A Novel Aptamer LL4A Specifically Targets Vemurafenib-Resistant Melanoma through Binding to the CD63 Protein
A Novel Aptamer LL4A Specifically Targets Vemurafenib-Resistant Melanoma through Binding to the CD63 Protein
复制标题
新型适体 LL4A 通过与 CD63 蛋白结合特异性靶向维莫非尼耐药黑色素瘤
DOI：
10.1016/j.omtn.2019.10.005
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发表时间：
2019-12-06
影响因子：
8.8
通讯作者：
Liu，Jing
中科院分区：
文献类型：
--
作者：
Li，Hui;Liu，Juan;Liu，Jing
黑色素瘤是一种高度侵袭性的肿瘤，预后较差，一半的黑色素瘤患者携带 BRAF 突变。 BRAF 抑制剂维莫非尼 (PLX4032) 已获得美国食品药品监督管理局 (FDA) 和欧洲药品管理局 (EMA) 批准用于治疗患有 BRAFV600E 突变的晚期黑色素瘤患者。然而，几乎所有患者的适应性耐药都阻碍了维莫非尼的疗效。在这项研究中，我们使用基于细胞的SELEX（通过指数富集的配体系统进化）策略，获得了一种针对维莫非尼耐药黑色素瘤细胞具有高亲和力和特异性的DNA适体（名为LL4）。优化的截短形式 (LL4A) 特异性结合对维莫非尼耐药的黑色素瘤细胞，解离常数在纳摩尔范围内，具有出色的稳定性和低毒性。同时，荧光成像证实，LL4A 在由维莫非尼耐药的黑色素瘤细胞形成的肿瘤中显着积累，但在体内相应亲代细胞形成的对照肿瘤中则没有显着积累。此外，使用pull-down测定结合液相色谱-串联质谱（LC-MS/MS）方法，将跨膜蛋白CD63鉴定为适体LL4A的结合靶标。 CD63 与 TIMP1 和 β1-整合素形成超分子复合物，激活核因子 кB (NF-кB) 和丝裂原激活蛋白激酶 (MAPK) 信号通路，并导致维莫非尼耐药。适配体 LL4A 可用于人类的诊断和治疗，以治疗靶向维莫非尼耐药的黑色素瘤。
Melanoma is a highly aggressive tumor with a poor prognosis, and half of all melanoma patients harbor BRAF mutations. A BRAF inhibitor, vemurafenib (PLX4032), has been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to treat advanced melanoma patients with BRAFV600Emutation. However, the efficacy of vemurafenib is impeded by adaptive resistance in almost all patients. In this study, using a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy, we obtained a DNA aptamer (named LL4) with high affinity and specificity against vemurafenib-resistant melanoma cells. Optimized truncated form (LL4A) specifically binds to vemurafenib-resistant melanoma cells with dissociation constants in the nanomolar range and with excellent stability and low toxicity. Meanwhile, fluorescence imaging confirmed that LL4A significantly accumulated in tumors formed by vemurafenib-resistant melanoma cells, but not in control tumors formed by their corresponding parental cellsin vivo. Further, a transmembrane protein CD63 was identified as the binding target of aptamer LL4A using a pull-down assay combined with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. CD63 formed a supramolecular complex with TIMP1 and β1-integrin, activated the nuclear factor кB (NF-кB) and mitogen-activated protein kinase (MAPK) signaling pathways, and contributed to vemurafenib resistance. Potentially, the aptamer LL4A may be used diagnostically and therapeutically in humans to treat targeted vemurafenib-resistant melanoma.