B型肝炎的感染是導致肝癌的一個主要致病因子，癌細胞也已被證實能快速地消耗葡萄糖並進行有氧的糖解作用來維持其細胞代謝，此機制稱為Warburg effect。 然而值得討論的是，在HBV病毒的感染機制中是否也牽涉Warburg effect。藉由免疫沉澱和質譜儀分析的方法，我們找到了參與Warburg effect重要的分子─PKM2，其會與HBV大表面蛋白結合。接著我們利用逆向的共同免疫沉澱和免疫螢光染色的技術，證實了它們兩者間的交互作用。再者，我們也找到了LHBs和PKM2之間的結合區域。我們進一步發現大量表現小表面蛋白 (為PKM2主要的結合區域)會減少細胞中PKM2的Oligomerization。接著，我們發現帶有B型肝炎病毒基因的細胞株1.3ES2其PKM2的下游分子c-myc具有高度的表現，且在帶有大型表面蛋白的穩定肝細胞株中磷酸化的STAT3和c-myc也有高度地活化。此外，我們也發現抑制PKM2和STAT3明顯地減少帶有大表面蛋白的細胞株中葡萄糖的消耗量和乳酸的生成量，證明了PKM2和STAT3在Warburg effect要扮演的重要角色。這項研究探討Warburg effect是如何影響HBV病毒造成肝癌的作用機制，並且我們認為HBV的大型表面蛋白會藉由與PKM2的結合促進Warburg effect。

Hepatitis B virus (HBV) is one of major etiologic factors for the development of hepatocellular carcinoma. It has been shown that cancer cells may accelerate glucose uptake and execute aerobic glycolysis, a phenomenon known as Warburg effect. Whether HBV plays a role in Warburg effect is not investigated. With the combination of affinity precipitation and mass spectrometry, we recently identified pyruvate kinase isoenzyme M2 (PKM2) as an interacting partner of HBV large surface protein (LHBs). Reciprocal immunoprecipitation and double immunofluorescence staining confirm the interaction between LHBs and PKM2 in hepatocytes. Moreover, we map the minimal interaction domains of PKM2 and LHBs. Overexpression of SHBs, which strongly binds to PKM2, significantly reduced oligomerization of PKM2 in the cell. Notably, we find that the expression of PKM2 downstream signaling molecule c-myc is elevated in hepatoma cells harboring a complete HBV genome (1.3ES2). Furthermore, overexpression of LHBs in hTERT-immortalized hepatocytes also promotes STAT3 phosphorylation and c-myc expression. Depletion of PKM2 or STAT3 significantly reduced glucose consumption and lactate production in LHBs-positive cells, indicating essential roles of PKM2 and STAT3 in Warburg effect. In conclusion, our study suggests a potential role of Warburg effect in HBV-mediated hepatocarcinogenesis. These data demonstrate that HBV LHBs promotes Warburg effect through binding to PKM2.

Abstract...........................................................................................................1
摘要.................................................................................................................2
1. Introduction.................................................................................................3
1.1 Hepatitis B virus (HBV) and hepatocellular carcinoma (HCC)........................3
1.2 The role of Pyruvate kinase M2 (PKM2).......................................................5
1.3 Warburg effect...........................................................................................7
2. Hypothesis and specific aims.......................................................................9
3. Materials and Methods...............................................................................10
4. Results.......................................................................................................15
4.1 HBV LHBs interacts with PKM2..................................................................15
4.2 Minimal interaction domains of LHBs and PKM2 are mapped....................16
4.3 Glucose consumption and lactate production is increased in LHBs-positive cells...............................................................................................................16
4.4 Activation of STAT3 and c-myc in LHBs-positive cells ..............................17
4.5 The roles of PKM2 and STAT3 on c-myc expression.................................18
4.6 SHBs affects oligomerization of PKM2......................................................18
4.7 PKM2 and STAT3 promote cell viability in LHBs cells................................19
4.8 LHBs promotes Warburg effect through PKM2 and STAT3.........................19
5. Discussion.................................................................................................21
6. Figures
Figure 1. HBV LHBs interacts with PKM2……..…………………….......................24
Figure 2. Subcellular interaction between LHBs and PKM2.……………………….25
Figure 3. Truncated LHBs and PKM2 fragments.…………………………………....26
Figure 4. Explore the binding region between LHBs and PKM2 in 293T cells…27
Figure 5. Explore the binding region between LHBs and PKM2 in NeHep-WT cells...............................................................................................................28
Figure 6. Glucose consumption is increased in LHBs-positive cells..............…29
Figure 7. Lactate production is increased in LHBs-positive cells………….….....30
Figure 8. Activation of p-STAT3 and c-myc in LHBs-positive cells………...…..31
Figure 9. Activation of c-myc in LHBs-positive cells….……..............…………..32
Figure 10. c-myc is down-regulated by STAT3 inhibitor STA-21.………………33
Figure 11. Expression level of c-myc is not reduced upon STAT-3 depletion..34
Figure 12. c-myc is regulated by PKM2………………………………………...........35
Figure 13. SHBs affects oligomerization of PKM2……………….....………….......36
Figure 14. PKM2 and STAT3 regulate cell viability in WT-LHBs cells……………37
Figure 15. PKM2 and STAT3 regulate cell viability in PreS2-LHBs cells………..38
Figure 16. PKM2 and STAT3 regulate glucose consumption in WT-LHBs cells.39
Figure 17. PKM2 and STAT3 regulate glucose consumption in PreS2-LHBs cells...............................................................................................................40
Figure 18. PKM2 and STAT3 regulate lactate production in WT-LHBs cells……41
Figure 19. PKM2 and STAT3 regulate lactate production in PreS2-LHBs cells..42
Figure 20. Current model……………………………………………………….............43
7. Reference...................................................................................................44
8. Tables........................................................................................................48

1 Glebe, D. & Bremer, C. M. The molecular virology of hepatitis B virus. Seminars in liver disease 33, 103-112, doi:10.1055/s-0033-1345717 (2013).
2 Seeger, C. & Mason, W. S. Hepatitis B virus biology. Microbiology and molecular biology reviews : MMBR 64, 51-68 (2000).
3 Lupberger, J. & Hildt, E. Hepatitis B virus-induced oncogenesis. World journal of gastroenterology : WJG 13, 74-81 (2007).
4 Takahashi, S. & Chayama, K. Integration of hepatitis B virus DNA and hepatocellular carcinoma. Journal of gastroenterology and hepatology 20, 1141-1142, doi:10.1111/j.1440-1746.2005.03940.x (2005).
5 Matsubara, K. [Hepatitis B virus: DNA integration and hepatocellular carcinoma]. Gan to kagaku ryoho. Cancer & chemotherapy 16, 530-541 (1989).
6 Prince, A. M. Hepatitis B virus and hepatocellular carcinoma: molecular biology provides further evidence for an etiologic association. Hepatology 1, 73-75 (1981).
7 Fernholz, D., Stemler, M., Brunetto, M., Bonino, F. & Will, H. Replicating and virion secreting hepatitis B mutant virus unable to produce preS2 protein. Journal of hepatology 13 Suppl 4, S102-104 (1991).
8 Wang, H. C., Huang, W., Lai, M. D. & Su, I. J. Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer science 97, 683-688, doi:10.1111/j.1349-7006.2006.00235.x (2006).
9 Hildt, E. & Hofschneider, P. H. The PreS2 activators of the hepatitis B virus: activators of tumour promoter pathways. Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer 154, 315-329 (1998).
10 Shen, F. C. et al. A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers. Journal of biomedical science 16, 84, doi:10.1186/1423-0127-16-84 (2009).
11 Noguchi, T., Inoue, H. & Tanaka, T. The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. The Journal of biological chemistry 261, 13807-13812 (1986).
12 Eigenbrodt, E., Basenau, D., Holthusen, S., Mazurek, S. & Fischer, G. Quantification of tumor type M2 pyruvate kinase (Tu M2-PK) in human carcinomas. Anticancer research 17, 3153-3156 (1997).
13 Mazurek, S., Boschek, C. B., Hugo, F. & Eigenbrodt, E. Pyruvate kinase type M2 and its role in tumor growth and spreading. Seminars in cancer biology 15, 300-308, doi:10.1016/j.semcancer.2005.04.009 (2005).
14 Tamada, M., Suematsu, M. & Saya, H. Pyruvate kinase M2: multiple faces for conferring benefits on cancer cells. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 5554-5561, doi:10.1158/1078-0432.CCR-12-0859 (2012).
15 Zhivotovsky, B. & Orrenius, S. The Warburg Effect returns to the cancer stage. Seminars in cancer biology 19, 1-3, doi:10.1016/j.semcancer.2008.12.003 (2009).
16 Martinez-Outschoorn, U. E. et al. Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect: implications for PET imaging of human tumors. Cell cycle 10, 2504-2520 (2011).
17 Zhou, C. F. et al. Pyruvate kinase type M2 is upregulated in colorectal cancer and promotes proliferation and migration of colon cancer cells. IUBMB life 64, 775-782, doi:10.1002/iub.1066 (2012).
18 Schneider, J. et al. Tumor M2-pyruvate kinase in lung cancer patients: immunohistochemical detection and disease monitoring. Anticancer research 22, 311-318 (2002).
19 Pottek, T., Muller, M., Blum, T. & Hartmann, M. Tu-M2-PK in the blood of testicular and cubital veins in men with testicular cancer. Anticancer research 20, 5029-5033 (2000).
20 Brinck, U., Eigenbrodt, E., Oehmke, M., Mazurek, S. & Fischer, G. L- and M2-pyruvate kinase expression in renal cell carcinomas and their metastases. Virchows Archiv : an international journal of pathology 424, 177-185 (1994).
21 Wang, C. et al. Inactivation of Spry2 accelerates AKT-driven hepatocarcinogenesis via activation of MAPK and PKM2 pathways. Journal of hepatology 57, 577-583, doi:10.1016/j.jhep.2012.04.026 (2012).
22 Kitamura, K. et al. Proliferative activity in hepatocellular carcinoma is closely correlated with glucose metabolism but not angiogenesis. Journal of hepatology 55, 846-857, doi:10.1016/j.jhep.2011.01.038 (2011).
23 Vander Heiden, M. G. et al. Evidence for an alternative glycolytic pathway in rapidly proliferating cells. Science 329, 1492-1499, doi:10.1126/science.1188015 (2010).
24 Anastasiou, D. et al. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 334, 1278-1283, doi:10.1126/science.1211485 (2011).
25 Rouzer, C. A. Spotlight. Chemical Research in Toxicology 25, 13-14, doi:10.1021/tx200539j (2012).
26 Hitosugi, T. et al. Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Science signaling 2, ra73, doi:10.1126/scisignal.2000431 (2009).
27 Lv, L. et al. Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Molecular cell 52, 340-352, doi:10.1016/j.molcel.2013.09.004 (2013).
28 Luo, W. et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell 145, 732-744, doi:10.1016/j.cell.2011.03.054 (2011).
29 Wong, N., De Melo, J. & Tang, D. PKM2, a Central Point of Regulation in Cancer Metabolism. International journal of cell biology 2013, 242513, doi:10.1155/2013/242513 (2013).
30 Goldberg, M. S. & Sharp, P. A. Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression. The Journal of experimental medicine 209, 217-224, doi:10.1084/jem.20111487 (2012).
31 Demaria, M. & Poli, V. PKM2, STAT3 and HIF-1alpha: The Warburg's vicious circle. Jak-Stat 1, 194-196, doi:10.4161/jkst.20662 (2012).
32 Gao, X., Wang, H., Yang, J. J., Liu, X. & Liu, Z. R. Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Molecular cell 45, 598-609, doi:10.1016/j.molcel.2012.01.001 (2012).
33 Yang, W. et al. Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480, 118-122, doi:10.1038/nature10598 (2011).
34 Wang, H. J. et al. JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism. Proceedings of the National Academy of Sciences of the United States of America 111, 279-284, doi:10.1073/pnas.1311249111 (2014).
35 Lee, J., Kim, H. K., Han, Y. M. & Kim, J. Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. The international journal of biochemistry & cell biology 40, 1043-1054, doi:10.1016/j.biocel.2007.11.009 (2008).
36 Kim, J. W. & Dang, C. V. Cancer's molecular sweet tooth and the Warburg effect. Cancer research 66, 8927-8930, doi:10.1158/0008-5472.CAN-06-1501 (2006).
37 Hsu, P. P. & Sabatini, D. M. Cancer cell metabolism: Warburg and beyond. Cell 134, 703-707, doi:10.1016/j.cell.2008.08.021 (2008).
38 Garber, K. Energy boost: the Warburg effect returns in a new theory of cancer. Journal of the National Cancer Institute 96, 1805-1806, doi:10.1093/jnci/96.24.1805 (2004).
39 Bayley, J. P. & Devilee, P. The Warburg effect in 2012. Current opinion in oncology 24, 62-67, doi:10.1097/CCO.0b013e32834deb9e (2012).
40 Feron, O. Pyruvate into lactate and back: from the Warburg effect to symbiotic energy fuel exchange in cancer cells. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 92, 329-333, doi:10.1016/j.radonc.2009.06.025 (2009).
41 Rattigan, Y. I. et al. Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment. Experimental cell research 318, 326-335, doi:10.1016/j.yexcr.2011.11.014 (2012).
42 Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029-1033, doi:10.1126/science.1160809 (2009).
43 Chen, Y., Cairns, R., Papandreou, I., Koong, A. & Denko, N. C. Oxygen consumption can regulate the growth of tumors, a new perspective on the Warburg effect. PloS one 4, e7033, doi:10.1371/journal.pone.0007033 (2009).
44 Lopez-Lazaro, M. The warburg effect: why and how do cancer cells activate glycolysis in the presence of oxygen? Anti-cancer agents in medicinal chemistry 8, 305-312 (2008).
45 Kondoh, H. Cellular life span and the Warburg effect. Experimental cell research 314, 1923-1928, doi:10.1016/j.yexcr.2008.03.007 (2008).
46 Chen, Z., Lu, W., Garcia-Prieto, C. & Huang, P. The Warburg effect and its cancer therapeutic implications. Journal of bioenergetics and biomembranes 39, 267-274, doi:10.1007/s10863-007-9086-x (2007).
47 Yang, W. & Lu, Z. Nuclear PKM2 regulates the Warburg effect. Cell cycle 12, 3154-3158, doi:10.4161/cc.26182 (2013).
48 Dang, C. V. PKM2 tyrosine phosphorylation and glutamine metabolism signal a different view of the Warburg effect. Science signaling 2, pe75, doi:10.1126/scisignal.297pe75 (2009).
49 Chong, C. L. et al. Dynamics of HBV cccDNA expression and transcription in different cell growth phase. Journal of biomedical science 18, 96, doi:10.1186/1423-0127-18-96 (2011).
50 Reid, Y., Gaddipati, J. P., Yadav, D. & Kantor, J. Establishment of a human neonatal hepatocyte cell line. In vitro cellular & developmental biology. Animal 45, 535-542, doi:10.1007/s11626-009-9219-0 (2009).
51 Semenova, G. & Chernoff, J. PKM2 enters the morpheein academy. Molecular cell 45, 583-584, doi:10.1016/j.molcel.2012.02.014 (2012).
52 Yang, W. et al. ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nature cell biology 14, 1295-1304, doi:10.1038/ncb2629 (2012).
53 Miller, D. M., Thomas, S. D., Islam, A., Muench, D. & Sedoris, K. c-Myc and cancer metabolism. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 5546-5553, doi:10.1158/1078-0432.CCR-12-0977 (2012).
54 Zhao, B. & Hu, M. Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells. Oncology letters 6, 1749-1755, doi:10.3892/ol.2013.1632 (2013).
55 Severino, P. et al. Solid lipid nanoparticles for hydrophilic biotech drugs: Optimization and cell viability studies (Caco-2 & HEPG-2 cell lines). European journal of medicinal chemistry 81C, 28-34, doi:10.1016/j.ejmech.2014.04.084 (2014).