利用整合基因體學來找尋能夠預測癌症轉移風險的生物標誌將有助於針對惡性腫瘤(例如肺癌)發展更好的治療策略。在本篇研究中，我們證明了腺苷酸激酶-4 (AK4)是一個與肺癌入侵與轉移極為相關的個基因。藉由分析肺癌病人的基因微陣列資料庫，我們發現相對於正常肺組織，AK4基因在肺腺癌的組織有明顯過渡表現的趨勢。我們進一步利用免疫組織染色發現高表現的AK4和肺癌病人的分期、復發、和不良的預後有顯著的正相關。在細胞與動物實驗中，抑制AK4的表現能降低肺癌細胞入侵轉移的能力；相反地，將AK4過度表現在低入侵轉移的肺癌細胞中能提昇其入侵與轉移的能力。我們接著利用基因微陣列分析發現ATF3 轉錄因子是AK4下游造成入侵轉移的重要調控因子。同時降低AK4與ATF3的表現能夠減低ATF3所造成的入侵抑制作用；另一方面，在AK4過度表現的細胞中強迫表達ATF3能夠降低由AK4所引起的細胞入侵。免疫組織染色進一步分析發現AK4 高ATF3低的病人比AK4 低ATF3 高病人有更為不良的預後。綜合上述，我們的研究結果發現了AK4所引起的肺癌惡化進程和復發是透過抑制ATF3轉錄因子而造成的入侵和轉移。

Using integrated “omics” approach to identify biomarkers that predict metastatic capacity might assist the development of better therapeutic strategies for aggressive cancers such as lung cancer. In this study, we show that adenylate kinase 4 (AK4) is a progression-associated gene in human lung cancer that promotes metastasis. Analysis of published microarray data showed that AK4 was upregulated in lung adenocarcinoma compared with normal cells. High AK4 expression was associated with advanced stage, disease recurrence and poor prognosis. Loss of AK4 expression suppressed the invasive potential of lung cancer cell lines, whereas AK4 overexpression promoted invasion in vitro and in vivo. Mechanistically, the transcription factor ATF3 was identified as a pivotal regulatory target of AK4. Simultaneous reduction in AK4 and ATF3 expression abolished the inhibitory effects of ATF3 on invasion. ATF3 overexpression in AK4-overexpressing cells limits invasion activity. Furthermore, patients with high AK4 and low ATF3 expression showed unfavorable outcomes compared with patients with low AK4 and high ATF3 expression. Taken together, our findings indicated that AK4 promotes malignant progression and recurrence by promoting metastasis in an ATF3-dependent manner.

Table of Content

Acknowledgement I
Table of Content II
摘要 IV
Abstract V
List of Figures VI
List of Tables VII
List of Tables VII
Chapter 1: Introduction 1
1.1 Lung cancer incidence and subtypes 1
1.2 Lung cancer genetic mutations and therapy 1
1.3 Integrated “omics” approach 3
1.4 Adenylate kinase 4 4
Chapetr 2: Materials and Methods 6
2.1 Specimens 6
2.2 Cell lines 6
2.3 Western blotting analysis 7
2.4 Invasion assay 7
2.5 Microarray analysis 8
2.6 Tissue array construction 9
2.7 Immunohistochemistry 10
2.8 Lentiviral shRNA and expression vectors 11
2.9 Animal studies 12
2.10 Statistical analysis 12
Chapter 3: Results 13
3.1 Identification of differentially expressed genes by microarray and MS-based proteomic profiling in CL1-5 invasive lung cancer cells 13
3.2 mRNA expression profiles of AK4 in lung cancer microarray datasets 14
3.3 Overexpression of AK4 in lung cancer correlated with advanced stage and poor prognosis 15
3.4 AK4 expression enhances the invasive ability of lung cancer cells 16
3.5 AK4 expression promotes metastasis in vivo 17
3.6 Gene expression profile analysis revealed ATF3 was a downstream target affected by AK4 18
3.7 ATF3 was a pivotal regulator of AK4-induced invasion 20
3.8 mRNA profiles of ATF3 in lung cancer patients 21
3.9 The combination of AK4 and ATF3 expression is a powerful prognostic predictor for lung cancer patients 22
3.10 Validation of AK4 and ATF3 expression in associated with prognosis using independent expression datasets. 23
3.11 Associations of AK4 and ATF3 expression with EGFR, KRAS and ALK status of NSCLC patients 24
3.12 AK4-recurrence signature identifies T-box transcription factor TBX2 is the most significantly activated upstream regulator in lung adenocarcinomas 25
Chapter 4: Discussion 27
References 35