Department of Oncology-Pathology, Karolinska Institutet, 171 64, Stockholm, Sweden.
Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Cell Death Dis. 2020 Oct 20;11(10):882. doi: 10.1038/s41419-020-03097-2.
Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival, however disease progression commonly ensues. In a previous study we identified afatinib and crizotinib in combination as a novel potential therapy for CMM independent of BRAF/NRAS mutation status. Herein, we elucidate the underlying mechanisms of the combination treatment effect to find biomarkers and novel targets for development of therapy that may provide clinical benefit by proteomic analysis of CMM cell lines and xenografts using mass spectrometry based analysis and reverse phase protein array. Identified candidates were validated using immunoblotting or immunofluorescence. Our analysis revealed that mTOR/Insulin signaling pathways were significantly decreased by the afatinib and crizotinib combination treatment. Both in vitro and in vivo analyses showed that the combination treatment downregulated pRPS6KB1 and pRPS6, downstream of mTOR signaling, and IRS-1 in the insulin signaling pathway, specifically ablating IRS-1 nuclear signal. Silencing of RPS6 and IRS-1 alone had a similar effect on cell death, which was further induced when IRS-1 and RPS6 were concomitantly silenced in the CMM cell lines. Silencing of IRS-1 and RPS6 resulted in reduced sensitivity towards combination treatment. Additionally, we found that IRS-1 and RPS6KB1 expression levels were increased in advanced stages of CMM clinical samples. We could demonstrate that induced resistance towards combination treatment was reversible by a drug holiday. CD171/L1CAM, mTOR and PI3K-p85 were induced in the combination resistant cells whereas AXL and EPHA2, previously identified mediators of resistance to MAPK inhibitor therapy in CMM were downregulated. We also found that CD171/L1CAM and mTOR were increased at progression in tumor biopsies from two matched cases of patients receiving targeted therapy with BRAFi. Overall, these findings provide insights into the molecular mechanisms behind the afatinib and crizotinib combination treatment effect and leverages a platform for discovering novel biomarkers and therapy regimes for CMM treatment.
目前用于治疗皮肤转移性恶性黑色素瘤（CMM）的方法可以改善患者的生存率，但疾病通常会进展。在之前的研究中，我们发现 afatinib 和 crizotinib 联合使用是一种新的潜在治疗方法，与 BRAF/NRAS 突变状态无关。在此，我们通过基于质谱的分析和反相蛋白阵列分析，用 CMM 细胞系和异种移植物进行蛋白质组学分析，阐明联合治疗效果的潜在机制，寻找生物标志物和新的治疗靶点，这些靶点可能通过治疗为患者提供临床获益。使用免疫印迹或免疫荧光验证了鉴定出的候选物。我们的分析表明，mTOR/胰岛素信号通路在 afatinib 和 crizotinib 联合治疗后显著降低。体外和体内分析均表明，联合治疗下调了 mTOR 信号下游的 pRPS6KB1 和 pRPS6 以及胰岛素信号通路中的 IRS-1，特别是使 IRS-1 的核信号失活。单独沉默 RPS6 和 IRS-1 对细胞死亡有类似的影响，当 CMM 细胞系中同时沉默 IRS-1 和 RPS6 时，这种影响进一步增强。沉默 IRS-1 和 RPS6 会降低对联合治疗的敏感性。此外，我们发现 IRS-1 和 RPS6KB1 的表达水平在 CMM 临床样本的晚期阶段增加。我们能够证明，通过药物休假期可以使联合治疗的诱导耐药性恢复。在联合耐药细胞中诱导了 CD171/L1CAM、mTOR 和 PI3K-p85，而先前鉴定为 CMM 中 MAPK 抑制剂治疗耐药的介质 AXL 和 EPHA2 下调。我们还发现，在接受 BRAFi 靶向治疗的两名患者的肿瘤活检样本中，在进展时 CD171/L1CAM 和 mTOR 增加。总的来说，这些发现为 afatinib 和 crizotinib 联合治疗效果的分子机制提供了新的见解，并为发现 CMM 治疗的新生物标志物和治疗方案提供了一个平台。
Zotero 插件
