越來越多證據表明了煙酰胺腺嘌呤核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）代謝對腫瘤細胞增殖和生存的重要性。最近一項研究證實了一個p53調節基因，TP53誘導的糖酵解和凋亡調節因數（TP53-inducible glycolysis and apoptosis regulator, TIGAR），在抑制凋亡和誘導NADPH產生方面的作用。抗凋亡蛋白B細胞淋巴瘤2（B-cell lymphoma 2, Bcl-2）之前被報導能夠通過還未確定的機制誘導NADPH的產生。在這篇論文研究中，我們假設Bcl-2與TIGAR耦合從而調節NADPH的產生和細胞生存，並且TIGAR可能成為一個促進細胞生長的新生存因數。這篇論文研究的目的是：1）檢測在NADPH代謝中Bcl-2與TIGAR間的生物聯繫；2）探究在哺乳動物系統中TIGAR調節的分子機制；3）研究TIGAR在體外和體內調節正常及腫瘤細胞生存的作用。 / 這篇論文的第一部分結果顯示在正常小鼠胚胎成纖維細胞（MEFs）和缺少功能性p53的人非小細胞肺癌（NSCLC）細胞中存在Bcl-2/TIGAR/NADPH這樣一個新的信號通路軸，重要抗凋亡蛋白Bcl-2與TIGAR耦合後，以一個特有並持久的方式調節NADPH代謝和細胞生長。用特異的小干擾RNA（siRNA）靶向抑制Bcl-2能夠在NSCLC細胞中抑制TIGAR/NADPH/生長信號軸。用小分子抑制劑ABT-737對Bcl-2進行藥理學抑制也能夠顯示相似的作用，而這個作用能夠通過過表達TIGAR被部分逆轉。而且，敲除TIGAR能夠降低Bcl-2誘導的NADPH產生和細胞生長，表明了TIGAR在介導TIGAR/NADPH/生長信號軸中的關鍵作用。 / 為了第二個目的，我們研究了Bcl-2對TIGAR的機制調節。我們發現過表達Bcl-2不能改變TIGAR的mRNA水平，但是能誘導轉錄後的TIGAR蛋白累積。有趣的是，TIGAR似乎能通過一個正反饋回路誘導Bcl-2的表達，這進一步促進了由Bcl-2誘導的TIGAR表達上調。除了Bcl-2，許多致癌基因或生長調節因數也被證實參與了TIGAR的調節，包括信號轉導和轉錄啟動因數3（signal transducer and activator of transcription 3, STAT3, Bcl-2的上有調節因數），表皮生長因數受體（epidermal growth factor receptor, EGFR）和肝細胞生長因數受體（hepatocyte growth factor receptor, c-Met）。 / 這篇論文的第三部分證實了TIGAR在缺少功能性p53細胞模型中的致癌作用。TIGAR過表達能夠誘導一些癌症的標誌性特徵，包括細胞代謝調節異常，細胞生長增加和凋亡減少。重要的是，TIGAR的過表達在MEFs和缺少功能性p53的NSCLC細胞中直接促成了腫瘤形成，促進了腫瘤的生長。但是功能性p53的存在無論在體外還是體內都能夠消除TIGAR這種生長促進和成瘤的作用。這些發現表明當p53功能消失時TIGAR能成為一個致癌基因，促進腫瘤形成。 / 總的來說，我們發現了在腫瘤中Bcl-2/TIGAR/NADPH這樣一條新的信號通路。Bcl-2通過增加TIGAR蛋白累積的轉錄後機制調節TIGAR。我們也證實了TIGAR受到多種致癌基因或生長調節因數的調節，包括STAT3，EGFR和c-Met。並且，我們闡明了當p53功能消失時TIGAR能成為一個致癌基因，促進細胞轉化和腫瘤形成。 / Emerging evidences reveal the importance of nicotinamide adenine dinucleotide phosphate (NADPH) metabolism for cancer cell proliferation and survival. A recent study demonstrated the role of a p53-regulated gene, TP53-inducible glycolysis and apoptosis regulator (TIGAR), in inhibiting apoptosis and inducing NADPH production. The anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) has previously been reported to induce NADPH generation through undefined mechanism. In this thesis study, we hypothesized that Bcl-2 is coupled to TIGAR for regulation of NADPH production and cell survival, and that TIGAR may serve as a novel survival factor contributing to cell growth. The objectives of the thesis study are: 1) to examine the biologic relationship between Bcl-2 and TIGAR in NADPH metabolism; 2) to investigate the molecular mechanisms of TIGAR regulation in mammalian systems; and 3) to examine the role of TIGAR in regulating normal and cancer cell survival in vitro and in vivo. / Findings in first part of the thesis revealed a novel signaling axis of Bcl-2/TIGAR/NADPH in normal mouse embryonic fibroblasts (MEFs) and human non-small cell lung cancer (NSCLC) cells lacking functional p53, coupling the major anti-apoptotic protein Bcl-2 to TIGAR regulation for NADPH metabolism and cell growth in a specific and sustained manner. Targeting of Bcl-2 by specific siRNA inhibited TIGAR/NADPH/growth axis in NSCLC cells. Pharmacologic inhibition of Bcl-2 by small molecule inhibitor ABT-737 also exhibited similar effects, which was partially reversed by the overexpression of TIGAR. In addition, TIGAR knockdown reduced Bcl-2-induced NADPH production and cell growth, implicating a critical role of TIGAR in mediating Bcl-2/NADPH/growth signaling axis. / For the second objective, we studied the mechanistic regulation of TIGAR by Bcl-2. We found that overexpression of Bcl-2 did not alter TIGAR mRNA expression but induced TIGAR protein accumulation post-transcriptionally. Interestingly, TIGAR seemed to induce Bcl-2 expression via a positive feedback loop, which may further contribute to the upregulation of TIGAR expression induced by Bcl-2. In addition to Bcl-2, a number of oncogene/growth regulators were demonstrated to be involved in TIGAR regulation, including signal transducer and activator of transcription 3 (STAT3, an upstream regulator of Bcl-2), epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met). / The third part of the thesis demonstrated the oncogenic role of TIGAR in cell models lacking functional p53. TIGAR overexpression induced several hallmark features of cancer including deregulated cell metabolism, increased cell growth and inhibited apoptosis. Strikingly, overexpression of TIGAR directly drove tumor formation and promoted tumor growth in MEFs and NSCLC cells lacking functional p53. The growth stimulatory and tumorigenic activities of TIGAR were abolished in the presence of functional p53 both in vitro and in vivo. These findings revealed TIGAR as an oncogene capable of driving tumorigenesis when p53 function is lost. / In summary, we have identified a novel signaling pathway of Bcl-2/TIGAR/NADPH in cancer. TIGAR is regulated by Bcl-2 via a post-transcriptional mechanism by enhancing TIGAR protein accumulation. We also demonstrated the regulation of TIGAR by multiple oncogene/growth regulators including STAT3, EGFR and c-Met. Furthermore, we have demonstrated TIGAR as an oncogene capable of driving cell transformation and tumorigenesis when p53 function is lost. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lam, Kai Yee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 158-176). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Declaration --- p.vi / Acknowledgements --- p.vii / Publications --- p.viii / Table of Content --- p.ix / List of Illustrations --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Overview of tumor metabolism --- p.1 / Chapter 1.1.1 --- Glucose metabolism --- p.4 / Chapter 1.1.2 --- Glutamine metabolism --- p.6 / Chapter 1.1.3 --- De novo fatty acid synthesis --- p.7 / Chapter 1.1.4 --- Advantages of metabolic reprogramming in tumor cells --- p.8 / Chapter 1.2 --- Mechanisms regulating metabolic reprogramming in cancers --- p.11 / Chapter 1.2.1 --- Tumor suppressor p53 --- p.11 / Chapter 1.2.2 --- Hypoxia-inducible factor 1 (HIF-1) --- p.13 / Chapter 1.2.3 --- PI3K/Akt/mTOR signaling pathway --- p.15 / Chapter 1.2.4 --- Oncogenic Myc --- p.16 / Chapter 1.2.5 --- Oncogenic Ras --- p.18 / Chapter 1.3 --- TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) --- p.22 / Chapter 1.3.1 --- TIGAR and oxidative stress --- p.25 / Chapter 1.3.2 --- TIGAR and carcinogenesis --- p.29 / Chapter 1.3.3 --- TIGAR and other diseases --- p.32 / Chapter 1.4 --- Hypotheses and Aims --- p.36 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- Chemicals and reagents --- p.38 / Chapter 2.1.2 --- Drugs --- p.41 / Chapter 2.1.3 --- Commercial detection kits --- p.41 / Chapter 2.1.4 --- Antibodies --- p.42 / Chapter 2.2 --- Cell culture --- p.43 / Chapter 2.3 --- Plasmids --- p.44 / Chapter 2.4 --- Transfection --- p.46 / Chapter 2.5 --- Retrovirus infection --- p.47 / Chapter 2.6 --- Drug treatment --- p.47 / Chapter 2.7 --- Cell viability assay --- p.48 / Chapter 2.8 --- Trypan blue exclusion assay --- p.49 / Chapter 2.9 --- NADPH assay --- p.49 / Chapter 2.10 --- Cell death detection ELISA --- p.50 / Chapter 2.11 --- Western blotting --- p.50 / Chapter 2.12 --- Reverse TranscriptionPolymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.13 --- Cell cycle analysis --- p.52 / Chapter 2.14 --- Transwell migration and matrigel invasion assays --- p.53 / Chapter 2.15 --- In vivo studies --- p.54 / Chapter 2.16 --- Histology and immunohistochemistry --- p.55 / Chapter 2.17 --- Statistical analysis --- p.56 / Chapter Chapter 3: --- Indentification of Novel Bcl-2/TIGAR/NADPH Signaling Axis / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Results --- p.60 / Chapter 3.2.1 --- Bcl-2-induced NADPH production and cell growth is associated with TIGAR upregulation in p53-null MEFs --- p.60 / Chapter 3.2.2 --- Identification of Bcl-2/TIGAR/NADPH signaling axis in p53-negative NSCLC cells --- p.63 / Chapter 3.2.3 --- Bcl-2 targeting reduces TIGAR expression and NADPH production in MEFs and NSCLC cells --- p.66 / Chapter 3.2.4 --- Pharmacologic intervention of Bcl-2 alters TIGAR levels in MEFs and NSCLC cells --- p.71 / Chapter 3.2.5 --- Bcl-2 targeting inhibits TIGAR expression, NADPH production and cell growth in Bcl-2-amplified small cell lung cancer (SCLC) cells --- p.76 / Chapter 3.2.6 --- Bcl-2-induced TIGAR expression is highly specific compared to other pro-survival proteins --- p.78 / Chapter 3.2.7 --- Bcl-2/TIGAR/NADPH signaling axis is functionally intact in p53-positive NSCLC cells --- p.80 / Chapter 3.3 --- Discussion --- p.83 / Chapter Chapter 4: --- Mechanisms of TIGAR Regulation / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.2 --- Results --- p.90 / Chapter 4.2.1 --- Bcl-2 regulates TIGAR by post-transcriptional mechanism --- p.90 / Chapter 4.2.2 --- Bcl-2-induced TIGAR expression is associated with upregulation of other pro-survival proteins --- p.94 / Chapter 4.2.3 --- STAT3 activation can serve as a biological signal for TIGAR induction --- p.97 / Chapter 4.2.4 --- Tyrosine kinase receptors contributes to TIGAR regulation --- p.100 / Chapter 4.2.5 --- The existence of TIGAR/Bcl-2 positive feedback loop --- p.102 / Chapter 4.3 --- Discussion --- p.104 / Chapter Chapter 5: --- TIGAR is Oncogenic / Chapter 5.1 --- Introduction --- p.107 / Chapter 5.2 --- Results --- p.109 / Chapter 5.2.1 --- Overexpression of TIGAR alters several hallmark features of cancer --- p.109 / Chapter 5.2.2 --- TIGAR drives tumor formation in vivo --- p.114 / Chapter 5.2.3 --- Tumorigenic activity of TIGAR is functionally counteracted by functional p53 --- p.123 / Chapter 5.3 --- Discussion --- p.134 / Chapter Chapter 6: --- Summary --- p.138 / Chapter Chapter 7: --- Future Prospective --- p.143 / Appendices --- p.148 / References --- p.158
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328243 |
| Date | January 2012 |
| Contributors | Lam, Kai Yee., Chinese University of Hong Kong Graduate School. Division of Medical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xviii, 176 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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