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Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines

在黑色素瘤細(xì)胞系中與 Encorafenib 和 Binimetinib 聯(lián)合治療期間與獲得性耐藥相關(guān)的分子改變

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.

黑色素瘤是最具侵襲性、最致命的皮膚癌形式。BRAF-MEK 抑制劑 (BRAFi/MEKi) 聯(lián)合療法是治療 BRAFV600 突變黑色素瘤的突破。然而，許多患者經(jīng)常對(duì)聯(lián)合治療產(chǎn)生耐藥性。我們研究的目的是描述對(duì) BRAFi/MEKi-s 獲得性耐藥背后的分子背景。在成功開發(fā)抗性細(xì)胞系后，我們研究了抗性細(xì)胞系的侵襲特性、基因和蛋白質(zhì)表達(dá)的變化以及“藥物假期”的影響。耐藥黑色素瘤細(xì)胞具有更高的侵襲潛力并獲得梭形結(jié)構(gòu)，并且許多癌癥相關(guān)蛋白在耐藥細(xì)胞中過度表達(dá)。此外，轉(zhuǎn)錄組分析表明，差異表達(dá)的基因在功能上與多種可能導(dǎo)致對(duì)抑制劑產(chǎn)生抗性的生物學(xué)功能相關(guān)。這些結(jié)果可能為進(jìn)一步了解 BRAFi/MEKi 耐藥性以及開發(fā)克服耐藥性的治療工具提供有價(jià)值的見解。

10層細(xì)胞工廠

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 抑制劑的聯(lián)合治療是晚期BRAF V600E/K患者的有希望的治療方法突變黑色素瘤。然而，獲得性耐藥在很大程度上限制了這種藥物組合的臨床療效。確定耐藥機(jī)制對(duì)于達(dá)成長期、持久的反應(yīng)至關(guān)重要。在這項(xiàng)研究中，我們開發(fā)了六種對(duì) BRAFi/MEKi 治療具有獲得性耐藥性的黑色素瘤細(xì)胞系，并確定了與耐藥性相關(guān)的分子改變。我們觀察到與敏感細(xì)胞相比，三種抗性細(xì)胞系的侵襲顯著增加。RNA 測序分析揭示了差異表達(dá)的基因，這些基因在功能上與多種生物學(xué)功能相關(guān)，包括上皮-間質(zhì)轉(zhuǎn)化、ROS 途徑和 KRAS 信號(hào)傳導(dǎo)。使用蛋白質(zhì)組分析儀陣列，檢測到幾種差異表達(dá)的蛋白質(zhì)，這些蛋白質(zhì)聚集成一個(gè)獨(dú)特的模式。E+BRes細(xì)胞。我們還觀察到，在停用抑制劑后，耐藥細(xì)胞的表現(xiàn)有所不同，其中五個(gè)根本沒有吸毒成癮，并且沒有表現(xiàn)出顯著增加的殺傷力；然而，一種抗性細(xì)胞系（WM1617 E+BRes）的活力顯著下降。我們選擇了三種耐藥細(xì)胞系來研究停藥后的蛋白質(zhì)表達(dá)變化。CapG、Enolase 2 和骨橋蛋白在“藥物假期”十天后在耐藥細(xì)胞中的表達(dá)模式相似，但 Snail 蛋白僅在 WM1617 E+BRes 中表達(dá)細(xì)胞，顯示出藥物依賴性表型，這可能與藥物成癮有關(guān)。我們的研究結(jié)果強(qiáng)調(diào)，黑色素瘤細(xì)胞使用幾種類型的抗性機(jī)制，包括改變不同蛋白質(zhì)的表達(dá)來繞過藥物治療。

5L高效搖瓶

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.

我們目前的數(shù)據(jù)提供了對(duì)差異表達(dá)基因的首次洞察，并提供了與黑色素瘤細(xì)胞系中 BRAFi/MEKi 抗性表型相關(guān)的蛋白質(zhì)表達(dá)模式。我們的研究結(jié)果有助于更深入地了解在 BRAF 突變黑色素瘤聯(lián)合治療期間導(dǎo)致獲得性耐藥的復(fù)雜機(jī)制的發(fā)展。然而，需要進(jìn)一步的研究來確定在 BRAFi/MEKi 治療期間負(fù)責(zé)治療逃逸的關(guān)鍵分子和信號(hào)通路，并防止黑色素瘤中出現(xiàn)獲得性耐藥性。

血清培養(yǎng)基瓶

關(guān)鍵詞： malignant melanoma,BRAFV600E,combination of encorafenib and binimetinib,BRAF and MEK inhibitors,drug resistance; proteome array,RNAseq,drug holiday,惡性黑色素瘤,布拉夫V600E,encorafenib 和 binimetinib 的組合,BRAF 和 MEK 抑制劑,耐藥性,蛋白質(zhì)組陣列,核糖核酸序列,藥物假期

來源：MDPI https://www.mdpi.com/2072-6694/13/23/6058/htm