title: Male-specific hepatitis B virus large surface protein variant W4P potentiates tumorigenicity and induces gender disparity.
abstract: BACKGROUND
  The underlying mechanisms of carcinogenesis and gender disparity in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remain unclear. Recently, we reported a novel HCC-related W4P/R mutation in the large surface protein (LHB) of HBV genotype C, which was found exclusively in male HCC patients.
  
  
  METHODS
  LHB sequences from a carrier (wild type; WT) and W4P variant LHB sequence from an HCC patient were cloned and used to generate NIH3T3 and Huh7 cell lines. Cell proliferation and in vitro tumorigenicity were assessed by cell growth and transformation assays. Male and female nude mice were injected with the cells to determine in vivo tumorigenicity. To confirm the effect of estrogen in W4P-mediated tumorigenicity, male mice were injected with estrogen and challenged with W4P-expressing cells. The serum levels of different cytokines from the mouse model and patients were analyzed by ELISA. A critical role of interleukin (IL)-6 signaling in W4P-mediated tumorigenicity was tested by inhibition of Jak2.
  
  
  RESULTS
  Although both WT and W4P variant LHBs enhanced cell proliferation by regulating the cell cycle and facilitated cell colony formation, the W4P variant demonstrated significantly higher activity. NIH3T3 cells expressing variant LHB, but not the WT, induced tumor in a nude mouse model. Tumor masses produced by variant LHB were significantly larger in male than female mice, and significantly reduced by estrogen. IL-6, but not tumor necrosis factor-α, was elevated in male mice harboring W4P-induced tumor, and was reduced by estrogen. IL-6 levels of HCC patients with the W4P variant were significantly higher than those of patients with WT LHB. W4P LHB induced higher production of IL-6 than WT LHB in cell lines, and the level was reduced by estrogen. The ability to reduce cell proliferation and colony formation of W4P LHB was hampered by inhibition of IL-6 signaling.
  
  
  CONCLUSIONS
  This study suggests that the W4P mutation during the natural course of chronic hepatitis B infection may contribute to HCC development, particularly in male patients, in an IL-6-dependent manner.
authors: Department of Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea. anny4015@gmail.com.
authors: Seoung-Ae
authors: SA
authors: Lee
authors: Department of Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea. wild0804@snu.ac.kr.
authors: Hong
authors: H
authors: Kim
authors: Department of Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea. 79wys@hanmail.net.
authors: You-Sub
authors: YS
authors: Won
authors: Department of Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea. lamseok@snu.ac.kr.
authors: Seung-Hyeok
authors: SH
authors: Seok
authors: Department of Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea. yirangna@gmail.com.
authors: YiRang
authors: Y
authors: Na
authors: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Korea. hanbo0810@khu.ac.kr.
authors: Han-Bo
authors: HB
authors: Shin
authors: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Korea. innks@khu.ac.kr.
authors: Kyung-Soo
authors: KS
authors: Inn
authors: Department of Microbiology and Immunology, Liver Research Institute, Cancer Research Institute and SNUMRC, College of Medicine, Seoul National University, Seoul, Korea. kbumjoon@snu.ac.kr.
authors: Bum-Joon
authors: BJ
authors: Kim
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002472:Cell Transformation, Viral; D004195:Disease Models, Animal; D004967:Estrogens / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D014644:Genetic Variation; D005838:Genotype; D006509:Hepatitis B / Q000150:complications / Q000821:virology; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D008113:Liver Neoplasms / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D012737:Sex Factors; D015398:Signal Transduction / Q000187:drug effects; D047368:Tumor Burden
chemical_list: D004967:Estrogens; D006514:Hepatitis B Surface Antigens; D015850:Interleukin-6
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