Division of Stem Cell Transplantation and Immunotherapy, 2nd Department of Medicine, Christian-Albrechts-University, Kiel, Germany.
Neoplasia. 2012 Mar;14(3):190-205. doi: 10.1593/neo.111636.
Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPβ-LIP. Thus, siRNA-mediated knockdown of C/EBPβ led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBPβ-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.
结直肠癌（CRC）中的致癌性 KRAS 突变与表皮生长因子受体（EGFR）靶向抗体（Ab）治疗无效相关。然而，持续激活的 KRAS（KRAS(G12V)）如何损害 EGFR-Ab 的效应机制尚不完全清楚。在这里，我们建立了同基因细胞系模型，以系统地研究 KRAS(G12V) 在 A431 异种移植模型中的肿瘤生长以及 EGFR-Ab 在体外的各种作用模式中的影响。KRAS(G12V) 通过刺激受体非依赖性下游信号转导，损害 EGFR-Ab 介导的生长抑制。KRAS(G12V) 还使肿瘤细胞对 EGFR-Ab 的 Fc 介导的效应机制（如补体依赖性细胞毒性（CDC）和抗体依赖性细胞介导的细胞毒性（ADCC））反应降低。CDC 和 ADCC 活性受损可能与 KRAS 突变型与野生型（wt）细胞中 EGFR 表达降低有关，而 KRAS4b 的小干扰 RNA（siRNA）介导的敲低可恢复 EGFR 表达。免疫组织化学实验也表明，KRAS 突变型与 KRAS 野生型结直肠样本相比，EGFR 表达降低。对 KRAS(G12V) 下调 EGFR 表达的潜在机制进行分析表明，六种不同的转录因子的活性显著降低。进一步的实验表明，CCAAT/增强子结合蛋白（C/EBP）家族通过上调抑制性家族成员 C/EBPβ-LIP 来抑制 EGFR 转录，从而参与调节 KRAS 突变型肿瘤细胞中的 EGFR 启动子活性。因此，siRNA 介导的 C/EBPβ 敲低导致 EGFR 表达增强和针对 KRAS 突变细胞的 Ab 介导的细胞毒性增强。综上所述，这些结果表明，KRAS(G12V) 信号诱导 C/EBPβ 依赖性 EGFR 表达抑制，从而损害 EGFR-Ab 的 Fc 介导的效应机制，并使 KRAS 突变型肿瘤细胞对这些治疗药物的敏感性降低。
