.nathanson{at}


Background & objectives Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC.

Design Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice.

Results ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis.

Conclusions These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.

  • liver cancer
  • calcium signaling
  • methylation
  • cell growth
  • apoptosis
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  • MTG and RMF contributed equally.

  • MHN and MFL contributed equally.

  • Contributors MTG and RMF: designed and performed experiments, analysed data and help to write the manuscript. AF, ACMLF, RCF, FOL and MCF: performed in vivo experiments and analyse data related to 5’-azacitidine treatments. MLS: performed immunohistochemistry in human tissues and analysed clinical data. EK: performed experiments and analysed data related to the ITPR3KO HepG2 cells. AM: processed DEN tissue samples and graded histological scores in HCC specimens. FG and YCC: designed and performed xenograft tumor assays in nude mice. MA: generated liver-specific ITPR3 knock out mice. JG, JJ and HZ: analysed RNA sequencing data. BN, JAG, CXL and PTV: identified and diagnosed human HCC specimens. AGO: performed in vivo calcium imaging experiments. MHN and MFL: supervised the project, formulated the hypothesis, designed experiments and edited the manuscript.

  • Funding This work was supported by grants from NIH (DK57751, DK34989, DK114041, DK112797 and OD20142 to MHN, and DK07356 to MHN and BN) and CNPq (300990/20147 to MFL). This work also was supported by the Gladys Phillips Crofoot Professorship.

  • Competing interests None declared.

  • Ethics approval All studies employing archived human liver specimens were approved by the local Human Investigation Committee at UFMG – Protocol # 71206617.8.0000.5149 and Yale University – Protocol # 1304011763). Animal studies were approved by the Institutional Animal Care and Use Committees at UFMG – Protocol #169/2014 and Yale University – Protocol # 2012-07602.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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