Broad-spectrum receptor tyrosine kinase inhibitors overcome de novo and acquired modes of resistance to EGFR-targeted therapies in colorectal cancer.
Broad-spectrum receptor tyrosine kinase inhibitors overcome de novo and acquired modes of resistance to EGFR-targeted therapies in colorectal cancer.
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DOI：
10.18632/oncotarget.26663
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发表时间：
2019-02-12
期刊：
影响因子：
--
通讯作者：
Singh, Bhuminder
中科院分区：
文献类型：
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作者：
Graves-Deal, Ramona;Bogatcheva, Galina;Singh, Bhuminder
人们越来越认识到，3D培养物比2D培养物更能预测体内治疗功效。使用人结肠直肠癌（CRC）细胞系HCA-7衍生物CC、SC和CC-CR的体外3D I型胶原培养物，我们先前鉴定出受体酪氨酸激酶（RTK）MET和罗恩的活化导致对EGF受体（EGFR）定向治疗性抗体西妥昔单抗的抗性。SC细胞中西妥昔单抗耐药的从头模式可以通过克唑替尼克服，克唑替尼是一种多RTK抑制剂，也靶向MET和罗恩。我们现在发现克唑替尼也能克服CC-CR细胞中获得性西妥昔单抗耐药。磷酸化RTK阵列分析显示，与西妥昔单抗敏感的CC对应物相比，SC和CC-CR细胞中几种RTK（包括MET和罗恩）的磷酸化增加。此外，其他多RTK抑制剂卡博替尼和BMS-777607有助于克服西妥昔单抗耐药性，如通过3D菌落生长和关键信号分子的活化状态所测量的。相反，添加RTK配体HGF和NRG 1诱导CC细胞中的西妥昔单抗耐药，这可以通过添加克唑替尼来阻断。我们通过流式细胞仪分析进一步确定了西妥昔单抗和克唑替尼协同作用的机制，并观察到西妥昔单抗耐药CRC 3D培养物中G1期细胞周期停滞增加。最后，我们证明了克唑替尼在体内克服了SC裸鼠异种移植物中的西妥昔单抗耐药性。因此，我们的工作表明，多重RTK抑制策略是一种有效的、广泛适用的策略，可以克服CRC对EGFR靶向治疗的耐药性，并突出了3D培养在这些研究中的相关性。含义声明：使用体外3D CRC培养物和体内CRC异种移植物，我们证明了用小分子抑制剂平行抑制多种RTK克服了CRC中EGFR导向疗法的从头和获得性抗性。
It is increasingly appreciated that 3D cultures are more predictive of in vivo therapeutic efficacy than 2D cultures. Using in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR, we previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab. The de novo mode of cetuximab resistance in SC cells could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. We now show that crizotinib also overcomes acquired cetuximab resistance in CC-CR cells. Phospho-RTK array analysis showed increased phosphorylation of several RTKs, including MET and RON, in SC and CC-CR cells compared to cetuximab-sensitive CC counterparts. Furthermore, other multi-RTK inhibitors cabozantinib and BMS-777607 helped overcome cetuximab resistance, as measured by 3D colony growth and activation state of key signaling molecules. Conversely, addition of RTK ligands HGF and NRG1 induced cetuximab resistance in CC cells, which could be blocked by addition of crizotinib. We further determined the mechanism of the cooperative effect of cetuximab and crizotinib by FACS analysis and observed increased cell cycle arrest in G1 phase in cetuximab-resistant CRC 3D cultures. Finally, we show that crizotinib overcomes cetuximab resistance in vivo in SC nude mice xenografts. Thus, our work shows that multi-RTK inhibition strategy is a potent, broadly applicable strategy to overcome resistance to EGFR-targeted therapeutics in CRC and highlights the relevance of 3D cultures in these studies. Statement of implication: Using in vitro 3D CRC cultures and in vivo CRC xenografts, we show that parallel inhibition of multiple RTKs with small molecule inhibitors overcomes de novo and acquired resistance to EGFR-directed therapies in CRC.