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系統識別號 U0026-2807201411115100
論文名稱(中文) 探討致癌因子參與抑制細胞自噬及丙型干擾素抗性
論文名稱(英文) Investigating the Involvement of Oncogenic Factors in Inducing Inhibition of Autophagy and IFN-γ Resistance
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 102
學期 2
出版年 103
研究生(中文) 江姿慧
研究生(英文) Tzu-Hui Chiang
學號 S96011029
學位類別 碩士
語文別 英文
論文頁數 66頁
口試委員 指導教授-林秋烽
口試委員-謝奇璋
口試委員-張志鵬
口試委員-邢中熹
中文關鍵字 免疫逃脫  丙型干擾素  細胞自噬  Bcl-2  mTOR  PTEN 
英文關鍵字 Immune escape  IFN-γ  Autophagy  Bcl-2  mTOR  PTEN 
學科別分類
中文摘要 自然殺手細胞及T細胞會藉由分泌丙型干擾素、穿孔素、顆粒溶解酶、CD95配體和腫瘤壞死因子相關誘導配體產生細胞毒性而減緩腫瘤生長。然而,癌細胞會藉由腫瘤免疫編輯進行免疫抑制而逃脫免疫系統的監控。基本上,丙型干擾素在宿主的免疫監控之中扮演重要角色。我們之前的研究指出丙型干擾素誘導的細胞自噬會幫助丙型干擾素的訊息傳遞。我們假設藉由致癌因子調控的細胞自噬會造成丙型干擾素抗性,這也許是一個腫瘤免疫逃脫的策略。在A549細胞中,丙型干擾素會活化JAK2/STAT1訊息傳遞並且誘導自噬作用,但卻無法在AS2細胞中順利進行。進一步在Atg5缺失的A549細胞中也證實自噬作用的抑制會造成丙型干擾素抗性。細胞失去自噬作用致使活性氧分子增加進而促成SHP2異常活化抑制丙型干擾素的訊息傳遞。除此之外,丙型干擾素誘導STAT1活化和自噬作用與p53的表現無關。值得注意的是異常的Bcl-2表現及mTOR訊息傳遞導致該細胞無法進行丙型干擾素所誘導的自噬作用和STAT1活化。此外,在AS2及PTEN缺失的A549細胞中證實PTEN缺失將增加Bcl-2及mTOR的表現造成活性氧分子增加使得SHP2異常活化進而破壞丙型干擾素的訊息傳遞。丙型干擾素在A549細胞會誘導細胞毒性而在AS2細胞卻不會。若在AS2細胞中抑制活性氧分子及SHP2活性可回復丙型干擾素所誘導的細胞毒性。另外在HEK293T細胞中過度表現mTOR及Bcl-2也會干擾丙型干擾素的訊息傳遞。這些發現指出異常的PTEN缺失及下游Bcl-2及mTOR活化的訊息傳遞不只抑制了自噬作用也造成了丙型干擾素抗性。這個結果證實了致癌基因抑制自噬作用可能會阻礙丙型干擾素的抗癌活性而在腫瘤生成之中扮演重要角色。
英文摘要 Nature killer and T cells produce interferon (IFN)-γ, perforin, granzymes, CD95 ligand, and tumor necrosis factor-related apoptosis-inducing ligand to retard tumor progression through cytotoxic induction. However, cancer cells may induce immunosuppression escape from immunosurveillance in the process of cancer immunoediting. Basically, IFN-γplays an important role in host immunosurveillance. Our previous findings showed that IFN-γ-induced autophagy facilitates IFN-γ signal transduction. In this study, we hypothesized that manipulating autophagy by oncogenic factors may cause IFN-γ resistance as a strategy for immune escape during tumorigenesis. IFN-γ activated Janus kinase/signal transducers and activator (JAK/STAT) signaling followed by an increase in autophagy in A549 human lung adenocarcinoma cells; however, either IFN-γ signaling or autophagy was defective in AS2 lung adenocarcinoma cells. As demonstrated in Atg5-deficient A549 cells, inhibition of autophagy resulted in IFN-γ resistance. Loss of autophagy increased reactive oxygen species (ROS)-activated Src homology domain containing phosphatase 2 (SHP2) activation that contributed to defect in IFN-γ signaling. In addition, IFN-γ induced STAT1 activation and autophagy independently of p53. Notably, excessive B-cell lymphoma 2 (Bcl-2) expression and mammalian target of rapamycin (mTOR) signaling determined cellular resistance to IFN-γ-induced autophagy as well as STAT1 activation. Moreover, decrease of phosphatase and tensin homolog (PTEN), the upstream of Bcl-2 and mTOR, also disrupted IFN-γ signaling that demonstrated in AS2 and PTEN-deficient A549 cells. Besides, PTEN decrease also caused increase ROS-activated SHP2. IFN-γ induced cytotoxicity effectively in A549 cells but not in AS2 cells. Inhibition of ROS and SHP2 reversed the effect of IFN-γ-induced cytotoxicity. Furthermore, overexpression of Bcl-2 and Akt which is the upstream of mTOR also interfered the IFN-γ signaling in the human embryonic kidney HEK293T cells. These findings indicate that oncogenic effects by aberrant PTEN expression and downstream signaling, Bcl-2 and mTOR, cause not only inhibition of autophagy but also IFN-γ resistance. These results provide evidence that oncogenic inhibition of autophagy may plays an essential role on tumorigenesis by interfering anticancer IFN-γ.
論文目次 Abstract in Chinese I
Abstract in English II
Acknowledgement III
Abbreviations IV
Contents VII
I. Introduction 1
I-1. Cancer immunoediting from immunosurveillance 1
I-2. IFN-γ 2
I-3. The anticancer activity of IFN-γ 3
I-4. Autophagy 4
I-5. The role of autophagy in cancer 4
I-6. The regulation between IFN-γ and autophagy 6
II. Study Objective and Specific Aims 7
II-1. Objective 7
II-2. Specific Aims 7
Specific Aim 1: To check the relationship between IFN-γ signaling and autophagy in several lines of non-small-cell lung cancer cells. 7
Specific Aim 2: To confirm the regulation of autophagy in IFN-γ signaling. 7
Specific Aim 3: To identify the possible oncogenic factors for autophagic regulation and IFN-γ resistance. 7
III. Materials and Methods 8
III-1. Cell Cultures 8
III-2. Reagents and Antibodies 8
III-3. Western Blotting 9
III-4. Immunostaining 9
III-5. Mitochondria and ROS detection 10
III-6. Luciferase reporter assay 10
III-7. Proliferation assay 11
III-8. Cytotoxicity assay 11
III-9. Plasmid transfection 11
III-10. RNA interference 12
III-11. Statistical Analysis 12
IV. Results 14
IV-1. IFN-γ Signaling is Disrupted Accompanied Inhibition of Autophagy 14
IV-2. Inhibition of Autophagy Interferes The IFN-γ Signal Transduction 14
IV-3. Loss of Autophagy Causes ROS-activated SHP2 Upregulation and Abrogates The IFN-γ Signaling 15
IV-4. IFN-γ Signaling is Independent of p53 15
IV-5. Excessive Bcl-2 and mTOR Expression Not Only Inhibits Autophagy But Also Disrupts IFN-γ Signaling 16
IV-6. PTEN Decrease is Followed by IFN-γ Signaling Disruption 17
IV-7. PTEN Decrease Interferes The IFN-γ Signaling 18
IV-8. PTEN Decrease Causes Upregulation of ROS-Activated SHP2 18
IV-9. Inhibition of ROS or SHP2 restores the IFN-γ anticancer activity 19
IV-10. Overexpression of Bcl-2 and Akt in Normal cell Interferes IFN-γ Signal Transduction 19
V. Discussion 21
VI. Conclusion and Implication 26
References 27
Figures and Figure Legends 33
Figure 1. Autophagy is induced after IFN-γ signaling in A549 cells but not in AS2. 33
Figure 2. Inhibition of autophagy interferes IFN-γ signaling. 35
Figure 3. Autophagy deficiency abrogates IFN-γ signaling by increased ROS-activated SHP2. 36
Figure 4. IFN-γ signaling is independent of p53. 38
Figure 5. Overexpression of Bcl-2 and mTOR retards IFN-γ signaling through inhibiting autophagy. 39
Figure 6. IFN-γ signaling is disrupted in cells with decreased PTEN. 41
Figure 7. PTEN decrease interferes IFN-γ signaling. 42
Figure 8. PTEN decrease causes defects in ICAM-1 expression. 43
Figure 9. Decrease of PTEN cause ROS increase followed by SHP2 activation. 44
Figure 10. Cells with decreased PTEN are resistant to IFN-γ-induced growth inhibition. 45
Figure 11. Cells with decreased PTEN are resistant to IFN-γ-induced cytotoxicity 46
Figure 12. Inhibition of SHP2 and ROS reverses the resistance of IFN-γ-induced growth inhibition and cytotoxicity in AS2 cells. 47
Figure 13.Overexpression of Bcl-2 and Akt interfere the IFN-γ signaling in HEK293T cells. 48
Figure 14.A hypothetical model for the mechanism that oncogenic factors cause IFN-γ resistance to anticancer activity by inhibiting autophagy. 49
Appendix 50
A. Materials 50
A-1 Chemicals 50
A-2 Antibodies 52
A-3 Kits 53
A-4 Consumables 53
A-5 Apparatus 54
B. Methods 55
B-1 Cell culture 55
B-2 Western blot 57
B-3 Lentiviral-based shRNA knockdown 59
B-4 Intracellular ROS assay 61
B-5 Overexpression 61
CURRICULUM VITAE 66
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