成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	陳以理 Chen, Yi-Li
論文名稱：	探討前梯度蛋白2調節肝癌發展及蕾莎瓦藥物之抗藥性 Anterior gradient 2 regulates endoplasmic reticulum stress in cancer progression and sorafenib-resistant hepatocellular carcinoma
指導教授：	陳政義 Chen, Cheng-Yi
學位類別：	碩士 Master
系所名稱：	醫學院 - 細胞生物與解剖學研究所 Institute of Cell Biology and Anatomy
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	43
中文關鍵詞：	蕾莎瓦、癌症發展、抗藥性、前梯度蛋白2 、內質網壓力途徑
外文關鍵詞：	sorafenib, cancer progression, drug resistance, AGR2, ER stress signaling
相關次數：	點閱：56 下載：8
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

肝細胞癌(HCC)佔肝癌總數的近80%，是全球第六大常見癌症，也是癌症相關死亡的第二大原因。其中蕾莎瓦(sorafenib)標靶治療晚期HCC患者的生存率仍不理想。不幸的是，到目前為止還沒有找到有效的生物標定物可以預測sorafenib在肝癌上的治療效果。為了實現這個目標，我們檢索了兩個數據庫，以找出和癌症發展和HCC存活率相關的潛在因子。在sorafenib耐藥性微陣列中，我們發現AGR2和HCC患者的總生存率以及非復發生存率具有高度相關性，這也與幾項臨床數據吻合。因此，我們假設sorafenib會調控AGR2相關的訊息途徑，並且AGR2的存在或許可以為肝癌細胞提供保護的能力。Sorafenib可能是透過轉譯後修飾而非轉錄調控來誘導AGR2分泌，並且AGR2在sorafenib敏感細胞中在其誘導的細胞生存、內質網壓力和細胞凋亡中都起到關鍵作用。除此之外，我們也建立了sorafenib抗藥性細胞，跟原本的母細胞相比，AGR2在抗藥性細胞中高度表達。我們認為sorafenib會降低細胞內AGR2的表現並同時促進其分泌到細胞外，從而去降低sorafenib敏感細胞中的內質網壓力與細胞存活。另一方面，AGR2在耐藥性細胞中的高表達則能夠穩定內質網壓力相關蛋白並產生更高的細胞存活率。在此項研究中，我們預計AGR2調控內質網壓力並影響細胞的癌症進展和sorafenib耐藥性。本研究的完成可以讓我們更加了解AGR2在sorafenib敏感性或抗藥性HCC中的作用，或許可用於開發預測性、早期診斷及預後因素等等。以建立專門治療HCC的標靶位點。

Hepatocellular carcinoma (HCC) represents almost 80% of total liver cancers, which is the sixth most common cancer and the second-highest cause of cancer-related deaths worldwide. The survival rate of advanced HCC patients with sorafenib targeted therapy is still unsatisfactory. Unfortunately, no useful biomarkers have been verified to predict the efficacy of sorafenib targeted therapy so far in HCC. To achieve this goal, we retrieved two databases to excavate potential factors implicated in cancer progression and survival rate in HCC. We have identified the anterior gradient 2 (AGR2) in the sorafenib-resistant microarray which was highly associated with overall survival and recurrence-free survival rates in HCC patients, and was also highly correlated with several clinical parameters. Therefore, we hypothesis that sorafenib treatment might regulate AGR2 pathways and presence protectable roles in sorafenib-resistance HCC. Sorafenib might induce AGR2 secretion via post-translational modification instead of transcriptional regulation, and AGR2 played a critical role in sorafenib-regulated cell viability, endoplasmic reticulum (ER) stress and induced cell apoptosis in sorafenib-sensitive cells. Additionally, we have established the sorafenib-resistant cells, and AGR2 was highly expressed in resistant cells compared with parental cells. We suggest that sorafenib decreased the intracellular AGR2 and contrarily induced AGR2 extracellular secretion, which reduced the regulation of ER stress and cell survival in sorafenib-sensitive cells. In addition, AGR2 was highly intracellular expressed in sorafenib-resistant cells, which in turn leaded to activation of Endoplasmic reticulum (ER) homeostasis and exhibition of higher cell survival. In the study, we anticipate that AGR2 regulates ER stress to influence cancer progression and sorafenib resistance in HCC. The accomplishment of this study can provide us an understanding the roles of AGR2 in both sorafenib-sensitive and sorafenib-resistant HCC, which can be applied to develop predictive, early diagnostic and prognostic factors for establishing novel therapeutic targets specifically for the treatment of HCC.

考試合格證明 I
Abstract II
中文摘要 IV
誌謝 V
目錄 VII
Introduction 1
Hepatocellular carcinoma (HCC) and sorafenib targeted therapy 1
AGR2 and cancers 1
Functions of AGR2 and related signaling pathways 2
AGR2 and ER stress relationship in HCC 4
Relationship between AGR2 and drug resistance 5
Hypothesis 6
Flow chart 7
Materials and Methods 8
Table 1. The list of chemicals and reagents in current work 8
Table 2. The list of chemicals and reagents used in current experiments 10
Table 3. The list of buffers utilized for protein lysis and pretreatment 11
Table 4. The list of buffers for Western blotting 11
Table 5. The list of chemicals and reagents for SDS-PAGE gel 12
Table 6. The list of antibodies utilized in Western blotting 13
Table 7. The list of chemicals and reagents utilized in RNA extraction 14
Table 8. Program settings and the list of chemicals utilized in RT-PCR 14
Table 9. Gene primer sequences and setting annealing temperatures 15
Results 16
AGR2 was relevant clinically in HCC 16
Sorafenib decreased cell viability and increased cell apoptosis in HCC 17
Sorafenib induced AGR2 secretion instead of transcriptional regulation 17
AGR2 regulated HCC cell viability and apoptosis 18
Sorafenib induced ER stress in HCC 18
AGR2 possesses diverse roles in sorafenib-resistant (SR) cells 19
AGR2 regulated ER stress-related mechanisms, cell viability and apoptosis in SR cells 21
Figures 24
Figure 1. Selection of the potential candidate genes and their association to the survival analysis  25
Figure 2. Clinical information of AGR2 expression 26
Figure 3. Sorafenib decreased cell viability and increased cell apoptosis in HCC 27
Figure 4. Sorafenib induced AGR2 secretion in HCC 29
Figure 5. AGR2-silenced regulated HCC cell viability and apoptosis 31
Figure 6. Treatment of soluble AGR2 reduced sorafenib-induced cell apoptosis in HCC 32
Figure 7. AGR2 involved in sorafenib-induced ER stress 34
Figure 8. Establishing sorafenib-resistant cell lines 36
Figure 9. Sorafenib-resistant (SR) cells possess lower ER stress under sorafenib treatment 38
Figure 10. Proposed model for investigating the role of AGR2 in sorafenib-sensitive and sorafenib-resistant HCCs  39
References 40
                                    

1. Ramesh, V., et al., Synthesis and biological evaluation of new rhodanine analogues bearing 2-chloroquinoline and benzo[h]quinoline scaffolds as anticancer agents. Eur J Med Chem, 2014. 83: p. 569-80.
2. Kudo, M., et al., Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. The Lancet, 2018. 391(10126): p. 1163-1173.
3. Tang, W., et al., The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther, 2020. 5(1): p. 87.
4. Zhu, Y.J., et al., New knowledge of the mechanisms of sorafenib resistance in liver cancer. Acta Pharmacol Sin, 2017. 38(5): p. 614-622.
5. Park, S. W., et al., The protein disulfide isomerase AGR2 is essential for production of intestinal mucus. Proceedings of the National Academy of Sciences, 2009. 106(17), 6950-6955.
6. Salmans, M. L., et al., The estrogen-regulated anterior gradient 2 (AGR2) protein in breast cancer: a potential drug target and biomarker. Breast Cancer Research, 2013. 15(2), 1-14.
7. Fritzsche, F.R., et al., Prognostic relevance of AGR2 expression in breast cancer. Clin Cancer Res, 2006. 12(6): p. 1728-34.
8. DiMaio, M.A., et al., Immunohistochemical panel for distinguishing esophageal adenocarcinoma from squamous cell carcinoma: a combination of p63, cytokeratin 5/6, MUC5AC, and anterior gradient homolog 2 allows optimal subtyping. Hum Pathol, 2012. 43(11): p. 1799-807.
9. Hong, X., et al., Effects of ER-resident and secreted AGR2 on cell proliferation, migration, invasion, and survival in PANC-1 pancreatic cancer cells. BMC Cancer, 2021. 21(1): p. 33.
10. Fessart, D., et al., Anterior Gradient-2 (AGR2) overexpression in colon cancer: a potential prognostic biomarker. bioRxiv, 2020
11. Park, K., et al., AGR2, a mucinous ovarian cancer marker, promotes cell proliferation and migration. Exp Mol Med, 2011. 43(2): p. 91-100.
12. Tian, S.B., et al., The prognostic value of AGR2 expression in solid tumours: a systematic review and meta-analysis. Sci Rep, 2017. 7(1): p. 15500.
13. Chung, K., et al., Serum AGR2 as an early diagnostic and postoperative prognostic biomarker of human lung adenocarcinoma. Cancer Biomark, 2011. 10(2): p. 101-7.
14. Tohti, M., et al., Serum AGR2 as a useful biomarker for pituitary adenomas. Clin Neurol Neurosurg, 2017. 154: p. 19-22.
15. Ramachandran, V., et al., Anterior gradient 2 is expressed and secreted during the development of pancreatic cancer and promotes cancer cell survival. Cancer Res, 2008. 68(19): p. 7811-8.
16. Bu, H., et al., The anterior gradient 2 (AGR2) gene is overexpressed in prostate cancer and may be useful as a urine sediment marker for prostate cancer detection. Prostate, 2011. 71(6): p. 575-87.
17. Fletcher, G.C., et al., hAG-2 and hAG-3, human homologues of genes involved in differentiation, are associated with oestrogen receptor-positive breast tumours and interact with metastasis gene C4.4a and dystroglycan. Br J Cancer, 2003. 88(4): p. 579-85.
18. Norris, A.M., et al., AGR2 is a SMAD4-suppressible gene that modulates MUC1 levels and promotes the initiation and progression of pancreatic intraepithelial neoplasia. Oncogene, 2013. 32(33): p. 3867-76.
19. Yuan, Z., et al., FOXA1 Promotes Cell Proliferation and Suppresses Apoptosis in HCC by Directly Regulating miR-212-3p/FOXA1/AGR2 Signaling Pathway. Onco Targets Ther, 2020. 13: p. 5231-5240.
20. Dahal Lamichane, B., et al., AGR2 is a target of canonical Wnt/beta-catenin signaling and is important for stemness maintenance in colorectal cancer stem cells. Biochem Biophys Res Commun, 2019. 515(4): p. 600-606.
21. Sommerova, L., et al., ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas. Cancers (Basel), 2020. 12(6).
22. Hrstka, R., et al., AGR2 oncoprotein inhibits p38 MAPK and p53 activation through a DUSP10-mediated regulatory pathway. Mol Oncol, 2016. 10(5): p. 652-62.
23. Hu, Z., et al., Knockdown of AGR2 induces cellular senescence in prostate cancer cells. Carcinogenesis, 2012. 33(6): p. 1178-86.
24. Gray, T.A., et al., Engineering a synthetic cell panel to identify signalling components reprogrammed by the cell growth regulator anterior gradient-2. Mol Biosyst, 2014. 10(6): p. 1409-25.
25. Maarouf, A., et al., Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapyinduced senescence escape. Int J Oncol, 2022. 60(1).
26. Yu, H., et al., Proteomic study explores AGR2 as pro-metastatic protein in HCC. Mol Biosyst, 2012. 8(10): p. 2710-8.
27. Sommerova, L., et al., Suppression of AGR2 in a TGF-beta-induced Smad regulatory pathway mediates epithelial-mesenchymal transition. BMC Cancer, 2017. 17(1): p. 546.
28. Fessart, D., et al., Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. Elife, 2016. 5.
29. Maurel, M., et al., Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation. EMBO Mol Med, 2019. 11(6).
30. Clarke, C., et al., The metastasis-inducing protein AGR2 is O-glycosylated upon secretion from mammary epithelial cells. Mol Cell Biochem, 2015. 408(1-2): p. 245-52.
31. Guo, H., et al., Tumor-secreted anterior gradient-2 binds to VEGF and FGF2 and enhances their activities by promoting their homodimerization. Oncogene, 2017. 36(36): p. 5098-5109.
32. Jia, M., et al., Pro-metastatic activity of AGR2 interrupts angiogenesis target bevacizumab efficiency via direct interaction with VEGFA and activation of NF-kappaB pathway. Biochim Biophys Acta Mol Basis Dis, 2018. 1864(5 Pt A): p. 1622-1633.
33. Dumartin, L., et al., ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation. Oncogene, 2017. 36(22): p. 3094-3103.
34. Hetz, C., The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol, 2012. 13(2): p. 89-102.
35. Bergstrom, J.H., et al., AGR2, an endoplasmic reticulum protein, is secreted into the gastrointestinal mucus. PLoS One, 2014. 9(8): p. e104186.
36. Higa, A., et al., Role of pro-oncogenic protein disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) in the control of endoplasmic reticulum homeostasis. J Biol Chem, 2011. 286(52): p. 44855-68.
37. Al-Shaibi, A.A., et al., Human AGR2 Deficiency Causes Mucus Barrier Dysfunction and Infantile Inflammatory Bowel Disease. Cell Mol Gastroenterol Hepatol, 2021. 12(5): p. 1809-1830.
38. Zhao, F., et al., Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2-/- mice. Dev Biol, 2010. 338(2): p. 270-9.
39. Luu, T.T., et al., Overexpression of AGR2 Is Associated With Drug Resistance in Mutant Non-small Cell Lung Cancers. Anticancer Res, 2020. 40(4): p. 1855-1866.
40. Li, J., et al., AGR2 is controlled by DNMT3a-centered signaling module and mediates tumor resistance to 5-Aza in colorectal cancer. Exp Cell Res, 2019. 385(1): p. 111644.
41. Zhang, Y., et al., miR-135b-5p enhances doxorubicin-sensitivity of breast cancer cells through targeting anterior gradient 2. J Exp Clin Cancer Res, 2019. 38(1): p. 26.
42. Liu, Q.G., et al., Knockdown of AGR2 induces cell apoptosis and reduces chemotherapy resistance of pancreatic cancer cells with the involvement of ERK/AKT axis. Pancreatology, 2018. 18(6): p. 678-688.
43. Kim, S.J., et al., Expression of anterior gradient 2 is decreased with the progression of human biliary tract cancer. Tohoku J Exp Med, 2014. 234(1): p. 83-8.
44. Ramachandran, V., et al., Anterior gradient 2 is expressed and secreted during the development of pancreatic cancer and promotes cancer cell survival. Cancer research, 2008. 68(19): p. 7811-7818.
45. Hrstka, R., et al., The pro-metastatic protein anterior gradient-2 predicts poor prognosis in tamoxifen-treated breast cancers. Oncogene, 2010. 29(34): p. 4838-47.
46. Mimura, N., et al., Blockade of XBP1 Splicing by Inhibition of IRE1α Is a Promising Therapeutic Option in Multiple Myeloma. Blood, 2011. 118(21): p. 133-133.
47. Zhou, T., et al., RACK1 modulates apoptosis induced by sorafenib in HCC cells by interfering with the IRE1/XBP1 axis. Oncol Rep, 2015. 33(6): p. 3006-14.

校內：立即公開
校外：立即公開

簡易檢索 / 詳目顯示

相關論文