臺灣博碩士論文加值系統

針對具有表皮生長因子受體(epidermal growth factor receptor, EGFR)突變的非小細胞肺癌中，目前最常使用的抗癌藥物為表皮生長因子受體酪胺酸激&;#37238;抑制劑-吉非替尼 (Gefitinib)，儘管此抑制劑具有顯著的治療效果，約30~40%病人在長期服用藥物的情況下，逐漸產生內生性抗藥性導致癌症的復發，目前在吉非替尼藥物引發的抗藥機制尚不完全清楚。
在實驗室先前的研究中，我們利用包含16,000條小髮夾核醣核酸(short hairpin RNA, shRNA)之干擾核醣核酸庫，針對激&;#37238;、磷酸&;#37238;、轉錄因子與去泛素酵素等多達3,000個基因挑出一個可能參與此獲得性吉非替尼抗藥性機制的候選基因 dual-specificity phosphatase 13 (DUSP13)，此基因會轉譯出兩個不同功能性的蛋白質：DUSP13A與DUSP13B。先前報導指出DUSP13A為新穎的apoptosis signal-regulating kinase 1 (ASK1)調控者；DUSP13B具有去磷酸&;#37238;之活性並且抑制下游mitogen-activated protein kinase (MAPK )訊息傳遞路徑的活化。目前為止，此二種蛋白質皆未被報導與肺癌抗藥性有關聯性。
為了了解DUSP13在獲得性吉非替尼抗藥性扮演的角色，專一性偵測DUSP13A與DUAP13B蛋白質表現為必須的步驟。由於目前市面上沒有合適的DUSP13A與DUSP13B之專一性抗體，我們透過免疫紐西蘭大白兔建立DUSP13A與DUSP13B之專ㄧ性抗體，希望能藉此更加了解何種DUSP13在抗藥性機制中扮演主要調控的角色。另一方面，我們利用活體外細胞聚落形成實驗 (in vitro colony forming assay) 觀察到給予吉非替尼作用時，剔除DUSP13基因的抗藥性細胞株形成聚落之能力明顯下降。接著分別利用西方墨點法與流式細胞儀觀察細胞凋亡標記poly ADP-ribose polymerase (PARP)、caspase3 與subG1 phase在給予吉非替尼作用的環境中表現量明顯上升。實驗室先前的研究發現當活體外剔除DUSP13基因的細胞株能夠明顯地回復對藥物之敏感性。另外，在活體動物皮下腫瘤接種實驗(in vivo mouse subcutaneous injection assay)中也觀察到剔除DUSP13基因之腫瘤在給予低濃度吉非替尼(10 mg/kg/day)治療後，其腫瘤生長明顯地受到抑制。
根據文獻指出，NRAS基因的突變可能促進ERK基因之表現，使抗藥性PC9/gef細胞株產生獲得性吉非替尼抗藥性。另一篇研究指出DUSP13B 蛋白質抑制MAPK 的活化。我們推測DUSP13B蛋白質可能參與調控MAPK路徑，藉此在獲得性吉非替尼抗藥性的PC9細胞中扮演一個重要的角色。

Gefitinib is an EGFR-tyrosine kinase inhibitor that has excellent antitumor activity in the advanced non-small-cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the dramatic response to such inhibitors, 30-40% of patients seem to have intrinsic resistance and ultimately get a rapid relapse. However, the underlying mechanisms of such resistance to gefitinib are not completely understood.
In our previous study, we used a pooled-based RNAi library screening which contains 16,000 shRNAs targeting all of known human kinases, phosphatases, and deubiquitinase to discover new candidates that confer acquired resistance to gefitinib. We identified a novel gene, dual-specificity phosphatase 13 (DUSP13), may be involved in the mechanism of acquired-gefitinib resistance. DUSP13 encodes two different functional proteins: DUSP13A and DUSP13B. Previous studies showed that DUSP13A functions as a novel regulator of apoptosis signal-regulating kinase 1 (ASK1) and DUSP13B has phosphatase activity that inactivates mitogen-activated protein kinase (MAPK) activation, but both of them have not been reported to involve in drug resistance of lung cancer.
To investigate the role of DUSP13 in acquired-gefitinib resistance, detecting the protein expression of DUSP13A and DUSP13B specifically is necessary. Due to there is neither commercially available anti-DUSP13A nor anti-DUSP13B antibody, we generated two specific antibodies that specifically react DUSP13A and DUSP13B proteins by New Zealand white rabbit immunization, and try to elucidate which protein plays the important role conferring acquired resistance to gefitinib. On the other hand, the in vitro colony formation assay showed that knockdown of DUSP13 inhibited colony forming ability in gefitinib treated gefitinib-resistance cells PC9. Two increased apoptosis markers, cleaved poly ADP-ribose polymerase (PARP) and cleaved caspase 3, assayed by Western blot and increase of subG1 phase assayed by flow cytometry are observed in the DUSP13 silenced gefitinib-resistance cells in presence of gefitinib.
The previous study in our laboratory found that knockdown of DUSP13 not only restored gefitinib sensitivity in vitro but also decreased in vivo tumor growth in low dose gefitinib (10 mg/kg/day) in the xenograft mouse model. Previous study indicated that the mechanism of acquired resistance to gefitinib in PC9/gef cells may be due to activating ERK pathway induced by NRAS mutation. Another study reported that DUSP13B inactivates MAPK activation. We suppose that DUSP13B may play an important role in MAPK pathway activation in the acquired gefitinib-resistance PC9 cells.

口試委員會審定書 I
致謝 II
中文摘要 III
Abstract V
1. Introduction 1
1.1 Lung adenocarcinoma 2
1.2 The mechanism of gefitinib resistance. 3
1.3 RNAi library screening 4
1.4 Protein tyrosine phosphatases (PTPs) family 5
1.5 Dual-specificity phosphatases 13 (DUSP13) 7
1.6 Preparation of target proteins for rabbit immunization 8
1.7 NSC663284, an inhibitor of dual-specificity phosphatase 13B 9
1.8 The previous investigation in laboratory 10
1.9 Specific aim 11
2. Materials and Methods 12
2.1 Cell lines and cell culture 13
2.2 High content RNAi screening in PC9 and PC9/gef cells. 13
2.3 Lentivirus production and transduction 14
2.4 Cell viability assay 14
2.5 Drug treatment 15
2.6 Cell cycle analysis 15
2.7 Dependent and independent colony formation 16
2.8 Plasmid construction 16
2.9 Quantitative real-time PCR 17
2.10 Competent cell preparation 18
2.11 IPTG induction 18
2.12 Protein purification 19
2.13 Coomassie Blue stain 19
2.14 Protein dialysis 20
2.15 Rabbit immunization 20
2.16 Specific antibody purification 21
2.17 Immunoblot 21
3. Results 23
3.1 DUSP13A/DUSP13B protein induction and recombinant protein purification. 24
3.2 Unpurified DUSP13B serum successfully recognizes the overexpressed DUSP13B protein in Escherichia coli cells. 26
3.3 Unpurified DUSP13A and DUSP13B serum successfully recognize the overexpressed DUSP13 in human cell lines. 28
3.4 Comparison of DUSP13A and DUSP13B mRNA expressions. 30
3.5 Knockdown of DUSP13 in PC9/gef-shLacZ cells can restore the gefitinib sensitivity. 31
3.6 Knockdown of DUSP13 in PC9/gef cells inhibits in vitro anchorage-independent and anchorage-dependent cell growth. 31
3.7 Cell sensitivity to NSC663284, a DUSP13B inhibitor, in PC9 and PC9/gef cells. 32
4. Discussion 34
5. Figures 41
Figure 1. DUSP13 isoforms. 42
Figure 2. Expression of DUSP13A and DUSP13B proteins by IPTG induction. 44
Figure 3. Purification of DUSP13A and DUSP13B proteins. 45
Figure 4. Optimization of DUSP13A protein purification. 46
Figure 5. DUSP13B protein quantification for New Zealand white rabbits immunization. 49
Figure 6. Specificity of first immunized DUSP13B serum from Rabbit-H with E. coli BL21 expressing DUSP13B protein. 50
Figure 7. Specificity of first immunized DUSP13B serum from Rabbit-L with E. coli BL21 expressing DUSP13B protein. 51
Figure 8. The detect limitation of forth immunized DUSP13B serum of Rabbit-H and Rabbit-L with E. coli BL21 expressing DUSP13B protein. 52
Figure 9. Anti-DUSP13B antibody successfully recognizes different DUSP13 isoforms but not DUSP13A. 53
Figure 10. Unpurified anti-DUSP13A antiserum recognizes DUSP13A. 56
Figure 11. The expression of DUSP13A and DUSP13B in PC9 and PC9/gef cells. 59
Figure 12. Knockdown of DUSP13 resensitize the gefitinib susceptibility in PC9/gef cells. 61
Figure 13. Knockdown of DUSP13 inhibits anchorage-dependent and anchorage-independent cell growth in PC9/gef cells. 64
Figure 14. NSC663284 sensitivity in PC9 and PC9/gef cell lines. 65
Figure 15. Drug sensitivity of NSC663284 and gefitinib in PC9 and PC9/gef cells. 66
6. References 67

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