Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines
在黑色素瘤细胞系中与 Encorafenib 和 Binimetinib 联合治疗期间与获得性耐药相关的分子改变
Melanoma is the most aggressive, deadliest form of skin cancer. Combined BRAF-MEK inhibitor (BRAFi/MEKi) therapy was a breakthrough in the treatment of melanoma with BRAFV600-mutations. However, many patients frequently develop drug resistance to the combinatory treatment. The aim of our study was to characterize the molecular background behind acquired resistance to BRAFi/MEKi-s. After the successful development of resistant cell lines, we investigated the invasion properties, changes in gene and protein expressions, as well as the effect of the “drug holiday” of the resistant cell lines. Drug-resistant melanoma cells had a higher invasive potential and acquired a spindle-like structure, and many cancer-related proteins were overexpressed in the resistant cells. Furthermore, transcriptome analysis revealed that differentially expressed genes are functionally linked to a variety of biological functions that may lead to resistance to the inhibitors. These results may offer valuable insight into further understanding of BRAFi/MEKi resistance, as well as to the development of therapeutic tools to overcome drug resistance.
黑色素瘤是最具侵袭性、最致命的皮肤癌形式。BRAF-MEK 抑制剂 (BRAFi/MEKi) 联合疗法是治疗 BRAFV600 突变黑色素瘤的突破。然而，许多患者经常对联合治疗产生耐药性。我们研究的目的是描述对 BRAFi/MEKi-s 获得性耐药背后的分子背景。在成功开发抗性细胞系后，我们研究了抗性细胞系的侵袭特性、基因和蛋白质表达的变化以及“药物假期”的影响。耐药黑色素瘤细胞具有更高的侵袭潜力并获得梭形结构，并且许多癌症相关蛋白在耐药细胞中过度表达。此外，转录组分析表明，差异表达的基因在功能上与多种可能导致对抑制剂产生抗性的生物学功能相关。这些结果可能为进一步了解 BRAFi/MEKi 耐药性以及开发克服耐药性的治疗工具提供有价值的见解。
Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.
使用 BRAF/MEK 抑制剂的联合治疗是晚期BRAF V600E/K患者的有希望的治疗方法突变黑色素瘤。然而，获得性耐药在很大程度上限制了这种药物组合的临床疗效。确定耐药机制对于达成长期、持久的反应至关重要。在这项研究中，我们开发了六种对 BRAFi/MEKi 治疗具有获得性耐药性的黑色素瘤细胞系，并确定了与耐药性相关的分子改变。我们观察到与敏感细胞相比，三种抗性细胞系的侵袭显着增加。RNA 测序分析揭示了差异表达的基因，这些基因在功能上与多种生物学功能相关，包括上皮-间质转化、ROS 途径和 KRAS 信号传导。使用蛋白质组分析仪阵列，检测到几种差异表达的蛋白质，这些蛋白质聚集成一个独特的模式。E+BRes细胞。我们还观察到，在停用抑制剂后，耐药细胞的表现有所不同，其中五个根本没有吸毒成瘾，并且没有表现出显着增加的杀伤力；然而，一种抗性细胞系（WM1617 E+BRes）的活力显着下降。我们选择了三种耐药细胞系来研究停药后的蛋白质表达变化。CapG、Enolase 2 和骨桥蛋白在“药物假期”十天后在耐药细胞中的表达模式相似，但 Snail 蛋白仅在 WM1617 E+BRes 中表达细胞，显示出药物依赖性表型，这可能与药物成瘾有关。我们的研究结果强调，黑色素瘤细胞使用几种类型的抗性机制，包括改变不同蛋白质的表达来绕过药物治疗。
Our current data offer the first insight into differentially expressed genes and provide protein expression patterns that are associated with a BRAFi/MEKi-resistant phenotype in melanoma cell lines. Our findings facilitate a more thorough understanding of the development of the complex mechanisms leading to acquired resistance during combined treatment in BRAF-mutant melanoma. However, further studies are needed to identify the key molecules and signalling pathways responsible for therapeutic escape during BRAFi/MEKi treatment and to prevent the initiation of acquired drug resistance in melanoma.
我们目前的数据提供了对差异表达基因的首次洞察，并提供了与黑色素瘤细胞系中 BRAFi/MEKi 抗性表型相关的蛋白质表达模式。我们的研究结果有助于更深入地了解在 BRAF 突变黑色素瘤联合治疗期间导致获得性耐药的复杂机制的发展。然而，需要进一步的研究来确定在 BRAFi/MEKi 治疗期间负责治疗逃逸的关键分子和信号通路，并防止黑色素瘤中出现获得性耐药性。
关键词： malignant melanoma,BRAFV600E,combination of encorafenib and binimetinib,BRAF and MEK inhibitors,drug resistance; proteome array,RNAseq,drug holiday,恶性黑色素瘤,布拉夫V600E,encorafenib 和 binimetinib 的组合,BRAF 和 MEK 抑制剂,耐药性,蛋白质组阵列,核糖核酸序列,药物假期