肺癌是全球癌症致死率最高的疾病，而腫瘤的轉移則是造成高死亡率的主要因素。細胞自噬系統可以清除不良損壞的胞器以對抗代謝壓力，促進細胞的存活，然而至今仍不清楚細胞自噬在癌症進程中所扮演的角色。在此項研究中我們發現一個調控細胞自噬的分子LC3A高量的表現在一部分的肺腺癌當中，並且和腫瘤的轉移程度呈現負相關。從各種實驗分析發現LC3A調控的細胞自噬，比起生長慢/高轉移的肺癌細胞，在生長快/低轉移的細胞中有較高的表現，然而如果抑制LC3A表現則會阻斷G1/S細胞週期的進行並使細胞生長變慢。比起生長慢的細胞，快速生長的細胞具有較高的耗氧率並且產生大量的ROS，如用奎寧抑制細胞自噬則會更進一步增加ROS的量。SOX2的表達會促進LC3A的表現並且加速細胞生長和降低轉移的能力。長期用奎寧抑制細胞自噬所篩選出的低量LC3A細胞會降低SOX2的表現，減緩細胞生長，以及增進轉移的能力。同樣的，在SOX2高表現且生長快速的細胞中減少LC3A的量會使細胞生長變慢並且促近轉移。綜合上述的研究結果，在癌症細胞中LC3A所調控的細胞自噬對SOX2調控的細胞生長非常重要，而且在肺線癌中LC3A有潛力可以當作一個預測病情的指標和治療的標的。

Lung cancer is the leading cause of cancer-related deaths in the world. Tumor metastasis is considered as the main cause that contributes to high mortality during lung cancer progression. Autophagy, a natural, self-devouring process, is crucial for mitigating metabolic stress and promotes cell survival. To date, the role of autophagy in cancer progression remains elusive. Here, we report that LC3A, an autophagy mediator, is highly expressed in a subgroup of lung adenocarcinoma, which is inversely associated with distant metastasis. Immunofluorescence staining and immunoblotting assays revealed that LC3A-mediated autophagy is more highly activated in the high-proliferative/low-invasive lung cancer cell than its low-proliferative/high-invasive counterpart. Clonogenic analysis and cell cycle assays showed that LC3A silencing attenuated cellular growth, causing G1/S cell cycle arrest in high-proliferative lung cancer cells. High-proliferative lung cancer cell exhibited a higher oxygen consumption rate (OCR) and showed a higher reactive oxygen species (ROS) levels compared to its low-proliferative counterpart; pharmacological inhibition of autophagy with chloroquine promoted the ROS production in high-proliferative lung cancer cells. SOX2 expression enhanced LC3A expression and promoted proliferation while inhibiting invasiveness in lung cancer cells. Pharmacological selection of LC3A-low cells with chloroquine not only attenuated SOX2 expression with decreased proliferation but also altered oncogenic properties with increased invasiveness in lung cancer cells. In agreement, knockdown of LC3A expression in SOX2-high proliferative cells enriched SOX2-low cells exhibiting decreased proliferation but increased invasiveness. Combined, our findings demonstrated that LC3A-mediated autophagy is essential for SOX2-induced oncogenic proliferation in lung cancer cells, with a potential to serve as a prognosis marker and therapeutic target in lung adenocarcinoma.

摘要 4
Abstract 5
致謝 7
Introduction 9
Lung cancer 9
Autophagy 9
SOX2 10
Mitochondria 11
Study aims 12
Materials and Methods 14
Cell lines 14
Quantitative Real-time Polymerase Chain Reaction (Q-PCR) 14
Clonogenic analysis 15
Cell tracking time-lapse microscopy 15
Matrigel invasion assay 16
Plasmid construction 16
Immunoblotting 17
Immunofluorescence staining 17
Lentiviral Infection 18
Flow cytometry analysis 18
Oxygen consumption rate (OCR) analysis 18
Public domain data analysis 19
Results 20
LC3A is expressed in lung adenocarcinoma and inversely associated with distant metastasis 20
LC3A is highly expressed in high proliferative lung cancer cells 20
LC3A silencing attenuates proliferation in lung cancer cells 21
Involvement of autophagy in ROS production 22
LC3A regulates mitochondrial dynamics 22
SOX2 mediates LC3A expression in lung cancer cells 23
LC3A expression determines distinct oncogenic properties in lung cancer cells 24
Discussion 26
Figures 30
References 45

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