2014-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/686367摘要：惡性黑色素細胞瘤是台灣最致命的皮膚癌症。在西方國家，百分之62.6的病人是第零或第一期，百分之23.1是第二期，相反地，台灣的病人當中，只有百分之40是第一或二期。手術是最重要的治療，然而轉移及復發很常見，對黑色素細胞轉移的病人而言，Dacarbazine是最有效的單一化學治療藥物，但是其藥物反應率也只有百分之15至25，因此化學治療僅被認為是一種緩解性的療法。 目前對轉移的黑色素細胞瘤的治療趨勢，已經往標靶治療移動。一個黑色素細胞瘤生物學很重要的里程碑是，已知約有一半的腫瘤具有活性型 BRAF 突變.雖然有BRAF 突變的腫瘤通常對 BRAF 抑制劑有反應，但是有一部份的腫瘤具有先天性或後天性的抗藥機轉。現在已有臨床試驗同時使用 BRAF 及 MEK 的抑制劑一起作治療，可惜的是仍然存在非 MAPK 訊息路徑相關的抗藥因子，所以仍可預見腫瘤復發。IGFR1, AKT or mTOR 等因子曾被報告過，但對於這些或其它因子如何改變腫瘤對藥物的反應仍然所知甚少。 如果要想辦法扭腫瘤的抗藥性，那就必須要了解抗藥性是如何產生。我們提出使用飛蛾跳躍子插入的突變形成，來當作篩選抗藥因子的工具，這樣的實驗平台包含通用性的啟動子及 Poly-A 序列，各可作為增加功能以及失去功能的突變篩選，我們可以每次操縱一個基因，並擴大這個篩選體達到全基因飽和、高通量研究，選擇經 BRAF及 MEK 抑制劑篩選而存活下來的腫瘤細胞，拿來分析其抗藥機轉。 在篩選抗藥基因的實驗當中，我們選擇了 SK-MEL-28 及 A375 兩種黑色素細胞瘤，兩者皆帶有 BRAF-V600E 突變，而且原本對 BRAF 抑制劑有敏感性，因此這兩個細胞株被選來作為一般黑色素細胞瘤的兩種代表，並且建成兩個獨立的突變庫。在加 BRAF抑制劑之前與之後，我們都用次世代定序來分析原本突變庫的大小，以及最後篩選出來的結果。我們只選擇在這兩個獨立突變庫都篩出來的共同熱點基因，才接受進一步的試管內分析及活體分析。 這些最後篩選出的基因，將以系統方法整合，最終的目標是: 獲得使腫瘤變得對標靶治療有感受性的臨床前資料，發展出新的藥物組合或是可用藥標的。 研究目標: 目標一: 建立黑色素細胞瘤之增加功能及失去功能的突變庫 目標二: 以正向基因篩選的方法找黑色素細胞瘤的抗藥基因 目標三: 以異種移植模式來確認抗藥性及證實有潛力的致敏者<br> Abstract: Malignant melanoma is the leading cause of death among skin cancers in Taiwan. In western countries, the majority of patients who present with melanoma have early stage disease, with 62.6% presenting with stage 0 or I disease, and an additional 23.1% with stage II disease. Conversely, only 40% are in stage I or II in Taiwan. Surgery is the most important treatment for malignant melanoma. However, metastasis and recurrence are frequent. Dacarbazine has been the most active single chemotherapeutic agent but the response rate only ranges from 15% to 25% in melanoma. Therefore, chemotherapy is considered a palliative measure. The trend of treating metastatic melanoma has shifted to target therapy. The activating mutations in BRAF can be found in 50% of all cutaneous melanomas, which is a landmark in the melanoma biology. Although the presence of an activating BRAF mutation generally correlates to the response to BRAF inhibitors, a significant proportion of BRAF mutated melanoma cell lines show signs of intrinsic or acquired resistance. Inhibition of both BRAF and MEK has been used as a new strategy in the clinical trial to override the resistance. However, the existence of MAPK pathway-independent factors predicts the tumor recurrence. Some resistant factors, such as IGFR1, AKT or mTOR, have been reported but how these factors and possibly others modulate the drug response is not yet understood. If efforts are made to reverse the resistance, it will be essential to understand how it occurs. We propose the PiggyBac transposon-mediated insertional mutagenesis for screening resistant factors. Transposon-based platform includes gain-of-function and loss-of-function screening by using transposons with universal promoter and poly-A sequence respectively. With this system, we can manipulate a gene at one time and expand the trapping pool to saturate genome-wide high-throughput screen. The mutagenized melanoma clones surviving the treatment of BRAF and MEK inhibitors will be analyzed for its resistance mechanism. For screening resistance, we have chosen SK-MEL-28 and A375 melanoma cell lines, both carrying BRAF-V600E mutation and sensitive to BRAF inhibitor. The two cell lines are adopted as representatives of general BRAF mutant melanoma and established as two independent mutagenesis libraries. Before and after BRAF inhibitor treatment, the next-generation sequencing is applied to sequence the baseline library and the screen result. Only the overlapped hits from SK-MEL-28 screening and A375 screening will be selected for in vitro and in vivo validation. The final results will be integrated with the knowledge-driven systematic approach. The eventual aim is to generate requisite pre-clinical data for identifying the novel drug combinations and druggable targets that can sensitize melanoma to the target therapy. Specific Aims: Aim 1. To establish the gain-of-function and loss-of-function insertional mutagenesis library of melanoma cell lines. Aim 2. Forward genetic screening for genes conferring drug resistance in melanoma. Aim 3. Xenograft model to confirm the resistance and verify the potential sensitizer for resistant melanoma.