臺灣博碩士論文加值系統

頭頸鱗狀細胞癌是一種高死亡率的疾病，在臨床病理中的細胞表現型具有異質化特性。癌症起始細胞在腫瘤中為一群極少數的細胞，能夠自我更新保持幹細胞特性，對癌症治療具有抵抗能力，並且伴隨表皮細胞間質轉化 (EMT) 發生。癌症治療中，消除癌症起始細胞能夠增加癌症治癒的機會。目前已有許多研究與標靶癌症起始細胞相關，且證實消除癌症起始細胞能夠減低腫瘤惡性程度。
本實驗室利用無血清癌症起始細胞篩選培養基分離出頭頸癌起始細胞球體（spheres），且這些頭頸癌起始細胞會過度表現CD133細胞表面標誌蛋白和增加表皮細胞間質轉化發生。CD133是一種表現在細胞膜上的醣蛋白，起初CD133為造血幹細胞的細胞表面標誌，近來研究發現CD133也能做為癌症起始細胞的細胞表面標誌。且証實CD133+細胞較CD133-細胞，有較高幹細胞特性、轉移、腫瘤生成能力和藥物排出能力。在臨床的數據中具有 CD133表現的病人在經由化療藥物治療後的預後較差。其他研究報告指出CD133與Src kinase有直接的交互作用 (direct interaction) 。 但目前在頭頸鱗狀細胞癌中CD133對癌症起始細胞的幹細胞特性調控及腫瘤生成能力的分子機制尚未了解。
本研究探討CD133能否調控的癌症起始細胞特性及其分子機轉，首先利用慢病毒載體 (Lentivirus vector) 在癌症起始細胞中降低CD133表現能夠降低其癌症起始細胞自我更新能力，以及削弱腫瘤生成能力及抑制細胞爬行能力。相反的，過度表現CD133會增進頭頸癌細胞株癌症起始細胞特性以及促進腫瘤生成能力及爬行能力。上皮間質細胞轉化，已知會促進細胞爬行能力以及癌症起始細胞特性，過度表現CD133會透過活化Src 激酶促進上皮間質細胞轉化，使用Src抑制劑 (PP2) 則能夠抑制CD133所促進的上皮間質細胞轉化。 利用點突變 (point mutation)方式，改變CD133與Src激酶的結合位置，實驗結果顯示CD133第852胺基酸從酪胺酸突變成苯丙胺酸 (Y852F)，能夠降低活化Src激酶的能力以及降低促進癌症起始細胞特性。
本研究證實CD133會透過Src調控頭頸癌癌症起始細胞自我更新及腫瘤生成能力和促進上皮間質細胞轉化，因此未來可將CD133和Src訊息傳遞路徑做為頭頸癌症治療的標的，以減少癌症起始細胞族群數量， 並進一步降低頭頸癌腫瘤惡性程度。

Head and Neck squamous cell carcinoma (HNSCC) is a lethal cancer with clinical, pathological, phenotypical and biological heterogeneity. In solid tumor, cancer initiating cells (CICs) being the rare population, are responsible for tumor growth, therapy resistance, and coupled with gain of epithelial-mesenchymal transition (EMT), have been identified. CD133 (prominin-1), a 5-transmembrane glycoprotein, is originally recognized as a hematopoietic stem cells marker, and has been considered as an important cell surface marker to represent the subpopulation of cancer-initiating cells. Previously, we have enriched a subpopulation of head and neck cancer initiating cells (HN-CICs) with up-regulation of CD133 and enhancement of EMT. Others demonstrate that Src kinase interacts with and phosphorylates the cytoplasmic domain of CD133. However, the physiological function of CD133/Src signaling in HNSCCs has not been uncovered.
In this study, I investigated whether CD133 could regulate cancer initiating cells properties and the molecular mechanism. Initially, down-regulation of CD133 significantly reduced the self-renewal ability and expression of stemness genes, and promoted the differentiation and apoptotic capability of HN-CICs. Additionally, knockdown of CD133 in HN-CICs also lessened both in vitro malignant properties including cell migration/cell invasiveness/anchorage independent growth, and in vivo tumor growth by nude mice xenotransplantation assay. In opposite, overexpression of CD133 enhanced the stemness properties and tumorigenic ability of HNSCCs. Lastly, up-regulation of CD133 increased phosphorylation of Src coupled with EMT transformation in HNSCCs, on the contrary, silence of CD133 or treatment of Src inhibitor inversely abrogated above phenotypic effects, which were induced by CD133 up-regulation in HNSCCs or HN-CICs. I also found point mutation of CD133Y852F could invert the Src activation. I present results suggested that CD133/Src signaling is a regulatory switch to gain of EMT and of stemness properties in HNSCC.
In the future, CD133/Src axis might be a potential therapeutic target for HNSCC by a potential therapeutic target for HNSCC by eliminating HN-CICs.

THESIS CONTENTS...II
LIST OF TABLES...V
LIST OF FIGURES...VI
ABSTRACT...9
CHAPTER I: INTRODUCTION...13
CHAPTERII: MATERIAL AND METHODS...26
CHAPTER III: Results...34
CHAPTER IV: DISSCUSION...45
References...99
VITA...109


CHAPTER
I: INTRODUCTION...13
Part 1. Head and neck squamous cell carcinoma (HNSCC)...13
1-1 Epidemiology, causes, and management of head and neck Squamous Cell Carcinoma (HNSCC)...13
Part 2. Cancer initiating cells (CICs)...14
2-1 Hierarchical versus stochastic model for explaining cancer initiating cells propagation and tumor functional heterogeneity...14
2-2 The cancer initiating cells or cancer stem cells hypothesis...15
2-3 Methodologies to isolate and identify cancer initiating cells...17
2-4 Implications and applications of CICs on cancer therapy...18
2-5 CD133/Prominin-1, a cancer initiating cells marker with unknown function in HNSCC...19
2-6 Cancer initiating cells and Epithelial-Mesenchymal Transition (EMT)...21
2-7 Src kinase, a proto-oncogene and an EMT inducer...22
Part3. Purpose and Specific Aims...24
II: MATERIAL AND METHODS...26
III: Results...34
3-1 Down-regulation of CD133 reduces stemness properties coupled with increased differentiation and apoptotic capabilities in HN-CICs...34
3-2 Targeting CD133 abrogates in vitro and in vivo malignant properties of HN-CICs...36
3-3 Overexpression of CD133 enhances stemness properties and tumorigenic potentials of HNSCCs...37
3-4 Overexpression of CD133 enhances Epithelial-Mesenchymal Transition...38
3-5 Src kinase is the downstream modulator of CD133 on regulating EMT in HNSCC and HN-CICs...39
3-6 CD133 downregulation and Src inhibition abrogate the p-Src activity and sphere formation ability in primary HN-CICs....41
3-7 To determine the downstream pathway which activated by CD133/Src signaling pathway...41
3-8 The role of Src binding site on CD133 inducing Src activation....42
IV: DISSCUSION...45
4.1. The functions of CD133 in CICs...45
4.2 CD133/Src axis regulates EMT in HNSCCs and HNCICs... 46
4.3 The role of CD133 in hypoxia condition...47
4.4 The Src activated mechanism via CD133...49
4.5 Activation of Src family kinase by CD133...50
4.6 Other molecular pathways in HNSCCs and HNCICs....50

LIST OF TABLES
TABLE PAGE
1. List of shRNAi Target Sequence...52
2. List of Cloning Primers...53
3. List of Antibodies...54
4. List of Material...56

LIST OF FIGURES
FIGURE 1.Establishment of CD133 knockdown HN-CICs by lentiviral-RNAi....58
2. The efficiency of CD133 down regulation by lentiviral-RNAi in HN-CICs...59
3. Knockdown of CD133 expression of HN-CICs reduces sphere formation efficiency...60
4. Depletion of CD133 expression of HN-CICs decreases stemness gene expression....61
5. Knockdown of CD133 expression of HN-CICs increases expression of differentiation marker CK18....62
6. Knockdown of CD133 expression of HN-CICs induces apoptosis...63
7. CD133 downregulation reduces migration ability of HN-CICs...64
8. CD133 knockdown reduces invasiveness ability of HN-CICs...65
9. Depletion of CD133 expression reduces clonogenicity of HN-CICs...66
10. CD133 knockdown impairs in vivo tumorigenic properties of HN-CICs....67
11. Establishment of CD133 overexpressing HNSCCs...68
12. Expression of CD133 in CD133-overexpressing HNSCCs...69
13. Overexpression of CD133 promotes sphere formation ability in HNSCCs....70
14. Overexpression of CD133 in HNSCCs increases side population cells...71
15. Overexpression of CD133 in HNSCCs promotes stemness gene expression and reduces differentiation marker expression...72
16. Overexpression of CD133 enhances invasiveness ability in HNSCCs...73
17. Overexpression of CD133 in HNSCCs increases anchorage-independent growth ability...74
18. Overexpression of CD133 enhances tumorigenic properties of HNSCCs...75
19. CD133-overexpressing HNSCCs show mesenchymal-like cell shape....76
21. Intracellular localization of vimentin in CD133-overexpressing HNSCCs....78
22. The expression of mesenchymal marker integrin αVβ6 in CD133-overexpressing HNSCCs....79
23. Knockdown of CD133 enhances the epithelial cell marker in HNCICs....80
24. Src kinase activation promotes EMT...81
25. Src kinase activation promotes sphere formation ability ....82
26. Src kinase activation promotes tumorigenicity in vitro....83
27. Src kinase activation promotes tumorigenicity in vivo...84
28. CD133 enhances Src activity...85
29 CD133 regulates Src activity...86
30. CD133/Src signaling regulates the mesenchymal transformation in HNSCCs....87
31. Inactivation of Src activity by Src inhibitor, PP2, inhibits tumorigenicity of HN-CICs in vivo...88
32. Src activity is require for expression of Oct-4 and Nanog...89
33. CD133 downregulation abrogates the p-Src activity in primary HNCICs....90
34. CD133 downregulation abrogates sphere formation ability in primary HNCICs....91
35. Src inhibition abrogates sphere formation ability in primary HNCICs....92
36. Overexpression of CD133 enhances HIF1α, HIF2α and Snail expression in HNSCCs....93
37. Establishment of CD133Y828F and CD133Y852F mutant constructs on Src interaction site....94
38. The Effect of CD133Y828F and CD133Y852F mutant on p-Src activity....95
39. CD133Y852F inverts the sphere formation ability....96
40. Schematic of the CD133/Src signaling pathways promoting EMT, stemness properties, and tumorigenicity of head and neck cancer initiating cells (HN-CICs)....97
Appendix Fig 1. Hierarchical versus stochastic model (15)....98

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