DISCUSSION

In this study, it was proved that LDHB suppression-mediated lactic acidosis induces mitochondrial respiratory dysfunction by inactivation of PDH in hepatoma cell lines. Thus, it was identified that several key functions: First, suppressed LDHB expression in hepatoma cell increased LDH5 activity and production and release. Accordingly, the increased extracellular lactate may be re-introduced back into the tumor cell and affect diverse cellular function.

Second, LDHB suppression is an upstream event of decreased mitochondrial respiration. Third, I clearly demonstrated that lactic acidosis formed by released lactate induced the mitochondrial respiratory dysfunction by inactivation of PDH. Finally, LDHB-suppression-induced PDH phosphorylation is mainly mediated by ERK activation.

Extracellular release of lactate and resultant microenvironmental acidification, even under aerobic conditions (i.e., aerobic glycolysis), is a common feature of cancer development.

Increased lactate levels often allows to predict for metastases and overall survival of patients, as shown by several studies (Holroyde et al., 1979; Walenta et al., 2000; Saraswathy et al., 2009). Lactate promotes tumor metastasis by inducing hyaluronan secretion from tumor-associated fibroblasts which generate a milieu favorable for migration (Rudrabhatla et al., 2006). Lactate itself also induces the tumor cell migration (Beckert et al., 2006; Baumann et al., 2009; Goetze et al., 2011). Lactate functions to stimulate production of VEGF, as well as a growth-promoting factors that assist in angiogenesis (Trabold et al., 2003). However, it remains unclear how lactate regulates mitochondrial respiratory function.

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PDC is the key enzyme of glucose metabolism which starts mitochondrial oxidation process, generating acetyl-CoA from pyruvate, the final of glycolysis. The regulation of PDH activity through phosphorylation is well known in particular in cancer cell. Several studied have shown that new mechanisms regulate in the activity of PDH. For example, it is known that activity of PDH regulated TPKL/GSK3β (Hoshi et al., 1996). Also, phosphorylation of tyrosine residue through PDK1 and PDP1 inhibits PDH in EGF-stimulated cells (Fan et al., 2014a; Fan et al., 2014b).

Several studies reported that PDKs play key roles in the metabolic alteration of cancer cell.

Expression of the PDK1 gene is upregulated by c-Myc and HIF-1α (Kim et al., 2006; Pardo et al., 2006; Dang et al., 2008). Interestingly, it was also proved that LDHB suppression-mediated lactic acidosis phosphorylated PDH through PDK activation by ERK without inducing PDK expression.

ERK pathway are critical in the regulation of several biological processes such as proliferation. In 2011, Grassian AR. et al. reported that ERK regulated PDH flux through PDK4 modulates cell proliferation (Grassian et al., 2011).

Our present study emphasize that aerobic glycolysis is not only achieved as an adaptive cellular strategy to meet energy deficit induced by tumoral hypoxia or mitochondrial respiratory defect, but also an active tumoral strategy to shut down mitochondrial respiratory function and induce aggressive tumoral properties.

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REFERENCES

1. Amann T, Maegdefrau U, Hartmann A, Agaimy A, Marienhagen J, Weiss TS, Stoeltzing O, Warnecke C, Scholmerich J, Oefner PJ, Kreutz M, Bosserhoff AK, Hellerbrand C: GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis. Am J Pathol 174: 1544-1552, 2009

2. Baumann F, Leukel P, Doerfelt A, Beier CP, Dettmer K, Oefner PJ, Kastenberger M, Kreutz M, Nickl-Jockschat T, Bogdahn U, Bosserhoff AK, Hau P: Lactate promotes glioma migration by TGF-beta2-dependent regulation of matrix metalloproteinase-2.

Neuro Oncol 11: 368-380, 2009

3. Beckert S, Farrahi F, Aslam RS, Scheuenstuhl H, Konigsrainer A, Hussain MZ, Hunt TK: Lactate stimulates endothelial cell migration. Wound Repair Regen 14: 321-324, 2006

4. Dang CV, Kim JW, Gao P, Yustein J: The interplay between MYC and HIF in cancer.

Nat Rev Cancer 8: 51-56, 2008

5. Drent M, Cobben NA, Henderson RF, Wouters EF, van Dieijen-Visser M: Usefulness of lactate dehydrogenase and its isoenzymes as indicators of lung damage or inflammation. Eur Respir J 9: 1736-1742, 1996

6. Fan J, Kang HB, Shan C, Elf S, Lin R, Xie J, Gu TL, Aguiar M, Lonning S, Chung TW, Arellano M, Khoury HJ, Shin DM, Khuri FR, Boggon TJ, Kang S, Chen J: Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding

37

and promotes the Warburg effect. J Biol Chem 289: 26533-26541, 2014a

7. Fan J, Shan C, Kang HB, Elf S, Xie J, Tucker M, Gu TL, Aguiar M, Lonning S, Chen H, Mohammadi M, Britton LM, Garcia BA, Aleckovic M, Kang Y, Kaluz S, Devi N, Van Meir EG, Hitosugi T, Seo JH, Lonial S, Gaddh M, Arellano M, Khoury HJ, Khuri FR, Boggon TJ, Kang S, Chen J: Tyr phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate the pyruvate dehydrogenase complex. Mol Cell 53: 534-548, 2014b

8. Goetze K, Walenta S, Ksiazkiewicz M, Kunz-Schughart LA, Mueller-Klieser W:

Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release. Int J Oncol 39: 453-463, 2011

9. Grassian AR, Metallo CM, Coloff JL, Stephanopoulos G, Brugge JS: Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation. Genes Dev 25: 1716-1733, 2011

10. Harris RA, Bowker-Kinley MM, Huang B, Wu P: Regulation of the activity of the pyruvate dehydrogenase complex. Adv Enzyme Regul 42: 249-259, 2002

11. Hiromasa Y, Fujisawa T, Aso Y, Roche TE: Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components. J Biol Chem 279: 6921-6933, 2004

12. Hitosugi T, Fan J, Chung TW, Lythgoe K, Wang X, Xie J, Ge Q, Gu TL, Polakiewicz RD, Roesel JL, Chen GZ, Boggon TJ, Lonial S, Fu H, Khuri FR, Kang S, Chen J:

38

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism. Mol Cell 44: 864-877, 2011

13. Holroyde CP, Axelrod RS, Skutches CL, Haff AC, Paul P, Reichard GA: Lactate metabolism in patients with metastatic colorectal cancer. Cancer Res 39: 4900-4904, 1979

14. Hoshi M, Takashima A, Noguchi K, Murayama M, Sato M, Kondo S, Saitoh Y, Ishiguro K, Hoshino T, Imahori K: Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

Proc Natl Acad Sci U S A 93: 2719-2723, 1996

15. Kim JH, Kim EL, Lee YK, Park CB, Kim BW, Wang HJ, Yoon CH, Lee SJ, Yoon G:

Decreased lactate dehydrogenase B expression enhances claudin 1-mediated hepatoma cell invasiveness via mitochondrial defects. Exp Cell Res 317: 1108-1118, 2011

16. Kim JW, Dang CV: Cancer's molecular sweet tooth and the Warburg effect. Cancer Res 66: 8927-8930, 2006

17. Kim JW, Tchernyshyov I, Semenza GL, Dang CV: HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 3: 177-185, 2006

18. Kolobova E, Tuganova A, Boulatnikov I, Popov KM: Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites. Biochem J 358: 69-77, 2001

39

19. Lewis BC, Prescott JE, Campbell SE, Shim H, Orlowski RZ, Dang CV: Tumor induction by the c-Myc target genes rcl and lactate dehydrogenase A. Cancer Res 60:

6178-6183, 2000

20. Pardo JV, Pardo PJ, Humes SW, M IP: Neurocognitive dysfunction in antidepressant-free, non-elderly patients with unipolar depression: alerting and covert orienting of visuospatial attention. J Affect Disord 92: 71-78, 2006

21. Rattigan YI, Patel BB, Ackerstaff E, Sukenick G, Koutcher JA, Glod JW, Banerjee D:

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment. Exp Cell Res 318: 326-335, 2012

22. Read RJ: Pushing the boundaries of molecular replacement with maximum likelihood.

Acta Crystallogr D Biol Crystallogr 57: 1373-1382, 2001

23. Robinson BH, McKay N, Goodyer P, Lancaster G: Defective intramitochondrial NADH oxidation in skin fibroblasts from an infant with fatal neonatal lacticacidemia.

Am J Hum Genet 37: 938-946, 1985

24. Roche TE, Baker JC, Yan X, Hiromasa Y, Gong X, Peng T, Dong J, Turkan A, Kasten SA: Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog Nucleic Acid Res Mol Biol 70: 33-75, 2001

25. Rudrabhatla SR, Mahaffey CL, Mummert ME: Tumor microenvironment modulates hyaluronan expression: the lactate effect. J Invest Dermatol 126: 1378-1387, 2006 26. Saraswathy S, Crawford FW, Lamborn KR, Pirzkall A, Chang S, Cha S, Nelson SJ:

40

Evaluation of MR markers that predict survival in patients with newly diagnosed GBM prior to adjuvant therapy. J Neurooncol 91: 69-81, 2009

27. Seifert F, Ciszak E, Korotchkina L, Golbik R, Spinka M, Dominiak P, Sidhu S, Brauer J, Patel MS, Tittmann K: Phosphorylation of serine 264 impedes active site accessibility in the E1 component of the human pyruvate dehydrogenase multienzyme complex. Biochemistry 46: 6277-6287, 2007

28. Taylor RW, Turnbull DM: Mitochondrial DNA mutations in human disease. Nat Rev Genet 6: 389-402, 2005

29. Trabold O, Wagner S, Wicke C, Scheuenstuhl H, Hussain MZ, Rosen N, Seremetiev A, Becker HD, Hunt TK: Lactate and oxygen constitute a fundamental regulatory mechanism in wound healing. Wound Repair Regen 11: 504-509, 2003

30. Vander Heiden MG, Cantley LC, Thompson CB: Understanding the Warburg effect:

the metabolic requirements of cell proliferation. Science 324: 1029-1033, 2009 31. Walenta S, Wetterling M, Lehrke M, Schwickert G, Sundfor K, Rofstad EK,

Mueller-Klieser W: High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers. Cancer Res 60: 916-921, 2000 32. Warburg O: On respiratory impairment in cancer cells. Science 124: 269-270, 1956 33. Yeaman SJ, Hutcheson ET, Roche TE, Pettit FH, Brown JR, Reed LJ, Watson DC,

Dixon GH: Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart. Biochemistry 17: 2364-2370, 1978

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핵심어: 간암세포주, 미토콘드리아 기능손상, 해당과정, LDHB (lactate dehydrogenase B), PDH (pyruvate dehydrogenase), Lactic acidosis, Hepatoma cell