Identification of MET and SRC Activation in Melanoma Cell Lines Showing Primary Resistance to PLX4032
Identification of MET and SRC Activation in Melanoma Cell Lines Showing Primary Resistance to PLX4032
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DOI：
10.1593/neo.111102
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发表时间：
2011-12-01
期刊：
影响因子：
4.8
通讯作者：
Rodolfo, Monica
中科院分区：
文献类型：
--
作者：
Vergani, Elisabetta;Vallacchi, Viviana;Rodolfo, Monica
PLX4032/vemurafenib是一种一流的小分子BRAF(V600E)抑制剂，对BRAF突变黑色素瘤患者具有临床活性。然而，在接受治疗的患者中会产生耐药性，需要采取策略来克服原始和获得性耐药性。为了探索PLX4032原发耐药的分子机制，我们研究了PLX4032对细胞增殖和信号的影响，研究了27个具有遗传特征的患者黑色素瘤细胞系。细胞对PLX4032的敏感性依赖于BRAFV600E，与其他黑色素瘤中常见的基因改变无关，如PTEN缺失、BRAF和MITF基因扩增。研究了两个对PLX4032缺乏敏感性且存在不同遗传改变的细胞系，作为初级抗性的模型。用MEK抑制剂UO126处理而不用PLX4032处理可抑制细胞生长和ERK激活。在CRAF被指示MEK-ERK信号交替激活的siRNA下调后，对PLX4032的抗性保持不变。多重连接依赖的探针扩增的基因特征和MALDI-TOF质谱仪分析磷酸化酪氨酸信号的分析表明，MET和SRC信号分别与MET、CTNNB1和CCND1基因的扩增有关。在MET扩增的黑色素瘤细胞中，PLX4032与药物或靶向MET的siRNA联合应用能有效地抑制细胞生长，减少细胞侵袭和迁移；在另一细胞系靶向SRC后也观察到类似的效果，表明MET和SRC信号在PLX4032原发耐药中发挥了作用。我们的结果支持黑色素瘤分子亚型分类的发展，以获得更有效的治疗。
PLX4032/vemurafenib is a first-in-class small-molecule BRAF(V600E) inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAFV600E and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies.