侵襲性泌尿道上皮癌的一個主要特徵是其複雜的染色體基因組改變。然而，哪些分子從基因組異變中驅動其異常表現，以及它們如何參與在腫瘤進程中仍然在很大程度上是未知的。在本研究中，首先藉由晶片式基因組雜交試驗（aCGH）對膀胱泌尿上皮癌基因組的異變情形做全面性的檢測。由結果中發現8q11.21此基因組擴增顯著發生在疾病特異性死亡和遠端轉移的患者中並且與患者的不良預後高度相關。在位於此位點的候選基因中，CEBPD mRNA表現與8q11.21的擴增最顯著相關，表明基因組擴增可驅動其表現。進一步證實CEBPD基因擴增和過度表達彼此具有高度相關性且兩者都與不利的臨床病理特徵和較差的患者臨床預後相關。值得注意的是，在aCGH數據集中發現了高頻率的CEBPD和C-MYC共擴增。為進一步釐清這些發現，我們檢測細胞株之中不同CEBPD表達水平時細胞的生物功能變化。CEBPD表達促進泌尿道上皮癌細胞生長，並且這種作用需要C-MYC參與。因此，我們檢視CEBPD與C-MYC之間可能的調控機制。結果顯示CEBPD表達可通過抑制基於蛋白酶體的蛋白降解增加C-MYC蛋白的穩定。此外，CEBPD的異位過表達導致葡萄糖消耗增加，乳酸生成增加，並且增強Hexokinase 2的表達。最後，本研究探討CEBPD在腫瘤轉移中的作用，CEBPD可透過調控MMP2增強細胞遷移和細胞侵襲能力。此外，CEBPD增強MMP2表現是透過直接結合於其啟動子區域所調控。綜合以上結果，本研究證明CEBPD基因擴增是驅動mRNA和蛋白質表達增加的機制。CEBPD增強MMP2介導正的細胞侵襲並增加C-MYC表現以改變葡萄糖代謝途徑賦予尿路上皮癌侵略性。

One major characteristic of aggressive urothelial carcinoma (UC) is its complex genomic alterations. However, what dysregulated-molecules drive from genomic aberrations and how they provide UC force to progress remain largely unknown. In this study, we performed array-based comparative genomic hybridization (aCGH) on 40 UC samples to comprehensively investigate genomic aberrations. Amplification of 8q11.21 was preferentially identified in patients who developed disease-specific death (53.8%) and distal metastasis (50.0%) but was barely detected in non-eventful cases (3.7% and 0%, respectively). Furthermore, amplification of 8q11.21 was significantly associated with poor outcomes in patients. Among these candidate genes harbored in 8q11.21, we identified CCAAT/enhancer binding protein delta (CEBPD) mRNA expression as most significantly associated with gain at 8q11.21, suggesting amplification-driven expression. By performing CEBPD-specific fluorescence in situ hybridization and immunohistochemistry, we confirmed that CEBPD amplification and overexpression were strongly related to each other (p<0.001). Moreover, both were associated with adverse clinic pathologic features and worse patient outcomes. Of note, we also discerned a high frequency of CEBPD and C-MYC co-amplification from the aCGH dataset. To extend these findings, we examined the biological effects with different CEBPD expression levels in human UC cell lines. CEBPD expression promotes UC cells growth and this effect is C-MYC dependent. Therefore, we investigated regulated mechanism between CEBPD and C-MYC. CEBPD expression is required for C-MYC protein stability by inhibiting proteasome-based degradation. Subsequently, we identified ectopic overexpression of CEBPD that leads to increased glucose consumption, lactate production and upregulated the expression of critical glycolytic genes Hexokinase 2 (HK2). Finally, we explored the role of CEBPD in tumor metastasis. Interestedly, CEBPD promoted cell migration and cell invasion ability in UC cells and these effects were attributed of Matrix Metalloproteinase 2 (MMP2). Moreover, expression of CEBPD significantly enhanced MMP2 expression and transcriptional activation by direct binding to its promoter region. Conclusively, our study demonstrates that CEBPD amplification is a mechanism driving increased mRNA and protein expression. CEBPD enhances MMP2-mediated cell invasiveness and increases C-MYC to alteration of glucose metabolic pathways that confers UC aggressiveness.

1. Aragon-Ching, J.B., Challenges and advances in the diagnosis, biology, and treatment of urothelial upper tract and bladder carcinomas. Urol Oncol, 2017. 35(7): p. 462-464.
2. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer Statistics, 2017. CA Cancer J Clin, 2017. 67(1): p. 7-30.
3. Alexandrov, L.B., et al., Mutational signatures associated with tobacco smoking in human cancer. Science, 2016. 354(6312): p. 618-622.
4. Burger, M., et al., Epidemiology and risk factors of urothelial bladder cancer. Eur Urol, 2013. 63(2): p. 234-41.
5. Muenyi, C.S., M. Ljungman, and J.C. States, Arsenic Disruption of DNA Damage Responses-Potential Role in Carcinogenesis and Chemotherapy. Biomolecules, 2015. 5(4): p. 2184-93.
6. Buttigliero, C., et al., Molecular biomarkers to predict response to neoadjuvant chemotherapy for bladder cancer. Cancer Treat Rev, 2017. 54: p. 1-9.
7. Blaveri, E., et al., Bladder cancer stage and outcome by array-based comparative genomic hybridization. Clin Cancer Res, 2005. 11(19 Pt 1): p. 7012-22.
8. Glaser, A.P., et al., The evolving genomic landscape of urothelial carcinoma. Nat Rev Urol, 2017. 14(4): p. 215-229.
9. Hurst, C.D., et al., Novel tumor subgroups of urothelial carcinoma of the bladder defined by integrated genomic analysis. Clin Cancer Res, 2012. 18(21): p. 5865-77.
10. Prat, E., et al., Comparative genomic hybridization analysis reveals new different subgroups in early-stage bladder tumors. Urology, 2010. 75(2): p. 347-55.
11. Simon, R., et al., Amplification pattern of 12q13-q15 genes (MDM2, CDK4, GLI) in urinary bladder cancer. Oncogene, 2002. 21(16): p. 2476-83.
12. Simon, R., et al., HER-2 and TOP2A coamplification in urinary bladder cancer. Int J Cancer, 2003. 107(5): p. 764-72.
13. Landschulz, W.H., et al., Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev, 1988. 2(7): p. 786-800.
14. Ramji, D.P. and P. Foka, CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J, 2002. 365(Pt 3): p. 561-75.
15. Agrawal, S., et al., The C/EBPdelta tumor suppressor is silenced by hypermethylation in acute myeloid leukemia. Blood, 2007. 109(9): p. 3895-905.
16. Radich, J.P., et al., Gene expression changes associated with progression and response in chronic myeloid leukemia. Proc Natl Acad Sci U S A, 2006. 103(8): p. 2794-9.
17. Musialik, E., et al., Comparison of promoter DNA methylation and expression levels of genes encoding CCAAT/enhancer binding proteins in AML patients. Leuk Res, 2014. 38(7): p. 850-6.
18. Chuang, C.H., et al., The combination of the prodrugs perforin-CEBPD and perforin-granzyme B efficiently enhances the activation of caspase signaling and kills prostate cancer. Cell Death Dis, 2014. 5: p. e1220.
19. Ko, C.Y., et al., Epigenetic silencing of CCAAT/enhancer-binding protein delta activity by YY1/polycomb group/DNA methyltransferase complex. J Biol Chem, 2008. 283(45): p. 30919-32.
20. Balamurugan, K., et al., The tumour suppressor C/EBPdelta inhibits FBXW7 expression and promotes mammary tumour metastasis. EMBO J, 2010. 29(24): p. 4106-17.
21. Hour, T.C., et al., Transcriptional up-regulation of SOD1 by CEBPD: a potential target for cisplatin resistant human urothelial carcinoma cells. Biochem Pharmacol, 2010. 80(3): p. 325-34.
22. Warburg, O., On the origin of cancer cells. Science, 1956. 123(3191): p. 309-14.
23. Dang, C.V., A. Le, and P. Gao, MYC-induced cancer cell energy metabolism and therapeutic opportunities. Clin Cancer Res, 2009. 15(21): p. 6479-83.
24. Prochownik, E.V. and Y. Li, The ever expanding role for c-Myc in promoting genomic instability. Cell Cycle, 2007. 6(9): p. 1024-9.
25. Dang, C.V., et al., The c-Myc target gene network. Semin Cancer Biol, 2006. 16(4): p. 253-64.
26. Popescu, N.C. and D.B. Zimonjic, Chromosome-mediated alterations of the MYC gene in human cancer. J Cell Mol Med, 2002. 6(2): p. 151-9.
27. Sauter, G., et al., c-myc copy number gains in bladder cancer detected by fluorescence in situ hybridization. Am J Pathol, 1995. 146(5): p. 1131-9.
28. Pan, J., et al., USP37 directly deubiquitinates and stabilizes c-Myc in lung cancer. Oncogene, 2015. 34(30): p. 3957-67.
29. Thomas, L.R. and W.P. Tansey, Proteolytic control of the oncoprotein transcription factor Myc. Adv Cancer Res, 2011. 110: p. 77-106.
30. Amati, B., Myc degradation: dancing with ubiquitin ligases. Proc Natl Acad Sci U S A, 2004. 101(24): p. 8843-4.
31. Fanjul-Fernandez, M., et al., Matrix metalloproteinases: evolution, gene regulation and functional analysis in mouse models. Biochim Biophys Acta, 2010. 1803(1): p. 3-19.
32. Yan, C. and D.D. Boyd, Regulation of matrix metalloproteinase gene expression. J Cell Physiol, 2007. 211(1): p. 19-26.
33. Szarvas, T., et al., Matrix metalloproteinases and their clinical relevance in urinary bladder cancer. Nat Rev Urol, 2011. 8(5): p. 241-54.
34. Vasala, K., P. Paakko, and T. Turpeenniemi-Hujanen, Matrix metalloproteinase-2 immunoreactive protein as a prognostic marker in bladder cancer. Urology, 2003. 62(5): p. 952-7.
35. Kanayama, H., et al., Prognostic values of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression in bladder cancer. Cancer, 1998. 82(7): p. 1359-66.
36. Miyata, Y., et al., Expression of metalloproteinase-2, metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 in transitional cell carcinoma of upper urinary tract: correlation with tumor stage and survival. Urology, 2004. 63(3): p. 602-8.
37. Huang, H.Y., et al., Skp2 overexpression is highly representative of intrinsic biological aggressiveness and independently associated with poor prognosis in primary localized myxofibrosarcomas. Clin Cancer Res, 2006. 12(2): p. 487-98.
38. Tang, D., G.S. Sivko, and J.W. DeWille, Promoter methylation reduces C/EBPdelta (CEBPD) gene expression in the SUM-52PE human breast cancer cell line and in primary breast tumors. Breast Cancer Res Treat, 2006. 95(2): p. 161-70.
39. Li, C.F., et al., HMDB and 5-AzadC Combination Reverses Tumor Suppressor CCAAT/Enhancer-Binding Protein Delta to Strengthen the Death of Liver Cancer Cells. Mol Cancer Ther, 2015. 14(11): p. 2623-33.
40. Welcker, M. and B.E. Clurman, FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer, 2008. 8(2): p. 83-93.
41. Miller, D.M., et al., c-Myc and cancer metabolism. Clin Cancer Res, 2012. 18(20): p. 5546-53.
42. Zhao, J., et al., Chromosomal imbalances in noninvasive papillary bladder neoplasms (pTa). Cancer Res, 1999. 59(18): p. 4658-61.
43. Fadl-Elmula, I., Chromosomal changes in uroepithelial carcinomas. Cell Chromosome, 2005. 4: p. 1.
44. Knowles, M.A., Molecular subtypes of bladder cancer: Jekyll and Hyde or chalk and cheese? Carcinogenesis, 2006. 27(3): p. 361-73.
45. Rogler, A., et al., Loss of MTUS1/ATIP expression is associated with adverse outcome in advanced bladder carcinomas: data from a retrospective study. BMC Cancer, 2014. 14: p. 214.
46. Stoehr, R., et al., Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer. Lab Invest, 2004. 84(4): p. 465-78.
47. Mahdy, E., et al., Chromosome 8 numerical aberration and C-MYC copy number gain in bladder cancer are linked to stage and grade. Anticancer Res, 2001. 21(5): p. 3167-73.
48. Kotake, T., et al., Detection of the c-myc gene product in urinary bladder cancer. Jpn J Cancer Res, 1990. 81(12): p. 1198-201.
49. Tanaka, T., et al., Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene. Embo j, 1997. 16(24): p. 7432-43.
50. Sterneck, E., et al., Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein delta. Proc Natl Acad Sci U S A, 1998. 95(18): p. 10908-13.
51. Litvak, V., et al., Function of C/EBPdelta in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals. Nat Immunol, 2009. 10(4): p. 437-43.
52. Liu, Y.W., et al., Inhibition of LPS-induced C/EBP delta by trichostatin A has a positive effect on LPS-induced cyclooxygenase 2 expression in RAW264.7 cells. J Cell Biochem, 2010. 110(6): p. 1430-8.
53. Borrelli, S., et al., Reciprocal regulation of p63 by C/EBP delta in human keratinocytes. BMC Mol Biol, 2007. 8: p. 85.
54. Wu, S.R., et al., CCAAT/enhancer-binding protein delta mediates tumor necrosis factor alpha-induced Aurora kinase C transcription and promotes genomic instability. J Biol Chem, 2011. 286(33): p. 28662-70.
55. Hsiao, Y.W., et al., CCAAT/enhancer binding protein delta in macrophages contributes to immunosuppression and inhibits phagocytosis in nasopharyngeal carcinoma. Sci Signal, 2013. 6(284): p. ra59.