於某些實性腫瘤中， 位於其中間區域的部分會因受到缺氧及失巢凋亡的逆境而誘導一系列的基因以避免細胞週期的抑制並促進細胞的持續生長。我們發現在缺氧及失巢凋亡的誘導下，CITED2可以避免肺癌細胞進入G1/S停滯。如果抑制HIF1α 則可以促進細胞週期及幫助細胞避免失巢凋亡；相反的，抑制HIF2α則會導致細胞進入停滯期及抑制非附著依賴性細胞生長，進而指出HIF1α及HIF2α 在肺癌中扮演不同的角色。此外，我們發現低氧及失巢凋亡會誘導CITED2，當抑制CITED2時會導致G1/S細胞週期停滯及促進失巢凋亡。染色質免疫沉澱技術和報告基因檢測法顯示HIF2α會作用於CITED2及促進其活性。我們進一步發現CITED2透過MYC/p300 mediated transactivation機制活化E2F3，進而促進細胞週期。Pearson分析進一步顯示CITED2與 HIF2α 的表現在非小細胞肺癌 (NSCLC) 中為正相關。Kaplan-Meier 分析顯示，CITED2表達量高的NSCLC病人癒後情形較差。我們的研究支持HIF2α-CITED2 在缺氧及失巢凋亡所調控的癌惡化中扮演重要角色，且此機制在非小細胞肺癌中可作為預後生物標記及治療標的。

In solid tumours, hypoxic and anoikis stresses, two common phenomena in the central region, induce a series of genes to prevent cell cycle arrest and promote continuous growth in certain types of cancer cells. Herein, we report that HIF2α responds to hypoxia and anoikis induction to induce CITED2, thus preventing lung cancer cells from cell cycle arrest. We observed that knockdown of HIF1α promoted cell cycle progression and enhanced anoikis resistance while HIF2α-silencing caused severe cell cycle arrest and inhibited anchorage-independent growth, suggesting distinct roles of HIF1α and HIF2α in lung cancer cells. We found that CITED2 was upregulated by both hypoxic and anoikis inductions while knockdown of CITED2 caused G1/S cell cycle arrest and promoted anoikis. Chromatin immunoprecipitation and promoter reporter analysis showed that HIF2α bonds to CITED2 promoter and enhances its activity. We further observed that hypoxia-mediated upregulation of CITED2 encouraged MYC/p300 mediated transactivation of E2F3 promoter, thus enhancing cell cycle progression. Pearson correlation analysis displayed that CITED2 was associated with HIF2α expression in NSCLC. Kaplan-Meier analysis revealed that CITED2 expression correlated with poor survival in patients with NSCLC. Thus, our findings support the notion that HIF2α-CITED2 axis plays a critical role in hypoxia- and anoikis- mediated cancer malignancy and molecules in this axis can serve as prognostic biomarkers and therapeutic targets in lung cancer treatment.

摘要 4
ABSTRACT 5
INTRODUCTION 6
LUNG CANCER 6
HYPOXIA 6
ANOIKIS 7
CITED2 8
STUDY AIMS: 9
MATERIALS AND METHODS 10
CELL CULTURE 10
LENTIVIRAL INFECTION 10
QUANTITATIVE REAL-TIME PCR (Q-PCR) 11
IMMUNOBLOTTING 11
SOFT AGAR ASSAY 11
CLONOGENIC ASSAY 12
CELL PROLIFERATION ASSAY 12
CELL-CYCLE ANALYSIS 12
RNA IN SITU HYBRIDIZATION AND TISSUE ARRAY 13
THREE-DIMENSIONAL (3D) SPHEROID ASSAY 13
CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY 13
LUCIFERASE REPORTER ASSAY 14
STATISTICAL ANALYSIS 15
TABLE 1: Q-PCR PRIMERS 15
TABLE 2: CHIP PRIMERS 16
RESULTS 17
HIF2Α CORRELATES WITH ADVANCED TUMOUR GRADE IN NSCLC. 17
HIF2Α MEDIATES HYPOXIA-INDUCED CELL GROWTH. 17
HIF2Α IS ESSENTIAL FOR HYPOXIA-INDUCED ANOIKIS RESISTANCE IN LUNG CANCER. 18
DISTINCT ROLES OF HIF1Α AND HIF2Α IN ANOIKIS RESISTANCE IN LUNG CANCER. 19
CITED2 MEDIATES HIF2Α-INDUCED ANOIKIS RESISTANCE. 20
HIF2Α BINDS CITED2 PROMOTER TO ENHANCE ITS EXPRESSION. 21
HYPOXIA INDUCES CITED2 TO BIND E2F3 PROMOTER, ENCOURAGING MYC-MEDIATED TRANSACTIVATION OF E2F3. 21
DISCUSSION 23
FIGURES 26
REFERENCES 42

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