組織蛋白乙醯化抑制劑Suberoylanilide hydroxamic acid (SAHA)可抑制多種癌細胞生長並被美國食品藥物監督管理局核淮用在癌症治療上。SAHA也可以抑制腫瘤血管生成。然而，SAHA能否抑制腫瘤淋巴血管生成並不清楚。本研究主題探討SAHA在乳癌細胞中抗淋巴血管生成的機制。本研究共分三部分，在第一部分中，我探討SAHA是否會影響淋巴管生成因子C(VEGF-C)的產生，減少淋巴血管細胞增生及移動至癌細胞。實驗結果發現，SAHA在多種乳癌細胞株中皆能有效抑制VEGF-C表現及分泌。我也發現SAHA能直接抑制VEGF-C表現是經由減少轉錄因子Sp1在VEGF-C啟動子-185/+38區域間的結合興活化。
第二部分，藉由表現淋巴血管主調控因子PROX1來建立一株具有淋巴血管特性的細胞株(FP01)，用來探討SAHA對淋巴血管生成的作用。實驗結果顯示，SAHA能有效抑制淋巴血管的增生及形成血管管狀結構的能力，並會減弱調控淋巴血管細胞重要的angiopoietin/Tie2訊息傳導路徑的作用。在啓動子實驗中顯示SAHA能經由轉錄抑制來降低Tie2的表現。另一方面，SAHA能快速增加泛素化酵素c-Cbl的表現使Tie2蛋白降解。抑制泛素化酵素c-Cbl能反轉SAHA對Tie2蛋白的降解作用。
第三部分，在乳癌細胞動物模式中證實SAHA能有效抑制乳癌生成，淋巴血管新生及轉移。
綜言之，SAHA不僅能抑制淋巴血管細胞的增生及形成淋巴血管管狀結構的能力，並能快速增加泛素化酵素c-Cbl的表現使Tie2進行降解，減弱調控淋巴血管細胞重要的angiopoietin/Tie2訊息傳導路徑。此外，SAHA亦能有效抑制乳癌細胞中淋巴管生成因子VEGFC表現及分泌。動物實驗也證實SAHA能有效抑制腫瘤形成、淋巴血管生成及淋巴轉移的能力。因此，在癌症治療上能利用SAHA來降低淋巴血管生成以抑制癌細胞轉移。

HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) exhibits anti-tumor effects on various types of human cancers and is now approved by U.S FDA for clinical cancer treatment. SAHA also suppresses tumor angiogenesis. However, the effect of SAHA on tumor lymphangiogeneis is unclear. My study focuses on anti-lymphangiogenic action of SAHA on breast cancers and contains three parts. In part one, I test whether SAHA affects the production of pro-lymphangiogenesis factor such as VEGF-C to reduce proliferation and migration of lymphatic endothelial cells toward cancer cells. I found that SAHA does-dependently inhibited the expression of VEGF-C in various breast cancer cell lines and the secretion of VEGF-C into conditioned medium was also suppressed. Furthermore, I cloned human VEGF-C gene promoter and demonstrated that SAHA directly suppressed VEGF-C transcription via the -185/+38 promoter region in a Sp1-mediated manner.
In part two, I aim to study the effect of SAHA on lymphatics endothelial cells (LECs). I established a lymphatic-like endothelial cell line (named as FP01) by overexpressing the master LEC transcription factor PROX1 in EA.hy926 endothelial cells. This cell lines showed similar gene expression pattern and phenotype of primarily cultured LECs. I found that SAHA can suppress proliferation, sprouting and tube formation of LECs. Moreover, SAHA could attenuate the angiopoietin/Tie signaling pathway which is important in the regulation of LEC function. The promoter activity assay revealed that SAHA down-regulated the expression of Tie2 through transcriptional repression. Interestingly, I also found that SAHA could quickly induce the expression of c-Cbl, the E3 ligase for Tie2 ubiquitination leading to Tie2 protein degradation. Knockdown of c-Cbl effectively reversed SAHA-induced Tie2 protein degradation.
In part three, I used breast cancer xenograft model to demonstrate whether SAHA could repress lymphangiogenesis and lymphatic metastasis in vivo. SAHA indeed inhibited tumor formation, lymphangiogenesis and metastasis in MDA-MB-231 luciferase-tagged xenograft model.
Taken together, SAHA not only suppresses proliferation, sprouting and tube formation of LECs and attenuates the Ang/Tie signaling in LECs by down-regulating Tie2 via transcriptional and post-transcriptional mechanism but also inhibits VEGF-C expression in breast cancer cells via transcriptional repression. Breast cancer xenograft model demonstrates that SAHA inhibits tumor formation, lymphangiogenesis and metastasis in vivo. Collectively, this drug exerts anti-lymphangiogenic activity in cancer treatment.

Pages
Chapter 1. Introduction 1

Chapter 2. Inhibition of lymphangiogenic factor VEGF-C expression and production by the histone deacetylase inhibitor suberoylanilide hydroxamic acid in breast cancer cells. 14

Chapter 3. Inhibition of proliferation, sprouting, tube formation and Tie2 signaling of lymphatic endothelial cells by histone deacetylase inhibitor SAHA.
36

Chapter 4. HDAC inhibitor suberoylanilide hydroxamic acid suppress lymphangiogenesis and metastasis in mouse model of breast cancer.
65

Chapter 5. References 86

Appendixes 94

1. Sarkies P, Sale JE: Cellular epigenetic stability and cancer. Trends Genet 28:118-27, 2012
2. Esteller M, Herman JG: Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol 196:1-7, 2002
3. Kouzarides T: Chromatin modifications and their function. Cell 128:693-705, 2007
4. Clayton AL, Hazzalin CA, Mahadevan LC: Enhanced histone acetylation and transcription: a dynamic perspective. Mol Cell 23:289-96, 2006
5. Marmorstein R: Structure and function of histone acetyltransferases. Cell Mol Life Sci 58:693-703, 2001
6. Marks P, Rifkind RA, Richon VM, et al: Histone deacetylases and cancer: causes and therapies. Nat Rev Cancer 1:194-202, 2001
7. Dokmanovic M, Clarke C, Marks PA: Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res 5:981-9, 2007
8. de Ruijter AJ, van Gennip AH, Caron HN, et al: Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 370:737-49, 2003
9. Xu WS, Parmigiani RB, Marks PA: Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 26:5541-52, 2007
10. Bolden JE, Peart MJ, Johnstone RW: Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 5:769-84, 2006
11. Witt O, Deubzer HE, Milde T, et al: HDAC family: What are the cancer relevant targets? Cancer Lett 277:8-21, 2009
12. Rosato RR, Grant S: Histone deacetylase inhibitors in clinical development. Expert Opin Investig Drugs 13:21-38, 2004
13. Rasheed WK, Johnstone RW, Prince HM: Histone deacetylase inhibitors in cancer therapy. Expert Opin Investig Drugs 16:659-78, 2007
14. Haberland M, Montgomery RL, Olson EN: The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet 10:32-42, 2009
15. Marks PA, Breslow R: Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat Biotechnol 25:84-90, 2007
16. Richon VM: Targeting histone deacetylases: development of vorinostat for the treatment of cancer. Epigenomics 2:457-65, 2010
17. Singh BN, Zhang G, Hwa YL, et al: Nonhistone protein acetylation as cancer therapy targets. Expert Rev Anticancer Ther 10:935-54, 2010
18. Dickinson M, Johnstone RW, Prince HM: Histone deacetylase inhibitors: potential targets responsible for their anti-cancer effect. Invest New Drugs 28 Suppl 1:S3-20, 2010
19. Insinga A, Monestiroli S, Ronzoni S, et al: Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway. Nat Med 11:71-6, 2005
20. Rosato RR, Almenara JA, Dai Y, et al: Simultaneous activation of the intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induces mitochondrial damage and apoptosis in human leukemia cells. Mol Cancer Ther 2:1273-84, 2003
21. Lindemann RK, Newbold A, Whitecross KF, et al: Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma. Proc Natl Acad Sci U S A 104:8071-6, 2007
22. Peart MJ, Tainton KM, Ruefli AA, et al: Novel mechanisms of apoptosis induced by histone deacetylase inhibitors. Cancer Res 63:4460-71, 2003
23. Shao Y, Gao Z, Marks PA, et al: Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A 101:18030-5, 2004
24. Rosato RR, Almenara JA, Cartee L, et al: The cyclin-dependent kinase inhibitor flavopiridol disrupts sodium butyrate-induced p21WAF1/CIP1 expression and maturation while reciprocally potentiating apoptosis in human leukemia cells. Mol Cancer Ther 1:253-66, 2002
25. Richon VM, Sandhoff TW, Rifkind RA, et al: Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci U S A 97:10014-9, 2000
26. Burgess A, Ruefli A, Beamish H, et al: Histone deacetylase inhibitors specifically kill nonproliferating tumour cells. Oncogene 23:6693-701, 2004
27. Eot-Houllier G, Fulcrand G, Magnaghi-Jaulin L, et al: Histone deacetylase inhibitors and genomic instability. Cancer Lett 274:169-76, 2009
28. Ruefli AA, Ausserlechner MJ, Bernhard D, et al: The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species. Proc Natl Acad Sci U S A 98:10833-8, 2001
29. Deroanne CF, Bonjean K, Servotte S, et al: Histone deacetylases inhibitors as anti-angiogenic agents altering vascular endothelial growth factor signaling. Oncogene 21:427-36, 2002
30. Kong X, Lin Z, Liang D, et al: Histone deacetylase inhibitors induce VHL and ubiquitin-independent proteasomal degradation of hypoxia-inducible factor 1alpha. Mol Cell Biol 26:2019-28, 2006
31. Chou CW, Chen CC: HDAC inhibition upregulates the expression of angiostatic ADAMTS1. FEBS Lett 582:4059-65, 2008
32. Wang Y, Wang SY, Zhang XH, et al: FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70. Biochem Biophys Res Commun 356:998-1003, 2007
33. Bali P, Pranpat M, Bradner J, et al: Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. J Biol Chem 280:26729-34, 2005
34. Gupta GP, Massague J: Cancer metastasis: building a framework. Cell 127:679-95, 2006
35. Achen MG, McColl BK, Stacker SA: Focus on lymphangiogenesis in tumor metastasis. Cancer Cell 7:121-7, 2005
36. Cao R, Bjorndahl MA, Religa P, et al: PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell 6:333-45, 2004
37. Mandriota SJ, Jussila L, Jeltsch M, et al: Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J 20:672-82, 2001
38. Lohela M, Bry M, Tammela T, et al: VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr Opin Cell Biol 21:154-65, 2009
39. Cueni LN, Detmar M: New insights into the molecular control of the lymphatic vascular system and its role in disease. J Invest Dermatol 126:2167-77, 2006
40. Alitalo K: The lymphatic vasculature in disease. Nat Med 17:1371-80, 2011
41. Augustin HG, Koh GY, Thurston G, et al: Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol 10:165-77, 2009
42. Duong T, Koopman P, Francois M: Tumor lymphangiogenesis as a potential therapeutic target. J Oncol 2012:204946, 2012
43. Norrmen C, Tammela T, Petrova TV, et al: Biological basis of therapeutic lymphangiogenesis. Circulation 123:1335-51, 2011
44. Tammela T, Alitalo K: Lymphangiogenesis: Molecular mechanisms and future promise. Cell 140:460-76, 2010
45. Skobe M, Hawighorst T, Jackson DG, et al: Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 7:192-8, 2001
46. Gou HF, Chen XC, Zhu J, et al: Expressions of COX-2 and VEGF-C in gastric cancer: correlations with lymphangiogenesis and prognostic implications. J Exp Clin Cancer Res 30:14, 2011
47. Tanaka T, Ishiguro H, Kuwabara Y, et al: Vascular endothelial growth factor C (VEGF-C) in esophageal cancer correlates with lymph node metastasis and poor patient prognosis. J Exp Clin Cancer Res 29:83, 2010
48. Ristimaki A, Narko K, Enholm B, et al: Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C. J Biol Chem 273:8413-8, 1998
49. Chilov D, Kukk E, Taira S, et al: Genomic organization of human and mouse genes for vascular endothelial growth factor C. J Biol Chem 272:25176-83, 1997
50. Marks PA, Breslow R: Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat Biotechnol 25:84-90, 2007
51. Duvic M, Vu J: Vorinostat: a new oral histone deacetylase inhibitor approved for cutaneous T-cell lymphoma. Expert Opin Investig Drugs 16:1111-20, 2007
52. Ellis L, Hammers H, Pili R: Targeting tumor angiogenesis with histone deacetylase inhibitors. Cancer Lett 280:145-53, 2009
53. Mattila MM, Ruohola JK, Karpanen T, et al: VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors. Int J Cancer 98:946-51, 2002
54. Flaherty KT: Sorafenib in renal cell carcinoma. Clin Cancer Res 13:747s-752s, 2007
55. Hanrahan EO, Heymach JV: Vascular endothelial growth factor receptor tyrosine kinase inhibitors vandetanib (ZD6474) and AZD2171 in lung cancer. Clin Cancer Res 13:s4617-22, 2007
56. Jimenez X, Lu D, Brennan L, et al: A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3. Mol Cancer Ther 4:427-34, 2005
57. Roberts N, Kloos B, Cassella M, et al: Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2. Cancer Res 66:2650-7, 2006
58. Rinderknecht M, Villa A, Ballmer-Hofer K, et al: Phage-derived fully human monoclonal antibody fragments to human vascular endothelial growth factor-C block its interaction with VEGF receptor-2 and 3. PLoS One 5:e11941, 2010
59. Stacker SA, Hughes RA, Williams RA, et al: Current strategies for modulating lymphangiogenesis signalling pathways in human disease. Curr Med Chem 13:783-92, 2006
60. Li L, Davie JR: The role of Sp1 and Sp3 in normal and cancer cell biology. Ann Anat 192:275-83, 2010
61. Wang L, Wei D, Huang S, et al: Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clin Cancer Res 9:6371-80, 2003
62. Safe S, Abdelrahim M: Sp transcription factor family and its role in cancer. Eur J Cancer 41:2438-48, 2005
63. Hung JJ, Wang YT, Chang WC: Sp1 deacetylation induced by phorbol ester recruits p300 to activate 12(S)-lipoxygenase gene transcription. Mol Cell Biol 26:1770-85, 2006
64. Wang SA, Chuang JY, Yeh SH, et al: Heat shock protein 90 is important for Sp1 stability during mitosis. J Mol Biol 387:1106-19, 2009
65. Li D, Marchenko ND, Moll UM: SAHA shows preferential cytotoxicity in mutant p53 cancer cells by destabilizing mutant p53 through inhibition of the HDAC6-Hsp90 chaperone axis. Cell Death Differ 18:1904-13, 2011
66. Maisonpierre PC, Suri C, Jones PF, et al: Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277:55-60, 1997
67. Yuan HT, Khankin EV, Karumanchi SA, et al: Angiopoietin 2 is a partial agonist/antagonist of Tie2 signaling in the endothelium. Mol Cell Biol 29:2011-22, 2009
68. Kim I, Kim JH, Moon SO, et al: Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Oncogene 19:4549-52, 2000
69. Murray BW, Padrique ES, Pinko C, et al: Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: enzymatic characterization of Tie2 and phospho-Tie2. Biochemistry 40:10243-53, 2001
70. Jones N, Chen SH, Sturk C, et al: A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function. Mol Cell Biol 23:2658-68, 2003
71. Jones N, Master Z, Jones J, et al: Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site mediates cell survival and migration. J Biol Chem 274:30896-905, 1999
72. Dellinger M, Hunter R, Bernas M, et al: Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice. Dev Biol 319:309-20, 2008
73. Shimoda H, Bernas MJ, Witte MH, et al: Abnormal recruitment of periendothelial cells to lymphatic capillaries in digestive organs of angiopoietin-2-deficient mice. Cell Tissue Res 328:329-37, 2007
74. Kajiya K, Kidoya H, Sawane M, et al: Promotion of lymphatic integrity by angiopoietin-1/Tie2 signaling during inflammation. Am J Pathol 180:1273-82, 2012

75. Pan MR, Chang TM, Chang HC, et al: Sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells. J Cell Sci 122:3358-64, 2009
76. Petrova TV, Makinen T, Makela TP, et al: Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J 21:4593-9, 2002
77. Wehrle C, Van Slyke P, Dumont DJ: Angiopoietin-1-induced ubiquitylation of Tie2 by c-Cbl is required for internalization and degradation. Biochem J 423:375-80, 2009
78. Deroanne CF, Bonjean K, Servotte S, et al: Histone deacetylases inhibitors as anti-angiogenic agents altering vascular endothelial growth factor signaling. Oncogene 21:427-36, 2002
79. Ugur HC, Ramakrishna N, Bello L, et al: Continuous intracranial administration of suberoylanilide hydroxamic acid (SAHA) inhibits tumor growth in an orthotopic glioma model. J Neurooncol 83:267-75, 2007
80. Muhlethaler-Mottet A, Meier R, Flahaut M, et al: Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells. Mol Cancer 7:55, 2008
81. Cheng HT, Hung WC: Inhibition of lymphangiogenic factor VEGF-C expression and production by the histone deacetylase inhibitor suberoylanilide hydroxamic acid in breast cancer cells. Oncol Rep 29:1238-44, 2013
82. Huang H, Bhat A, Woodnutt G, et al: Targeting the ANGPT-TIE2 pathway in malignancy. Nat Rev Cancer 10:575-85, 2010
83. Oliner J, Min H, Leal J, et al: Suppression of angiogenesis and tumor growth by selective inhibition of angiopoietin-2. Cancer Cell 6:507-16, 2004
84. Rosen, L.S. et al. First-in-human dose-escalation safety and PK trial of a novel intravenous humanized monoclonal CovX body inhibiting angiopoietin 2.J.Clin. Oncol. Abstr. 28: 2524-, 2010
85. Shimamoto G, Gegg C, Boone T, et al: Peptibodies: A flexible alternative format to antibodies. MAbs 4:586-91, 2012
86. Beck A, Reichert JM: Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. MAbs 3:415-6, 2011
87. Herbst RS, Hong D, Chap L, et al: Safety, pharmacokinetics, and antitumor activity of AMG 386, a selective angiopoietin inhibitor, in adult patients with advanced solid tumors. J Clin Oncol 27:3557-65, 2009
88. Eccles S, Paon L, Sleeman J: Lymphatic metastasis in breast cancer: importance and new insights into cellular and molecular mechanisms. Clin Exp Metastasis 24:619-36, 2007
89. Zijlstra A, Mellor R, Panzarella G, et al: A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction. Cancer Res 62:7083-92, 2002
89. Zijlstra A, Mellor R, Panzarella G, et al: A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction. Cancer Res 62:7083-92, 2002
90. Bergers G, Hanahan D: Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 8:592-603, 2008
91. Drummond DC, Noble CO, Kirpotin DB, et al: Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 45:495-528, 2005
92. Johnstone RW, Licht JD: Histone deacetylase inhibitors in cancer therapy: is transcription the primary target? Cancer Cell 4:13-8, 2003
93. Rahmani M, Yu C, Dai Y, et al: Coadministration of the heat shock protein 90 antagonist 17-allylamino- 17-demethoxygeldanamycin with suberoylanilide hydroxamic acid or sodium butyrate synergistically induces apoptosis in human leukemia cells. Cancer Res 63:8420-7, 2003
94. Rahmani M, Reese E, Dai Y, et al: Cotreatment with suberanoylanilide hydroxamic acid and 17-allylamino 17-demethoxygeldanamycin synergistically induces apoptosis in Bcr-Abl+ Cells sensitive and resistant to STI571 (imatinib mesylate) in association with down-regulation of Bcr-Abl, abrogation of signal transducer and activator of transcription 5 activity, and Bax conformational change. Mol Pharmacol 67:1166-76, 2005
95. Gryder BE, Sodji QH, Oyelere AK: Targeted cancer therapy: giving histone deacetylase inhibitors all they need to succeed. Future Med Chem 4:505-24, 2012