||探討新穎微管抑制劑 MPT0B098 作用於缺氧下所引發的上皮細胞轉殖成間質細胞之成效
||Study the Effect of Novel Microtubule Inhibitor MPT0B098 on Epithelial to Mesenchymal Transition under Hypoxia Condition
||Institute of Molecular Medicine
Epithelial to mesenchymal transition (EMT)
microtubule inhibitor MPT0B098
上皮間質轉換（EMT）起先是在胎胚發育時期被發現。而近年來得知其與腫瘤細胞從良性轉化成惡性腫瘤有關。此外，越來越多的證據顯示經由缺氧誘導因子（HIF）可以調節一些引發上皮細胞轉殖成間質細胞的訊息路徑。我們實驗室先前找到一個新穎的微管抑制劑—7-芳基-二氫吲哚-1-苯磺胺酰胺（MPT0B098），其不同於其他微管抑制劑，它藉由調節人類抗原 R （HuR）來抑制缺氧誘導因子-1α 的核醣核酸以及蛋白質的表現 。本研究米進一步探討 MPT0B098 對EMT的進行及機轉之影響。由於細胞型態的變化是EMT 發生的過程很重要標誌，我們首先觀察到缺氧環境下細胞的型態有明顯的變化，同時 MPT0B098 可以有效抑制細胞從上皮細胞轉化成間質細胞型態。從這裡我們得知 MPT0B098 對於調控 EMT 來說是很有潛力的。接下來我們也發現 MPT0B098 確實能夠抑制 EMT 相關的蛋白。另外，MPT0B098 相較於其他微管抑制劑有著更加顯著抑制效果。在缺氧的環境下，我們觀察到 OECM-1 細胞有高度的侵略性以及移動力，而在 MPT0B098 的影響下可以降低細胞的移動能力。接著探討 MPT0B098 的機制，我們發現轉化生長因子-β活化的 Smads 訊息傳遞訊號能有效的被 MPT0B098 隨著濃度變化給抑制。並且在深入研究後我們發現 MPT0B098 是透過抑制細胞因子轉化生長因子-β 訊息核糖核酸以及蛋白質的表現量。由於在缺氧環境下缺氧誘導因子-1α能夠和細胞因子轉化生長因子-β驅動子結合，並且調控他們的轉錄能力，於是我們擬進一步探討 MPT0B098是否是透過缺氧誘導因子-1α來調控細胞因子轉化生長因子-β。結果顯示直接降低缺氧誘導因子-1α轉譯表現量對細胞因子轉化生長因子-β 沒有影響。接著我們又進一步探討 MPT0B098 在常氧下對細胞因子轉化生長因子-β的影響，結果顯示在常氧環境下 MPT0B098 的抑制效果仍然存在。綜此，本研究首次闡明一個新穎的微管抑制劑藉由調控細胞因子轉化生長因子-β訊息傳遞路徑來抑制缺氧下所引發的上皮間質轉化。
Epithelial to mesenchymal transition (EMT) is first recognized as a characteristic of embryogenesis in development. Recently, it has been involved in tumor transformation from benign to malignant tumor and cancer metastasis. Moreover, evidence has demonstrated that regulation of EMT signaling pathways is associated with HIF-1α, under hypoxia condition. We recently discovered a novel microtubule inhibitor, 7-aryl-indoline-1-benzene-sulfonamide (MPT0B098). Distinguished from other microtubule inhibitors, MPT0B098 destabilizes the HIF-1α mRNA under hypoxia by inhibiting human antigen R (HuR) translocation from the nucleaus to cytoplasm. Therefore, the objections of this study were to identify the idea and possible underlying mechanism of MPT0B098 on EMT inhibition under hypoxia. We firstly observed that MPT0B098 could inhibit a change of OEC-M1 cells from epithelial to mesenchymal morphologies under hypoxia, indicating that MPT0B098 potentially modulated the process of EMT. Furthermore, our data showed that the protein levels of mesenchymal markers, including N-cadherin, twist, slug and vimentin, were down-regulated in a concentration-dependent manner in OEC-M1 cells treated with various concentrations of MPT0B098 under hypoxia. In addition, MPT0B098 exerted a stronger inhibitory activity against EMT-related proteins than other microtubule inhibitors. Furthermore, we observed that the migratory ability of OEC-M1cells decreased in a concentration-dependent manner when the cells are treated with various concentrations of MPT0B098 under hypoxia. To investigate the mechanism of MPT0B098 action against EMT, we found that TGF-β-induced phosphorylation of receptor-associated Smads were effectively blocked through decreasing TGF-β mRNA and protein expression when cells treating with MPT0B098 in a concentration-dependent manner under hypoxia. Since the interplay between HIF-1α and TGF-βis well-known, we then knocked down HIF-1α expression under hypoxia. Surprisingly, the inhibitory activity of HIF-1α-targeted siRNA on canonical TGF-β signaling cascade is no difference between cells treated with or without MPT0B098 treatment. Moreover, we also demonstrated that MPT0B098 could inhibit TGF-βs protein expressions in a concentration-dependent manner under a normoxia condition. Taken together, results from this study may provide a novel insight into mechanism of action of microtubule inhibitor for inhibiting EMT.
Chinese Abstract II
English Abstract IV
1. Metastasis 1
1-1 Hypoxia 2
1-2 Epithelial-to-Mesenchymal Transition (EMT) 3
1-3 Transforming growth factor‑β (TGF-β) signaling 3
1-4 TGF‑β signaling in EMT 4
1-5 Transcriptional regulation of EMT by Smads in TGF-β signaling 5
1-5-1 Snail family transcription factors 5
1-5-2 ZEB family transcription factors 6
1-5-3 Helix-loop-helix family factors 6
1-6 Non-Smad signaling in TGF-β-induced EMT 7
1-7 Head and Neck Cancer 7
2. Microtubule-targeted anticancer drugs 8
Specific aims 9
Materials and Methods 10
1. Chemicals and antibodies 10
2. Cell lines and culture conditions 10
3. Cell viability assay 11
4. Western blot analysis 11
5. Invasion assay 11
6. Wound healing assay 12
7. Immunofluorescent analysis of F-actin 12
8. RT-PCR 12
9. Hypoxia inducible factor 1 alpha (HIF-1α) knockdown 13
10. Statistical Analysis 13
1. Anti-proliferative activity of microtubule inhibitors in OEC-M1 cells 14
2. The effect of MPT0B098 on hypoxia-induced EMT process in OEC-M1 cells 14
3. MPT0B098 is more potent than other microtubule inhibitors to inhibit the expression of EMT-related protein 14
4. Hypoxia promotes migration and invasion of OEC-M1 cells 15
5. MPT0B098 inhibits cell migration and the expression of F-actin and p-FAK under hypoxic-condition 16
6. MPT0B098 does not affect the invasion of OEC-M1 cells 17
7. MPT0B098 down-regulates TGF-β-induced phosphorylation of receptor-associated Smads by decreasing TGF-β mRNA and protein levels 17
8. HIF-1α knockdown causes no effect on TGF-β-induced phosphorylation of receptor-associated Smads in OEC-M1 cells 18
9. The inihbitory effect of MPT0B098 on TGF- β-induced Smad signaling under a normoxia condition 19
1. Brodland, D.G. and J.A. Zitelli, Mechanisms of metastasis. Journal of the American Academy of Dermatology, 1992. 27(1): p. 1-8.
2. Liotta, L. and W. Stetler-Stevenson, Principles of molecular cell biology of cancer: cancer metastasis. Cancer: principles and practice of oncology, 1993. 1: p. 134-149.
3. Chaffer, C.L. and R.A. Weinberg, A perspective on cancer cell metastasis. Science, 2011. 331(6024): p. 1559-1564.
4. Fidler, I.J., The pathogenesis of cancer metastasis: the'seed and soil'hypothesis revisited. Nature Reviews Cancer, 2003. 3(6): p. 453-458.
5. Yang, M.-H. and K.-J. Wu, TWIST activation by hypoxia inducible factor-1 (HIF-1). Cell Cycle, 2008. 7(14): p. 2090-2096.
6. Thompson, E.W. and E.D. Williams, EMT and MET in carcinoma—clinical observations, regulatory pathways and new models. Clinical and Experimental Metastasis, 2008. 25(6): p. 591-592.
7. Hill, R.P., D.T. Marie-Egyptienne, and D.W. Hedley. Cancer stem cells, hypoxia and metastasis. in Seminars in radiation oncology. 2009. Elsevier.
8. Kudo-Saito, C., et al., Cancer metastasis is accelerated through immunosuppression during Snail-induced EMT of cancer cells. Cancer cell, 2009. 15(3): p. 195-206.
9. Wang, Y., et al., [Relationship between epithelial-mesenchymal transition and lung metastasis in hepatocellular carcinoma]. Zhonghua wai ke za zhi [Chinese journal of surgery], 2008. 46(21): p. 1624-1627.
10. Brabletz, T., et al., Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and β-catenin. Cells Tissues Organs, 2005. 179(1-2): p. 56-65.
11. Thiery, J.P., Epithelial–mesenchymal transitions in tumour progression. Nature Reviews Cancer, 2002. 2(6): p. 442-454.
12. Lamouille, S., J. Xu, and R. Derynck, Molecular mechanisms of epithelial–mesenchymal transition. Nature Reviews Molecular Cell Biology, 2014. 15(3): p. 178-196.
13. Barar, J., Targeting Tumor Microenvironment: The Key Role of Immune System. BioImpacts: BI, 2012. 2(1): p. 1.
14. Whiteside, T., The tumor microenvironment and its role in promoting tumor growth. Oncogene, 2008. 27(45): p. 5904-5912.
15. Brown, J.M. and A.J. Giaccia, The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer research, 1998. 58(7): p. 1408-1416.
16. Bennewith, K.L. and S. Dedhar, Targeting hypoxic tumour cells to overcome metastasis. BMC cancer, 2011. 11(1): p. 504.
17. Ruas, J.L. and L. Poellinger. Hypoxia-dependent activation of HIF into a transcriptional regulator. in Seminars in cell & developmental biology. 2005. Elsevier.
18. Schipani, E. Hypoxia and HIF-1α in chondrogenesis. in Seminars in cell & developmental biology. 2005. Elsevier.
19. Loboda, A., A. Jozkowicz, and J. Dulak, HIF-1 and HIF-2 transcription factors—similar but not identical. Molecules and cells, 2010. 29(5): p. 435-442.
20. Holmquist-Mengelbier, L., et al., Recruitment of HIF-1α and HIF-2α to common target genes is differentially regulated in neuroblastoma: HIF-2α promotes an aggressive phenotype. Cancer cell, 2006. 10(5): p. 413-423.
21. Wenger, R.H., Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. The FASEB journal, 2002. 16(10): p. 1151-1162.
22. Talbot, L.J., S.D. Bhattacharya, and P.C. Kuo, Epithelial-mesenchymal transition, the tumor microenvironment, and metastatic behavior of epithelial malignancies. International journal of biochemistry and molecular biology, 2012. 3(2): p. 117.
23. Epstein, F.H., et al., Role of transforming growth factor β in human disease. New England Journal of Medicine, 2000. 342(18): p. 1350-1358.
24. Meulmeester, E. and P. ten Dijke, The dynamic roles of TGF‐β in cancer. The Journal of pathology, 2011. 223(2): p. 206-219.
25. Wendt, M.K., M. Tian, and W.P. Schiemann, Deconstructing the mechanisms and consequences of TGF-β-induced EMT during cancer progression. Cell and tissue research, 2012. 347(1): p. 85-101.
26. Koinuma, D., et al., Promoter‐wide analysis of Smad4 binding sites in human epithelial cells. Cancer science, 2009. 100(11): p. 2133-2142.
27. Moustakas, A. and C.H. Heldin, Signaling networks guiding epithelial–mesenchymal transitions during embryogenesis and cancer progression. Cancer science, 2007. 98(10): p. 1512-1520.
28. Hurd, T.W., et al., Direct interaction of two polarity complexes implicated in epithelial tight junction assembly. Nature cell biology, 2003. 5(2): p. 137-142.
29. Xu, J., S. Lamouille, and R. Derynck, TGF-β-induced epithelial to mesenchymal transition. Cell research, 2009. 19(2): p. 156-172.
30. Peinado, H., D. Olmeda, and A. Cano, Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nature Reviews Cancer, 2007. 7(6): p. 415-428.
31. Nieto, M.A., The snail superfamily of zinc-finger transcription factors. Nature reviews Molecular cell biology, 2002. 3(3): p. 155-166.
32. Barrallo-Gimeno, A. and M.A. Nieto, The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development, 2005. 132(14): p. 3151-3161.
33. Cano, A., et al., The transcription factor snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nature cell biology, 2000. 2(2): p. 76-83.
34. Batlle, E., et al., The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature cell biology, 2000. 2(2): p. 84-89.
35. Hemavathy, K., et al., Human Slug is a repressor that localizes to sites of active transcription. Molecular and cellular biology, 2000. 20(14): p. 5087-5095.
36. Hoot, K.E., et al., Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression. The Journal of clinical investigation, 2008. 118(8): p. 2722.
37. Ju, W., et al., Deletion of Smad2 in mouse liver reveals novel functions in hepatocyte growth and differentiation. Molecular and cellular biology, 2006. 26(2): p. 654-667.
38. Morita, T., T. Mayanagi, and K. Sobue, Dual roles of myocardin-related transcription factors in epithelial–mesenchymal transition via slug induction and actin remodeling. The Journal of cell biology, 2007. 179(5): p. 1027-1042.
39. Postigo, A.A., et al., Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. The EMBO journal, 2003. 22(10): p. 2453-2462.
40. Shirakihara, T., M. Saitoh, and K. Miyazono, Differential regulation of epithelial and mesenchymal markers by δEF1 proteins in epithelial–mesenchymal transition induced by TGF-β. Molecular biology of the cell, 2007. 18(9): p. 3533-3544.
41. Postigo, A.A., Opposing functions of ZEB proteins in the regulation of the TGFβ/BMP signaling pathway. The EMBO journal, 2003. 22(10): p. 2443-2452.
42. Massari, M.E. and C. Murre, Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Molecular and cellular biology, 2000. 20(2): p. 429-440.
43. Chen, Z.-F. and R.R. Behringer, twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes & development, 1995. 9(6): p. 686-699.
44. El Ghouzzi, V., et al., Mutations of the TWIST gene in the Saethre-Chotzene syndrome. Nature genetics, 1997. 15(1): p. 42-46.
45. Howard, T.D., et al., Mutations in TWIST, a basic helix–loop–helix transcription factor, in Saethre-Chotzen syndrome. Nature genetics, 1997. 15(1): p. 36-41.
46. Yang, J., S.A. Mani, and R.A. Weinberg, Exploring a new twist on tumor metastasis. Cancer research, 2006. 66(9): p. 4549-4552.
47. Yang, J., et al., Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell, 2004. 117(7): p. 927-939.
48. Ansieau, S., et al., Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence. Cancer cell, 2008. 14(1): p. 79-89.
49. Zhang, Y.E., Non-Smad pathways in TGF-β signaling. Cell research, 2009. 19(1): p. 128-139.
50. Valcourt, U., et al., TGF-β and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Molecular biology of the cell, 2005. 16(4): p. 1987-2002.
51. Derynck, R. and Y.E. Zhang, Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature, 2003. 425(6958): p. 577-584.
52. Polanska, H., et al., Clinical significance of head and neck squamous cell cancer biomarkers. Oral oncology, 2013.
53. Ramos, M., S. Benavente, and J. Giralt, Management of squamous cell carcinoma of the head and neck: updated European treatment recommendations. 2010.
54. Siegel, R., et al., Cancer statistics, 2014. CA: a cancer journal for clinicians, 2014. 64(1): p. 9-29.
55. Li, J.Z., et al., Hypoxia in head and neck squamous cell carcinoma. ISRN otolaryngology, 2012. 2012.
56. Dunst, J., et al., Tumor volume and tumor hypoxia in head and neck cancers. Strahlentherapie und Onkologie, 2003. 179(8): p. 521-526.
57. Nordsmark, M., M. Overgaard, and J. Overgaard, Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiotherapy and oncology, 1996. 41(1): p. 31-39.
58. Brizel, D.M., et al., Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck. International Journal of Radiation Oncology* Biology* Physics, 1997. 38(2): p. 285-289.
59. Vaupel, P., Blood flow and oxygenation status of head and neck carcinomas, in Oxygen Transport to Tissue XIX. 1997, Springer. p. 89-95.
60. Mattern, J., et al., Association of resistance‐related protein expression with poor vascularization and low levels of oxygen in human rectal cancer. International journal of cancer, 1996. 67(1): p. 20-23.
61. Movsas, B., et al., Increasing levels of hypoxia in prostate carcinoma correlate significantly with increasing clinical stage and patient age. Cancer, 2000. 89(9): p. 2018-2024.
62. Höckel, M., et al., Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer research, 1996. 56(19): p. 4509-4515.
63. Toustrup, K., et al. Hypoxia gene expression signatures as prognostic and predictive markers in head and neck radiotherapy. in Seminars in radiation oncology. 2012. Elsevier.
64. Nogales, E., Structural insights into microtubule function. Annual review of biochemistry, 2000. 69(1): p. 277-302.
65. Perez, E.A., Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Molecular cancer therapeutics, 2009. 8(8): p. 2086-2095.
66. Cheng, Y.-C., et al., MPT0B098, a novel microtubule inhibitor that destabilizes the hypoxia-inducible factor-1α mRNA through decreasing nuclear–cytoplasmic translocation of RNA-binding protein HuR. Molecular cancer therapeutics, 2013. 12(7): p. 1202-1212.
67. Jiang, J., Y.-l. Tang, and X.-h. Liang, A new vision of hypoxia promoting cancer progression. Cancer biology & therapy, 2011. 11(8): p. 714-723.
68. Nien, C.-Y., et al., 5-Amino-2-aroylquinolines as highly potent tubulin polymerization inhibitors. Journal of medicinal chemistry, 2010. 53(5): p. 2309-2313.
69. Pirozzi, G., et al., Epithelial to mesenchymal transition by TGFβ-1 induction increases stemness characteristics in primary non small cell lung cancer cell line. PLoS One, 2011. 6(6): p. e21548.
70. Stehbens, S.J., A. Akhmanova, and A. Yap, Microtubules and cadherins: a neglected partnership. Frontiers in bioscience (Landmark edition), 2008. 14: p. 3159-3167.
71. Chaudary, N. and R.P. Hill, Hypoxia and metastasis. Clinical Cancer Research, 2007. 13(7): p. 1947-1949.
72. Stricker, J., T. Falzone, and M.L. Gardel, Mechanics of the F-actin cytoskeleton. Journal of biomechanics, 2010. 43(1): p. 9-14.
73. van Nimwegen, M.J. and B. van de Water, Focal adhesion kinase: a potential target in cancer therapy. Biochemical pharmacology, 2007. 73(5): p. 597-609.
74. Parsons, J.T., et al., Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement. Oncogene, 2000. 19(49).
75. Xu, D., et al., Matrix metalloproteinase-9 regulates tumor cell invasion through cleavage of protease nexin-1. Cancer research, 2010. 70(17): p. 6988-6998.
76. Hung, S.-P., et al., Hypoxia-Induced Secretion of TGF-β1 in Mesenchymal Stem Cell Promotes Breast Cancer Cell Progression. Cell transplantation, 2013. 22(10): p. 1869-1882.
77. Shtokalo, D., et al., Insights from the HuR-interacting transcriptome: ncRNAs, ubiquitin pathways, and patterns of secondary structure dependent RNA interactions. Molecular Genetics and Genomics, 2012. 287(11-12): p. 867-879.
78. Zhang, H., et al., PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells. American Journal of Physiology-Cell Physiology, 2014. 306(7): p. C648-C658.
79. Dulbecco, R., et al., Functional changes of intermediate filaments in fibroblastic cells revealed by a monoclonal antibody. Proceedings of the National Academy of Sciences, 1983. 80(7): p. 1915-1918.
80. Evans, R.M. and L.M. Fink, An alteration in the phosphorylation of vimentin-type intermediate filaments is associated with mitosis in cultured mammalian cells. Cell, 1982. 29(1): p. 43-52.
81. Bierie, B. and H.L. Moses, TGF-β and cancer. Cytokine & growth factor reviews, 2006. 17(1): p. 29-40.
82. Copple, B.L., Hypoxia stimulates hepatocyte epithelial to mesenchymal transition by hypoxia‐inducible factor and transforming growth factor‐β‐dependent mechanisms. Liver International, 2010. 30(5): p. 669-682.
83. Dong, C., et al., Microtubule binding to Smads may regulate TGFβ activity. Molecular cell, 2000. 5(1): p. 27-34.
84. Massagué, J., TGFβ signalling in context. Nature reviews Molecular cell biology, 2012. 13(10): p. 616-630.
85. Teppo, S., et al., The hypoxic tumor microenvironment regulates invasion of aggressive oral carcinoma cells. Experimental cell research, 2013. 319(4): p. 376-389.
86. Yang, X., et al., Inactivation of lysyl oxidase by β-aminopropionitrile inhibits hypoxia-induced invasion and migration of cervical cancer cells. Oncology reports, 2013. 29(2): p. 541-548.
87. Friedl, P. and K. Wolf, Tumour-cell invasion and migration: diversity and escape mechanisms. Nature Reviews Cancer, 2003. 3(5): p. 362-374.
88. Schaller, M.D., Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions. Journal of Cell Science, 2010. 123(7): p. 1007-1013.
89. Deryugina, E.I. and J.P. Quigley, Matrix metalloproteinases and tumor metastasis. Cancer and Metastasis Reviews, 2006. 25(1): p. 9-34.
90. Jordan, M.A. and L. Wilson, Microtubules as a target for anticancer drugs. Nature Reviews Cancer, 2004. 4(4): p. 253-265.
91. Chen, X.-m., et al., Colchicine-induced apoptosis in human normal liver L-02 cells by mitochondrial mediated pathways. Toxicology in Vitro, 2012. 26(5): p. 649-655.
92. Von Hoff, D. The taxoids: same roots, different drugs. in Seminars in oncology. 1997.
93. Markman, M., Managing taxane toxicities. Supportive care in cancer, 2003. 11(3): p. 144-147.
94. Fojo, A.T. and M. Menefee. Microtubule targeting agents: basic mechanisms of multidrug resistance (MDR). in Seminars in oncology. 2005. Elsevier.