探討TIMP-3在EGFR抑制劑治療神經母細胞瘤中的調節機制

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陳婷軒(Ting-Xuan Chen) 查詢紙本館藏

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生命科學系

論文名稱

探討TIMP-3在EGFR抑制劑治療神經母細胞瘤中的調節機制
(Exploring the regulation mechanism of TIMP-3 in the EGFR inhibitor treatment of neuroblastoma)

相關論文

★ 探討化合物 Y 抑制神經母細胞瘤增生之效果	★ 探討化合物Y抑制神經母細胞瘤轉移之效果
★ 探討硒代胱氨酸於神經母細胞瘤之治療潛力

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至系統瀏覽論文 (2026-8-31以後開放)

摘要(中)

Neuroblastoma (NB) is the most common extracranial solid tumor in children, with high-risk NB patients facing a survival rate of less than 50%. Therefore, a novel therapeutic avenue for NB is imperative. Notably, EGFRs, ErbB1 and ErbB2, are highly expressed in NB cells, and activating these receptors significantly promotes the proliferation of NB cells. Targeting ErbB1 and ErbB2 may be a potential treatment for NB. The preliminary data showed that the ErbB1 and ErbB2 dual inhibitor, CL -387785, exerts notable inhibitory effects on NB cell proliferation, migration, and invasion. Additionally, transcriptomic analysis via qPCR array showed that TIMP3 was upregulated in CL-387785-treated cells. In this study, I would like to examine the therapeutic efficacy of CL-387785 in vivo and further explore the regulatory mechanism of TIMP3 in NB. By the mouse subcutaneous xenograft and tail-vein metastasis model, I found that CL-387785 effectively inhibits the tumor growth and the liver metastasis of NB cells. After EGF treatments, the mRNA expression of TIMP-3 was inhibited, while DNMT1 and EZH2 were induced. Conversely, treatments of the epigenetic regulation inhibitors, 5-Azacytidine (5-Aza), Tazemetostat (EPZ-6438), and Trichostatin A (TSA), upregulated the TIMP3 mRNA level. These in-vitro findings suggest that the EGFR pathway likely regulates TIMP3 both transcriptionally and epigenetically. However, further investigations are warranted to elucidate the precise mechanism through which EGFR modulates TIMP3 expression and consequently impacts NB progression.

摘要(英)

關鍵字(中)

★ 神經母細胞瘤
★ 表觀遺傳學

關鍵字(英)

★ Neuroblastoma
★ TIMP3
★ CL-387,785
★ EGFR signaling
★ Epigenetics

論文目次

中文摘要 i
Abstract ii
Acknowledgments iii
Table of Contents iv
List of Figures vi
List of Tables vii
I. Introduction 1
1-1 Neuroblastoma 1
1-1-1 Staging System 2
1-1-2 Treatment strategies 3
1-2 Epidermal growth factor receptor (EGFR) 4
1-3 Tissue inhibitors of metalloproteinases 3 (TIMP3) 5
1-4 Rationale 7
II. Materials and methods 8
2-1 Cell culture 8
2-1-1 SK-N-BE(2)-C 8
2-1-2 SH-SY5Y 8
2-1-3 HEK293T 8
2-2 Western blot 8
2-2-1 Cell harvesting 8
2-2-2 SDS Polyacrylamide-gel-electrophoresis (SDS-PAGE) 8
2-2-3 Protein transfer 9
2-2-4 Blocking and antiboby binding 9
2-2-5 Membrane striping 10
2-3 Real-time quantitative polymerase chain reaction (RT-qPCR) 10
2-3-1 RNA extraction 10
2-3-2 Reverse transcription 10
2-3-3 Real-time quantitative polymerase chain reaction 11
2-4 Vector Construction 11
2-4-1 PCR amplification of insert 11
2-4-2 Restriction Enzyme Digestion 12
III. Results 15
3-1 Metastasis models for examining the effect of CL-387785 on NB metastasis 15
3-2 The xenograft mouse model shows that CL inhibits NB cell proliferation. 16
3-3 EGFR treatment inhibit TIMP3 expression 16
3-4 TIMP3 expression is influenced by EZH2-mediated histone modification 16
3-5 5-Aza, EPZ-6438, TSA treatment induced TIMP3 expression . 17
3-6 Luciferase reporter assay vector construction 18
3-7 Luciferase reporter assay 18
IV. Discussion 20
V. References 24
VI. Tables and Figures 28

參考文獻

1. Johnsen JI, Dyberg C, Wickström M: Neuroblastoma-A Neural Crest Derived Embryonal Malignancy. Frontiers in molecular neuroscience 2019, 12:9.
2. Maris JM: Recent advances in neuroblastoma. The New England journal of medicine 2010, 362(23):2202-2211.
3. Chung C, Boterberg T, Lucas J, Panoff J, Valteau-Couanet D, Hero B, Bagatell R, Hill-Kayser CE: Neuroblastoma. Pediatric blood & cancer 2021, 68 Suppl 2(Suppl 2):e28473.
4. Gomez RL, Ibragimova S, Ramachandran R, Philpott A, Ali FR: Tumoral heterogeneity in neuroblastoma. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 2022, 1877(6):188805.
5. Epp S, Chuah SM, Halasz M: Epigenetic Dysregulation in MYCN-Amplified Neuroblastoma. International journal of molecular sciences 2023, 24(23):17085.
6. Hansford LM, Thomas WD, Keating JM, Burkhart CA, Peaston AE, Norris MD, Haber M, Armati PJ, Weiss WA, Marshall GM: Mechanisms of embryonal tumor initiation: distinct roles for MycN expression and MYCN amplification. Proceedings of the National Academy of Sciences of the United States of America 2004, 101(34):12664-12669.
7. Maris JM, Matthay KK: Molecular Biology of Neuroblastoma. Journal of Clinical Oncology 1999, 17(7):2264-2264.
8. Ruiz-Pérez MV, Henley AB, Arsenian-Henriksson M: The MYCN Protein in Health and Disease. Genes 2017, 8(4):113.
9. Sokol E, Desai AV: The Evolution of Risk Classification for Neuroblastoma. Children 2019, 6(2):27.
10. Monclair T, Brodeur GM, Ambros PF, Brisse HJ, Cecchetto G, Holmes K, Kaneko M, London WB, Matthay KK, Nuchtern JG et al: The International Neuroblastoma Risk Group (INRG) staging system: an INRG Task Force report. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2009, 27(2):298-303.
11. Cohn SL, Pearson AD, London WB, Monclair T, Ambros PF, Brodeur GM, Faldum A, Hero B, Iehara T, Machin D et al: The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2009, 27(2):289-297.
12. Swift CC, Eklund MJ, Kraveka JM, Alazraki AL: Updates in Diagnosis, Management, and Treatment of Neuroblastoma. RadioGraphics 2018, 38(2):566-580.
13. Pinto NR, Applebaum MA, Volchenboum SL, Matthay KK, London WB, Ambros PF, Nakagawara A, Berthold F, Schleiermacher G, Park JR et al: Advances in Risk Classification and Treatment Strategies for Neuroblastoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2015, 33(27):3008-3017.
14. Hero B, Simon T, Spitz R, Ernestus K, Gnekow AK, Scheel-Walter HG, Schwabe D, Schilling FH, Benz-Bohm G, Berthold F: Localized infant neuroblastomas often show spontaneous regression: results of the prospective trials NB95-S and NB97. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2008, 26(9):1504-1510.
15. Laprie A, Michon J, Hartmann O, Munzer C, Leclair MD, Coze C, Valteau-Couanet D, Plantaz D, Carrie C, Habrand JL et al: High-dose chemotherapy followed by locoregional irradiation improves the outcome of patients with international neuroblastoma staging system Stage II and III neuroblastoma with MYCN amplification. Cancer 2004, 101(5):1081-1089.
16. Yu AL, Gilman AL, Ozkaynak MF, London WB, Kreissman SG, Chen HX, Smith M, Anderson B, Villablanca JG, Matthay KK et al: Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. The New England journal of medicine 2010, 363(14):1324-1334.
17. Orofiamma LA, Vural D, Antonescu CN: Control of cell metabolism by the epidermal growth factor receptor. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2022, 1869(12):119359.
18. Bogdan S, Klämbt C: Epidermal growth factor receptor signaling. Current Biology 2001, 11(8):R292-R295.
19. Rude Voldborg B, Damstrup L, Spang-Thomsen M, Skovgaard Poulsen H: Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Annals of Oncology 1997, 8(12):1197-1206.
20. Yamaoka T, Ohba M, Ohmori T: Molecular-Targeted Therapies for Epidermal Growth Factor Receptor and Its Resistance Mechanisms. International journal of molecular sciences 2017, 18(11):2420.
21. Baselga J: Why the Epidermal Growth Factor Receptor? The Rationale for Cancer Therapy. The Oncologist 2002, 7(S4):2-8.
22. Zheng C, Shen R, Li K, Zheng N, Zong Y, Ye D, Wang Q, Wang Z, Chen L, Ma Y: Epidermal growth factor receptor is overexpressed in neuroblastoma tissues and cells. Acta biochimica et biophysica Sinica 2016, 48(8):762-767.
23. Goji J, Nakamura H, Ito H, Mabuchi O, Hashimoto K, Sano K: Expression of c-ErbB2 in human neuroblastoma tissues, adrenal medulla adjacent to tumor, and developing mouse neural crest cells. The American journal of pathology 1995, 146(3):660-672.
24. Discafani CM, Carroll ML, Floyd MB, Hollander IJ, Husain Z, Johnson BD, Kitchen D, May MK, Malo MS, Minnick AA et al: Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with In Vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785). Biochemical Pharmacology 1999, 57(8):917-925.
25. Fan D, Kassiri Z: Biology of Tissue Inhibitor of Metalloproteinase 3 (TIMP3), and Its Therapeutic Implications in Cardiovascular Pathology. Frontiers in Physiology 2020, 11:661.
26. Arpino V, Brock M, Gill SE: The role of TIMPs in regulation of extracellular matrix proteolysis. Matrix Biology 2015, 44-46:247-254.
27. Lee WT, Wu PY, Cheng YM, Huang YF: Tissue Inhibitor of Metalloproteinase 3: Unravelling Its Biological Function and Significance in Oncology. International journal of molecular sciences 2024, 25(6):3191.
28. Nagase H, Visse R, Murphy G: Structure and function of matrix metalloproteinases and TIMPs. Cardiovascular Research 2006, 69(3):562-573.
29. Bachman KE, Herman JG, Corn PG, Merlo A, Costello JF, Cavenee WK, Baylin SB, Graff JR: Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers. Cancer research 1999, 59(4):798-802.
30. Gu P, Xing X, Tänzer M, Röcken C, Weichert W, Ivanauskas A, Pross M, Peitz U, Malfertheiner P, Schmid RM et al: Frequent loss of TIMP-3 expression in progression of esophageal and gastric adenocarcinomas. Neoplasia (New York, NY) 2008, 10(6):563-572.
31. Masson D, Rioux-Leclercq N, Fergelot P, Jouan F, Mottier S, Théoleyre S, Bach-Ngohou K, Patard JJ, Denis MG: Loss of expression of TIMP3 in clear cell renal cell carcinoma. European journal of cancer (Oxford, England : 1990) 2010, 46(8):1430-1437.
32. Wild A, Ramaswamy A, Langer P, Celik I, Fendrich V, Chaloupka B, Simon B, Bartsch DK: Frequent methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene in pancreatic endocrine tumors. The Journal of clinical endocrinology and metabolism 2003, 88(3):1367-1373.
33. Yun J, Park MH, Son DJ, Nam KT, Moon DB, Ju JH, Hwang OK, Choi JS, Kim TH, Jung YS et al: IL-32 gamma reduces lung tumor development through upregulation of TIMP-3 overexpression and hypomethylation. Cell death & disease 2018, 9(3):306.
34. Su CW, Lin CW, Yang WE, Yang SF: TIMP-3 as a therapeutic target for cancer. Therapeutic advances in medical oncology 2019, 11:1758835919864247.
35. Xu C, Hou Z, Zhan P, Zhao W, Chang C, Zou J, Hu H, Zhang Y, Yao X, Yu L et al: EZH2 regulates cancer cell migration through repressing TIMP-3 in non-small cell lung cancer. Medical oncology (Northwood, London, England) 2013, 30(4):713.
36. Yang X, Du WW, Li H, Liu F, Khorshidi A, Rutnam ZJ, Yang BB: Both mature miR-17-5p and passenger strand miR-17-3p target TIMP3 and induce prostate tumor growth and invasion. Nucleic acids research 2013, 41(21):9688-9704.
37. Yang Z, Yuan Z, Fan Y, Deng X, Zheng Q: Integrated analyses of microRNA and mRNA expression profiles in aggressive papillary thyroid carcinoma. Molecular medicine reports 2013, 8(5):1353-1358.
38. Gan R, Yang Y, Yang X, Zhao L, Lu J, Meng QH: Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3. Cancer gene therapy 2014, 21(7):290-296.
39. Du L, Ma S, Wen X, Chai J, Zhou D: Oral squamous cell carcinoma cells are resistant to doxorubicin through upregulation of miR‑221. Molecular medicine reports 2017, 16(3):2659-2667.
40. Li W, Yi J, Zheng X, Liu S, Fu W, Ren L, Li L, Hoon DSB, Wang J, Du G: miR-29c plays a suppressive role in breast cancer by targeting the TIMP3/STAT1/FOXO1 pathway. Clinical epigenetics 2018, 10:64.
41. Lu R, Wang X, Chen ZF, Sun DF, Tian XQ, Fang JY: Inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon cancer cells. The Journal of biological chemistry 2007, 282(16):12249-12259.
42. Samudio-Ruiz SL, Hudson LG: Increased DNA methyltransferase activity and DNA methylation following Epidermal Growth Factor stimulation in ovarian cancer cells. Epigenetics 2012, 7(3):216-224.
43. Shah MY, Calin GA: MicroRNAs miR-221 and miR-222: a new level of regulation in aggressive breast cancer. Genome medicine 2011, 3(8):56.
44. Zhang C, Zhang J, Hao J, Shi Z, Wang Y, Han L, Yu S, You Y, Jiang T, Wang J et al: High level of miR-221/222 confers increased cell invasion and poor prognosis in glioma. Journal of translational medicine 2012, 10:119.
45. Su C-W, Chang Y-C, Chien M-H, Hsieh Y-H, Chen M-K, Lin C-W, Yang S-F: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis. Cell death & disease 2019, 10(11):793.

指導教授

吳沛翊(Pei-Yi Wu)

審核日期

2024-8-22

推文