臺灣博碩士論文加值系統

結直腸癌(colorectal cancer)是位於全世界女性第二及男性第三常見的癌症，根據研究，多數第三期(stage III)前的患者能透過手術或手術結合化療治癒，一旦形成癌症轉移發展為第四期(stage IV)之患者通常存活率低於10%。因此我們想要找出結直腸癌細胞發生轉移之前可幫助診斷之基因生物標記，達到早期診斷和治療的目標。我們透過分析GEO (Gene Expression Omnibus)資料庫中不同時期之結直腸癌病患的微陣列基因表現(gene expression microarray，又稱GEM)數據，比對正常組織與原發性腫瘤間及原發性與轉移性腫瘤之間基因表現的差異後，鑑定出一個在結直腸癌轉移時表現會上升的ENPEP基因：胺基胜肽酶A (Aminopeptidase A，又稱APA)。在細胞實驗中我們發現APA大量表現會促進大腸癌SW480細胞的移動能力；但當透過RNA干擾作用於大腸癌HT29細胞中抑制APA表現後，細胞移動能力隨之降低，並可能與調控上皮細胞間質轉化(epithelial-mesenchymal transition，又稱EMT)之轉錄因子TWIST相關。過去文獻發現TWIST的不正常表現可促進結直腸癌轉移並使乳腺上皮細胞具有幹細胞特性，。因此，我們也探究結直腸癌中APA所誘發癌細胞移動能力和TWIST表現之相關性，及對於癌症進程的影響，我們發現於臨床二及三期病人檢體APA的mRNA表現上升且與TWIST的mRNA表現趨勢有一致的現象，利用細胞株實驗也證實APA確實與TWIST的mRNA和蛋白表現量有高度的相關性，表示APA與TWIST的表現上升可能形成較具惡性的癌細胞。此外我們以細胞的非貼附性生長與聚球實驗實驗中發現，APA大量表現會促進癌細胞轉型及癌症幹細胞生成，提高腫瘤新生和轉移之潛能。APA為為調節腎素-血管收縮素系統(renin–angiotensin system，又稱RAS)之重要酵素，其酵素活性被指出與血管新生作用相關，所以我們將影響APA酵素活性作用的重要位點進行突變，發現當APA酵素活性被抑制後會降低APA所造成的促進癌細胞惡性能力，包括：細胞移動、細胞非貼附性生長、癌症幹細胞生成及其抗對藥物之抗藥性，後續又以活體動物腫瘤生成實驗發現一致性的結果。我們也發現APA活性對於癌細胞的調控依賴TWIST進行作用。此外我們利用組織微陣列切片進行免疫組織染色) 分析臨床檢體中APA及TWIST的蛋白表現，發現APA表現較高的病患也有較高的TWIST表現。最後，根據SurvExpress分析同時表現APA及TWIST之大量病患mRNA圖譜(profiles)顯示，同時表現APA及TWIST確實對病患存活具有重要的影響，顯示APA有潛力當作結直腸癌的預後因子。本研究證明APA為一具有高潛能的結直腸癌早期標記。臨床上已有針對抑制APA酵素活性藥物，未來若以此類藥物或開發新的APA活性抑制劑，或許能提供一個減緩結直腸癌進程的有效治療方法。

Colorectal cancer (CRC) is the second most diagnosed cancer in females and third in males worldwide. Studies have shown that most CRC patients can be cured by surgery or surgery combined with chemotherapy before in the third stage (stage III) of cancer. In case of metastasis, patients in stage IV CRC have poor survival rate, usually less than 10 percent. We aimed to identify a gene biomarker for metastasis prediction to achieve early diagnosis and early treatment. We performed data analysis of Gene Expression Omnibus (GEO) microarray database comparing primary and metastatic colorectal cancers to search metastasis-related genes. Aminopeptidase A (APA; also known ENPEP) was identified from our analysis and characterized as a cell motility effector by gene ontology analysis. We have confirmed that overexpression of APA increased mobility in SW480. On the other hand, down-regulation of APA by RNA interference in HT29 cells significantly reduced the cells mobility. Our results indicated that APA induced cell migratory ability was also related to a process of epithelial-mesenchymal transition (EMT) and EMT transcription factor, TWIST. Previous studies have shown that TWIST is a key regulator on metastatic CRC and transforming mammary epithelial cells to breast cancer stem cells. Hence, we explored the role of TWIST in APA-induced cell motility and cancer progression. Our data demonstrated that higher APA mRNA levels were observed in patients in advanced stages of cancer, which were consistent with TWIST mRNA levels. We also observed TWIST induction in APA-overexpressing SW480 cells and TWIST down-regulation in APA knocked down HT29 cells, suggesting that increasing APA and TWIST expression may promote the degree of malignancy in CRC cells. Moreover, APA overexpression in CRC cells induced anchorage-independent capability and sphere formation indicating a high potential of tumorigenesis or metastasis. APA is a key regulator in renin– angiotensin system (RAS) and its enzymatic activity was linked with angiogenesis. Thus, mutation of APA at the critical site of enzymatic activity was made to study the relationship between APA activity and cancer malignancy. Both APA silencing and impaired APA activity by mutation were found to reduce migratory capacity, cancer anchorage, stemness properties, and drug resistance in vitro and in vivo. We therefore suggest that APA enzymatic activity affects tumor initiation and cancer malignancy in a TWIST-dependent manner. We used immunohistochemical staining (IHC) to comapre APA-TWIST co-expression in tissue array, and found significant correlation between APA and TWIST.
Finally, according to data from SurvExpress analyses of TWIST1 and APA mRNA expression profiles, high APA and TWIST expression are positively correlated with poor CRC prognosis. Taken together APA may act as a potential biomarker for prediction of metastasis. To employ clinical available APA inhibitor or develop new drugs targeting on APA activity in tumorigenesis inhibition, may provide an effective treatment to curtail the progression of CRC.

中文摘要---------------------------------------------------------------- I
Abstract --------------------------------------------------------------- III
List of Abbreviations---------------------------------------------------- V
Contents ---------------------------------------------------------------VI
Chapter 1 Introduction---------------------------------------------------1
1.1 Colorectal cancer ----------------------------------------------------1
1.2 Carcinogenesis pathways in colorectal cancer------------------------1
1.3 Metastasis-----------------------------------------------------------2
1.4 Cancer stem cells ---------------------------------------------------4
1.5 Aminopeptidase A ---------------------------------------------------4
1.6 Specific aims --------------------------------------------------------5
Chapter 2 Materials and Methods ---------------------------------------6
2.1 Cell cultures --------------------------------------------------------6
2.2 Microarray expression analysis--------------------------------------6
2.3 RNA extraction, RT-PCR and real-time RT-PCR ----------------------7
2.4 DNA extraction -----------------------------------------------------8
2.5 Plasmid construction -----------------------------------------------8
2.6 Transient transfection ----------------------------------------------9
2.7 Lentivirus production and infection----------------------------------9
2.8 Anchorage-independent (soft-agar) colony formation assays -------10
2.9 Clonogenic assays -------------------------------------------------11
2.10 Western blot analyses---------------------------------------------11
2.11 Subcellular fractionation ------------------------------------------12
2.12 Cell migration and invasion assays --------------------------------12
2.13 Wound healing assays --------------------------------------------12
2.14 Immunofluoresence assays ---------------------------------------13
2.15 Biopsy samples ---------------------------------------------------13
2.16 Immunohistochemical analysis ------------------------------------13
2.17 Luciferase reporter constructs and luciferase assays --------------14
2.18 Flow cytometry analysis ------------------------------------------14
2.19 ALDH activity assays ---------------------------------------------15
2.20 APA enzymatic activity assays ------------------------------------15
2.21 Sphere formation assays ------------------------------------------15
2.22 Animal experiments ----------------------------------------------16
2.23 Statistical analyses -----------------------------------------------16
Chapter 3 Results------------------------------------------------------18
3.1 Elevated expression of Aminopeptidase A (ENPEP) with CRC disease progression and cell mobility in vitro -------------------------------------18
3.2 Aminopeptidase A expression was correlation with tumor malignancy-19
3.3 Aminopeptidase A upregulates TWIST during cancer progression ----19
3.4 Knockdown of Aminopeptidase A impaired the cell migration ability
and TWIST expression --------------------------------------------------20
3.5 Aminopeptidase A expression promotes the formation of cancer stem- like cells--------------------------------------------------------------------20
3.6 Depletion of Aminopeptidase A prevented sphere formation and stemness function ----------------------------------------------------------------21
3.7 Aminopeptidase A activity is important for cell migration induction---21
3.8 Aminopeptidase A mutation at metabolic activity site impairs cell
mobility ----------------------------------------------------------------22
3.9 TWIST induction and TWIST-induced phenotypes are dependents on Aminopeptidase A activity ----------------------------------------------22
3.10 APA-regulated TWIST expression via NF-κB translocation ----------23
3.11 TWIST knockdown in APA-overexpressing cells minimizes cell motion and CSC-like properties induced by enzymatic activity ----------------------23
3.12 Aminopeptidase A expression affects tumorigenesis in vivo --------24
3.13 Aminopeptidase A expression in CT26 cells enhances the cell malignancies and tumorigenesis in vivo-----------------------------------------------24
3.14 APA protein expression are correlates with TWIST protein expression in clinical patients---------------------------------------------------------25
3.15 APA expression affects patient prognoses--------------------------25
3.16 mRNA levels of ENPEP are correlate with APA protein level ---------26
3.17 APA expression may upregulated the G-protein couple receptor signaling pathway in CRC---------------------------------------------------------26
3.18 Aminopeptidase A increase EGFR related legends and dependent on its enzymaticactivity--------------------------------------------------------27
Chapter 4 Discussion----------------------------------------------------28
Figures and Tables ------------------------------------------------------ 33
Figure 1. Increased Aminopeptidase A expression is associated with cell
motility. -----------------------------------------------------------------33
Figure 2. Aminopeptidase A expression is increased with CRC progression
and promotes cancer malignancy. ---------------------------------------34
Figure 3. Positive correlation between APA and TWIST expression in CRC
cell lines and patients. --------------------------------------------------35
Figure 4. Knockdown of APA decreases the cell mobility and TWIST
expression in HT29 cells. -----------------------------------------------36
Figure 5. Stemness properties are enhanced in APA-overexpressed SW480 cells. -------------------------------------------------------------------------37
Figure 6. Expression of CD44/CD24 validation in APA-overexpressing SW480 cells.--------------------------------------------------------------------38
Figure 7. Sphere formation ability is impaired in APA-depleted HT29 cells. --------------------------------------------------------------------------39
Figure 8. Knockdown of APA decreases the stemness properties in HT29 cells. --------------------------------------------------------------------------40
Figure 9. Inhibition of APA activity by CPRECESIC reduces the migratory capability in SW480 cells. ----------------------------------------------------------41
Figure 10. APA enzymatic activity is required for anchorage-independent capability. ----------------------------------------------------------------42
Figure 11. APA enzymatic activity is important to regulate TWIST expression. ---------------------------------------------------------------------------43
Figure 12. Protein stability of Snail is not regulated by APA in CRC cells. ---44
Figure 13. APA enzymatic activity was important to proliferation rate and drug resistance capability in SW480 cells. --------------------------------------45
Figure 14. APA enhances the expression of TWIST may through induction of NFκB p65 translocation and activation in colon cancer. --------------------------46
Figure 15. APA-mediated cell migration is impaired in TWIST knockdown cells. ---------------------------------------------------------------------------47
Figure 16. Stemness properties are impaired by APA activity mutation or TWIST inhibition. ----------------------------------------------------------------48
Figure 17. APA expression and its enzymatic activity revealed enhanced tumorigenicity in vivo. ----------------------------------------------------49
Figure 18. APA induced cell malignancies was also found in a mouse in vitro model by expressed the mouse APA in CT26 cells. -------------------------------50
Figure 19. Overexpression of mouse APA in CT26 cells enhances tumorigenicity and cell metastasis in vivo. -----------------------------------------------51
Figure 20. The IHC analysis of correlation between APA and TWIST expression on paired CRC samples. -----------------------------------------------------52
Figure 21. Double-positive expression in APA/TWIST was correlated with poor prognoses in CRC patients. ----------------------------------------------53
Figure 22.The analyses of correlation between ENPEP and APA expression on paired CRC samples. -----------------------------------------------------54
Figure 23. Signaling pathway validation of of APA overexpression by microarray analysis and Ingenuity Pathways Analysis (IPA). --------------------------55
Figure 24. Validation of up-regulation factors of microarray profiles by real-time qPCR. -------------------------------------------------------------------56
Figure 25. To test the effective concentration of Afatinib in CRC cells. ----57
Figure 26.APA-overexpression may increase EGFR downstream signaling
in an enzymatic activity dependent manner. ------------------------------58
Table 1. List of fragment amplification primers used in this study. ---------59
Table 2. List of sequencing primers used in this study.--------------------60
Table 3. List of RT-qPCR primers used in this study. ----------------------61
Table 4. List of shRNA used in gene knockdown by lentivirus production.
--------------------------------------------------------------------------62
Table 5. List of Antibodies used in this study. -----------------------------63
Table 6. Gender analysis of CRC patients in terms of APA or TWIST
expression levels (IHC). --------------------------------------------------64
Table 7. APA/TWIST co-expression levels (IHC) for colorectal cancer patients according to gender. -----------------------------------------------------65
Table 8. Clinicopathologic characteristics of patients that APA expression related to prognosis in colorectal cancer tissue array.-----------------------------66
Table 9. Clinicopathologic characteristics of patients that validation of APA and TWIST co-expression levels in colorectal cancer tissue array. -------------70
Appendices ------------------------------------------------------------- 73
References-------------------------------------------------------------- 77
Publications------------------------------------------------------------- 83

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69-90.
2. Pino MS, Chung DC. The chromosomal instability pathway in colon cancer. Gastroenterology. 2010;138(6):2059-2072.
3. Nazemalhosseini Mojarad E, Kuppen PJ, Aghdaei HA, Zali MR. The CpG island methylator phenotype (CIMP) in colorectal cancer. Gastroenterol Hepatol Bed Bench. 2013;6(3):120-128.
4. Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology. 2010;138(6):2073-2087.e2073.
5. Bae JM, Kim JH, Kang GH. Molecular Subtypes of Colorectal Cancer and Their Clinicopathologic Features, With an Emphasis on the Serrated Neoplasia Pathway. Arch Pathol Lab Med. 2016;140(5):406-412.
6. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature. 1997;386(6625):623-627.
7. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature. 1998;396(6712):643-649.
8. Barber TD, McManus K, Yuen KW, et al. Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers. Proc Natl Acad Sci U S A. 2008;105(9):3443-3448.
9. Thiagalingam S, Laken S, Willson JK, et al. Mechanisms underlying losses of heterozygosity in human colorectal cancers. Proc Natl Acad Sci U S A. 2001;98(5):2698-2702.
10. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61(5):759-767.
11. Haigis KM, Kendall KR, Wang Y, et al. Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nat Genet. 2008;40(5):600-608.
12. Baker SJ, Fearon ER, Nigro JM, et al. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science. 1989;244(4901):217-221.
13. Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science. 1995;268(5215):1336-1338.
14. Thiagalingam S, Lengauer C, Leach FS, et al. Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat Genet. 1996;13(3):343-346.
15. Samuels Y, Velculescu VE. Oncogenic mutations of PIK3CA in human cancers. Cell Cycle. 2004;3(10):1221-1224.
16. Kartheuser A, West S, Walon C, et al. The genetic background of familial adenomatous polyposis. Linkage analysis, the APC gene identification and mutation screening. Acta Gastroenterol Belg. 1995;58(5-6):433-451.
17. Behrens J, Jerchow BA, Wurtele M, et al. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science. 1998;280(5363):596-599.
18. Cadigan KM, Waterman ML. TCF/LEFs and Wnt signaling in the nucleus. Cold Spring Harb Perspect Biol. 2012;4(11).
19. McGrath JP, Capon DJ, Smith DH, et al. Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene. Nature. 1983;304(5926):501-506.
20. Omidifar NM, Geramizadeh BM, Mirzai MM. K-ras Mutation in Colorectal Cancer, A Report from Southern Iran. Iran J Med Sci. 2015;40(5):454-460.
21. Downward J. Ras signalling and apoptosis. Curr Opin Genet Dev. 1998;8(1):49-54.
22. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011;11(11):761-774.
23. Li W, Qiu T, Zhi W, et al. Colorectal carcinomas with KRAS codon 12 mutation are associated with more advanced tumor stages. BMC Cancer. 2015;15:340.
24. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin. 2007;57(1):43-66.
25. Urosevic J, Garcia-Albeniz X, Planet E, et al. Colon cancer cells colonize the lung from established liver metastases through p38 MAPK signalling and PTHLH. Nat Cell Biol. 2014;16(7):685-694.
26. Christiansen JJ, Rajasekaran AK. Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res. 2006;66(17):8319-8326.
27. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 2003;425(6958):577-584.
28. Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol. 2003;15(6):740-746.
29. Yang J, Mani SA, Donaher JL, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004;117(7):927-939.
30. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 2009;119(6):1429-1437.
31. Gulhati P, Bowen KA, Liu J, et al. mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res. 2011;71(9):3246-3256.
32. Javle MM, Gibbs JF, Iwata KK, et al. Epithelial-mesenchymal transition (EMT) and activated extracellular signal-regulated kinase (p-Erk) in surgically resected pancreatic cancer. Ann Surg Oncol. 2007;14(12):3527-3533.
33. Puisieux A, Brabletz T, Caramel J. Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol. 2014;16(6):488-494.
34. Mani SA, Guo W, Liao MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133(4):704-715.
35. Theodoropoulos PA, Polioudaki H, Agelaki S, et al. Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer. Cancer Lett. 2010;288(1):99-106.
36. Iinuma H, Watanabe T, Mimori K, et al. Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes' stage B and C colorectal cancer. J Clin Oncol. 2011;29(12):1547-1555.
37. Vermeulen L, Sprick MR, Kemper K, Stassi G, Medema JP. Cancer stem cells--old concepts, new insights. Cell Death Differ. 2008;15(6):947-958.
38. Nanus DM, Engelstein D, Gastl GA, et al. Molecular cloning of the human kidney differentiation antigen gp160: human aminopeptidase A. Proc Natl Acad Sci U S A. 1993;90(15):7069-7073.
39. Glenner GG, Mc MP, Folk JE. A mammalian peptidase specific for the hydrolysis of N-terminal alpha-L-glutamyl and aspartyl residues. Nature. 1962;194:867.
40. Li L, Wu Q, Wang J, Bucy RP, Cooper MD. Widespread tissue distribution of aminopeptidase A, an evolutionarily conserved ectoenzyme recognized by the BP-1 antibody. Tissue Antigens. 1993;42(5):488-496.
41. Migaud M, Durieux C, Viereck J, Soroca-Lucas E, Fournie-Zaluski MC, Roques BP. The in vivo metabolism of cholecystokinin (CCK-8) is essentially ensured by aminopeptidase A. Peptides. 1996;17(4):601-607.
42. Zini S, Fournie-Zaluski MC, Chauvel E, Roques BP, Corvol P, Llorens-Cortes C. Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release. Proc Natl Acad Sci U S A. 1996;93(21):11968-11973.
43. Rozenfeld R, Reaux A, Iturrioz X, et al. Aminopeptidase A, generating one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, plays a key role in central control of blood pressure. Proc West Pharmacol Soc. 2003;46:39-44.
44. Teranishi J, Ishiguro H, Hoshino K, Noguchi K, Kubota Y, Uemura H. Evaluation of role of angiotensin III and aminopeptidases in prostate cancer cells. Prostate. 2008;68(15):1666-1673.
45. Ino K, Nagasaka T, Okamoto T, et al. Expression of aminopeptidase A in human gestational choriocarcinoma cell lines and tissues. Placenta. 2000;21(1):63-72.
46. Suganuma T, Ino K, Shibata K, et al. Regulation of aminopeptidase A expression in cervical carcinoma: role of tumor-stromal interaction and vascular endothelial growth factor. Lab Invest. 2004;84(5):639-648.
47. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol. 2010;17(6):1471-1474.
48. Ridley AJ, Schwartz MA, Burridge K, et al. Cell migration: integrating signals from front to back. Science. 2003;302(5651):1704-1709.
49. Sabbah M, Emami S, Redeuilh G, et al. Molecular signature and therapeutic perspective of the epithelial-to-mesenchymal transitions in epithelial cancers. Drug Resist Updat. 2008;11(4-5):123-151.
50. Huang EH, Hynes MJ, Zhang T, et al. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res. 2009;69(8):3382-3389.
51. Kim YH, Kim G, Kwon CI, Kim JW, Park PW, Hahm KB. TWIST1 and SNAI1 as markers of poor prognosis in human colorectal cancer are associated with the expression of ALDH1 and TGF-beta1. Oncol Rep. 2014;31(3):1380-1388.
52. Marchio S, Lahdenranta J, Schlingemann RO, et al. Aminopeptidase A is a functional target in angiogenic blood vessels. Cancer Cell. 2004;5(2):151-162.
53. Goto Y, Hattori A, Mizutani S, Tsujimoto M. Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A. J Biol Chem. 2007;282(51):37074-37081.
54. Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene. 2010;29(34):4741-4751.
55. Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H. NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene. 2007;26(5):711-724.
56. Criswell TL, Arteaga CL. Modulation of NFkappaB activity and E-cadherin by the type III transforming growth factor beta receptor regulates cell growth and motility. J Biol Chem. 2007;282(44):32491-32500.
57. Li CW, Xia W, Huo L, et al. Epithelial-mesenchymal transition induced by TNF-alpha requires NF-kappaB-mediated transcriptional upregulation of Twist1. Cancer Res. 2012;72(5):1290-1300.
58. Lin Q, Taniuchi I, Kitamura D, et al. T and B cell development in BP-1/6C3/aminopeptidase A-deficient mice. J Immunol. 1998;160(10):4681-4687.
59. Soares KC, Foley K, Olino K, et al. A preclinical murine model of hepatic metastases. J Vis Exp. 2014(91):51677.
60. Daub H, Weiss FU, Wallasch C, Ullrich A. Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature. 1996;379(6565):557-560.
61. Gomez I, Pena C, Herrera M, et al. TWIST1 is expressed in colorectal carcinomas and predicts patient survival. PLoS One. 2011;6(3):e18023.
62. Valdes-Mora F, Gomez del Pulgar T, Bandres E, et al. TWIST1 overexpression is associated with nodal invasion and male sex in primary colorectal cancer. Ann Surg Oncol. 2009;16(1):78-87.
63. Franco HL, Casasnovas J, Rodriguez-Medina JR, Cadilla CL. Redundant or separate entities?--roles of Twist1 and Twist2 as molecular switches during gene transcription. Nucleic Acids Res. 2011;39(4):1177-1186.
64. Ruppenthal RD, Nicolini C, Filho AF, et al. TWIST1 promoter methylation in primary colorectal carcinoma. Pathol Oncol Res. 2011;17(4):867-872.
65. Khan MA, Chen HC, Zhang D, Fu J. Twist: a molecular target in cancer therapeutics. Tumour Biol. 2013;34(5):2497-2506.
66. Vichalkovski A, Gresko E, Hess D, Restuccia DF, Hemmings BA. PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage. Oncogene. 2010;29(24):3554-3565.
67. Cheng GZ, Zhang WZ, Sun M, et al. Twist is transcriptionally induced by activation of STAT3 and mediates STAT3 oncogenic function. J Biol Chem. 2008;283(21):14665-14673.
68. Hong J, Zhou J, Fu J, et al. Phosphorylation of serine 68 of Twist1 by MAPKs stabilizes Twist1 protein and promotes breast cancer cell invasiveness. Cancer Res. 2011;71(11):3980-3990.
69. Li J, Zhou BP. Activation of beta-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC Cancer. 2011;11:49.
70. Yang MH, Wu KJ. TWIST activation by hypoxia inducible factor-1 (HIF-1): implications in metastasis and development. Cell Cycle. 2008;7(14):2090-2096.
71. Sosic D, Olson EN. A new twist on twist--modulation of the NF-kappa B pathway. Cell Cycle. 2003;2(2):76-78.
72. Bai D, Ueno L, Vogt PK. Akt-mediated regulation of NFkappaB and the essentialness of NFkappaB for the oncogenicity of PI3K and Akt. Int J Cancer. 2009;125(12):2863-2870.
73. Yan W, Fu Y, Tian D, et al. PI3 kinase/Akt signaling mediates epithelial-mesenchymal transition in hypoxic hepatocellular carcinoma cells. Biochem Biophys Res Commun. 2009;382(3):631-636.
74. Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem. 1997;272(36):22642-22647.
75. Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science. 2001;294(5545):1337-1340.
76. Kubota R, Numaguchi Y, Ishii M, et al. Ischemia-induced angiogenesis is impaired in aminopeptidase A deficient mice via down-regulation of HIF-1alpha. Biochem Biophys Res Commun. 2010;402(2):396-401.
77. Kuwai T, Kitadai Y, Tanaka S, et al. Expression of hypoxia-inducible factor-1alpha is associated with tumor vascularization in human colorectal carcinoma. Int J Cancer. 2003;105(2):176-181.
78. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003;3(10):721-732.
79. Antczak C, De Meester I, Bauvois B. Ectopeptidases in pathophysiology. Bioessays. 2001;23(3):251-260.
80. Peer WA. The role of multifunctional M1 metallopeptidases in cell cycle progression. Ann Bot. 2011;107(7):1171-1181.
81. Tsujimoto M, Goto Y, Maruyama M, Hattori A. Biochemical and enzymatic properties of the M1 family of aminopeptidases involved in the regulation of blood pressure. Heart Fail Rev. 2008;13(3):285-291.
82. Aminopeptidase A is a functional target in angiogenic blood vessels.
83. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012;12(4):278-287.
84. Mentzel S, Assmann KJ, Dijkman HB, et al. Inhibition of aminopeptidase A activity causes an acute albuminuria in mice: an angiotensin II-mediated effect? Nephrol Dial Transplant. 1996;11(11):2163-2169.
85. Reaux A, Iturrioz X, Vazeux G, et al. Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure. Biochem Soc Trans. 2000;28(4):435-440.