2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/646858摘要：肺癌是台灣以及世界各國最嚴重的癌症死亡原因。表皮生長素受體(EGFR)是ErbB受體家族的一員。EGFR受到活化，可促進抗凋亡、細胞增生、血管新生、侵襲及轉移。近年來EGFR抑制劑，小分子tyrosine kinase inhibitor (TKI)對某些肺癌病人有明顯的療效，但使用數個月後就產生抗藥性。有報導指出癌幹細胞(cancer stem cells, CSCs)與抗凋亡有關，但是此現象的機制並不清楚。我們在EGFR-TKI抗藥性細胞株篩檢，初步研究發現對EGFR- TKI抗藥性的細胞有較高IL-8之表現；轉殖IL-8除了促進腫瘤細胞表現較多CSCs之特性外，也使得EGFR-TKI敏感性的細胞產生抗藥性，而減少細胞凋亡；而這些轉殖IL-8之細胞內的抗凋亡蛋白(Bcl-2)較不易受到gefitinib抑制，這會促使癌細胞更容易存活。我們也發現，以IL-8中和抗體(neutralizing antibody)抑制IL-8活性後，可使EGFR-TKI 抗藥性之細胞中的癌幹細胞轉變成敏感性。此外，微小核醣核酸(microRNA)是體內的非蛋白質製造(non-protein coding)之小核醣核酸，是重要之基因調節因子。最近之證據顯示，微小核醣核酸之異常表現與腫瘤形成、病患預後及化療反應有關。微小核醣核酸可調控IL-8的表現，反之，IL-8也可活化下游的訊息傳遞來促進或減少某些微小核醣核酸的形成。我們先前利用微小核醣核酸微陣列技術，發現miR-146b (一種轉移抑制基因)在抗藥性細胞株內的表現量有大幅減少的趨勢，並且報導也指出當外加miR-146b後，可以降低IL-8的表現量，但是目前不知道確切的機轉。因此我們也很想知道miR-146b是否能夠直接結合到IL-8的3’-UTR來抑制IL-8的表現以及是否會影響癌幹細胞的族群大小。在此計畫，我們希望找出IL-8造成EGFR-TKI的抗藥性是否與IL-8促進癌幹細胞特性有關。透過調控IL-8的上游微小核醣核酸，研究此微小核醣核酸及其調控的IL-8在EGFR-TKI藥物敏感性的角色。並且了解此微小核醣核酸以及IL-8表現量與臨床使用EGFR TKI 效果之關連性。最終期望能對EGFR-TKI 抗藥性之病患的治療指出一條新的方向。<br> Abstract: Lung cancer is the leading cause of cancer deaths around the world. Activation of EGFR stimulates anti-apoptosis, cell proliferation, angiogenesis, invasion and metastasis. EGFR tyrosine kinase inhibitors (TKIs) have been developed for treatment of non-small cell lung cancer. Despite initial responses to these EGFR inhibitors, tumor ultimately developed resistance. Some studies reported that cancer stem cells propagated under EGFR-TKI treatment, but the mechanism is not known. We used microarray to screen the EGFR TKI resistance-related genes in the paired gefitinib sensitive cell lines (PC9 or HCC827) and resistant cell lines (PC9/gef or HCC827/gef) and found IL-8 was overexpressed in the gefitinib resistant cells. And, we demonstrated that cancer stem cells sub-population expanded after IL-8 transfection to PC9 cell (named as PC9/IL-8) and conferred resistance to gefitinib. The Bcl-2 expression in PC9/IL-8 was kept unchanged after gefitinib treatment, which may contribute to a part of EGFR TKI resistance in PC9/IL-8. Furthermore, treatment of IL-8 neutralizing antibody against IL-8 activity, ALDH+ sub-population from PC9/gef became sensitive to gefitinib treatment.The microRNAs are a new class of small non-protein-coding RNAs that can act as endogenous RNA interference. Recent studies indicate that aberrant expressions of microRNAs are relevant with tumorigenesis, prognosis and the response to chemotherapy. MicroRNAs would post-transcriptionally regulate IL-8 expression; in the other hand, IL-8 is able to up-regulate or repress the expression of microRNAs through activating down-stream mediators. MiR-146b, an invasion suppressor identified by a microRNA microarray screening, is down-regulated in gefitinib-resistant cell lines, and ectopic miR-146b decreases IL-8 level without known mechanism. We are interested in studying the role of miR-146b in EGFR TKI resistance, the effect of miR-146b on IL-8-3’-UTR, and the change of stem cell population after miR-146b over-expression.In the project, we would like to find out whether IL-8 results in EGFR-TKI resistance through activating cancer stem cells. And, we hope the reversal of EGFR-TKI resistance and inhibition of cancer cell stemness could be accomplished after repression of IL-8 activity. Finally, we also like to examine the axis of miR-146b-IL8-stemness, and investigate the clinical relevance of miR-146b or IL-8 in EGFR-TKI efficacy. This will bring a new era to the treatment of lung cancer.Our specific aims:1. Confirm that IL-8 regulates the EGFR-TKI resistance through increasing cancer stem cells population.2. Explore the regulation of IL-8 by miR-146b.3. Investigate the reversal of EGFR TKI resistance after over-expression of miR-146b and repression of IL-8 activity.4. Investigate the clinical relevance of IL-8 and miR-146b in lung cancer patients.肺癌IL-8幹細胞EGFR-TKI 抗藥性Lung CancerIL-8stem cellEGFR-TKI resistance