| 摘要: | 黑色素瘤是最具侵襲性的皮膚癌，標靶藥物 Vemurafenib (PLX4032)為 BRAFV600E突變的黑色素瘤之抑制劑。然而，黑色素瘤患者在經過 PLX4032 治療後出現了不良反應，包括抗藥性黑色素瘤及繼發性腫瘤。大多數的繼發性腫瘤有 21%-60% 屬於 RAS 突變，而增加 BRAF-CRAF 異二聚體的表現以激活 MEK/ERK 通路。PLX8394 為另一種新型 BRAFV600E 的抑制劑，可以抑制 BRAF-CRAF 異二聚體的表現。BRAFV600E抑制劑造成的繼發性腫瘤進展及機制尚不明確。因此，我們想要了解 BRAFV600E 抑製劑治療的黑色素瘤釋放的外泌體是否影響正常角質細胞及表皮細胞的繼發性腫瘤進展。在這次研究中，我們將 PLX4032 或 PLX8394 處理的角質細胞、上皮細胞、黑色素瘤細胞和 PLX4032 抗藥性黑色素瘤細胞所收集的條件培養基 (CM)處理角質細胞和上皮細胞，進行細胞的功能測定。從結果中觀察到用 PLX4032 處理的黑色素瘤細胞增加了角質細胞的遷移以及表皮細胞致瘤性。重要的是，與親代黑色素瘤細胞相比，經過PLX4032 處理的抗藥性黑色素瘤細胞的 CM 培養的角質細胞遷移能力顯著增加。相反，
角質細胞的遷移不受 PLX8394 處理的親代或抗性黑色素瘤細胞的 CM 培養的影響。
外泌體可以運輸物質，包括 DNA、RNA、miRNA 和蛋白質。我們從黑色素瘤的 CM 中純化了外泌體，並發現了角質細胞遷移能力增加與黑色素瘤細胞的外泌體轉移有關。此外，用 PLX4032 處理的黑色素瘤釋放出的外泌體增加了角質細胞的 BRAF-CRAF 異二聚體表達。最後，我們還從轉染了miR-567 的黑色素瘤細胞收集 CM，並觀察到轉染 miR-567 的黑色素瘤可以降低角質細胞的遷移能力。
綜合上述結果，PLX4032 處理的黑色素瘤細胞的外泌體增強了角質細胞的遷移能力和 BRAF-CRAF 異二聚體表達，相反地， PLX8394 處理的黑色素瘤細胞的外泌體與PLX4032 相比可以降低角質細胞的遷移及 BRAF-CRAF異二聚體表達趨勢。這些結果代表黑色素瘤釋放出來的外泌體在繼發性腫瘤進展中扮演關鍵角色。；
Melanoma is the most aggressive form of skin cancer. Vemurafenib (PLX4032) is an inhibitor of the V600E mutant form of BRAF gene (BRAFV600E) for the treatment of melanoma. However, melanoma patients treated with PLX4032 cause adverse reactions including resistance and the formation of secondary tumor. Most of the secondary tumors have 21%-60% RAS mutations, which enhances the BRAF-CRAF heterodimer formation to activate the MEK/ERK pathway. PLX8394, a novel BRAFV600E inhibitor, could inhibit BRAF-CRAF interaction. BRAFV600E inhibitor-induced secondary tumor development are not fully defined. Therefore, we hypothesized that exosomes released from melanoma treated with BRAFV600E inhibitors may affect the progression of secondary tumors which are originated from normal keratinocytes, epidermal cells.
In this study, the conditioned medium (CM) collected from keratinocytes, epithelial, melanoma cells and PLX4032-resistant melanoma cells treated with BRAFV600E inhibitor,
PLX4032 or PLX8394, were used to treat keratinocyte and epithelial cells to perform cell-based functional assay. The results showed that melanoma cells treated with PLX4032 enhanced the migration, anchorage-independent growth of keratinocyte or epidermal cells. Importantly, the migrative ability of keratinocyte cells cultured with the CM from resistant melanoma cells treated with PLX4032 was increased significantly as compared to parental melanoma cells. In
contrast, tumorigenesis of keratinocyte cells was not affected by cultured with the CM from parental or resistant melanoma cells treated with PLX8394.
Exosomes can transport cargo, including DNA, RNA, miRNA and proteins. We purified exosomes from CM and revealed the mechanism of the enhanced migration activity on keratinocytes are linked to exosomes transferred from melanoma cells. Moreover, exosomes derived from melanoma treated with PLX4032 increase BRAF-CRAF heterodimer expression of keratinocytes. Finally, we also collected CM from melanoma cells overexpressed miR-567 and observed that melanoma overexpression of miR-567 could decrease migration activity on keratinocytes.
In conclusion, exosomes derived from melanoma cells treated with PLX4032 enhanced migrative ability and BRAF-CRAF heterodimer expression of keratinocytes, in contrast, exosomes derived from melanoma cells treated with PLX8394 decreased migrative ability and BRAF-CRAF heterodimer expression of keratinocytes compared to melanoma cells treated with PLX4032. These results suggest that exosomes released from melanoma play a key role in secondary tumors progression. |
