探討丙酮酸激酶M2之非代謝酵素功能在肺癌及胰臟癌進成中所扮演的角色

Non-metabolic Function of PKM2 in the Progression of Lung Cancer and Pancreatic Cancer

楊奕婕(Yi-Chieh Yang)

指導教授 ： 歐大諒

共同指導教授 ： 華國泰

國立臺灣大學/醫學院/腫瘤醫學研究所/博士(2017年)

https://doi.org/10.6342/NTU201700692

摘要

代謝途徑的變異是癌細胞的重要指標之一，目前已知癌細胞並不需仰賴週遭氧氣濃度即產生有氧糖解作用 (aerobic glycolysis) 生成能量和大量乳酸 (lactate)，又稱瓦伯格效應 (Warburg effect)。PKM2 (Pyruvate kinase M2；M2型丙酮酸激酶) 已知為糖解作用中重要的速率決定步驟之關鍵酵素，癌細胞可透過多種訊息傳遞途徑進而調控其酵素活性，已被證實在調控癌細胞之代謝變異中扮演重要的角色。然而，除了已知的醣代謝酵素活性調控之外，近年來許多文獻指出M2型丙酮酸激酶 (PKM2) 具有多種細胞核內非代謝功能 (nonmetabolic function)，可做為一蛋白激酶 (protein kinase) 接受細胞生長訊息以磷酸化下游蛋白，促使癌細胞分裂並且抵抗環境壓力。
表皮細胞生長因子受器 (EGFR) 突變常被發現於非小細胞肺癌中，並開啟致癌的訊息傳遞路徑。表皮細胞生長因子受器酪胺酸激脢的抑制劑 (EGFR-TKI)已用於病人治療多年，但長期使用後常會造成抗藥性；開發抵抗藥物抗藥性的新方式是迫切必須的。本研究的第一部分，發現了細胞質中M2型丙酮酸激酶 (PKM2) 新穎的非代謝功能。在細胞質中，M2型丙酮酸激酶 (PKM2) 可作為一佐監護蛋白 (cochaperone) 透過與熱休克蛋白90 (heat shock protein 90; HSP90) 共同作用，以達到穩定表皮細胞生長因子受器 (EGFR) 的表現量；研究中也發現，細胞中主要是透過低酵素活性的二聚體 (dimeric) PKM2來達到穩定EGFR的表現，若給予細胞活化劑使PKM2成為四聚體 (tetramer)，則會減短 EGFR蛋白的酵素半衰期，穩定效果大幅降低。在突變病人中也觀察到PKM2與EGFR表現量呈正相關，並且PKM2高表現會與造成較差病人的無疾病存活期 (disease free survival)。本研究發現了PKM2在細胞質中非代謝酵素的功能，同時也提供了對抗EGFR-TKI抗藥性的新策略。
另一方面，腫瘤的長大的過程中因為血管生成的缺乏，使得中心癌細胞處於缺乏葡萄糖 (glucose) 的環境，對於此環境造成的代謝壓力，癌細胞需要有其適應方式才能繼續存活。在本研究的第二部分，我們發現葡萄糖缺乏會促使細胞質中的PKM2 進入細胞核，增加癌幹細胞族群以對抗代謝壓力。當細胞缺乏葡萄糖時，會活化腺苷單磷酸活化蛋白激酶 (AMP-activated protein kinase; AMPK)，透過 Ran (Ras-related nuclear protein) 將PKM2同時帶入細胞核中。細胞核中大量累積的PKM2蛋白會進一步與八聚體結合轉錄因4 (octamer-binding transcription factor 4; OCT4) 共同活化下游與癌幹細胞相關的基因表現。除此之外，動物實驗結果指出核內PKM2可促進癌細胞的轉移能力。本部分研究指出細胞質中的AMPK會協同PKM2進入細胞核以增加癌幹細胞族群，並進一步幫助癌細胞對抗葡萄糖缺乏誘發之代謝壓力。
有別於過去已知PKM2在細胞質中糖解作用的代謝酵素角色，本研究在不同細胞模式提供PKM2細胞質與細胞核中新穎之非代謝功能。透過了解此多樣的機制，能夠提供針對PKM2設計的抗癌藥物更全方面的思考。

關鍵字

表皮細胞生長因子受器 ； 丙酮酸激?M2 ； 表皮細胞生長因子受器酪胺酸激?的抑制劑 ； 癌幹細胞 ； 腺?單磷酸活化蛋白激? ； 葡萄糖缺乏

並列摘要

Metabolic alteration is one of the hallmarks of cancer cells. In contrast to normal cells, cancer cells had been reported to highly rely on aerobic glycolysis even the oxygen concentration is sufficient. This phenomenon is also called “Warburg effect”. Pyruvate kinase isoenzyme M2 (PKM2) is one of the rate-limiting enzymes in the glycolysis process, which catalyzes PEP into ATP and pyruvate. In addition to the well-established metabolic function, PKM2 works as a protein kinase in cancer cells were revealed in the past years. Both metabolic and nonmetabolic functions of PKM2 provide the advantage for the growth of cancer cells.
The secondary mutation of EGFR resulted in drug resistance is one of the most critical issues in lung cancer therapy. In the first part of this study, we report that PKM2 stabilizes mutant EGFR protein and sustain cell survival signaling in lung cancer cells. The depletion of PKM2 leads to proteasome degradation of EGFR and significantly inhibited the tumor growth of EGFR mutant cells in xenograft model. Mechanistically, PKM2 directly interacts with mutant EGFR and stabilize EGFR by maintaining its binding with HSP90 and co-chaperones. Stabilization of EGFR is relied on dimeric PKM2, and the protein half-life of mutant EGFR was decreased when PKM2 was forced to form tetramer after treating activators. Clinical levels of PKM2 positively correlate with mutant EGFR expression and correlate with patient outcome.
Most of solid tumors suffer from glucose limited since the less amounts of vessels into tumor core. In the second part of the study, we found the nuclear translocation of the glycolic key enzyme, PKM2, in response to glucose deprivation in cancer cells. We demonstrate that glucose deprivation stimulates the AMPK activation and results in the nuclear translocation of PKM2 through Ran protein. Nuclear PKM2 then binds with OCT4 to promote the expression of cancer stemness-related genes. Thus, the nuclear PKM2 is able to enrich the cancer stem cell population. Furthermore, the nuclear PKM2 promotes cancer metastasis in an orthotopic xenograft model. Our findings provide first evidence that the cytosolic nutrient sensor AMPK works as a cooperator to help PKM2 carry out its nonmetabolic function in nucleus, and that is necessary for repopulation of cancer cells against and adapt to the metabolic stress.
These results reveal a previously undescribed non-glycolysis function of PKM2 in cytoplasm and nucleus, and that provide a novel insight for the scientists to develop the drugs which target on PKM2.

並列關鍵字

EGFR ； PKM2 ； EGFR TKIs ； glucose deprivation ； cancer stem cell ； AMPK

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