臺灣博碩士論文加值系統

靈芝蛋白(Ling Zhi-8, LZ-8)為從靈芝(Ganoderma lucidum)純化之免疫調節蛋白，具有抑制腫瘤增生和調節人類免疫系統之作用。在本研究中，我們藉由基因工程大量表達與純化LZ-8 (rLZ-8)；在小鼠肺癌模式中，我們發現rLZ-8有效地抑制於小鼠體內的肺癌增長與轉移現象，且可以增加的肺癌小鼠的存活率。在第一章中，我們主要探討rLZ-8如何抑制肺癌細胞的移動性和轉移能力。我們證實rLZ-8可以透過干擾與影響粘著斑激酶(Focal adhesion kinase, FAK)的活性，有效地誘導上皮間質細胞轉化(epithelial-mesenchymal transition, EMT)的改變，致使抑制腫瘤轉移之能力。在體外試驗中，我們發現藉由抑制FAK的活性使調控EMT的轉錄因子Slug表現量下降後，可增強E-cadherin蛋白表達並抑制癌細胞的移動能力。在機制探討上，我們證實廢除FAK的蛋白表現與活性可以誘導MDM2和p21的表達，並致使形成MDM2/p21/Slug複合體，導致Slug被MDM2進行泛素化作用而降解。這項研究確定了rLZ-8抗腫瘤轉移之能力，即是通過負調控FAK的活性進而誘導Slug的泛素化和降解作用，導致改變EMT和細胞移動能力。除此之外，臨床報導中指出，肺癌細胞之上皮生長因子受器(epidermal growth factor receptor, EGFR)不正常突變與增強表現，易造成腫瘤之惡化及轉移現象；在第二章中，我們主要探討rLZ-8抑制肺癌增生過程中，rLZ-8和EGFR的相關性。我們發現rLZ-8會降低EGFR表現量並誘導肺癌細胞凋亡。rLZ-8有效地抑制一般型與突變型的EGFR表達，並抑制EGFR的活性與其下游信號傳導途徑，如AKT和ERK1/2。此外，我們證明rLZ-8會與EGFR交互作用並誘導EGFR內吞作用和活化其降解訊號。功能研究顯示rLZ-8可以誘導EGFR的1045酪胺酸位點進行磷酸化並驅使泛素化粘接酶CBL與EGFR形成EGFR/CBL複合體，致使EGFR被泛素化與降解。為驗證rLZ-8於臨床之應用價值，我們進一步分析rLZ-8與臨床用藥順鉑(cisplatin)合併使用之功效。不論在一般型與突變型的EGFR肺癌細胞中，我們發現rLZ-8皆可以強化順鉑誘導的細胞凋亡現象。這項研究更確定rLZ-8抑制肺癌增殖之能力，即是通過活化肺癌細胞的EGFR降解機制進而促進肺癌細胞走向細胞凋亡。本研究內容主要描述rLZ -8抑制肺癌增殖與轉移能力之機制乃是以干擾細胞黏著能力並誘導一般型與突變型的EGFR之降解路徑為標靶，成功證實rLZ -8的新穎抗癌機制，並闡明rLZ-8是為一具有潛力開發作為新型癌症治療之藥物。

Ling Zhi-8 (LZ-8), a protein from Ganoderma lucidum, is related with inhibiting tumorigenesis and enhancing human immune system. In this study, we find that recombinant LZ-8 (rLZ-8) suppresses lung cancer progression and metastasis as well as increases the survival rate in Lewis lung carcinoma cell (LLC1)-bearing mice. In Chapter 1, we focus on how rLZ-8 suppresses lung cancer cells mobility and metastasis. We demonstrate that rLZ-8 effectively induces changes in epithelial-to-mesenchymal transition (EMT) by interfering with focal adhesion kinase (FAK) functions, resulting in suppression of tumor metastasis. Functional studies reveal that downregulating Slug expression by abolishing FAK activity enhances E-cadherin expression and represses cancer cell mobility. Mechanistically, we demonstrate that inhibition of FAK induces MDM2 and p21 expression and enhances formation of MDM2/p21/Slug complex, resulting in ubiquitin-dependent Slug degradation. This study identify a novel mechanism for rLZ-8 anti-metastasis activity, namely the regulation of EMT and cell mobility via the negative modulation of FAK, leading to the ubiquitination and degradation of Slug. In Chapter 2, since abnormal EGFR expression is associated with human lung cancer, we examined the correlation of rLZ-8 and EGFR during the rLZ-8-suppressed lung tumorigenesis. We find that rLZ-8 induces apoptosis as well as rLZ-8 downregulates the wild-type (wt) and mutated (mut) EGFR expression, and inhibits basal and EGF-induced EGFR downstream signaling pathways, involving AKT and ERK1/2. Furthermore, we demonstrate that rLZ-8 binds to EGFR and triggers EGFR endocytosis and degradation signalings. We evaluate the mechanisms of rLZ-8-induced downregulation of EGFR. Functional studies reveal that rLZ-8-induced phosphorylation of EGFR at tyrosine 1045 (Y1045) leads to binding between EGFR and Cbl and that EGFR ubiquitination results in its proteasome degradation. To verify the clinical application of rLZ-8, we find that combination of rLZ-8 and cisplatin synergistically inhibits viability of lung cancer cells with wt- and mut-EGFR. Taken together, these findings suggest that rLZ-8 may have potential as a therapeutic agent for the treatment of lung cancer.

中文摘要...............................................I
ABSTRACT..............................................II
ABBREVIATIONS.........................................III
CONTENTS...............................................V
LITERATURE REVIEW.....................................1
1. Ganoderma lucidum..................................1
2. Fungal immunoregulatory proteins, Ling Zhi-8.......1
3. Lung cancer........................................2
4. Epithelial-to-mesenchymal transition and tumor metastasis............................................3
5. Epidermal growth factor receptor (EGFR)............4
6. Focal adhesion kinase (FAK)........................5
7. Endoplasmic reticulum stress.......................6
8. Cell death: necrosis, apoptosis and autophagy......7
MATERIALS AND METHODS.................................9
1. Cell lines.........................................9
2. Preparation of the recombinant Ling Zhi-8 (rLZ-8) protein...............................................9
3. Reagents and antibodies............................10
4. Cell viability assay...............................10
5. Western blot analysis..............................11
6. Cell cycle assay...................................11
7. Apoptosis assay....................................11
8. Fluorescence and Confocal Laser Microscopy.........12
9. Immunoprecipitation of EGFR or Slug for the in vitro ubiquitination assays.................................13
10. In vitro cell migration and invasion Transwell® assays................................................13
11. Wound closure assays..............................14
12. Determination of cell morphology..................14
13. Cell adhesion assay...............................14
14. Cell re-adhesion assay............................14
15. Plasmids and Transfection.........................15
16. Lentivirus production.............................15
17. Nuclear protein extraction........................16
18. Tumor metastasis study in vivo....................16
19. In vivo LLC1 tumour-bearing mouse model...........17
20. Statistical analysis..............................17
CHAPTER 1.............................................19
Focal adhesion kinase blockage controls cancer cell mobility by inducing Slug degradation during Ling Zhi-8-mediated suppression of lung tumor metastasis.........19
1.1. Abstract.........................................19
1.2. Introduction.....................................20
1.3. Results..........................................22
1.3.1. rLZ-8 reduces the metastatic potential of LLC1 cell-bearing mice.....................................22
1.3.2. rLZ-8 inhibits cell mobility and alters the expression of EMT-related markers in vitro............22
1.3.3. rLZ-8 suppresses FAK activity and FAK downstream signalings via interfering cell adhesion..............23
1.3.4. rLZ-8 reduces the expression of the EMT-related transcriptional factor Slug...........................24
1.3.5. rLZ-8 reduces Slug expression via accelerated ubiquitin-dependent proteasome-mediated degradation...25
1.3.7. rLZ-8 enhances the conjugation of MDM2 and p21 on Slug, and knockdown of MDM2 interferes with the rLZ-8-induced degradation of Slug...........................26
1.4. Discussion.......................................28
1.5. Figures..........................................32
Figure 1-1. rLZ-8 inhibits metastasis formation and increases the survival rate in LLC1-bearing mice......32
Figure 1-2. rLZ-8 changes the expression of EMT markers and suppresses the migratory and invasive properties of NSCLC.................................................34
Figure 1-3. rLZ-8 changes the formation of protrusions and the destruction of actin filaments................36
Figure 1-4. rLZ-8 suppresses FAK activity via binding to filopodia and interfering the focal adhesion..........38
Figure 1-5. rLZ-8-inhibition of FAK reduces the expression of Slug but induces E-cadherin expression..40
Figure 1-6. rLZ-8 induces ubiquitination-dependent proteasomal degradation of Slug.......................42
Figure 1-7. rLZ-8 enhances the formation of a MDM2/p21/Slug complex, and MDM2-shRNA (Sh-MDM2) abolishes the rLZ-8-induced degradation of Slug in CL1-5 cells..44
Figure 1-8. Abolishment of FAK induces p21 expression.46
Figure 1-9. The proposed model for the role of LZ-8 in Slug degradation and cell mobility....................48
1.6. Table............................................49
Table 1-1. Protein differentially expressed in lung tumor of mice received rLZ-8................................49

CHAPTER 2.............................................50
Ling Zhi-8 inhibits epidermal growth factor receptor signaling in lung cancer by inducing the casitas b-lineage lymphoma–mediated degradation of EGFR.........50
2.1. Abstract.........................................50
2.2. Introduction.....................................51
2.3. Results..........................................53
2.3.1. rLZ-8 suppresses EGFR-dependent proliferation in NSCLC.................................................53
2.3.2. rLZ-8 inhibits auto-phosphorylation of EGFR and downregulates EGFR expression in NSCLC cells..........54
2.3.3. rLZ-8 suppresses tumourigenesis and reduces EGFR protein levels in LLC1 cell-bearing male C57BL6 mice in vitro and in vivo.....................................54
2.3.4. rLZ-8 binds to EGFR to promote its clathrin-mediated endocytosis..................................55
2.3.5. rLZ-8 induces EGFR degradation via the ubiquitin proteasome pathway....................................57
2.3.6. rLZ-8 induces EGFR degradation signalling and enhances the conjugation of Cbl and EGFR..............58
2.3.7. Knockdown of c-Cbl and Cbl-b recovers rLZ-8-induced degradation of EGFR...........................59
2.3.8. Accumulation of rLZ-8 in ER triggers ER stress signalling and autophagy molecules, as well as EGFR involves in rLZ-8-induced autophagy...................60
2.3.9. Combinations of cisplatin with rLZ-8 synergistically induce apoptosis and inhibit growth in gefitinib-resistant lung cells........................61
2.4. Discussion.......................................63
2.5. Figures..........................................67
Figure 2-1. Depletion of EGFR suppresses proliferation of lung cancer cells.....................................67
Figure 2-2. rLZ-8 inhibits cell viability and induces apoptosis in lung cancer cells........................68
Figure 2-3. rLZ-8 inhibits autophosphorylation of EGFR and suppresses EGFR expression........................70
Figure 2-4. rLZ-8 does not affect the expression of TGFβR, integrin β1, Her2 and FGFR.....................72
Figure 2-5. rLZ-8 inhibits tumour growth in LLC1-allograft mice in vitro and in vivo...................73
Figure 2-6. rLZ-8-induced EGFR endocytosis and degradation depends on clathrin.......................75
Figure 2-7. rLZ-8 interacts with EGFR.................77
Figure 2-8. Combination of C225 (cetuximab) and rLZ-8 synergistically downregulates EGFR expression.........79
Figure 2-9. rLZ-8 enhances the ubiquitination-dependent proteasome-mediated degradation of EGFR in NSCLCs.....80
Figure 2-10. rLZ-8 induces the phosphorylation of EGFR (Y1045) and Cbl (Y731) and enhances the conjugation of Cbl and EGFR..........................................82
Figure 2-11. Knockdown of c-Cbl/Cbl-b interferes with/disturbs the rLZ-8-induced degradation of EGFR...84
Figure 2-12. rLZ-8 accumulates in endoplasmic reticulum (ER) and triggers ER stress and autophagy.............86
Figure 2-13. Combinations of cisplatin and rLZ-8 synergistically inhibit growth in NSCLC cells.........88
Figure 2-14. The proposed model for the role of LZ-8 in EGFR degradation......................................90
CONCLUSION............................................91
REFERENCES............................................93
APPENDIX..............................................101

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