對酪氨酸激酶抑制劑具有抗藥性之非小細胞肺癌代謝靈活性及多倍體不穩定性之探討

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题名:	對酪氨酸激酶抑制劑具有抗藥性之非小細胞肺癌代謝靈活性及多倍體不穩定性之探討 Molecular Insights into Metabolic Flexibility and Ploidy Instability in the Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer Cells
作者:	黃中玉 HUANG, CHUNG-YU
贡献者:	醫學生物科技博士學位學程高淑慧
关键词:	非小細胞肺癌;酪胺酸激酶抑制劑抗藥性;表皮生長因子接受器;代謝靈活性;單羧基運輸蛋白1;氧化磷酸化;糖解作用;多核細胞;巨大細胞;癌細胞融合;融合基因 non-small cell lung cancer (NSCLC);TKIs-resistance;epidermal growth factor receptor (EGFR);metabolic flexibility;monocarboxylate transporter 1 (MCT-1);AZD3965、multinuclear cell、giant cell;cancer cell fusion;DYSF;TIMP3;ADAMTS15;fusogene
日期:	2021-01-12
上传时间:	2021-05-31 12:12:50 (UTC+8)
摘要:	自從表皮生長因子接受器 (epidermal growth factor receptor, EGFR) 被發現在非小細胞肺癌 (non-small cell lung cancer，NSCLC) 中有基因擴增 (amplification) 與突變 (mutation) 後，提供了新的標靶治療 (target therapy) 的方向。最常見的和NSCLC相關的EGFR突變為外顯子 (exon) 19和外顯子20。這些突變會造成EGFR過度活化 (hyperactivation)，並導致癌細胞增生 (proliferation)。這些具有EGFR突變的病人適合優先使用EGFR酪胺酸激酶抑制劑 (tyrosine kinase inhibitors，TKIs) 的標靶治療，例如艾瑞莎 (Iressa、gefitinib)。然而，在NSCLC的病人中常會發生治療所產生的抗藥性 (resistance)。先前的研究大多注重在探討TKIs抗藥性的機制，只有少部分的研究再探討具有TKIs感受性 (sensitive) 和具有TKIs抗藥性的NSCLC之間的代謝靈活性之差異。首先，我們發現具有TKIs抗藥性的NSCLC (Ire) 細胞藉由增加粒線體的生物能量 (bioenergetics) 以及粒線體的呼吸複合體 (mitochondrial respiratory complexes) 的活性，使代謝方式從原本的糖解作用 (glycolysis) 轉換為有氧磷酸化作用 (oxidative phosphorylation)。另外，Ire細胞顯著地表現單羧基運輸蛋白1 (monocarboxylate transporter 1，MCT-1)，MCT-1蛋白可協助細胞將乳酸運輸進細胞質。因此，我們推測可以藉由抑制MCT-1的表現來影響Ire細胞存活。結果顯示MCT-1的抑制劑AZD3965的治療會降低Ire細胞的細胞增生、存活率 (viability)、移行 (migration)、以及粒線體的生物能量。總結，這些結果顯示Ire細胞傾向於利用有氧磷酸化作用而非糖解作用，並且抑制MCT-1的表現可能有助於治療具有TKIs抗藥性的NSCLC。此外，我們也發現在Ire細胞中具有大量的巨大多核細胞 (giant multinuclear cell)。由於細胞週期的停止 (arrest)，先前研究大多認為巨大的多核細胞是一種正在死亡的細胞。但近年來卻發現此類多核細胞雖然處於休眠但依然長期存活並具有代謝活性。多核細胞可以藉由停止細胞分裂而非細胞凋亡 (apoptosis) 的方式來對抗抗癌藥物，之後在適當的環境下重新細胞分裂出具有抗藥性的子細胞。並且許多研究發現除了抗藥性外，多核細胞大多發生在有轉移或復發 (recurrence) 的病人。因此，部分學者認為多核細胞是一種新的癌症幹細胞 (stem cells) 。目前雖然已有許多研究專注於多核癌細胞和抗藥性之間的關係，但多核細胞的形成原因尚不明瞭。因此，我們嘗試尋找相關的融合基因 (fusogenes) 來做為治療的目標。首先我們發現在PE089細胞和Ire細胞中都有多核細胞的存在。我們也發現在PE089細胞給予 EGF、gefitinib、AZD3965和oligomycin後，多核細胞的比例有增加的趨勢。接著我們利用了核糖核酸測序 (RNAseq) 分析具有TKIs感受性的細胞和抗藥性細胞之間各種基因表量的差異，並從中挑出了幾個目標基因，包含ADAMTS15 (編碼disintegrin-like and metalloprotease with thrombospondin type 1 motif 15蛋白)、TIMP3 (編碼TIMP3蛋白) 以及DYSF (編碼Dysferlin蛋白)。根據這些結果，我們認為藥物的給予會增加具有TKIs感受性細胞(PE089)的多核細胞，但是對具有TKIs抗藥性的細胞並無顯著影響。因此，我們推測多核細胞可能與細胞產生抗藥性有關。 Since the discovery of epidermal growth factor receptor (EGFR) amplified and mutation in the non-small cell lung cancer (NSCLC), it provides a new concept for target therapy. Most of NSCLC-related EGFR mutations are found in exon 19 and exon 21. These mutations cause the hyperactivation of the EGFR, and it leads to promoting cancer proliferation. The patients acquiring EGFR mutations are superior to treat with target therapy of EGFR tyrosine kinase inhibitor (TKI), such as gefitinib. However, therapeutic resistant perpetually happens in NSCLC. Previous studies have concentrated on the mechanisms of gefitinib-resistance, but no further investigation have examined the flexible metabolism between gefitinib-sensitive and gefitinib-resistant NSCLC. First, we found that gefitinib-resistant NSCLC (Ire) cells have shifted the metabolism from glycolysis to oxidative phosphorylation (OXPHOS) by massively increase the mitochondrial bioenergetics and mitochondrial complex activities. Additionally, Ire cells represent a significant expression of monocarboxylate transporter 1 (MCT-1), which transports lactate into cytosol. Therefore, we speculated that inhibiting the expression of MCT-1 might afford a new strategy to against the TKI resistance. The treatment of MCT-1 inhibitor, AZD3965, attenuated cell proliferation, cell viability, cell migration, and mitochondrial bioenergetics in Ire cells. Collectively, these results demonstrated that Ire cells tended to undergo OXPHOS rather than glycolysis and inhibiting the expression of MCT-1 might be a potential therapeutic strategy for gefitinib-resistant NSCLC. On the other hand, we found that higher levels of giant multinuclear cells in Ire cells. Previous researches demonstrated that giant multinuclear polyploidy cells are dying cells due to the cell cycle arrest. However, recent studies showed that the giant multinuclear polyploidy cells might be the dormant cells with persistent viability and metabolic activation. Multinuclear cells represent cell cycle arrest rather than commit apoptosis to against the anticancer drug treatment. They start to cell division to generate anticancer drug-resistant daughter cells after in the presence of moderate anti-cancer treatment. Furthermore, multinuclear cells are correlated with resistance and are found in patients with metastasis and recurrence. Thus, some researchers considered multinuclear cells as a new type of cancer stem cells. Although many studies focused on the correlation between multinuclear cells and drug-resistance, the mechanism of multinuclear cells development remains unclear. Thus, we wanted to search the tentative fusogenes to be the possible therapeutic target. First, we found that multinuclear cells existed in both PE089 cells and Ire cells. We also have found that the numbers of multinuclear cells are increase in PE089 after EGF, gefitinib, AZD3965, and oligomycin treatment. Thus, we analyzed the alternative gene expressions between TKIs-sensitive cells and TKIs-resistant NSCLC by RNAseq. We further analyzed three target genes, including ADAMTS15 (encoded a disintegrin-like and metalloprotease with thrombospondin type 1 motif 15), TIMP3 (encoded tissue inhibitor of metalloproteinases 3), and DYSF (encoded dysferlin). Based on these results, we suggested that drug-treatment might increase the ratio of multinuclear cells in TKI-sensitive cells but not in TKI-resistant cells. Thus, we deduced that multinuclear cell might have correlation with acquiring drug-resistant.
描述:	博士指導教授:高淑慧委員:劉柯俊委員:林詠峯委員:張壯榮委員:蕭哲志
数据类型:	thesis
显示于类别:	[醫學生物科技博士學位學程] 博碩士論文

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