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研究生:劉欣怡
研究生(外文):Hsin-Yi Liu
論文名稱:探討核內M2型丙酮酸激酶誘導癌細胞復育以抵抗葡萄糖缺乏逆境
論文名稱(外文):Nuclear pyruvate kinase M2 induces tumor repopulation to strive against glucose depletion stress
指導教授:郭明良郭明良引用關係
指導教授(外文):Min-Liang Kuo
口試委員:張震東華國泰蕭宏昇
口試委員(外文):Geen-Dong ChangKuo-Tai HuaMichael Hsiao
口試日期:2015-07-22
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:77
中文關鍵詞:M2型丙酮酸激酶癌細胞復育癌症幹細胞葡萄糖缺乏腺苷單磷酸活化蛋白激酶
外文關鍵詞:pyruvate kinase M2 (PKM2)tumor repopulationcancer stem cellsAMP-activated protein kinase (AMPK)
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癌細胞的代謝主要倚賴大量的有氧糖解作用 (aerobic glycolysis) 來產生能量,又稱為瓦伯格效應 (Warburg effect)。然而,由於癌細胞的大量增生及周遭血管的缺乏,使得癌細胞時常處於葡萄糖缺乏的狀況,因此癌細胞需要對於這樣的環境壓力有所因應才能存活下來。在先前的文獻中指出,癌細胞在葡萄糖缺乏的狀況下會影響瓦伯格效應、糖質新生作用、細胞遷移能力、內質網壓力及癌症幹細胞特性。針對癌症幹細胞與葡萄糖缺乏的關聯性,先前研究指出葡萄糖缺乏會促進腦癌幹細胞特性,且腦癌幹細胞會藉由葡萄糖攝取的提升使其較容易存活。然而,有其他文獻提出較不同的概念,側群細胞 (side population cells) 被認為是具有癌症幹細胞特性的細胞族群,文獻指出葡萄糖缺乏會減少測群细胞的數目。因此,葡萄糖缺乏對於癌症幹細胞的影響仍待釐清,且其詳細的分子機制也並不清楚。在本研究中,我們發現葡萄糖缺乏會促進癌細胞球體形成 (sphere formation) 能力及癌症幹細胞標記基因的表現,如CD133、CD44、EPCAM、NANOG、NOTCH1、OCT4和SOX2等。PKM2 (Pyruvate kinase M2;M2型丙酮酸激酶) 為糖解作用中催化速率限制步驟的關鍵酵素,我們也進一步確認PKM2會參與此葡萄糖缺乏反應,我們將細胞抑制或過度表現PKM2皆會影響葡萄糖缺乏誘發的癌症幹細胞特性。近年來有許多文獻指出,PKM2除了在糖解作用扮演的角色外,也能進入細胞核中作為蛋白質激酶以調控基因表現。因此,我們分析了葡萄糖缺乏下PKM2在細胞中的分布狀況,利用免疫螢光染色及核質分離技術,我們證實葡萄糖缺乏會促進PKM2的入核。此外,AMPK (AMP-activated protein kinase;腺苷單磷酸活化蛋白激酶) 為感應細胞能量的關鍵調控者,我們發現細胞在葡萄糖缺乏下會活化AMPK進而影響PKM2的tyrosine (酪胺酸) 磷酸化並促進PKM2的入核。事實上,我們觀察到只有部分的細胞在葡萄糖缺乏下有PKM2入核的現象,因此我們推論葡萄糖缺乏僅促進少數細胞族群的PKM2入核,進而增加這群細胞的癌細胞特性,使其能適應葡萄糖缺乏逆境並導致癌細胞復育。我們將處理葡萄糖逆境的細胞分離出CD133+ 和CD133- 兩群細胞,實驗結果觀察到僅CD133+的細胞有PKM2入核的現象,CD133- 的細胞則無。總和以上,有別於PKM2在糖解作用的角色,我們證實核內PKM2對於葡萄糖缺乏誘發之癌症幹細胞特性的影響,並進一步促進癌細胞復育 (cancer repopulation) 以幫助癌細胞對抗此代謝壓力。

The reprogramming of cancer metabolism is recognized as the Warburg effect, which demonstrates that cancer cells rely on aerobic glycolysis for energy generation. However, cancer cells are generally glucose deprived due to rapid proliferation and poor vascularization, hence cancer cells are forced to cope with glucose depletion stress and survive. Previous studies revealed that cancer cells manipulate the Warburg effect, gluconeogenesis pathway, migration ability, ER stress and cancer stem cell phenotypes in response to glucose depletion. Focus on the correlation between cancer stem cells and glucose depletion, it has been reported that the brain tumor initiation cell (BTIC) phenotypes are enhanced during glucose depletion, and BTICs preferentially survive under glucose depletion through enhancing glucose uptake. Nevertheless, some reports have opposite suggestions that glucose starvation causes a rapid depletion of side population (SP) cells, which are stem-like cells within cancer cells. Hence, the role of glucose depletion in affecting cancer stem cells largely remains unclear. Herein, we observed that glucose depletion enhanced the sphere formation ability and up-regulated the expression of cancer stem cell markers like CD133, CD44, EPCAM, NANOG, NOTCH1, OCT4 and SOX2. We confirmed that PKM2 (pyruvate kinase M2), the glycolytic key enzyme, plays an important role in this response. By knockdown PKM2, cancer stemness gene expression and sphere formation ability which were enhanced by glucose depletion were abolished. Recent evidences reveal that PKM2 not only plays a role in glycolysis, but also acts as a protein kinase or transcriptional coactivator in the nucleus. Thus we analyzed the distribution of PKM2 during glucose depletion. By immunofluorescence staining and cell fractionation, we confirmed that glucose depletion induced PKM2 nuclear translocation. Besides, AMPK is a key regulator of energy homeostasis. We dissect that AMPK, which is activated by glucose depletion, interacted with PKM2 and regulated its Tyr105 phosphorylation, resulting in the nuclear translocation of PKM2. In fact, we observed that only a small fraction of cancer cells was nuclear PKM2 accumulated. Thus we proposed that glucose depletion induced PKM2 nuclear translocation and cancer stem cell properties in a small population of cancer cells, which could preferentially survive and lead to cancer repopulation. We certainly found that the sorted CD133 positive subpopulations within cancer cells were nuclear PKM2 enriched, whereas CD133 negative cells were not. Collectively, we demonstrated a new role of nuclear PKM2 on glucose depletion-induced cancer stem cell properties and cancer repopulation, which helps cancer cells to thrive against this metabolic stress.

口試委員審定書 ………………………………………………………….……..…. Ⅰ
中文摘要 …………………………………………………………….….………….. Ⅱ
Abstract …………………………………………………………………………….... Ⅳ

Chapter 1.Introduction …………………………………………………..…….....… 1
1.1 Cancer metabolism and microenvironment
1.2 In response to glucose depletion in cancer cells
1.3 Cancer stem cells (CSCs) and glucose depletion
1.4 Dual role of AMP-activated protein kinase (AMPK) on cancer progression
1.5 Pyruvate kinase isoform M2 (PKM2) and cancers
1.6 Non-metabolic functions of nuclear PKM2 in cancer cells
1.7 Motivation and purpose

Chapter 2.Materials and methods ……………………………………………… 10
2.1 Cell culture
2.2 Glucose depletion and drug treatment
2.3 Transfection and lentivirus infection
2.4 RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
2.5 Western blotting analysis
2.6 Sphere formation assay
2.7 Immunofluorescence staining and confocal microscopic analysis
2.8 Cell fractionation assay
2.9 Flow cytometry and cell sorting
2.10 Immunoprecipitation
2.11 Site-directed mutagenesis
2.12 Statistical analysis

Chapter 3.Results ………………………………………………………..… 19
3.1 Glucose depletion induces cancer cell repopulation and cancer stemness
3.2 Glucose depletion enhances cancer stemness through PKM2
3.3 PKM2 translocates into nucleus in response to glucose depletion
3.4 The nuclear translocation of PKM2 is accompanied with AMPK under glucose depletion and it depends on AMPK activity
3.5 Glucose depletion increases physiological interaction between PKM2 and AMPK
3.6 Glucose depletion promotes p-PKM2 Y105 nuclear accumulation through AMPK activity

Chapter 4.Discussion ………........................................................….... 29

Chapter 5.Figures and figure legends ………...........................…..….… 36
Figure 1.Cancer stemness increases during glucose depletion
Figure 2.PKM2 affects cancer stemness under glucose depletion
Figure 3.Nuclear PKM2 accumulates under glucose depletion
Figure 4.Treatment of AMPK activator and inhibitor affect nuclear translocation of PKM2 and AMPK
Figure 5.Physiological interaction between PKM2 and AMPK is induced by glucose depletion
Figure 6.AMPK regulates PKM2 phosphorylation at Tyr105 in response to glucose depletion
Figure 7.Working model
Figure 8.ERK inhibitor and FGFR1 inhibitor treatments do not influence PKM2 Tyr105 phosphorylation and nuclear translocation
Figure 9.SAICAR do not alter PKM2 nuclear translocation during glucose depletion

Chapter 6.Tables ………..........................................................…..... 69
Table 1.The primer sequences for RT-qPCR used in the current study

Chapter 7.References …….......................................................……. 72


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