摘要: 在全球癌症發病率與死亡率中,肺癌致死率始終高居第一。近來,利用個人化醫療雖然延長了病患的存活率,但是整體的死亡率卻還是無法有效降低。當非小細胞肺癌細胞的表皮生長因子受體活化後會誘發一連串的細胞功能改變,包括細胞增生、轉移侵襲、抑制細胞凋亡以及對傳統化療藥物產生抗藥性。因此過去10 年來發展出許多標靶藥物,像是gefitinib、erlotinib 以及afatinib。這些標靶藥物一開始都能夠有效毒殺帶有表皮生長因子受體活化型突變的癌細胞;但是,癌細胞最後都會產生次發性突變,像是常見的T790M 點突變、c-Met 擴增或是PI3KCA 突變,進而造成標靶藥物失效。然而,除了這些次發性突變造成抗藥性外,有些後天性的抗藥性機制,目前仍然是不知道原因。 細胞自噬作用(autophagy)本來是一種細胞自我保護的機制,可是在一些研究中卻發現這機制會造成化療藥物、放射線治療與標靶藥物無法有效殺死癌細胞。為了研究細胞自噬作用與表皮生長因子受體抑制劑 (EGFR TKI) 抗藥性之間的關連性,我們比較了gefitinib 抗藥性和gefitinib 敏感性細胞間自噬作用的差異性。我們首先發現具有gefitinib 抗藥性的細胞株(PC9/gef 或是HCC827/gef) 會有較強的細胞自噬作用。隨著投與自噬作用抑制劑在gefitinib 抗藥性細胞,gefitinib 產生的細胞毒殺能力會被大幅提高外;利用小片段核糖核酸干擾劑 (siLC3B)也著實提升了gefitinib 的細胞毒殺效用。 我們假設細胞自噬作用能夠賦予癌細胞對EGFR TKIs 產生抗藥性,因此想要探討細胞自噬作用造成EGFR TKIs 抗藥性的機制為何。我們利用cDNA 微陣列以及microRNA 微陣列分析比較EGFR TKI 敏感性與EGFR TKI 抗藥性細胞之間的基因以及microRNA 的差異性。也發現了STC2基因和miR-204 的表現量,與EGFR TKI 抗藥性或是細胞自噬作用之間存在著一定的關連性。以STC2 為例,我們在臨床檢體分析中發現具有STC2 高表現量的病患,無惡化存活期或是最終的存活期都是較低。利用細胞株進行的實驗也可以觀察到, gefitinib 誘發的caspase-3 活化、粒線體膜電位降低或是細胞內鈣離子的波動都會被STC2 抑制。整體來說,內質網具有調控細胞內鈣離子以及影響膜蛋白PERK 活化的能力。其中,控透過內質網活化的膜蛋白 (PERK)則具有啟動細胞自噬作用的能力。目前的研究中雖然可以同時觀察到PERK 活化與STC2 大量表現,但是沒有任何研究證實STC2 可以影響autophagy 或是直接活化PERK。而我們首先再gefitinib 抗藥性細胞中觀察到,抑制STC2 後可以達到抑制細胞自噬作用的目的,這讓我們亟欲知道STC2 對於細胞自噬作用的調控機制為何,這又和EGFR TKI 抗藥性有何關聯性存在。過去幾年,一些研究指出微小核糖核酸(microRNA)會影響細胞自噬作用。透過microRNA 微陣列分析結果,我們也挑選出miR-204,因為它具有調控細胞自噬作用關鍵蛋白-LC3B 的能力。 此計劃中,我們首要分析肺癌病人檢體分析來確認臨床上細胞自噬作用與EGFR TKI 抗藥性的關連性。再者,STC2 與miR-204 對於EGFR TKI 抗藥性與細胞自噬作用的影響。最終,透過調控STC2 或是mir-204 又是否可以促使EGFR TKI 抗藥性癌細胞回復敏感性。目標:1、探討STC2 如何調控細胞自噬作用與EGFR TKI 抗藥性是否有關連性存在。2、微小核糖核酸 miR-204 如何抑制細胞自噬作用,這作用又是否和EGFR TKI 抗藥性息息相關。3、檢測臨床檢體並分析STC2 與miR-204 表現量和細胞自噬作用強弱的關連性;此關連性又是否和EGFR TKI 抗藥性相關。
Abstract: Lung cancer is the leading cause of cancer death worldwide. Recently, personalized cancer therapy haschanged the survival of lung cancer patients, but not mortality. Mutation of epidermal growth factor receptor (EGFR) activates the signaling pathway mediating a variety of events including cellular proliferation, migration, invasion, blockade of apoptosis, and chemoresistance in non-small-cell lung cancer (NSCLC). A number of EGFR tyrosine kinase inhibitors (TKIs) have been developed in the last decade, such as gefitinib, erlotinib and afatinib. These EGFR TKIs are very effective against EGFR-activating mutations in the tyrosine kinase (TK) domain, but eventually cancer cells develop resistance to EGFR TKIs by secondary mutation(T790M), c-Met amplification, PIK3CA mutations etc. However, a few mechanisms about acquired resistance to EGFR TKI resistance are not well understood. Autophagy, as a cytoprotective response, has been reported to confer resistance to treatment such as chemotherapy, radiotherapy and target therapy. To investigate the correlation between autophagy and EGFR TKI resistance, we found that gefitinib-resistant cancer cell lines (PC9/gef or HCC827/gef) exhibit higher level of autophagy than parental gefitinib-sensitive cell lines (PC9 or HCC827) in our preliminary studies. Herein, we also evaluated the synergic effect of combining gefitinib and the pharmacological inhibitor of autophagy (3-methyladenine, 3-MA), and we showed that 3-MA enhanced gefitinib-induced cytotoxicity in gefitinib-resistant cells. Furthermore, knockdown of LC3B (an autophagy regulator) also reverted EGFR TKI resistance. These results indicated that autophagy is critical in the blockade of EGFR TKI-induced cytotoxicity. We suggest that activation of autophagy confers resistance against EGFR TKIs and we want tostudy the mechanism of autophagy-induced resistance to EGFR-TKIs. We did cDNA microarray and microRNA microarray screenings using paired EGFR TKI sensitive and resistant lung cancer cell lines. And we found stanniocalcin-2 (STC2) and miR-204 are putative links between EGFR TKI resistance and autophagy. Recently, we showed that STC2 was up-regulated in gefitinib-resistant cell lines and tumor specimens from TKI-acquired resistant patients. There is a concomitant increased STC2 mRNA expression and decreased progression-free or overall survival in patients. We found STC2 conferred resistance against gefitinib in TKI-sensitive cell lines by inhibiting gefitinib-induced caspase-3 activation, mitochondria membrane potential collapse, and calcium fluctuation. Recently, Endoplasmic reticulum (ER)-dependent activation of PERK pathway was demonstrated to induce activation of autophagy. However, the direct relationship of autophagy and STC2 is still understood. It is the first time, we showed that knockdown of STC2 resulted in suppression of autophagy in gefitinib-resistant cells. And we want to elucidate the role of STC2 in the autophagy-induced EGFR TKI resistance. In the past few years, mounting evidence indicates microRNAs contribute significantly to autophagy in cancers. In our screenings, we identified the putative EGFR TKI resistance-related microRNA- miR-204, which directlytargets LC3B. In the project, we'd like to confirm the clinical relevance of autophagy and EGFR TKI resistance in lung cancer patients. We'll determine the role of STC2 and miR-204 in EGFR TKI resistance and autophagy, and investigate the reversal of EGFR TKI resistance after repression of STC2 or overexpression of miR-204.Our specific aims:(1) Elucidate the mechanism involved in STC2-mediated autophagy and its effect on EGFR TKIresistance.(2) Elucidate the role of miR-204 in inhibition of autophagy and its effect on EGFR TKI resistance.(3) Confirm the role of STC2 and miR-204 in autophagy induced EGFR TKI resistance in clinicalspecimens