探討黑色素細胞瘤經dabrafenib及trametinib合併治療抗性之分子機轉

Identification of Molecular Mechanism of Acquired Resistance to Dabrafenib and Trametinib Co-treatment in Melanoma

陳奕璇(Yi-Xuan Chen)

指導教授 ： 張正琪

國立臺灣大學/牙醫專業學院/口腔生物科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.11097

摘要

對於BRAF有突變之已轉移黑色素細胞瘤，使用Dabrafenib (D) 及Trametinib (T) 合併治療是最新的標靶治療。雖然此種合併治療之臨床益處已被證實，但多數病人依舊產生抗藥性，然而其抗藥的分子機轉仍然未知。細胞實驗中，我們藉由將A375細胞暴露在逐漸提高的兩者藥物濃度的培養液中，以建立此兩者藥物單獨及合併治療之抗藥株(A375DR、A375TR、A375DTR)，並使用Western Blot及MTT assay來確認其抗藥性。此外我們利用基因晶片、重組蛋白、拮抗劑及knockdown系統找尋下游標的。而在動物實驗中，我們使用了nude mice，藉由打入A375細胞並每日灌食dabrafenib及trametinib來建立活體的抗藥株材料，當其腫瘤復發並長至1200 mm3時便犧牲，收集其血清及腫瘤，透過qRT-PCR、ELISA及IHC的方式進行後續研究。
研究結果顯示在抗藥株中內皮素-1的表現量大幅上升，此現象在合併使用兩種藥物的抗藥株(A375DTR)更為顯著，且進一步抑制內皮素-1的表現後，可使A375DTR的抗藥性消失。在A375DTR抗藥株中，Akt磷酸化的表現量透過內皮素-1/內皮素接受器B的訊息傳遞而上升。此外不論抑制Akt的磷酸化或阻斷內皮素接受器B皆使A375DTR的抗藥性回復。總結來說，我們推測內皮素-1及Akt的活化可能參與了黑色素細胞瘤經dabrafenib及trametinib合併治療後抗性之分子機轉，因此內皮素-1可能是合併使用此兩種標靶藥物產生抗性後的治療標的。

關鍵字

dabrafenib ； 抗藥性 ； 內皮素-1 ； 黑色素細胞癌 ； trametinib

並列摘要

The combination of dabrafenib and trametinib is a targeted therapy for BRAF-mutated metastatic melanoma. Although this combination therapy has proven clinical benefits in cutaneous melanoma, some patients still develop resistance. However, the mechanism of the drug resistance is still unknown. In vitro, we established A375 drug resistant clones (A375DR, A375TR, and A375DTR cells) by chronically exposing A375 cells to dabrafenib and/or trametinib, and further confirmed the drug resistance by Western Blot and MTT assay. Besides, we found the downstream target(s) in drug resistance by microarray, recombinant protein, antagonists and knockdown system. In vivo, we also established A375 resistant tumors by oral gavage of dabrafenib and trametinib every day in a nude mice xenograft model. When the mice developed relapsed tumors after initial regression, we collected tumor and blood samples for further real-time RT-PCR, ELISA and immunohistochemistry studies.
We demonstrated that endothelin-1 (ET-1) was increased in A375 drug resistance clones, especially A375DTR cells, and the drug resistance ability were decreased by ET-1 shRNA knockdown. Phosphorylation of Akt was increased through ET-1-ETBR signaling in A375DTR cells. Furthermore, inhibition of Akt phosphorylation or blockade of ETBR reversed the drug resistance ability of A375DTR cells. In conclusion, we demonstrated the molecular mechanism of acquired resistance to dabrafenib and trametinib co-treatment in cutaneous melanoma involves ET-1 and Akt activation. ET-1 may be a potential therapeutic target for acquired resistance to dabrafenib and trametinib co-treatment in cutaneous melanoma.

並列關鍵字

dabrafenib ； drug resistance ； endothelin-1 ； melanoma ； trametinib

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