慢性B型肝炎感染是形成肝癌的主要危險因子之一。在此研究中，我們提出實驗證據指出病毒大表面抗原蛋白(viral large surface antigen, LHBS)促進肝細胞帶有超倍體(hyperploidy)進而助於肝細胞癌化(hepatocarcinogenesis)。首先，我們發現帶有HBV基因組的兩種肝癌細胞株HepG2.2.15和1.3ES2，其細胞基因組數量都有增加。透過觀察細胞有絲分裂進程，我們發現其帶有多倍體的細胞是來自於有絲分裂期的胞質分裂失敗(cytokinesis)所導致。我們發現當肝癌細胞被轉染含有HBV全基因組(complete HBV genome)的質體，會增加胞質分裂失敗的機率；但在被轉染含有敲除HBV基因組(HBV-knockout genome)以及被轉染含有敲除病毒表面抗原基因組(HBsAg-deficient genome)的細胞不會增加胞質分裂失敗的比例。下一步，我們發現單獨表現病毒大表面抗原蛋白就足以增加肝細胞基因組數。我們的實驗結果指出，表現LHBS蛋白的細胞會帶著細胞間期(interphase)產生的DNA損傷進入有絲分裂期(mitosis)，造成接下來的染色體結構異常(chromosome structure aberration)，以及形成之後的染色質橋(chromatin bridge)。此外，我們發現在有LHBS表現的細胞中，polo-like kinase 1(Plk1)激酶蛋白高度活化，使細胞藉由Plk1的調控繞過G2/M檢查點，將未解除的DNA損傷帶入有絲分裂期。我們發現，利用小分子抑制劑抑制Plk1的活性，可有選擇性地對表現LHBS蛋白的細胞造成細胞毒殺、阻止其非貼附性生長(anchorage-independent growth)能力、以及限制其在小鼠異體腫瘤(xenograft tumorigenesis)的生長。最後，我們對慢性B肝患者肝切片進行組織免疫螢光染色，並利用影像細胞計數(image cytometry)量測其肝細胞核大小、分析與LHBS蛋白表現的關係，在13位病患組織切片中，有10位帶有LHBS的肝細胞核大於未表現LHBS蛋白的肝細胞。此外，在兩種HBV實驗動物模式(土撥鼠實驗動物模式、嵌合人類肝細胞小鼠實驗動物模式)的肝組織切片，我們也發現相似的實驗分析結果。總結的說，這些結果揭示LHBS蛋白影響肝細胞形成超倍體的角色，也指出表現在肝細胞中的LHBS蛋白可以做為慢性B肝感染患者的癌前病變標記。

Chronic Hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC). In this study we provide evidence that viral large surface antigen (LHBS) promotes hepatocyte hyperploidy and thereby contributes hepatocarcinogenesis. First, increased ploidy content was detected in two HBV-positive hepatoma cell lines HepG2.2.15 and 1.3ES2. Through monitoring mitotic progression, we found that polyploid cells are derived from cells failed in cytokinesis. Cells transfected with complete HBV genome increased cytokinesis failure, which effect was not detected in cells transfected with complete HBV-knockout and HBsAg-deficient mutant. Next, we showed that expression of viral LHBS alone is sufficient to promote polyploidy through the induction of cytokinesis failure. Our data indicated that LHBS-expressing cells persevered pre-mitotic DNA damages into mitosis, created subsequent chromosome structural aberrations, and then increased chromatin bridge formation. To explain persisted DNA damages and successive mitosis entry, we found that polo-like kinase 1 hyperactivated and expressed in LHBS cells, which could bypass G2/M checkpoint in the presence of unresolved DNA damages. Notably, inhibition of polo-like kinase 1 not only caused a significant cytotoxicity in LHBS-expressing cells, but also blocked anchorage-independent growth and thereby restrain LHBS-induced xenograft tumorigenesis. Finally, we measured hepatocyte DNA content in cirrhotic livers and analyzed by image cytometry. LHBS-positive hepatocytes displayed increased genome content in 10 out of 13 patients with chronic hepatitis B. Similar outcomes were also observed from two animal model (woodchuck hepatitis B and HBV-infected mice with chimeric humanized liver). Taken together, these results demonstrate the role of LHBS in the development of hepatocyte hyperploidy. In conclusion, our data indicate that viral LHBS may serve a pre-malignant indicator in patients with chronic HBV infection.

Contents ---------------------------------------------------------------------------------------------- 1
Abstract ----------------------------------------------------------------------------------------------- 2
中文摘要-----------------------------------------------------------------------------------------------------------3
Introduction -------------------------------------------------------------------------------------------4
1. Hepatitis B virus--------------------------------------------------------------------------------4
2. Polyploidy, aneuploidy in development and cancer----------------------------------------8
3. Hepatocyte polyploidization------------------------------------------------------------------9
4. Liver cell dysplasia and hepatocellular carcinoma---------------------------------------10
5. Large HBV Surface antigen (LHBS) and its clinical relevance-------------------------11
Specific Aims ---------------------------------------------------------------------------------------14
Materials and Methods-----------------------------------------------------------------------------15
Results -----------------------------------------------------------------------------------------------20
1. HBV-positive hepatocytes displayed increased genome content-----------------------20
2. Viral LHBS increased hepatocyte ploidy through the induction of cytokinesis failure------21
3. LHBS induces aberrant anaphase chromatin bridges-------------------------------------22
4. DNA damages persist into mitosis in LHBS cells----------------------------------------23
5. Upregulation of Plk1 and Cdk1 override the G2/M checkpoint in LHBS cells------23
6. Inhibition of Plk1 blocked LHBS-mediated clonogenic cell survival and xenograft
tumorigenesis-----------------------------------------------------------------------------------------------25
7. Clinical relevance of viral LHBS expression on Chronic HBV patients--------------26
Discussions ------------------------------------------------------------------------------------------27
1. Hepatocyte hyperploidy and its roles in hepatocellular carcinoma---------------------27
2. Plk1 and its potential roles in HBV-related HCC-----------------------------------------28
3. The oncogenic role of HBV LHBS protein------------------------------------------------29
4. Clinical prospect and possible aspects in this study--------------------------------------30
References -------------------------------------------------------------------------------------------31
Figures -----------------------------------------------------------------------------------------------38
Tables -------------------------------------------------------------------------------------------------------------61
Appendix -------------------------------------------------------------------------------------------------------- 63
CV-----------------------------------------------------------------------------------------------------------------64

1 Seeger, C. & Mason, W. S. Molecular biology of hepatitis B virus infection. Virology 479-480, 672-686, doi:10.1016/j.virol.2015.02.031 (2015).
2 World Health Organization. Hepatitis B Fact Sheet, <http://www.who.int/en/news-room/fact-sheets/detail/hepatitis-b> (2018 ).
3 Kim, G. A. et al. HBsAg seroclearance after nucleoside analogue therapy in patients with chronic hepatitis B: clinical outcomes and durability. Gut 63, 1325-1332, doi:10.1136/gutjnl-2013-305517 (2014).
4 Chau, K. H., Hargie, M. P., Decker, R. H., Mushahwar, I. K. & Overby, L. R. Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc. Hepatology 3, 142-149 (1983).
5 Mommeja‐Marin, H., Mondou, E., Blum, M. R. & Rousseau, F. Serum HBV DNA as a marker of efficacy during therapy for chronic HBV infection: analysis and review of the literature. Hepatology 37, 1309-1319 (2003).
6 Lucifora, J. & Protzer, U. Attacking hepatitis B virus cccDNA–The holy grail to hepatitis B cure. Journal of hepatology 64, S41-S48 (2016).
7 Chang, J., Guo, F., Zhao, X. & Guo, J. T. Therapeutic strategies for a functional cure of chronic hepatitis B virus infection. Acta Pharm Sin B 4, 248-257, doi:10.1016/j.apsb.2014.05.002 (2014).
8 Ringelhan, M., O'Connor, T., Protzer, U. & Heikenwalder, M. The direct and indirect roles of HBV in liver cancer: prospective markers for HCC screening and potential therapeutic targets. J Pathol 235, 355-367, doi:10.1002/path.4434 (2015).
9 Arzumanyan, A., Reis, H. M. & Feitelson, M. A. Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma. Nat Rev Cancer 13, 123-135, doi:10.1038/nrc3449 (2013).
10 Kondo, Y., Ninomiya, M., Kakazu, E., Kimura, O. & Shimosegawa, T. Hepatitis B surface antigen could contribute to the immunopathogenesis of hepatitis B virus infection. ISRN gastroenterology 2013 (2013).
11 Op den Brouw, M. L. et al. Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus. Immunology 126, 280-289, doi:10.1111/j.1365-2567.2008.02896.x (2009).
12 Tu, T., Budzinska, M. A., Shackel, N. A. & Urban, S. HBV DNA Integration: Molecular Mechanisms and Clinical Implications. Viruses 9, 75, doi:10.3390/v9040075 (2017).
13 Lau, C. C. et al. Viral-human chimeric transcript predisposes risk to liver cancer development and progression. Cancer Cell 25, 335-349, doi:10.1016/j.ccr.2014.01.030 (2014).
14 Tu, T., Budzinska, M. A., Vondran, F. W. R., Shackel, N. A. & Urban, S. Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles. J Virol, JVI. 02007-02017, doi:10.1128/JVI.02007-17 (2018).
15 Otto, S. P. The evolutionary consequences of polyploidy. Cell 131, 452-462, doi:10.1016/j.cell.2007.10.022 (2007).
16 Storchova, Z. & Pellman, D. From polyploidy to aneuploidy, genome instability and cancer. Nat Rev Mol Cell Biol 5, 45-54, doi:10.1038/nrm1276 (2004).
17 Davoli, T. & de Lange, T. The causes and consequences of polyploidy in normal development and cancer. Annu Rev Cell Dev Biol 27, 585-610, doi:10.1146/annurev-cellbio-092910-154234 (2011).
18 Santaguida, S. & Amon, A. Short- and long-term effects of chromosome mis-segregation and aneuploidy. Nat Rev Mol Cell Biol 16, 473-485, doi:10.1038/nrm4025 (2015).
19 Gordon, D. J., Resio, B. & Pellman, D. Causes and consequences of aneuploidy in cancer. Nat Rev Genet 13, 189-203, doi:10.1038/nrg3123 (2012).
20 Gupta, S. Hepatic polyploidy and liver growth control. Semin Cancer Biol 10, 161-171, doi:10.1006/scbi.2000.0317 (2000).
21 Margall-Ducos, G., Celton-Morizur, S., Couton, D., Bregerie, O. & Desdouets, C. Liver tetraploidization is controlled by a new process of incomplete cytokinesis. Journal of Cell Science 120, 3633-3639, doi:10.1242/jcs.016907 (2007).
22 Gentric, G. & Desdouets, C. Polyploidization in liver tissue. Am J Pathol 184, 322-331, doi:10.1016/j.ajpath.2013.06.035 (2014).
23 Celton-Morizur, S., Merlen, G., Couton, D. & Desdouets, C. Polyploidy and liver proliferation: central role of insulin signaling. Cell Cycle 9, 460-466, doi:10.4161/cc.9.3.10542 (2010).
24 Toyoda, H. et al. Changes to hepatocyte ploidy and binuclearity profiles during human chronic viral hepatitis. Gut 54, 297-302, doi:10.1136/gut.2004.043893 (2005).
25 Studach, L. et al. Polo-like kinase 1 activated by the hepatitis B virus X protein attenuates both the DNA damage checkpoint and DNA repair resulting in partial polyploidy. J Biol Chem 285, 30282-30293, doi:10.1074/jbc.M109.093963 (2010).
26 Park, Y. N. Update on precursor and early lesions of hepatocellular carcinomas. Arch Pathol Lab Med 135, 704-715, doi:10.1043/2010-0524-RA.1 (2011).
27 Anthony, P. P., Vogel, C. L. & Barker, L. F. Liver cell dysplasia: a premalignant condition. J Clin Pathol 26, 217-223 (1973).
28 Kim, H. et al. Large liver cell change in hepatitis B virus-related liver cirrhosis. Hepatology 50, 752-762, doi:10.1002/hep.23072 (2009).
29 Gripon, P., Le Seyec, J., Rumin, S. & Guguen-Guillouzo, C. Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity. Virology 213, 292-299, doi:10.1006/viro.1995.0002 (1995).
30 Le Seyec, J., Chouteau, P., Cannie, I., Guguen-Guillouzo, C. & Gripon, P. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol 73, 2052-2057 (1999).
31 Yan, H. et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1, e00049, doi:ARTN e0004910.7554/eLife.00049 (2012).
32 Bruss, V. Hepatitis B virus morphogenesis. World J Gastroenterol 13, 65-73 (2007).
33 Pollicino, T., Cacciola, I., Saffioti, F. & Raimondo, G. Hepatitis B virus PreS/S gene variants: pathobiology and clinical implications. Journal of hepatology 61, 408-417, doi:10.1016/j.jhep.2014.04.041 (2014).
34 Tseng, T. C. & Kao, J. H. Clinical utility of quantitative HBsAg in natural history and nucleos(t)ide analogue treatment of chronic hepatitis B: new trick of old dog. J Gastroenterol 48, 13-21, doi:10.1007/s00535-012-0668-y (2013).
35 Chiang, C. J., Yang, Y. W., You, S. L., Lai, M. S. & Chen, C. J. Thirty-year outcomes of the national hepatitis B immunization program in Taiwan. JAMA 310, 974-976, doi:10.1001/jama.2013.276701 (2013).
36 Ni, Y. H. & Chen, D. S. Hepatitis B vaccination in children: the Taiwan experience. Pathol Biol (Paris) 58, 296-300, doi:10.1016/j.patbio.2009.11.002 (2010).
37 Volz, T. et al. The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus. Journal of hepatology 58, 861-867, doi:10.1016/j.jhep.2012.12.008 (2013).
38 Churin, Y., Roderfeld, M. & Roeb, E. Hepatitis B virus large surface protein: function and fame. Hepatobiliary Surg Nutr 4, 1-10, doi:10.3978/j.issn.2304-3881.2014.12.08 (2015).
39 Hildt, E., Saher, G., Bruss, V. & Hofschneider, P. H. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology 225, 235-239, doi:10.1006/viro.1996.0594 (1996).
40 Meyer, M. et al. Hepatitis B virus transactivator MHBst: activation of NF-kappa B, selective inhibition by antioxidants and integral membrane localization. The EMBO journal 11, 2991-3001 (1992).
41 Hildt, E., Munz, B., Saher, G., Reifenberg, K. & Hofschneider, P. H. The PreS2 activator MHBst of hepatitis B virus activates c‐raf‐1/Erk2 signaling in transgenic mice. The EMBO journal 21, 525-535 (2002).
42 Wang, H. C. et al. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. American Journal of Pathology 163, 2441-2449, doi:Doi 10.1016/S0002-9440(10)63599-7 (2003).
43 Hadziyannis, S., Gerber, M. A., Vissoulis, C. & Popper, H. Cytoplasmic Hepatitis-B Antigen in Ground-Glass Hepatocytes of Carriers. Archives of Pathology 96, 327-330 (1973).
44 Fan, Y. F. et al. Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. J Gastroenterol Hepatol 15, 519-528 (2000).
45 Hung, J.-H. et al. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-κB and pp38 mitogen-activated protein kinase. Journal of Biological Chemistry 279, 46384-46392 (2004).
46 Yang, J. C. et al. Enhanced expression of vascular endothelial growth factor-A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis. Hepatology 49, 1962-1971, doi:10.1002/hep.22889 (2009).
47 Wang, H. C. et al. Hepatitis B virus pre-S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes. Hepatology 41, 761-770, doi:10.1002/hep.20615 (2005).
48 Hsieh, Y.-H. et al. Hepatitis B virus pre-S2 mutant surface antigen induces degradation of cyclin-dependent kinase inhibitor p27Kip1 through c-Jun activation domain-binding protein 1. Molecular Cancer Research 5, 1063-1072 (2007).
49 Wang, H. C., Huang, W., Lai, M. D. & Su, I. J. Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Sci 97, 683-688, doi:10.1111/j.1349-7006.2006.00235.x (2006).
50 Su, I. J., Wang, H. C., Wu, H. C. & Huang, W. Y. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection. J Gastroenterol Hepatol 23, 1169-1174, doi:10.1111/j.1440-1746.2008.05348.x (2008).
51 Tsai, H. W. et al. Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma. Oncotarget 7, 27724-27734, doi:10.18632/oncotarget.8388 (2016).
52 Chou, Y. C. et al. Evaluation of transcriptional efficiency of hepatitis B virus covalently closed circular DNA by reverse transcription-PCR combined with the restriction enzyme digestion method. J Virol 79, 1813-1823, doi:10.1128/JVI.79.3.1813-1823.2005 (2005).
53 Sells, M. A., Chen, M. L. & Acs, G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 84, 1005-1009 (1987).
54 Yen, T. T. et al. Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability. Oncotarget 7, 23346-23360, doi:10.18632/oncotarget.8109 (2016).
55 Reid, Y., Gaddipati, J. P., Yadav, D. & Kantor, J. Establishment of a human neonatal hepatocyte cell line. In Vitro Cell Dev Biol Anim 45, 535-542, doi:10.1007/s11626-009-9219-0 (2009).
56 Bissig, K. D., Le, T. T., Woods, N. B. & Verma, I. M. Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model. Proc Natl Acad Sci U S A 104, 20507-20511, doi:10.1073/pnas.0710528105 (2007).
57 Shih, Y. M. et al. Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice. Sci Rep 5, 15259, doi:10.1038/srep15259 (2015).
58 Chen, C. C. et al. Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA. Gene Ther 14, 11-19, doi:10.1038/sj.gt.3302846 (2007).
59 Yuh, C. H., Chang, Y. L. & Ting, L. P. Transcriptional regulation of precore and pregenomic RNAs of hepatitis B virus. J Virol 66, 4073-4084 (1992).
60 Nielsen, C. F. et al. PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis. Nat Commun 6, 8962, doi:10.1038/ncomms9962 (2015).
61 Giam, M. & Rancati, G. Aneuploidy and chromosomal instability in cancer: a jackpot to chaos. Cell Div 10, 3, doi:10.1186/s13008-015-0009-7 (2015).
62 Burrell, R. A. et al. Replication stress links structural and numerical cancer chromosomal instability. Nature 494, 492-496, doi:10.1038/nature11935 (2013).
63 Timofeev, O., Cizmecioglu, O., Settele, F., Kempf, T. & Hoffmann, I. CDC25 phosphatases are required for timely assembly of CDK1/cyclin B at the G2/M transition. Journal of Biological Chemistry, jbc. M109. 096552 (2010).
64 de Gooijer, M. C. et al. The G2 checkpoint—a node‐based molecular switch. FEBS open bio 7, 439-455 (2017).
65 Hyun, S. Y., Hwang, H. I. & Jang, Y. J. Polo-like kinase-1 in DNA damage response. BMB Rep 47, 249-255 (2014).
66 Hall-Jackson, C. A., Cross, D. A. E., Morrice, N. & Smythe, C. ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK. Oncogene 18, 6707-6713, doi:DOI 10.1038/sj.onc.1203077 (1999).
67 Sarkaria, J. N. et al. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59, 4375-4382 (1999).
68 Huang, S. N. & Chisari, F. V. Strong, sustained hepatocellular proliferation precedes hepatocarcinogenesis in hepatitis B surface antigen transgenic mice. Hepatology 21, 620-626 (1995).
69 Celton-Morizur, S. & Desdouets, C. Polyploidization of liver cells. Adv Exp Med Biol 676, 123-135 (2010).
70 Celton-Morizur, S., Merlen, G., Couton, D., Margall-Ducos, G. & Desdouets, C. The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents. J Clin Invest 119, 1880-1887 (2009).
71 Margall-Ducos, G., Celton-Morizur, S., Couton, D., Bregerie, O. & Desdouets, C. Liver tetraploidization is controlled by a new process of incomplete cytokinesis. J Cell Sci 120, 3633-3639, doi:10.1242/jcs.016907 (2007).
72 Kudryavtsev, B. N., Kudryavtseva, M. V., Sakuta, G. A. & Stein, G. I. Human hepatocyte polyploidization kinetics in the course of life cycle. Virchows Arch B Cell Pathol Incl Mol Pathol 64, 387-393 (1993).
73 Duncan, A. W. et al. The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 467, 707-710, doi:10.1038/nature09414 (2010).
74 Duncan, A. W. et al. Frequent aneuploidy among normal human hepatocytes. Gastroenterology 142, 25-28, doi:10.1053/j.gastro.2011.10.029 (2012).
75 Roessler, S. et al. Integrative genomic identification of genes on 8p associated with hepatocellular carcinoma progression and patient survival. Gastroenterology 142, 957-966 e912, doi:10.1053/j.gastro.2011.12.039 (2012).
76 Kusano, N. et al. Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinomas: their relationship to clinicopathological features. Hepatology 29, 1858-1862, doi:10.1002/hep.510290636 (1999).
77 Fujiwara, T. et al. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437, 1043-1047, doi:10.1038/nature04217 (2005).
78 Andreassen, P. R., Lohez, O. D., Lacroix, F. B. & Margolis, R. L. Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1. Mol Biol Cell 12, 1315-1328, doi:10.1091/mbc.12.5.1315 (2001).
79 Pellegrino, R. et al. Oncogenic and tumor suppressive roles of polo-like kinases in human hepatocellular carcinoma. Hepatology 51, 857-868, doi:10.1002/hep.23467 (2010).
80 Sun, W. et al. High expression of polo-like kinase 1 is associated with early development of hepatocellular carcinoma. Int J Genomics 2014, 312130, doi:10.1155/2014/312130 (2014).
81 Studach, L. L. et al. Polo-like kinase 1 inhibition suppresses hepatitis B virus X protein-induced transformation in an in vitro model of liver cancer progression. Hepatology 50, 414-423, doi:10.1002/hep.22996 (2009).
82 Steegmaier, M. et al. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Curr Biol 17, 316-322, doi:10.1016/j.cub.2006.12.037 (2007).
83 Haupenthal, J. et al. Reduced efficacy of the Plk1 inhibitor BI 2536 on the progression of hepatocellular carcinoma due to low intratumoral drug levels. Neoplasia 14, 410-419 (2012).
84 Shirakawa, J. et al. Insulin signaling regulates the FoxM1/PLK1/CENP-a pathway to promote adaptive pancreatic β cell proliferation. Cell metabolism 25, 868-882. e865 (2017).
85 Smith, L. et al. The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation. Sci Rep 7, 16115, doi:10.1038/s41598-017-16394-2 (2017).
86 Chisari, F. V. et al. Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59, 1145-1156 (1989).
87 Wu, J. et al. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology 49, 1132-1140, doi:10.1002/hep.22751 (2009).
88 Su, I. J. et al. The emerging role of hepatitis B virus pre-S2 deletion mutant proteins in HBV tumorigenesis. Journal of biomedical science 21, 98, doi:10.1186/s12929-014-0098-7 (2014).
89 Chen, B. F. et al. High prevalence and mapping of pre-S deletion in hepatitis B virus carriers with progressive liver diseases. Gastroenterology 130, 1153-1168, doi:10.1053/j.gastro.2006.01.011 (2006).
90 Chen, C. H. et al. Pre-S deletion and complex mutations of hepatitis B virus related to advanced liver disease in HBeAg-negative patients. Gastroenterology 133, 1466-1474, doi:10.1053/j.gastro.2007.09.002 (2007).
91 Mathai, A. M. et al. Type II ground-glass hepatocytes as a marker of hepatocellular carcinoma in chronic hepatitis B. Human pathology 44, 1665-1671, doi:10.1016/j.humpath.2013.01.020 (2013).
92 Wang, H. C. et al. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol 163, 2441-2449, doi:10.1016/S0002-9440(10)63599-7 (2003).
93 Hung, J. H. et al. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase. J Biol Chem 279, 46384-46392, doi:10.1074/jbc.M403568200 (2004).
94 Hsieh, Y. H. et al. Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis. J Pathol 236, 337-347, doi:10.1002/path.4531 (2015).
95 Koo, J. S. et al. Predictive value of liver cell dysplasia for development of hepatocellular carcinoma in patients with chronic hepatitis B. J Clin Gastroenterol 42, 738-743, doi:10.1097/MCG.0b013e318038159d (2008).
96 Libbrecht, L., Craninx, M., Nevens, F., Desmet, V. & Roskams, T. Predictive value of liver cell dysplasia for development of hepatocellular carcinoma in patients with non-cirrhotic and cirrhotic chronic viral hepatitis. Histopathology 39, 66-73 (2001).
97 Lee, R. G., Tsamandas, A. C. & Demetris, A. J. Large cell change (liver cell dysplasia) and hepatocellular carcinoma in cirrhosis: matched case-control study, pathological analysis, and pathogenetic hypothesis. Hepatology 26, 1415-1422, doi:10.1002/hep.510260607 (1997).
98 Borzio, M. et al. Liver cell dysplasia is a major risk factor for hepatocellular carcinoma in cirrhosis: a prospective study. Gastroenterology 108, 812-817 (1995).