||Investigate the Role of the B55δ Regulatory Subunit of Protein Phosphatase 2A in the Tumorigenesis and Metastasis of Colorectal Cancer
||Institute of Molecular Medicine
B55δ regulatory subunit
大腸直腸癌為世界第三常見及最容易造成死亡的癌症排行第三，大多數的大腸直腸癌病人發生轉移的現象與其不良的預後相關，因此找尋大腸直腸癌過程中的生物標記物對於大腸直腸癌病人的預後診斷以及標靶治療相當重要。蛋白質磷酸酶2A型(PP2A)為一種絲氨酸/蘇胺酸磷水解酶，已知具有抑癌的功能。其完全酶組成主要由結構蛋白A次單元，催化蛋白C次單元以及種類多樣的調節性B次單元所組成，其中B次單元可決定PP2A完全酶的受質選擇性以及其在細胞中的座落位置。調節次單元可以分為四個家族B (B55或PR55)，B’ (B56或PR61)，B’’ (PR72)和B’’’ (PR93或PR110)。而B55δ次單元屬於B55家族成員並由PPP2R2D基因所編碼出來的蛋白，在許多文獻中已經指出其在細胞週期的有絲分裂中扮演重要的調控者。也有文獻指出在肝癌中，PP2A-B55δ可以透過對於小分子核糖核酸-133b (microRNA-133b)的調控來促進肝癌病人化學治療的效果；在胃癌中，PP2A-B55δ可能透過抑制哺乳動物雷帕黴素靶蛋白通路(mTOR)來促進胃癌腫瘤的生長及轉移。然而，其在大腸及其他種癌症中扮演什麼角色目前仍不清楚。在數據庫資料搜尋，我們發現B55δ在大腸直腸癌的腫瘤組織中相較於正常組織都是降低表現的，然而高表現的B55δ卻造成大腸直腸癌病人的存活率下降。綜合上述，我們假設B55δ可能在大腸直腸癌的進程中扮演不同的角色。首先，我們分析了B55δ在七個不同的大腸癌細胞株中的表現。接著我們建立過度表現B55δ或以核糖核酸干擾法將B55δ表現減低之細胞株去探討B55δ是否會調控腫瘤細胞的增生。從結果我們發現，B55δ的過度表現會在沒有影響到細胞存活率的情況下抑制HCT116及SW620細胞株的生長。出乎意料的是，在細胞群落形成實驗(Colony formation assay)的結果顯示，B55δ降低表現時也會去抑制SW620的細胞複製形成群落的能力。此外，B55δ在HCT116和SW620的細胞中降低表現時，細胞形態呈現拉長的、紡錘狀的數目增加；反之，過度表達B55δ時則會減少這類型態的細胞數。從細胞遷移(Transwell migration assay)及侵襲實驗(Transwell invasion assay)的結果顯示，B55δ在HCT116和SW620的細胞中大量表達時會增加細胞爬行及侵襲的能力，相反的，降低B55δ的表達則會顯著的降低細胞爬行和侵襲的能力。為了更清楚了解B55δ影響細胞爬行及侵襲能力是否透過上皮-間質細胞轉換(epithelial-mesenchymal transition, EMT)的調控。在我們探究的EMT分子標記中，我們發現在HCT116以及SW620細胞中B55δ的大量表現會造成E-cadherin表現降低及Snail表現增加，而B55δ降低表現時會導致E-cadherin的增加，有趣的是，在SW620細胞中也造成Snail的表現增加。綜合以上的結果，我們發現B55δ能夠抑制HCT116及SW620細胞的生長，但會促進HCT116和SW620細胞的遷移和侵襲能力以及E-cadherin的喪失，但可能並不是只有透過Snail來造成。綜合我們的結果，B55δ可能在大腸直腸癌進展的早期階段抑制腫瘤發生，但是在晚期可能透過上皮-間質細胞轉換的調控來促進轉移。
Colorectal cancer (CRC) is the third most common type of cancer and the third most common cause of cancer deaths in the world. CRC patients with distant metastases at the time of diagnosis are associated with worse prognosis. Therefore, it is urgent to find more biomarkers and therapeutic targets for improving detection, diagnosis, and prognosis and survival for patients with CRC. Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase in eukaryotic cells and mostly acts as a tumor suppressor in human cancer. The PP2A holoenzyme is composed of a structural A subunit, a catalytic C subunit, and a variable regulatory B subunit. The regulatory B subunits determine the substrate specificity and subcellular localization of PP2A. The regulatory B subunits have been categorized into four families, B (B55 or PR55), B' (B56 or PR61), B” (PR72) and B'” (PR93 or PR110). The B55δ subunit, a member of B55 family and encoded by the PPP2R2D gene, plays a crucial role in regulating mitosis in the cell cycle. Data mining of expression profiles of B55δ showed differential expression levels of B55δ in different types of cancer, and reduced expression levels of B55δ were found in tumor parts as compared to that of the normal counterparts in datasets of CRC specimens. However, higher expression levels of B55δ in tumor tissues were associated with reduced survival rate in a subset of CRC patients. Additionally, differential expression levels of B55δ were found in different CRC cell lines. We hypothesized that B55δ may play a dual role in colorectal tumor progression. In agreement with the known role of B55δ in cell cycle, growth curve analysis showed that stable B55δ overexpression reduced proliferation of both HCT116 and SW620 cells compared to cells with vector only. B55δ overexpression or knockdown did not affect steady-state viability of HCT116 and SW620 cells. Similarly, using colony formation assay, both HCT116 and SW620 cells with B55δ overexpression showed reduced colony number compared to that of cells with vector only. Unexpectedly, the colony number of SW620 cells with stable knockdown of B55δ expression was also reduced compared to that of cells with vector only. Furthermore, B55δ overexpression increased the number of both SW620 and HCT116 cells with epithelial-like morphology, and B55δ knockdown increased the number of SW620 and HCT116 cells with mesenchymal morphology as compared to that of cells stably expressing vector. However, transwell migration assay showed that B55δ overexpression increased the migration ability, whereas B55δ knockdown reduced the migration ability of both HCT116 and SW620 cells. In addition, transwell invasion assay showed that B55δ overexpression increased the invasion ability, whereas B55δ knockdown reduced the invasion ability of SW620 cells. We further analyzed several EMT markers to elucidate whether B55δ regulates molecular changes of EMT of SW620 cells. The data showed that E-cadherin is negatively regulated by B55δ in both HCT116 and SW620 cells, but Snail is both negatively and positively regulated by B55δ in SW620 cells. In summary, our results showed that B55δ inhibits proliferation of CRC HCT116 and SW620 cells, but down-regulates the level of E-cadherin and promotes migration and invasion of CRC cells. Our finding suggests that it may suppress tumorigenesis in early stages of CRC progression, but promote metastasis in later stages of CRC progression.
List of Contents IX
List of Figures XI
List of Tables XII
List of Abbreviations XIII
Colorectal cancer (CRC) 15
Protein phosphatase 2A (PP2A) 16
The structure of PP2A 16
The B subunit of PP2A 17
The B55δ regulatory subunit of protein phosphatase 2A 17
Cell cycle 18
PP2A-B55δ regulates mitosis of cell cycle 19
Epithelia-Mesenchymal Transition (EMT) in cancer metastasis 20
Table 1. Molecular markers of epithelial and mesenchymal cell types 21
Materials and Methods 24
Antibodies and Reagents 25
Table 2. Primary and secondary antibodies applied in Western blotting 26
DNA constructs 26
Table 3. List of lentiviral shRNA constructs 27
Reagents of DNA cloning 27
Cell culture and cell lines 27
Table 4. Cell lines used in this thesis and the characteristics of each cell line below 29
Retrovirus and Lentivirus preparation 31
Retrovirus and Lentivirus transduction 32
Western blotting 33
Cell proliferation assay 33
Transwell® migration assay 33
Transwell® invasion assay 34
Colony formation assay 34
The B55δ regulatory subunit may have dual roles in tumor progression of CRC 36
The protein levels of B55δ vary among several colorectal cancer cell lines 36
Stable overexpression or knockdown of expression of B55δ did not affect the levels of PP2A-Aα/β and PP2Ac in both SW620 and HCT116 cells 37
B55δ downregulates cell proliferation in both SW620 and HCT116 cells 37
Stable overexpression or knockdown of B55δ affects morphology of SW620 and HCT116 cells 38
B55δ promotes motility of colorectal cancer cells 38
B55δ enhances the invasion ability of colorectal cancer cells 38
B55δ negatively regulates the level of E-cadherin in SW620 and HCT116 cells by up-regulating Snail 39
The morphology of SW620 and HCT116 cells regulated by B55δ overexpression or knockdown is not consistent with changes of their motility regulated by B55δ 43
B55δ negatively regulates expression of E-cadherin but has a dual role in regulating expression of Snail 43
The role of B55δ in colorectal cancer progression 45
B55δ as a biomarker and therapeutic target of CRC 45
1. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2019. CA Cancer J Clin, 2019. 69(1): p. 7-34.
2. Schluter, K., et al., Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential. Am J Pathol, 2006. 169(3): p. 1064-73.
3. Armaghany, T., et al., Genetic alterations in colorectal cancer. Gastrointest Cancer Res, 2012. 5(1): p. 19-27.
4. Pino, M.S. and D.C. Chung, The chromosomal instability pathway in colon cancer. Gastroenterology, 2010. 138(6): p. 2059-72.
5. Polakis, P., The adenomatous polyposis coli (APC) tumor suppressor. Biochim Biophys Acta, 1997. 1332(3): p. F127-47.
6. Kinzler, K.W. and B. Vogelstein, Lessons from hereditary colorectal cancer. Cell, 1996. 87(2): p. 159-70.
7. Lane, D.P. and S. Benchimol, p53: oncogene or anti-oncogene? Genes Dev, 1990. 4(1): p. 1-8.
8. Vogelstein, B., et al., Genetic alterations during colorectal-tumor development. N Engl J Med, 1988. 319(9): p. 525-32.
9. Geiersbach, K.B. and W.S. Samowitz, Microsatellite instability and colorectal cancer. Arch Pathol Lab Med, 2011. 135(10): p. 1269-77.
10. Ogino, S., et al., Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer. Gut, 2007. 56(11): p. 1564-71.
11. Shen, L., et al., Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer. Proc Natl Acad Sci U S A, 2007. 104(47): p. 18654-9.
12. Janssens, V., J. Goris, and C. Van Hoof, PP2A: the expected tumor suppressor. Curr Opin Genet Dev, 2005. 15(1): p. 34-41.
13. Janssens, V. and J. Goris, Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J, 2001. 353(Pt 3): p. 417-39.
14. Lechward, K., et al., Protein phosphatase 2A: variety of forms and diversity of functions. Acta Biochim Pol, 2001. 48(4): p. 921-33.
15. Schonthal, A.H., Role of serine/threonine protein phosphatase 2A in cancer. Cancer Lett, 2001. 170(1): p. 1-13.
16. Virshup, D.M., Protein phosphatase 2A: a panoply of enzymes. Curr Opin Cell Biol, 2000. 12(2): p. 180-5.
17. Xu, Y., et al., Structure of the protein phosphatase 2A holoenzyme. Cell, 2006. 127(6): p. 1239-51.
18. McCright, B., et al., The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem, 1996. 271(36): p. 22081-9.
19. Hemmings, B.A., et al., alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry, 1990. 29(13): p. 3166-73.
20. Walter, G., et al., Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein. Proc Natl Acad Sci U S A, 1989. 86(22): p. 8669-72.
21. Groves, M.R., et al., The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell, 1999. 96(1): p. 99-110.
22. Ruediger, R., et al., Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus. Mol Cell Biol, 1992. 12(11): p. 4872-82.
23. Ruediger, R., et al., Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens. J Virol, 1994. 68(1): p. 123-9.
24. Xing, Y., et al., Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell, 2006. 127(2): p. 341-53.
25. Ogris, E., D.M. Gibson, and D.C. Pallas, Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen. Oncogene, 1997. 15(8): p. 911-7.
26. Tolstykh, T., et al., Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits. Embo j, 2000. 19(21): p. 5682-91.
27. Wu, J., et al., Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo j, 2000. 19(21): p. 5672-81.
28. Yu, X.X., et al., Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen. Mol Biol Cell, 2001. 12(1): p. 185-99.
29. Wei, H., et al., Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p. J Biol Chem, 2001. 276(2): p. 1570-7.
30. Arino, J., et al., Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes. Proc Natl Acad Sci U S A, 1988. 85(12): p. 4252-6.
31. Green, D.D., S.I. Yang, and M.C. Mumby, Molecular cloning and sequence analysis of the catalytic subunit of bovine type 2A protein phosphatase. Proc Natl Acad Sci U S A, 1987. 84(14): p. 4880-4.
32. Stone, S.R., J. Hofsteenge, and B.A. Hemmings, Molecular cloning of cDNAs encoding two isoforms of the catalytic subunit of protein phosphatase 2A. Biochemistry, 1987. 26(23): p. 7215-20.
33. Sontag, J.M. and E. Sontag, Regulation of cell adhesion by PP2A and SV40 small tumor antigen: an important link to cell transformation. Cell Mol Life Sci, 2006. 63(24): p. 2979-91.
34. Letourneux, C., G. Rocher, and F. Porteu, B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK. Embo j, 2006. 25(4): p. 727-38.
35. Ito, A., et al., A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. Embo j, 2000. 19(4): p. 562-71.
36. Seeling, J.M., et al., Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science, 1999. 283(5410): p. 2089-91.
37. Chen, W., et al., Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell, 2004. 5(2): p. 127-36.
38. Lai, T.Y., et al., The B56gamma3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1. Oncotarget, 2016. 7(4): p. 4542-58.
39. Seshacharyulu, P., et al., Phosphatase: PP2A structural importance, regulation and its aberrant expression in cancer. Cancer Lett, 2013. 335(1): p. 9-18.
40. Hegarat, N., et al., PP2A/B55 and Fcp1 regulate Greatwall and Ensa dephosphorylation during mitotic exit. PLoS Genet, 2014. 10(1): p. e1004004.
41. Tuck, C., et al., Robust mitotic entry is ensured by a latching switch. Biol Open, 2013. 2(9): p. 924-31.
42. Zhuang, Q., et al., Protein phosphatase 2A-B55delta enhances chemotherapy sensitivity of human hepatocellular carcinoma under the regulation of microRNA-133b. J Exp Clin Cancer Res, 2016. 35: p. 67.
43. Yu, S., et al., PPP2R2D, a regulatory subunit of protein phosphatase 2A, promotes gastric cancer growth and metastasis via mechanistic target of rapamycin activation. Int J Oncol, 2018. 52(6): p. 2011-2020.
44. Cunningham, C.E., et al., Therapeutic relevance of the protein phosphatase 2A in cancer. Oncotarget, 2016. 7(38): p. 61544-61561.
45. Schafer, K.A., The cell cycle: a review. Vet Pathol, 1998. 35(6): p. 461-78.
46. Saya, H., Molecular mechanism of tumorigenesis by cell cycle regulation and new therapeutical approaches. Japanese Journal of Neurosurgery, 2005. 14(6): p. 373-378.
47. Kim, Y.T. and M. Zhao, Aberrant cell cycle regulation in cervical carcinoma. Yonsei Med J, 2005. 46(5): p. 597-613.
48. Stewart, M.P., et al., Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature, 2011. 469(7329): p. 226-30.
49. Guttinger, S., E. Laurell, and U. Kutay, Orchestrating nuclear envelope disassembly and reassembly during mitosis. Nat Rev Mol Cell Biol, 2009. 10(3): p. 178-91.
50. Belmont, A.S., Mitotic chromosome structure and condensation. Curr Opin Cell Biol, 2006. 18(6): p. 632-8.
51. Walczak, C.E., S. Cai, and A. Khodjakov, Mechanisms of chromosome behaviour during mitosis. Nat Rev Mol Cell Biol, 2010. 11(2): p. 91-102.
52. Lu, L., M.S. Ladinsky, and T. Kirchhausen, Cisternal organization of the endoplasmic reticulum during mitosis. Mol Biol Cell, 2009. 20(15): p. 3471-80.
53. Zaal, K.J., et al., Golgi membranes are absorbed into and reemerge from the ER during mitosis. Cell, 1999. 99(6): p. 589-601.
54. Wurzenberger, C. and D.W. Gerlich, Phosphatases: providing safe passage through mitotic exit. Nat Rev Mol Cell Biol, 2011. 12(8): p. 469-82.
55. Dephoure, N., et al., A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A, 2008. 105(31): p. 10762-7.
56. Nurse, P., Universal control mechanism regulating onset of M-phase. Nature, 1990. 344(6266): p. 503-8.
57. Bollen, M., D.W. Gerlich, and B. Lesage, Mitotic phosphatases: from entry guards to exit guides. Trends Cell Biol, 2009. 19(10): p. 531-41.
58. De Wulf, P., F. Montani, and R. Visintin, Protein phosphatases take the mitotic stage. Curr Opin Cell Biol, 2009. 21(6): p. 806-15.
59. Trinkle-Mulcahy, L. and A.I. Lamond, Mitotic phosphatases: no longer silent partners. Curr Opin Cell Biol, 2006. 18(6): p. 623-31.
60. Wlodarchak, N. and Y. Xing, PP2A as a master regulator of the cell cycle. Crit Rev Biochem Mol Biol, 2016. 51(3): p. 162-84.
61. Manchado, E., et al., Targeting mitotic exit leads to tumor regression in vivo: Modulation by Cdk1, Mastl, and the PP2A/B55alpha,delta phosphatase. Cancer Cell, 2010. 18(6): p. 641-54.
62. Lindqvist, A., V. Rodríguez-Bravo, and R.H. Medema, The decision to enter mitosis: feedback and redundancy in the mitotic entry network. The Journal of cell biology, 2009. 185(2): p. 193-202.
63. Tang, Z., T.R. Coleman, and W.G. Dunphy, Two distinct mechanisms for negative regulation of the Wee1 protein kinase. The EMBO journal, 1993. 12(9): p. 3427-3436.
64. Mueller, P.R., T.R. Coleman, and W.G. Dunphy, Cell cycle regulation of a Xenopus Wee1-like kinase. Molecular biology of the cell, 1995. 6(1): p. 119-134.
65. Harvey, S.L., et al., Cdk1-dependent regulation of the mitotic inhibitor Wee1. Cell, 2005. 122(3): p. 407-420.
66. Hoffmann, I., et al., Phosphorylation and activation of human cdc25‐C by cdc2‐‐cyclin B and its involvement in the self‐amplification of MPF at mitosis. The EMBO journal, 1993. 12(1): p. 53-63.
67. Larue, L. and A. Bellacosa, Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways. Oncogene, 2005. 24(50): p. 7443-54.
68. Li, L. and W. Li, Epithelial-mesenchymal transition in human cancer: comprehensive reprogramming of metabolism, epigenetics, and differentiation. Pharmacol Ther, 2015. 150: p. 33-46.
69. Liao, T.T. and M.H. Yang, Revisiting epithelial-mesenchymal transition in cancer metastasis: the connection between epithelial plasticity and stemness. Mol Oncol, 2017. 11(7): p. 792-804.
70. Lee, J.Y. and G. Kong, Roles and epigenetic regulation of epithelial-mesenchymal transition and its transcription factors in cancer initiation and progression. Cell Mol Life Sci, 2016. 73(24): p. 4643-4660.
71. Kalluri, R. and R.A. Weinberg, The basics of epithelial-mesenchymal transition. J Clin Invest, 2009. 119(6): p. 1420-8.
72. Huber, M.A., N. Kraut, and H. Beug, Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol, 2005. 17(5): p. 548-58.
73. Davis, F.M., et al., Targeting EMT in cancer: opportunities for pharmacological intervention. Trends Pharmacol Sci, 2014. 35(9): p. 479-88.
74. Flatmark, K., et al., Twelve colorectal cancer cell lines exhibit highly variable growth and metastatic capacities in an orthotopic model in nude mice. Eur J Cancer, 2004. 40(10): p. 1593-8.
75. Leibovitz, A., et al., Classification of human colorectal adenocarcinoma cell lines. Cancer Res, 1976. 36(12): p. 4562-9.
76. Hewitt, R.E., et al., Validation of a model of colon cancer progression. J Pathol, 2000. 192(4): p. 446-54.
77. Slater, C., J.A. De La Mare, and A.L. Edkins, In vitro analysis of putative cancer stem cell populations and chemosensitivity in the SW480 and SW620 colon cancer metastasis model. Oncol Lett, 2018. 15(6): p. 8516-8526.
78. Liu, Y.J., et al., Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell, 2015. 160(4): p. 659-672.
79. Polacheck, W.J., I.K. Zervantonakis, and R.D. Kamm, Tumor cell migration in complex microenvironments. Cell Mol Life Sci, 2013. 70(8): p. 1335-56.
80. Batlle, E., et al., The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol, 2000. 2(2): p. 84-9.
81. Serrano-Gomez, S.J., M. Maziveyi, and S.K. Alahari, Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol Cancer, 2016. 15: p. 18.
82. Chang, R., P. Zhang, and J. You, Post-translational modifications of EMT transcriptional factors in cancer metastasis. Open Life Sciences, 2016. 11(1): p. 237-243.
83. Walsh, C.T., S. Garneau-Tsodikova, and G.J. Gatto, Jr., Protein posttranslational modifications: the chemistry of proteome diversifications. Angew Chem Int Ed Engl, 2005. 44(45): p. 7342-72.
84. Pejaver, V., et al., The structural and functional signatures of proteins that undergo multiple events of post-translational modification. Protein Sci, 2014. 23(8): p. 1077-93.
85. Saitoh, M., Epithelial-mesenchymal transition is regulated at post-transcriptional levels by transforming growth factor-beta signaling during tumor progression. Cancer Sci, 2015. 106(5): p. 481-8.
86. Zhou, B.P., et al., Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol, 2004. 6(10): p. 931-40.
87. Gonzalez, D.M. and D. Medici, Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal, 2014. 7(344): p. re8.
88. Puisieux, A., T. Brabletz, and J. Caramel, Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol, 2014. 16(6): p. 488-94.
89. Onder, T.T., et al., Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res, 2008. 68(10): p. 3645-54.
90. Wang, J., et al., The transcription repressor, ZEB1, cooperates with CtBP2 and HDAC1 to suppress IL-2 gene activation in T cells. Int Immunol, 2009. 21(3): p. 227-35.
91. Zhang, P., Y. Sun, and L. Ma, ZEB1: at the crossroads of epithelial-mesenchymal transition, metastasis and therapy resistance. Cell Cycle, 2015. 14(4): p. 481-7.
92. Shouse, G., et al., Novel B55alpha-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest. Oncotarget, 2016. 7(38): p. 61081-61092.
93. Ruvolo, P.P., et al., The protein phosphatase 2A regulatory subunit B55alpha is a modulator of signaling and microRNA expression in acute myeloid leukemia cells. Biochim Biophys Acta, 2014. 1843(9): p. 1969-77.
94. Yang, Y., et al., Reactivating PP2A by FTY720 as a novel therapy for AML with C-KIT tyrosine kinase domain mutation. J Cell Biochem, 2012. 113(4): p. 1314-22.
95. Chung, V., et al., Safety, Tolerability, and Preliminary Activity of LB-100, an Inhibitor of Protein Phosphatase 2A, in Patients with Relapsed Solid Tumors: An Open-Label, Dose Escalation, First-in-Human, Phase I Trial. Clin Cancer Res, 2017. 23(13): p. 3277-3284.
96. Chang, K.E., et al., The protein phosphatase 2A inhibitor LB100 sensitizes ovarian carcinoma cells to cisplatin-mediated cytotoxicity. Mol Cancer Ther, 2015. 14(1): p. 90-100.
97. Gordon, I.K., et al., Protein Phosphatase 2A Inhibition with LB100 Enhances Radiation-Induced Mitotic Catastrophe and Tumor Growth Delay in Glioblastoma. Mol Cancer Ther, 2015. 14(7): p. 1540-1547.
98. Ho, W.S., et al., PP2A inhibition with LB100 enhances cisplatin cytotoxicity and overcomes cisplatin resistance in medulloblastoma cells. Oncotarget, 2016. 7(11): p. 12447-63.