2015-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/686540摘要：BRAF抑制劑是轉移型黑色素細胞瘤治療上的一個基石，vemurafenib是一BRAF抑制劑，在台灣已於2013年核准上市，針對具有V600E突變的晚期腫瘤可以使用。臨床有效療可以達80%，然而大多數人無可避免地在治療一年內產生抗藥性。為了研究其抗藥機轉，我們已經執行了飛蛾跳躍子突變形成篩選的研究，找到MITF與TYRP1是兩個可能調控BRAF抑制劑之敏感性的因子。MITF是一個轉錄因子，可以調控TYRP1等基因。MITF過去已經知道與BRAF抑制劑的抗藥性有關，但其機制仍不清楚，有一個可能是，MITF相關的抗藥性有部分是經由TYRP1所造成，另外，TYRP1可能是一個比MITF更好的標靶去克服抗藥性的問題，因為MITF是轉錄因子，有許多其他的功能，將MITF抑制會影響到正常細胞的功能。我們的研究顯示，在具有BRAF-V600E突變的SK-MEL-28黑色素細胞株中將TYRP1移除，會使得黑色素細胞瘤對BRAF抑制劑的感受性增加(即指更有效)，我們也發現，將TYRP1移除並不會改變主要的MAPK訊息路徑，或者是替代的AKT-mTOR路徑。由於TYRP1是一種參與真黑色素生成的DHICA oxidase酵素，減少TYRP1表現除了會減少黑色素的產生之外，理論上也會造成活性氧物種的增加。因此，我們假設TYRP1的移除後，透過增加氧化壓力的增加，而使細胞較易被BRAF抑制劑所殺死。為了驗證我們的假說，我們將會測量實驗中氧化壓力的變化，並且執行一個「援救」的實驗，加不同的活性氧物種清道夫。另外，我們也會做臨床腫瘤檢體染色，與腫瘤的小鼠移植實驗，來證實經體外實驗發現的結果。<br> Abstract: BRAF inhibitor is the cornerstone for the treatment of metastatic melanoma. InTaiwan, the BRAF inhibitor, vemurafenib, has been approved in 2013 for late-stagemelanoma carrying V600E mutation and demonstrated a substantial benefit. The clinicalresponse rate of vemurafenib is about 80% but resistance inevitably occurs within one year inmost patients. To explore the mechanism of resistance to BRAF inhibitor, we have done thepiggyBac transposon mutagenesis screening. MITF (microphthalmia-associated transcriptionfactor) and TYRP1 (tyrosinase-related protein 1) are identified as potential regulators ofsensitivity to BRAF inhibitor. MITF is a transcription factor that can regulate TYRP1 and theother genes. MITF is known to associate with resistance to BRAF inhibition but themechanism is unknown. There is a possibility that MITF modulate the sensitivity to BRAFinhibition through regulating TYRP1. In addition, TYRP1 may be a better target to overcomedrug resistance than MITF since MITF exerted a more diverse function in normal cells. Ourstudy showed that depletion of TYRP1 in BRAF-V600E mutant SK-MEL-28 melanomasensitized the cells to BRAF inhibition. We also found that TYRP1 depletion did notsignificantly alter the canonical MAPK pathway or the alternative AKT-mTOR pathway.Given that TYRP1 is a DHICA oxidase enzyme in the eumelanin synthesis pathway, depletionof TYRP1 in melanoma will decrease the melanin production as well as increase of reactiveoxygen species (ROS) theoretically. Thus, we hypothesize that depletion in TYRP1 canincrease the oxidative stress which make the melanoma cells vulnerable to BRAF inhibition.To testify our hypothesis, we will measure the oxidative stress and perform a rescue studywith different ROS scavengers. Clinical melanoma samples and the animal xenograft studywill also be used to confirm the in vitro findings.