摘要:非小細胞肺癌為造成癌症死亡的首要原因,而傳統的化學治療對患者的療效有限。近來,標靶治療的進展已使部分癌症患者獲得有效的治療。尤其是具有EGFR 基因突變的肺癌,使用EGFR tyrosine kinase inhibitors (TKIs)藥物常能達到顯著的治療反應。然而,病患的腫瘤最終將隨著EGFR TKI 的使用而產生抗藥性,使得藥物反應只能維持約10 到12 個月。這種續發抗藥性產生的影響使得學界迫切希望瞭解造成抗藥性的分子機轉,以進而尋求解決之道。目前已知造成 EGFR TKI 續發抗藥性的機轉包括EGFR 基因產生T790M 突變與MET 基因的複製。上皮-間質轉化(epithelial-to-mesenchymal transition,EMT)為一胚胎發育相關的過程,已被發現與癌症進展與藥物抗藥性有重要的影響。許多研究也顯示EMT 與EGFR TKI 的抗藥性有關。最近,我們研究團隊證實EMT 調節因子中的Slug,在續發EGFR TKI 抗藥性的肺癌PC9/IR 細胞中出現過度活化的現象,並且證實此活化引發EGFR TKI 的抗藥性。為了研究造成EGFR TKI 抗藥性的其他機轉,我們將另一具EGFR TKI 感受性的肺癌細胞株HCC827 持續暴露於gefitinib 中,因而分離出續發性抗藥性的細胞株HCC827/IR。雖然此抗藥性細胞株明顯展現了EMT 的現象,但包括Slug在內的許多EMT 調節因子並未出現活化情形。這些結果顯示HCC827/IR 細胞的EMT與EGFR TKI 抗藥性應是由其他分子機轉造成。微小核醣核酸(microRNA)為一群不進行蛋白質轉錄的RNA,可以調節數以百計的標的基因,並調控許多生物功能。我們利用HCC827/IR 的細胞模型,以microRNA微陣列篩選與續發性抗藥性相關的microRNA。其中,miR-503 為可能的標的。miR-503在HCC827/IR 細胞的表現,相較於具EGFR TKI 感受性的HCC827 親代細胞下降約三倍左右。miR-503 為miR-15/107 微小核醣核酸基因群的一員,而這一群的微小核醣核酸近來被發現在癌細胞的許多生物功能扮演了重要的調控角色。我們發現將HCC827/IR細胞過度活化miR-503 可以減弱EMT,以及與EMT 相關的細胞行為(如細胞活動與侵犯),顯現了miR-503 在肺癌細胞中尚不為人知的功能。透過細胞與動物的研究,我們將探討miR-503 在調控肺癌EMT、細胞凋零、與EGFR TKI 抗藥性的角色,並尋找miR-503 調控的標的基因。透過這些研究,我們期待能尋找新的治療模式,以克服肺癌細胞對EGFR TKI 治療產生的抗藥性。
Abstract: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death, andconventional chemotherapeutic drugs are only modestly effective in advanced disease. Recentadvances with targeted therapies have provided a marked benefit to subsets of patients whosetumors harbor specific genetic abnormalities. In particular, cancers with EGFR mutationsachieve marked and durable response to treatment with the EGFR tyrosine kinase inhibitors(TKIs). However, despite this initial response, patients with NSCLCs containing EGFRmutations acquire resistance to EGFR inhibitors, with the median time to disease progressionabout 10-12 months. The development of drug resistance that invariably occurs aftertreatment with EGFR TKIs has spurred efforts to understand the biology underlying resistanceand to identify therapeutic strategies to overcome it.Currently known genetic changes underlying acquire resistance to EGFR TKIs include asecondary mutation of T790M in EGFR and MET amplification. Epithelial-to-mesenchymaltransition (EMT), an embryonic program characterized by the loss of proteins involved in celljunction and the expression of mesenchymal markers, is associated with progression andresistance to chemotherapeutic drugs in cancer. Studies also support the association of EMTand EGFR TKI resistance in EGFR-mutant NSCLC. Recently, our group demonstrated thatSNAI2 (Slug), but not other EMT activators, was over-expressed in acquiredgefitinib-resistant cell line PC9/IR, and that SNAI2 (Slug) caused resistance to gefitinib inthis cell model. To study other mechanisms of EGFR TKI resistance, we have developedanother cell-based system by culturing gefitinib-sensitive HCC827 cells in the presence ofincreasing concentration of gefitinib and were able to isolate resistant cell line HCC827/IR.Interestingly, despite that this gefitinib-resistant cell line have the transition to mesenchymalphenotype, the EMT activators SNAI2 (Slug) and others were not up-regulated comparedwith the parental gefitinib-sensitive cells, indicating that other mechanisms would be involvedin the acquisition of EMT and resistance to EGFR TKIs.MicroRNAs, a class of non-protein coding RNAs, can post-transcriptionally regulatehundreds of their target gene and control a wide range of biological functions. We havescreened the microRNAs which are up-regulated or down-regulated with the development ofEGFR TKI resistance in the HCC827/IR cell model by microRNA low density array. Of thescreened microRNAs, miR-503 represents one of the candidate, which is 3-folddown-regulated in HCC827/IR cells compared with the parental cells. MiR-503 is a memberof the miR-15/107 group of microRNA genes, and this group is increasing appreciated to playkey functions in human cancer. We found that over-expressing of miR-503 in HCC827/IRcells attenuated EMT and EMT-related events such as cell migration and invasion, disclosingthe unrevealed role of microRNA in lung cancer. Through studies of cell lines and animalmodels, we will investigate the role of miR-503 in the regulation of EMT, anti-apoptosis, andEGFR TKI resistance in lung cancer. And we will identify the target genes that are regulatedby miR-503 to exhibit the biological functions. Through the study of epigenetic generegulation, we hope to find the ways to overcome resistance to EGFR TKIs and to suggest anew treatment strategy in advanced NSCLC.