肺癌是世界上死亡率最高的癌症之一。在肺癌病患中，表皮生長因子受體（Epidermal growth factor receptor; EGFR）的突變已被廣泛使用於肺癌標靶治療和生物標記（biomarker）上。其中，透過阻斷腫瘤生長訊息傳遞來抑制癌細胞
之標靶藥物：表皮生長因子受體的酪胺酸激酶抑制劑（EGFR-tyrosine kinase inhibitor; EGFR-TKI）多年來成功治癒帶有 EGFR 突變的肺癌病人。儘管如此，部份治療初期狀況良好的病人卻開始產生抗藥性，且抗藥性越來越頻繁出現。由於癌細胞生長迅速之下造成實質腫瘤增大，使得腫瘤中心遠離供血血管而產生低
氧壓力，低氧是腫瘤快速生長時必須面對的一種微環境（ Hypoxia
microenvironment）。然而，低氧微環境與 EGFR-TKI 篩選如何調控腫瘤異質性
以促進癌細胞增生、存活和表皮間葉轉變（Epithelial-Mesenchymal transition; EMT）依然未知。我們的研究發現氧氣感應因子脯胺醯羥化酶（Prolyl hydroxylase 3;PHD3）在 EGFR-TKI 抗性的細胞株中的表現量和 EGFR-TKI 敏感株相比明顯降低，同時產生EMT及增生能力的下降。PHD3的缺失與抗藥性產生和癌症存活的關係仍須進一步探索，在此研究中我們賦予 PHD3在肺癌中與其他癌症完全
不同的角色，為肺癌進程中由低氧微環境調控之細胞增生和存活提供了新的見解。

Lung cancer is the leading cause of cancer deaths in the world. Mutations of the epidermal growth factor receptor (EGFR) have served as therapy targets and biomarkers in lung adenocarcinoma cancer patients. EGFR-tyrosine kinase inhibitors (EGFR-TKIs), suppressing tumor growth via blocking survival signal, have achieved successful survival outcomes in patients harboring EGFR mutations. Nonetheless, resistance to EGFR-TKIs will inevitably occur. Hypoxia, a critical tumor microenvironment, limits oxygen supply in the center of solid
tumors as their sizes increase. However, how hypoxia interacts with EGFR-TKI selection to generate cancer cell heterogeneity, thus affecting cancer cell proliferation and epithelial-to-mesenchymal transdifferentiation (EMT), is unclear. In this study, we found that the oxygen sensor Prolyl hydroxylase 3 (PHD3) is highly downregulated in EGFR-TKI selected resistant cells, harboring EMT feature and lower proliferation ability. The effect of PHD3 loss in
EGFR-TKI tolerance and cancer cell survival is underway. In conclusion, we have identified new roles of PHD3 in lung cancer, which may provide novel insights into hypoxia-mediated cancer in proliferation and survival of neoplasia.

摘要
Abstract
目錄
Introduction..............1
Lung cancer...............1
Target therapy for lung adenocarcinoma................1
Epidermal growth factor receptor (EGFR)...............2
EGFR-TKI resistance.........................3
SOX2 in stem cell and cancer plasticity...............4
Hypoxia microenvironment....................5
Prolyl hydroxylase in hypoxia regulation .............5
Materials and methods ................................7
Chemicals andreagents.................................7
Cell culture..........................................7
Hypoxia microenvironment .............................8
Lentiviral transfection...............................8
Chromatin immunoprecipitation (ChIP) .................9
Plasmid Construction..................................9
Quantitative–PCR (qPCR)..............................10
Clonogenic Assay.....................................11
Flow cytometry analysis .............................12
Immunoblotting ......................................12
Statistical Analysis.................................13
Results .............................................14
PHD3 expression is regulated during ESC differentiation and cytokine stimulation in lung cancer cells.....................14
PHD3 expression is downregulated by TGF-b stimulation in lung cancer cells. .....15
PHD3 expression is regulated under the development of EGFR-TKI resistance in lung cancer cells.....................16
PHD3 expression is regulated by epigenetic modification..............17
PHD3 regulates cell cycle progression...............18
PHD3 antagonizes HIF-1α-mediated cell growth arrest..........19
PHD3 predicts a poor survival in lung adenocarcinoma.........20
Conclusion..........................................21
Discussions ........................................22
PHD3 in EGFR-mutated lung cancer and EGFR-TKI selection......22
PHD3 maintains cancer stemness and inhibits EMT..............23
PHD3 serves as a biomarker in lung adenocarcinoma............24
HIF-1α and glycolysis metabolism....................25
HIF-1α and anchorage-independent growth.............27
Figures ............................................29
References .........................................55
Abbreviation .......................................60

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