- 學位論文
探討突變型EGFR透過粒線體代謝轉變促進腫瘤生成
Study of Activating Mutant EGFR Driven Tumor Initiation through Mitochondria-Mediated Metabolism Shift
摘要
腫瘤生成(tumor initiation)為基因變異所致，癌細胞中的基因突變可大致分成驅動突變(driver mutations)及乘客突變(passenger mutations)，其中驅動突變具有生長優勢，可不受正常細胞增殖、分化等限制，持續擴增成癌細胞，帶有此特性的基因稱作癌症驅動基因(cancer driver genes)。在非小細胞肺癌中，約有2/3病人帶有驅動基因變異，在表皮生長因子受體(epidermal growth factor receptor, EGFR)變異中，又以exon 19 deletion或是exon 21 L858R mutation所占比例最高。粒線體與癌症有密切關聯，已有研究指出細胞在腫瘤生成時會發生代謝途徑重組(metabolic reprogramming)而與正常細胞有所差異，可做為治療標的。因此，我們想了解在突變型EGFR驅動的腫瘤生成，有哪些粒線體蛋白參與在細胞代謝的改變。 首先，在NIH3T3細胞株過表現EGFR L858R篩選出穩定表現的細胞，並確認這些細胞有較好的細胞增生及群落形成能力，已有研究指出EGFR突變之癌細胞會使其下游蛋白表現上調，但在我們的細胞模型及動物模型這些下游蛋白並沒有顯著改變，因此，我們推測腫瘤生成時細胞會先改變細胞代謝讓自己增生不經由典型的癌細胞EGFR訊息傳遞。為了進一步了解哪些蛋白與細胞代謝改變有關，我們萃取NIH3T3過表現EGFR L858R及對照組的粒線體蛋白進行蛋白質體(proteome)分析，根據結果挑選差異倍數(fold change)大於1.5及小於0.67之蛋白，分析其路徑多與細胞週期及代謝相關，我們也在氧化磷酸化(oxidative phosphorylation)及糖解(glycolysis)功能性實驗中，發現EGFR L858R組別在這兩種代謝途徑的使用與對照組相比皆有顯著差異。 同時，在過表現EGFR L858R之細胞及帶有轉殖基因(transgene)的動物模型中，皆有看到一部分EGFR L858R轉位(translocation)到粒線體的情形且正常細胞與癌細胞的蛋白分子量有所差異，因此，我們推測轉位到粒線體的EGFR L858R可能與其他蛋白作用，參與細胞代謝改變，未來將更進一步探討此交互關係與機制。
並列摘要
Tumor initiation has been reported due to genetic alterations. These mutations in cancer cell genome can be grouped into driver mutations and passenger mutations. Only driver mutations confer growth advantages to grow abnormally, invade and metastasize, and those genes with characteristics of driver mutations are known as cancer driver genes. It is reported that two-thirds of the NSCLC patients harbor driver genes mutation. The majority of EGFR mutation belongs to exon 19 deletion and exon 21 L858R mutation. Mitochondria play an important role in cancer. From tumor initiation to metastasis, metabolic reprogramming has been considered as one of hallmark, which could be another strategy for cancer treatment. Therefore, our aim is to explore the role of mitochondrial proteins modulated metabolism shift of EGFR L858R driven tumor initiation. First, we perform overexpression on NIH3T3, and select cells with EGFR L858R expression. These cells have better proliferation and colony expansion properties. Some studies have indicated EGFR downstream proteins are up-regulated in cancer cells. However, in our study, the expression level of EGFR downstream protein is not significantly different in both in vitro and in vivo models. Therefore, we suggest that cell will change their metabolism first to supply cell proliferation through a non-canonical EGFR signaling pathway. To understand which mitochondrial proteins involved in the metabolic change, we extracted mitochondrial proteins of NIH3T3-EGFR L858R and the control for proteomic analysis. Enriched networks of fold change greater than 1.5 and less than 0.67 proteins are most linked to cell cycle and metabolism. Also, in some oxidative phosphorylation and glycolysis functional assay, it is significantly different between NIH3T3-EGFR L858R and the control. Additionally, there is EGFR L858R translocated to mitochondria in EGFR L858R -overexpressed cells and EGFR L858R transgenic mice. The molecular weight of EGFR L858R in mitochondria is different between cancer cells and normal cells. Therefore, we suggest mitochondrial EGFR L858R may interact with other proteins for modulating metabolism at tumor initiation. In the future, we will investigate the mechanism and interaction of mitochondrial EGFR L858R and some potential proteins.