探討BRAF抑制劑透過細胞間訊息誘導腫瘤形成之研究
中文摘要
標靶藥物Vemurafenib (PLX4032)是小分子抑制劑，作為抑制BRAFV600E基因突變來治療黑色素瘤，晚期黑色素瘤患者使用PLX4032治療後，提升患者整體存活率，然而，在臨床上發現15%-30%的患者平均治療10週後產生良性或惡性皮膚腫瘤的第二癌症，大多發生皮膚性的鱗狀上皮細胞癌（cutaneous squamous cell carcinoma, cSCC）和角化棘皮瘤（keratoacanthoma, KA），目前BRAF抑制劑誘導第二癌症發展機制尚不清楚。因此我們研究想解析是否用PLX4032治療黑色素瘤會釋放訊息在細胞間，影響表皮細胞誘發腫瘤形成。我們利用PLX4032治療黑色素瘤細胞收集的條件培養基(conditioned-medium, CM )來進行實驗，實驗的結果觀察到表皮細胞經過條件培養基培養會促使細胞增生，透過上調表皮細胞MAPK / ERK信號路徑。先前研究發現，細胞外囊泡（extracellular vesicles, EVs）是奈米大小的膜狀結構，在腫瘤微環境中作為細胞間訊息傳遞，調控細胞基因表現和蛋白質功能的表達。接下來，我們想要解析PLX4032治療黑色素瘤細胞釋放出的EVs是否影響表皮細胞增生，因此我們先從條件培養基中分離出EVs，透過蛋白質分析、奈米顆粒追蹤分析和穿透式電子顯微鏡實驗證明EVs的物理性質及特徵，確定完全純化出EVs。並透過螢光顯微鏡觀察表皮細胞對EVs的攝取, 建立可以觀察及追蹤黑色素瘤細胞株釋放的EVs。以上結果得到，PLX4032誘發表皮細胞增生，可能受黑色素瘤釋放EVs在細胞間訊息傳遞的影響。

Study of the intercellular communication involving in BRAF inhibitor-induced tumorigenesis
Abstract
Vemurafenib (PLX4032) is a small molecule inhibitor of the V600E mutant form of BRAF gene used in the treatment of melanoma. The treatment of PLX4032 in metastatic melanoma with BRAF mutation ensures the clinical improvement of the cancer. However, previous studies showed that 15-30% of the patients with PLX4032 treatment developed secondary benign or malignant skin tumors after an average of ten weeks from start of treatment. Cutaneous squamous cell carcinoma (cSCC) and keratoacanthomas (KA) were the majority of skin tumors presenting in the patients. The mechanism of PLX4032-induced secondary tumor development is not well defined. We asked whether the intercellular communication, especially factors released from melanoma treated with PLX4032 affects epidermal cells to promote the formation of the secondary tumors. In this study, we utilized the conditioned medium (CM) collected from melanoma cells treated with PLX4032 to investigate our hypothesis. Our results found that cell proliferation and cell survival increased in epidermal cells with CM when compared with cells without CM. Interestingly, MAPK/ERK signaling pathway was enhanced in CM-treated cells. Recent studies showed that extracellular vesicles (EVs) have been described as the important mediators of cell-to-cell communication in tumor microenvironment and regulate various physiological and pathological processes of receiver cells. We further want to ask whether the EVs released from melanoma treated with PLX4032 can influence epidermal cells to promote the formation of the secondary tumors. Here, the physicochemical characterization of EVs were checked by western blot, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM) to confirm that EVs were isolated successfully. Here, we established the stable melanoma cell lines for visualization and tracking of the released EVs, and the uptake of the EVs by the epidermal cells was observed by fluorescent microscopy. Our results indicated that PLX4032 treated melanoma cells may affect other normal cells in the tumor microenvironments survival through intercellular communication.

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