臺灣博碩士論文加值系統

爾必得舒(Cetuximab)為癌症的標靶藥物之一，其藉著抑制癌症表皮生長因子受體，進而達到抑制下游訊息傳導路徑活化，此藥物已被廣泛用來治療頭頸癌(HNSCC)，但有些病人卻產生抗藥性導致預後不如預期。為了釐清頭頸癌細胞產生抗藥性的原因，我們在體外篩選出對爾必得舒產生抗藥性的頭頸癌細胞株，並利用RNA-Sequence方法發現在抗藥性細胞株中會高度表達Snail 以及Lymphotoxin beta (LTβ)。Snail是造成癌細胞上皮間質細胞轉化的重要誘導物之一，並且透過實驗發現會調節細胞激素LTβ。由於有文獻指出腫瘤細胞在上皮間質細胞轉化過程中會伴隨著抗藥性的產生，因此我們猜測Snail-LTβ路徑是造成抗藥性的原因之一。 首先我們使用shRNA技術降低抗藥性植株中Snail或是LTβ表現，結果發現皆能有效增加爾必得舒對於抗藥株的毒殺作用。目前LTβ在腫瘤細胞中扮演的角色仍不清楚，過往認為LTβ會在樹突細胞的細胞膜上與LTβ形成α1β2 heterodimer，並結合到其他細胞表面上的LTβ receptor。這樣的結合會活化NFκB訊息路徑，而在我們的實驗中也發現爾必得舒抗藥株的TNF，IL6等NFκB訊息路徑的下游基因表現高於非抗藥性的細胞。由於NFκB訊息路徑與細胞增生作用相關，因此我們認為Snail- LTβ 路徑是透過NFB使得頭頸癌抗藥株能夠抵抗爾必得舒的毒殺作用。最後我們發現在頭頸癌抗藥株中同時給予NFB抑制劑處理能大幅增加細胞對於爾必得舒的敏感度，並且在小鼠動物模型中，同時給予NFκB抑制劑以及爾必得舒，對於腫瘤有著最好的抑制效果。此研究結果可以提供爾必得舒抗藥性的患者另一種有效的治療方式。

Cetuximab, a monoclonal antibody against the epidermal growth factor receptor EGFR extracellular domain, has been approved for the treatment of head and neck squamous cell carcinomas (HNSCC). However, many patients acquire resistance after Cetuximab treatment. To clarify the reason of resistance response in HNSCC, we established Cetuximab-resistant cell line in vitro. By RNA-sequencing, we found Snail and Lymphotoxin beta (LTβ) highly expressed in Cetuximab-resistant cell line. Our previously results indicated Snail, an important inducer of epithelial-mesenchymal transtition (EMT), regulates cytokines expression in cancer cells, such as LTβ. Other studies indicated that cancer cells acquire drug resistance in the process of EMT. Therefore, we proposed Snail- LTβ axis drives Cetuximab resistance. Our results showed knock-down of Snail or LTβ increased cytotoxic effect of Cetuximab in resistance cells. It demonstrated Snail- LTβ axis plays a major role in Cetuximab resistance of HNSCC. However, the function of LTβ in cancer cell remains elusive. Previous studies show LTβ bind lymphotoxin-alpha to form heterotrimer- LTα1β2 on dendritic cell surface then bind to LTβ receptor in other cells which activated NFKB pathway. Our data found that Cetuximab resistance cells expressed higher NFKB downstream genes than wild-type cancer cells. We thought NFκB signaling is important for HNSCC acquired Cetuximab-resistant. We combined NFκB inhibitor and Cetuximab treatment showed great synergistic effect for Cetuximab-resistant cells. Taken together, this study presents a therapeutic option by enhancing Cetuximab effectiveness for Cetuximab-resistant patients.

Contents
English abstract…………………………………………………………..…...1
Chinese abstract.................................................................................................2
I. Introduction……………………………………………………………........3
1-1 Head and neck cancer…………………………………………………….....3
1-2 Epidermal growth factor receptor (EGFR)………………………………....3
1-3 EGFR expression in head and neck cancer………………………………....4
1-4 EGFR-targeted therapies and resistance………………………………….....5
1-5 Epithelial-mesenchymal transition (EMT)……………………………….....5
1-6 Lymphotoxin Beta (LTβ)…………………………………………………....6
1-7 LTβR-NFκB signaling…………………………………………………..…..7
II. Materials and Methods……………………………………………..…….8
2-1 Cell lines and plasmids…………………………………………………..…..8
2-2 Protein extraction and western blot analysis………………………………...8
2-3 Reverse transcription and quantitative PCR analysis…………………….....9
2-4 PCR, sequencing, and mutational analysis…………………………………10
2-5 Luciferase activity assay……………………………………………………10
2-6 Nuclear/cytosolic fractionation……………………………………………..11
2-7 Electrophoretic mobility shift assay (EMSA)………………………………11
2-8 Cell viability assay………………………………………………………….12
2-9 NFB activity Assays……………………………………………………….12
2-10 Statistical analysis……………………………………………………….....12
III. Results…………………………………………………………………….…13
3-1 EMT regulator Snail contributes to Cetuximab resistance of HNSCC……..13
3-2 Snail induces Lymphotoxinβ expression……………………………….…...14
3-3 LTβ is critical for Snail-induced Cetuximab resistance………………….........14
3-4 LTβ induces downstream NFκB pathway in Cetuximab resistance cells……..15
3-5 Knock-down of LTβ in Cetuximab resistance cells reduces NFκB signaling...16
3-6 LTβ contributes to EGFR signaling in Cetuximab resistant cells………..……16
3-7 Potential drug therapy for Cetuximab resistance cells……………………...…18
IV. Discussion…………………………………………………………...………..19
V. References……………………………………………………………………..22
VI. Figures...............................................................................................................26
Figure1. EMT regulator Snail contributes to Cetuximab resistance of HNSCC….…26
Figure2. Snail induces Lymphotoxinβ expression…………………………………..28
Figure3. LTβ is critical for Snail-induced Cetuximab resistance……………………30
Figure4. LTβ induces downstream NFkB pathway in Cetuximab resistance cells..…32
Figure5. Knock-down of LTβ in Cetuximab resistance cells attenuated NFB signaling……………………………………………………………………………...35
Figure6. LTβ contributes to EGFR signaling in Cetuximab resistant cells………….39
Figure7. Potential drug therapy for Cetuximab resistance cells…………………..…41
VII.Tables…………………………………………………………………………43
VIII. Supplemental Figures………………………………………………………..47

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