惡性腫瘤進展演變是眾多基因變異累積的結果，其中腫瘤細胞週期失調以及免疫脫逃的特性是促成腫瘤進展的兩大原因。KLF4是一個轉錄因子，它的生理功能可以抑制細胞週期G1/S期的進程，但是KLF4在不同部位的人類癌症中扮演著不同的角色，在頭頸鱗狀上皮癌的角色目前仍不清楚。第三號誘餌受體(Decoy receptor 3, DcR3)是腫瘤壞死因子受器超家族的成員之ㄧ，能透受體中和或非受體中和作用，調控宿主的免疫機能。高度DcR3表達在許多人類癌症已知與較差的預後相關，但其作用機制仍不清楚。本研究分析KLF4及DcR3在頭頸鱗狀上皮癌的表達狀況，以及它們在腫瘤進展演變中扮演的角色。
經由免疫組織染色我們發現，在正常及癌前病變上皮中存在層次特異性的KLF4表現，但是62例頭頸鱗狀上皮癌中有42例(67.7%)KLF4蛋白表現有下降的現象；值得注意的是，另20例(32.3%)具有持續KLF4表達的頭頸鱗狀上皮癌，卻有較差的存活率(p=0.005)，這個現象在晚期病患特別明顯。我們接著在SAS細胞株中建立KLF4過度表現的穩定轉植株(stable clones)，發現KLF4過度表現之後顯著造成穩定轉植株細胞移行與侵犯能力的增加，多重抗藥特性的出現，以及皮下腫瘤形成能力(in vivo tumorigenicity)的上升。以免疫組織染色分析56例頭頸鱗狀上皮癌之DcR3表達，結果顯示有21例(37.5%)為高DcR3表達，有顯著較差存活率，顯示DcR3是一個不良預後因子。我們過去的研究發現DcR3可調控體外(in vitro)巨噬細胞的活化，降低第二型主要組織相容性複合體(MHC class II, MHC-II)的表現。為了進一步研究DcR3是否透過調控腫瘤伴隨巨噬細胞來促進腫瘤進展，我們首先建立CT26-DcR3穩定轉殖株。與載體轉殖株相比較，我們發現DcR3穩定轉殖株的皮下腫瘤生長較為快速，同時有較多的腫瘤伴隨巨噬細胞浸潤現象。我們接著建立以CD68啟動子(promoter)表現DcR3的DcR3轉殖鼠，來進一步研究DcR3對腫瘤進展的影響。分析DcR3轉殖鼠的巨噬細胞，可以發現IL-10、IL-1ra、YM1，以及精氨酸酶(arginase)活性的增加；而IL-12、TNF-?恁BIL-6, NO，以及MHC-II的表現則有下降現象。此外，CT26腫瘤皮下生長及擴散能力在DcR3轉殖鼠都發現顯著上升，而精氨酸酶抑制劑nor-NOHA，以及組蛋白去乙醯抑制劑(histone deacetylase inhibitor) sodium valproate，可以抑制此一現象。
綜上所述，KLF4與DcR3在頭頸鱗狀上皮癌的高度表達，可以促進腫瘤進展演變，但透過不同的調控機制。其中持續KLF4表達直接促成侵犯及抗藥特性的增加，代表對於此類腫瘤治療藥物劑量與合併治療的重要性。另一方面，高度DcR3表達可透過間接調控腫瘤伴隨巨噬細胞的方式促進腫瘤進展，而組蛋白去乙醯抑制劑的合併使用將可能增加現有標準治療的效果。因此，我們的研究將能對未來臨床病患預後分期以及發展個人化癌症治療的概念，提供重要的訊息。

Cancer progression involves multiple complicated mechanisms caused by an accumulation of genetic alterations. Cell cycle dysregulation and immune evasion are two important cancer hallmarks leading to progression. Krüppel-like factor 4 (KLF4) is a transcription factor that physiologically inhibits cell cycle G1/S progression, but has contrasting roles in different human cancers. Its role in head and neck squamous cell carcinoma (HNSCC) has not been elucidated. Decoy receptor 3 (DcR3) is a member of the tumor necrosis factor receptor superfamily that can modulate host immunity through both decoy and non-decoy functions. Up-regulation of DcR3 correlates with poor prognosis in various human cancers, but the mechanisms involved remain unclear. In this study, the expression of KLF4 and DcR3 in HNSCC was evaluated, and their roles in tumor progression were investigated.
KLF4 expression was evaluated by immunohistochemistry (IHC), showing compartment-specific expression in normal and dysplastic epithelia. Forty-two (67.7%) of 62 HNSCC had decreased expression, while the other 20 (32.3%) had persistent KLF4 expression that independently correlated with worse disease-specific survival (p=0.005), especially in those with advanced disease. Similarly, enforced expression of KLF4 in cell lines SAS demonstrated increased in vitro migration/invasion abilities, multi-drug resistance, and in vivo tumorigenicity. The IHC of DcR3 in 56 HNSCCs demonstrated high DcR3 expression in 21 (37.5%) samples that independently correlated with poor survival (p=0.016). Because DcR3 is capable of modulating macrophage differentiation with down-regulated MHC-II expression in vitro, this study investigated whether DcR3 promotes tumor progression via TAMs. CT26-DcR3 stable transfectants were first established. In subcutaneous tumor growth assay, DcR3-transfectants grew faster and attracted TAMs infiltration compared to vector control clones. CD68 promoter-driven DcR3 transgenic (Tg) mice were next generated. Macrophages isolated from DcR3-Tg mice displayed higher levels of IL-10, IL-1ra, YM1, and arginase activity, while IL-12, TNF-?? IL-6, NO, and MHC-II expressions were down-regulated. Both tumor growth and spreading abilities of CT26 cells were significantly enhanced in DcR3-Tg mice, but could be abolished by arginase inhibitor nor-NOHA and histone deacetylase (HDAC) inhibitor sodium valproate.
In conclusion, persistent KLF4 expression and overexpression of DcR3 contribute to tumor progression in HNSCC via different control mechanisms. Persistent KLF4 expression in HNSCC directly confers aggressiveness and drug resistance, indicating the importance of treatment dosage and combination. On the other hand, DcR3 overexpression modulates TAM differentiation in vivo which contributes to tumor progression, while HDAC inhibitor is promising in potentiating current standard therapy for tumors with high DcR3 expression. These results provide valuable information for improving clinical prognostication and future personalized cancer treatment.

CONTENTS
PAGE NO.
ACKNOWLEDGEMENTS ………………………………………………………… 3
CONTENT ………………………………………………………………………….. 4
ABSTRACT (English) ……………………………………………………………. 8
ABSTRACT (Chinese) …………………………………………………………… 10
ABBREVIATIONS ………………………………………………………………… 12
CHAPTER 1 INTRODUCTION
1.1 Cancer in Taiwan and treatment dilemma ………………………………. 13
1.2 Heterogeneous controls in cancer progression ……………...…………. 13
1.3 Krüppel-like factor 4 (KLF4) ………………………………………………. 14
1.3.1 Diverse physiological functions of KLF4 ………………………...….. 14
1.3.2 KLF4 as a context-dependent gene in human cancer ……………. 15
1.4 Decoy receptor 3 (DcR3) ………………..………………………………... 15
1.4.1 Roles of DcR3 in immunomodulation ………………………………. 15
1.4.2 Roles of DcR3 in human diseases and cancers ………………….. 16
1.4.3 Tumor-associated macrophage (TAM) ……………………………… 17
1.4.4 DcR3 modulates macrophage differentiation ………………………. 18
1.5 Aims of the project …………………………………………………………. 18
CHAPTER2 MATERIALS AND METHODS
2.1 Reagents and kits …………………………………………………………. 19
2.2 Patients and tissues ………………………………………………………. 26
2.3 Immunohistochemistry (IHC) and scoring ………………………………. 26
2.4 Protein extraction and Western blot analysis ………………………….. 27
2.5 RNA purification and real-time PCR analysis …………………………. 28
2.6 Cell culture, plasmid construction and stable transfection …………… 28
2.7 Cell migration and invasion assays …………………………………….. 29
2.8 Chemosensitivity assay ………………………………………………….. 29
2.9 Subcutaneous tumor growth assay …………………………………….. 30
2.10 Tail vein injection metastasis assay ……………………………………. 30
2.11 Generation of DcR3-Tg mice ……………………………………………. 30
2.12 Isolation of peritoneal macrophages and TAMs ………………………. 31
2.13 Flow cytometry and enzyme-lined immunosorbent assay …………… 32
2.14 Gelatin zymography ……………………………………………………… 32
2.15 Arginase and nitric oxide assay ………………………………………… 32
2.16 Statistical analysis ………………………………………………………... 33
CHAPTER 3 PERSISTENT KLF4 EXPRESSION PREDICTS PROGRESSION AND POOR PROGNOSIS OF HEAD AND NECK SQUAMOUS CELL CARCINOMA
3.1 Introduction ………………………………………………………………… 34
3.1.1 KLF4 and cell cycle regulation ……………………………………… 34
3.1.2 Role of KLF4 in HNSCC …………………………………………….. 34
3.2 Results …………………………………………………………………….. 35
3.2.1 Decreased KLF4 expression in a majority of HNSCC …………… 35
3.2.2 Persistent KLF4 expression is associated with poor prognosis
in HNSCC ……………………………………………………………… 36
3.2.3 KLF4 overexpression increases migration and invasion
abilities of HNSCC cells ……………………………………………… 37
3.2.4 KLF4 overexpression enhances tumorigenicity of HNSCC
cells in vivo …………………………………………………………….. 38
3.2.5 High KLF4 expression induces multi-drug resistance in
HNSCC cells ………………………………………………………….. 38
3.3 Discussion ……………………………………………………………....... 39
3.3.1 KLF4 expression in normal epithelium and HNSCC ………………. 39
3.3.2 Oncogenic role of persistent KLF4 expression in HNSCC ……….. 39
3.3.3 Hypothetic model for pleiotropic functions of KLF4 ……………….. 40
CHAPTER4 DECOY RECEPTOR 3 ENHANCES TUMOR PROGRESSION VIA INDUCTION OF TUMOR-ASSOCIATED MACROPHAGES
4.1 Introduction ………………………………………………………………… 42
4.2 Results ……………………………………………………………………… 43
4.2.1 Poor prognostic impact of high DcR3 expression in HNSCC ……. 43
4.2.2 DcR3 induced TAM infiltration and promoted tumor growth ……… 44
4.2.3 Generation and characterization of CD68 promoter-driven
DcR3-Tg mice …………………………………………………………. 44
4.2.4 DcR3 modulated macrophage differentiation in DcR3-Tg mice …. 45
4.2.5 Subcutaneous tumor growth was enhanced by DcR3 ……………. 46
4.2.6 DcR3 enhanced tumor spreading in vivo …………………………… 47
4.2.7 DcR3-enhanced tumor growth was abolished by
nor-NOHA and VPA …………………………………………………… 47
4.3 Discussion …………………………………………………………………. 48
4.3.1 Macrophage/TAM differentiation modulated by DcR3 in vivo ……. 48
4.3.2 DcR3-modulated TAMs contribute to tumor progression ………… 49
4.3.3 Therapeutic implications …………………………………………….. 50
CHAPTER 5 DISCUSSION AND CONCLUSIONS
5.1 Contribution of KLF4 and DcR3 to HNSCC progression ……………. 51
5.2 Oncogenic functions of KLF4 …………………………………..………… 52
5.3 Protumoral effect of DcR3 ……………………………………………….. 53
5.4 Conclusions ……………………………………………………………….. 54
REFERENCES ……………………………………………………………………. 56
FIGURE AND TABLES …………………………………………………………... 68
PUBLICATIONS ………………………………………………………………….. 98

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