在台灣，肺癌是目前致死率最高的癌症，而轉移是造成高死亡率的原因，然而目前細胞自噬系統在癌症轉移中所扮演的角色尚不清楚。自噬系統是細胞清除損壞胞器以及蛋白質的代謝系統，而在我們的研究中發現一個與自噬系統相關的重要分子─LC3A於肺腺癌中是大量表現的，並且與癌症轉移呈負相關。為了瞭解LC3A在癌症中的角色，我們在LC3A高表現的肺癌細胞中抑制它的表達，發現抑制LC3A會使得癌細胞停留在G1/S階段，進而減緩生長速度，若細胞經自體吞噬抑制劑─奎寧處理，也會有相似的結果，顯示LC3A會影響細胞的生長。長期處理奎寧產生的抗藥株會增強轉移的能力、降低生長速率以及LC3A的表現，同樣地，長期抑制LC3A會導致轉移能力的上升，顯示受LC3A調控的自噬機制會負調節轉移能力。另外我們發現在肺腺癌中LC3A和SOX2的表現呈正相關，表現外源的SOX2會促進LC3A的表達，若抑制LC3A會減少SOX2的表現量，顯示SOX2與LC3A有交互正回饋的關係。相對於低LC3A生長緩慢的細胞，高LC3A快速生長的細胞有較高的耗氧率以及活性氧物種。長期抑制LC3A會降低耗氧率，顯示自噬系統會去調控粒線體的活性。TOM20螢光染色發現生長快速的細胞會有更多小片段形狀的粒線體，顯示大量粒線體在進行分裂程序。綜合上述的結果，LC3A調控的自噬系統會調節粒線體的活性以及癌細胞的塑性，因此LC3A具有作為肺腺癌生物標記以及治療標靶的潛力。

Lung cancer is the leading cause of neoplasia-related morbidity and now ranks as the first cause of cancer-associated death in Taiwan. Metastasis has been attributed as the main cause of high mortality rate in lung cancer during cancer progression; however, the role of autophagy, a self-degradative metabolic process, in cancer metastasis is elusive. In this study, we found that LC3A, a key molecule involved in autophagy, was highly expressed in lung adenocarcinoma but reversely correlated with metastatic potential. To understand the role of LC3A in oncogenesis of lung cancer, LC3A was knocked down in LC3A-positive lung cancer cells, followed by various functional assays. We observed that LC3A-silencing caused G1/S cell cycle arrest, leading to the decreased cell proliferation. Pharmacological inhibition of autophagy with chloroquine attenuated cell growth in LC3A-positive lung cancer cells, indicating the involvement of LC3A in cell proliferation. Intriguingly, long-term treatment of LC3A-positive lung cancer cells with chloroquine endowed cells with the chloroquine resistance and increased migration and invasion abilities while cell proliferation, barrier properties and LC3A expression were decreased in these resistant cells. Consistently, long-term silencing of LC3A enhanced migration and invasion in lung cancer cells, indicating a negative interplay between LC3A-mediated autophagy and cancer cell invasion. Correlation analysis showed that LC3A expression was associated with SOX2 levels in primary lung adenocarcinoma. Ectopic expression of SOX2 enhanced LC3A levels whereas LC3A knockdown attenuated SOX2 expression, suggesting the presence of a positive feedback loop between LC3A and SOX2. We found that LC3A-high/fast-proliferating cells contained higher oxygen consumption rate (OCR) and released more reactive oxygen species (ROS) compared to their LC3A-low/slow-proliferating counterparts. Long-term silencing of LC3A inhibited OCR, demonstrating the participation of LC3A-mediated autophagy in regulation of mitochondrial activity. Immunofluorescence imaging with antibodies against TOM20 revealed that the fragmented phenotype of mitochondria is dominant in LC3A-high/fast-proliferating lung cancer cells, indicating a high mitochondrial fission process in these cells. These findings support the notion that LC3A-mediated autophagy regulates mitochondrial activity and determines cancer plasticity with the potential to serve as a predictive biomarker for lung cancer progression.

摘要 3
Abstract 4
致謝 6
Introduction 7
Lung cancer 7
Autophagy 7
SOX2 signaling 8
Mitochondria 9
Study aims 10
Materials and Methods 12
Cell Culture 12
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) 12
Quantitative Real-time Polymerase Chain Reaction (Q-PCR) 12
Cell Proliferation Assay 13
Clonogenic Analysis 14
Doubling time 14
Lentiviral Infection 14
Immunoblotting 15
Immunofluorescence Staining 15
TCGA and GEO database statistics 16
Electric Cell-substrate Impedance Sensor (ECIS) Analysis 17
Live Cell Migration Assay 17
Matrigel Invasion Assay 17
Flow Cytometry Analysis 18
Measurement of mitochondrial oxygen consumption rate 18
Results 20
LC3A is highly expressed in lung adenocarcinoma, correlating with metastatic-free survival. 20
LC3A regulates cell growth. 20
LC3A levels affect cancer plasticity 21
SOX2 regulates LC3A expression. 23
Autophagy regulates oxygen consumption and ROS release. 23
Discussion 25
Figure 28
Referances 44

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