2017-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/686324摘要：BRAF抑制劑是目前治療具BRAF突變黑色素細胞瘤遠端轉移之基石，由於長期使用BRAF抑制 劑後會有存活訊號之再活化的現象，因此可預期腫瘤再發，臨床上，的確大部分的病人在一年之內即 產生腫瘤抗藥性。為了克服抗藥性，找尋抗藥與非抗藥腫瘤細胞有何不同，成為了一個很重要的課題。 我們發現在抗BRAF抑制劑的細胞中，粒線體變得更碎裂，而這是由於DRP1第616位置胺基酸之磷 酸化，可能導致粒線體自噬並幫助腫瘤渡過治療的壓力。另外，由活性氧化物所造成的氧化壓力，在 抗藥細胞中是遠超過非抗藥細胞，這可能與粒線體之過度碎裂化有關。因此，在我們的計畫中，我們 將確認不同的抗藥細胞皆有相似的粒線體動態平衡偏移與過度的粒線體氧化壓力，接著藉由減少内在 TYRP1及外部給予氧化壓力藥物elesclomol，來看氧化壓力對細胞抗藥性的影響。由於粒線體碎裂造 成的自噬已知道可以清除與回收由氧化壓力造成的不健康粒線體，我們將試著藉由量化PARKIN的粒 線體位移，來證明抗藥細胞的mitophagy是否有增加的情況。計畫的最終目的是想克服抗藥性，因此 會將目標鎖定在粒線體動態平衡與自噬，藉由小干擾RNA來減少DRP1及藥物FK506來逆轉粒線體 過度碎裂的情況。而針對粒線體自睦，將用chloroquine抑制整體的自睦作用，以及用mDIVI來減少 粒線體自身的自噬，預期有可能藉此減低腫瘤抗藥性。最後，我們會在小鼠腫瘤異種移植的模式上， 驗證矯正粒線體動態平衡是否可以逆轉黑色素細胞抗藥性。<br> Abstract: BRAF inhibitor is the cornerstone for the treatment of metastatic BRAF-mutant melanoma. The reactivation of pro-survival signals under long-term BRAF inhibitor exposure predicts the tumor relapse and most patients developed tumor resistance within one year. To overcome the drug resistance, it is mandatory to search for the targets that differentially express between resistant and non-resistant cancer cells. For the melanoma resistant to BRAF inhibitor, we have found that the mitochondria tend to be more fragmented. The fission of mitochondria is mediated by phosphorylation of DRP1 at serine 616 and preconditioned to mitophagy, which may help cancer survival against the treatment. Besides, the oxidative stress mediated by reactive oxygen species in drug resistant cells significantly exceeds that in the non-resistant cells. The excessive oxidative stress may be caused by mitochondrial hyperfragmentation. In this grant proposal, we will confirm that different resistant cells have the same features of skewed mitochondrial dynamics and exaggerated mitochondria-derived oxidative stress in BRAF inhibitor-resistant cells. The impact of increased oxidative stress on resistant cells by depleting TYRP1 endogenously or providing pro-oxidant elesclomol exogenously will be examined. Because mitochondria fission-affiliated mitophagy has been known to remove and recycle the unhealthy mitochondria induced by oxidative stress, we will quantify and confirm the increased mitophagy in resistant cells by characterizing PARKIN translocation to mitochondria. To conquer the resistance to BRAF inhibitor, we will target the mitochondrial dynamics and mitophagy. For mitochondrial dynamics, we will reverse the mitochondria hyperfragmentation through DRP1 knockdown by siRNA and drug modulation by FK506. In addition, we will inhibit overall autophagy by chloroquine and inhibit mitophagy by mDIVI. These interventions are expected to reduce the drug resistance. Finally, we will test whether interfering mitochondrial dynamics will reverse the drug resistance to BRAF inhibitor in a xenograft mouse model.BRAF抑制劑抗藥性粒線體動態平衡氧化壓力TYRP1DRP1PARKIN粒線體自噬BRAF inhibitordrug resistancePARKINmitophagy