黃素腺嘌呤二核苷酸(Flavin adenine dinucleotide, FAD)是經由核黃素(riboflavin)代謝形成的產物，其會作為輔因子參與在核酸、蛋白質、脂質的合成以及細胞能量形成的檸檬酸循環(TCA cycle)和電子傳遞鏈等生化反應中；黃素腺嘌呤二核苷酸合成酶(Flavin adenine dinucleotide synthetase 1, FLAD1)是細胞內負責將黃素腺嘌呤單核苷酸(FMN)催化為黃素腺嘌呤二核苷酸的酵素，在先前的研究中，FLAD1在多種的癌症病人身上有高表達的情況，並且和病患的較差預後有相關性。本篇研究希望能調查高表達FLAD1在癌症中所扮演的角色，首先透過癌症資料庫: cBioportal、Oncomine、Human protein atlas分析出多種不同癌症病患中FLAD1在基因體和蛋白質層面的表現量都比一般人高，另外分析Prognoscan、KM plotter資料庫，可以得知FLAD1高表達的情況在多種癌症中都導致病患較差的預後，尤其在乳癌病患上，FLAD1的表達不但和患者生存率相關，也和患者的遠端轉移及復發有正相關性。為了調查FLAD1在乳癌進展中的角色，本研究中使用RNA干擾技術分別建立穩定抑制FLAD1的兩種乳癌細胞株(MCF7、MDA-MB-231)，並測定了對照組細胞(mock和vector組)與FLAD1抑制乳癌細胞之間的差異，在細胞生長測試、MTT測試、細胞群落能力實驗中，FLAD1抑制的MCF7以及MDA-MB-231細胞都表現出較差的增殖能力，而在傷口癒合以及細胞爬行觀察實驗中，FLAD1抑制的MDA-MB-231細胞表現出較差的細胞爬行能力，MCF7細胞上則無明顯差異；在產酸能力測試中，FLAD1抑制和對照組細胞並沒有明顯差異，顯示FLAD1抑制不會影響細胞糖酵解作用效率，有趣的是，以補充核黃素的細胞培養液培養FLAD1抑制的乳癌細胞，其增殖能力會恢復，揭示乳癌細胞中高表達FLAD1可能與調整細胞內FAD代謝量以利細胞快速增生有關。

Flavin adenine dinucleotide (FAD) is one of the derivative from riboflavin. It participates in mutiple organic compounds formation like amino acid, carbohydrate, fatty acid and nucleotide as an important cofactor. Flavin adenine dinucleotide synthetase 1 (FLAD1) takes part in hydrolyzing FAD to form flavin mononuleotide (FMN) and catalyzing the adenylation of FMN to form FAD. In previous studies, the high level of FLAD1 was detected in various cancers and was associated with poor prognosis of gastric cancer patients. The purpose of this study was to investigate the effects of FLAD1 overexpression in cancer. Analysis of cBioPortal, Oncomine and The Human Protein Atlas mega databases showed that various cancer patients especially breast cancer patients had higher FLAD1 expression in DNA, RNA and protein levels than healthy people. From Prognoscan and KM plotter databases, the high-expressed FLAD1 was related to the unfavorable survival rates in breast cancer. To study the effect of FLAD1 in breast cancer, stable FLAD1 knockdown breast cancer cell lines (MCF7 and MDA-MB-231) were established by using RNAi technique. FLAD1 knockdown cells showed a significant down-regulation of tumor proliferation compared with mock- and vector-transfected cells in cell growth assay, colony formation assay and MTT assay. The result of wound healing assay indicated the decreased tumor migration in FLAD1 knockdown MDA-MB-231 cells. The ability of aerobic glycolysis did not change in FLAD1 knockdown cells which was confirmed by culture medium acidification assay. Interestingly, the proliferation ability of FLAD1 knockdown cells was rescued by culture medium with 10 µM riboflavin. It seems the function of FLAD1 to regulate the amount of FAD in cells may help breast cancer cells proliferation and promote the progression of breast cancer cells.

Keywords: bioinformation database, breast cancer, Flavin adenine dinucleotide, Flavin adenine dinucleotide synthetase 1 (FLAD1), FLAD1 knockdown, riboflavin

1. Hanahan, D. and R.A. Weinberg, Hallmarks of cancer: the next generation. Cell, 2011. 144(5): p. 646-74.
2. Weinberg, F., et al., Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci U S A, 2010. 107(19): p. 8788-93.
3. Huber, V., et al., Cancer acidity: An ultimate frontier of tumor immune escape and a novel target of immunomodulation. Semin Cancer Biol, 2017. 43: p. 74-89.
4. Martin, G.S., Cell signaling and cancer. Cancer Cell, 2003. 4(3): p. 167-74.
5. Steven, A., S.A. Fisher, and B.W. Robinson, Immunotherapy for lung cancer. Respirology, 2016. 21(5): p. 821-33.
6. Alexandrov, L.B., et al., Signatures of mutational processes in human cancer. Nature, 2013. 500(7463): p. 415-21.
7. Unterleuthner, D., et al., Cancer-associated fibroblast-derived WNT2 increases tumor angiogenesis in colon cancer. Angiogenesis, 2020. 23(2): p. 159-177.
8. Viallard, C. and B. Larrivee, Tumor angiogenesis and vascular normalization: alternative therapeutic targets. Angiogenesis, 2017. 20(4): p. 409-426.
9. Collaborators, G.B.D.C.o.D., Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet, 2018. 392(10159): p. 1736-1788.
10. Global Burden of Disease Cancer, C., et al., Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-years for 32 Cancer Groups, 1990 to 2015: A Systematic Analysis for the Global Burden of Disease Study. JAMA Oncol, 2017. 3(4): p. 524-548.
11. Waks, A.G. and E.P. Winer, Breast Cancer Treatment: A Review. JAMA, 2019. 321(3): p. 288-300.
12. Yu, K.D., et al., Effect of Adjuvant Paclitaxel and Carboplatin on Survival in Women With Triple-Negative Breast Cancer: A Phase 3 Randomized Clinical Trial. JAMA Oncol, 2020. 6(9): p. 1390-1396.
13. Forouzanfar, M.H., et al., Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis. Lancet, 2011. 378(9801): p. 1461-84.
14. Kwa, M.J. and S. Adams, Checkpoint inhibitors in triple-negative breast cancer (TNBC): Where to go from here. Cancer, 2018. 124(10): p. 2086-2103.
15. Samanta, D., et al., Chemotherapy induces enrichment of CD47(+)/CD73(+)/PDL1(+) immune evasive triple-negative breast cancer cells. Proc Natl Acad Sci U S A, 2018. 115(6): p. E1239-E1248.
16. Narod, S.A. and L. Salmena, BRCA1 and BRCA2 mutations and breast cancer. Discov Med, 2011. 12(66): p. 445-53.
17. Baretta, Z., et al., Effect of BRCA germline mutations on breast cancer prognosis: A systematic review and meta-analysis. Medicine (Baltimore), 2016. 95(40): p. e4975.
18. Warburg, O., On the origin of cancer cells. Science, 1956. 123(3191): p. 309-14.
19. Denko, N.C., Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nat Rev Cancer, 2008. 8(9): p. 705-13.
20. Helmlinger, G., et al., Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med, 1997. 3(2): p. 177-82.
21. Rofstad, E.K., et al., Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res, 2006. 66(13): p. 6699-707.
22. Weinhouse, S., On respiratory impairment in cancer cells. Science, 1956. 124(3215): p. 267-9.
23. Lu, H., R.A. Forbes, and A. Verma, Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis. J Biol Chem, 2002. 277(26): p. 23111-5.
24. Ancey, P.B., C. Contat, and E. Meylan, Glucose transporters in cancer - from tumor cells to the tumor microenvironment. FEBS J, 2018. 285(16): p. 2926-2943.
25. Tseng, P.L., et al., The decrease of glycolytic enzyme hexokinase 1 accelerates tumor malignancy via deregulating energy metabolism but sensitizes cancer cells to 2-deoxyglucose inhibition. Oncotarget, 2018. 9(27): p. 18949-18969.
26. Sun, Q., et al., Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth. Proc Natl Acad Sci U S A, 2011. 108(10): p. 4129-34.
27. Kosugi, M., et al., MUC1-C oncoprotein regulates glycolysis and pyruvate kinase M2 activity in cancer cells. PLoS One, 2011. 6(11): p. e28234.
28. De Milito, A. and S. Fais, Tumor acidity, chemoresistance and proton pump inhibitors. Future Oncol, 2005. 1(6): p. 779-86.
29. Lin, C.C., et al., Loss of the respiratory enzyme citrate synthase directly links the Warburg effect to tumor malignancy. Sci Rep, 2012. 2: p. 785.
30. Tseng, P.L., et al., Decreased succinate dehydrogenase B in human hepatocellular carcinoma accelerates tumor malignancy by inducing the Warburg effect. Sci Rep, 2018. 8(1): p. 3081.
31. Dang, L., K. Yen, and E.C. Attar, IDH mutations in cancer and progress toward development of targeted therapeutics. Ann Oncol, 2016. 27(4): p. 599-608.
32. Koivunen, P., et al., Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem, 2007. 282(7): p. 4524-4532.
33. Akram, M., Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys, 2014. 68(3): p. 475-8.
34. Barile, M., et al., Riboflavin transport and metabolism in humans. J Inherit Metab Dis, 2016. 39(4): p. 545-57.
35. Bafunno, V., et al., Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flx1p carrier in FAD export. J Biol Chem, 2004. 279(1): p. 95-102.
36. Dourado, D., M. Swart, and A.T.P. Carvalho, Why the Flavin Adenine Dinucleotide (FAD) Cofactor Needs To Be Covalently Linked to Complex II of the Electron-Transport Chain for the Conversion of FADH2 into FAD. Chemistry, 2018. 24(20): p. 5246-5252.
37. Papa, S., et al., The oxidative phosphorylation system in mammalian mitochondria. Adv Exp Med Biol, 2012. 942: p. 3-37.
38. Jin, C., et al., Riboflavin Transporters RFVT/SLC52A Mediate Translocation of Riboflavin, Rather than FMN or FAD, across Plasma Membrane. Biol Pharm Bull, 2017. 40(11): p. 1990-1995.
39. Yonezawa, A. and K. Inui, Novel riboflavin transporter family RFVT/SLC52: identification, nomenclature, functional characterization and genetic diseases of RFVT/SLC52. Mol Aspects Med, 2013. 34(2-3): p. 693-701.
40. Giancaspero, T.A., et al., Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain. Biochem Biophys Res Commun, 2015. 465(3): p. 443-9.
41. Hu, P., et al., FLAD1 is up-regulated in Gastric Cancer and is a potential prediction of prognosis. Int J Med Sci, 2020. 17(12): p. 1763-1772.
42. Leone, P., et al., Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding. Int J Mol Sci, 2019. 20(24).
43. Galluccio, M., et al., Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase. Protein Expr Purif, 2007. 52(1): p. 175-81.
44. Muru, K., et al., FLAD1-associated multiple acyl-CoA dehydrogenase deficiency identified by newborn screening. Mol Genet Genomic Med, 2019. 7(9): p. e915.
45. Yamada, K., et al., Flavin adenine dinucleotide synthase deficiency due to FLAD1 mutation presenting as multiple acyl-CoA dehydrogenation deficiency-like disease: A case report. Brain Dev, 2019. 41(7): p. 638-642.
46. Garcia-Villoria, J., et al., FLAD1, encoding FAD synthase, is mutated in a patient with myopathy, scoliosis and cataracts. Clin Genet, 2018. 94(6): p. 592-593.
47. Auranen, M., et al., Patient with multiple acyl-CoA dehydrogenation deficiency disease and FLAD1 mutations benefits from riboflavin therapy. Neuromuscul Disord, 2017. 27(6): p. 581-584.
48. Yildiz, Y., et al., Post-mortem detection of FLAD1 mutations in 2 Turkish siblings with hypotonia in early infancy. Neuromuscul Disord, 2018. 28(9): p. 787-790.
49. Ye, C., et al., Multiple novel hepatocellular carcinoma signature genes are commonly controlled by the master pluripotency factor OCT4. Cell Oncol (Dordr), 2020. 43(2): p. 279-295.
50. Mitra, R., et al., Prediction of postoperative recurrence-free survival in non-small cell lung cancer by using an internationally validated gene expression model. Clin Cancer Res, 2011. 17(9): p. 2934-46.
51. Mei, M., et al., Significant Diagnostic and Prognostic Value of FLAD1 and Related MicroRNAs in Breast Cancer after a Pan-Cancer Analysis. Dis Markers, 2021. 2021: p. 6962526.
52. Wu, S., et al., The Functional Impact of Alternative Splicing on the Survival Prognosis of Triple-Negative Breast Cancer. Front Genet, 2020. 11: p. 604262.
53. Chae, Y.C., et al., Landscape of the mitochondrial Hsp90 metabolome in tumours. Nat Commun, 2013. 4: p. 2139.
54. McBride, H.M., M. Neuspiel, and S. Wasiak, Mitochondria: more than just a powerhouse. Curr Biol, 2006. 16(14): p. R551-60.
55. Kumar, P., A. Nagarajan, and P.D. Uchil, Analysis of Cell Viability by the MTT Assay. Cold Spring Harb Protoc, 2018. 2018(6).
56. Prabst, K., et al., Basic Colorimetric Proliferation Assays: MTT, WST, and Resazurin. Methods Mol Biol, 2017. 1601: p. 1-17.
57. Comsa, S., A.M. Cimpean, and M. Raica, The Story of MCF-7 Breast Cancer Cell Line: 40 years of Experience in Research. Anticancer Res, 2015. 35(6): p. 3147-54.
58. Vander Heiden, M.G., L.C. Cantley, and C.B. Thompson, Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 2009. 324(5930): p. 1029-33.
59. Bezawork-Geleta, A., et al., Mitochondrial Complex II: At the Crossroads. Trends Biochem Sci, 2017. 42(4): p. 312-325.
60. Sharma, P., et al., Crystal structure of an assembly intermediate of respiratory Complex II. Nat Commun, 2018. 9(1): p. 274.
61. Zhang, J. and Q. Zhang, Using Seahorse Machine to Measure OCR and ECAR in Cancer Cells. Methods Mol Biol, 2019. 1928: p. 353-363.
62. Santoro, V., et al., SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism. Oncotarget, 2020. 11(8): p. 801-812.
63. Bartys, N., R. Kierzek, and J. Lisowiec-Wachnicka, The regulation properties of RNA secondary structure in alternative splicing. Biochim Biophys Acta Gene Regul Mech, 2019. 1862(11-12): p. 194401.
64. Lee, Y. and D.C. Rio, Mechanisms and Regulation of Alternative Pre-mRNA Splicing. Annu Rev Biochem, 2015. 84: p. 291-323.