表皮生长因子受体（EGFR）配体 Amphiregulin 和 Epiregulin 的交叉抑制以及成纤维细胞生长因子受体 3（FGFR3）信号的去抑制导致野生型 KRAS 肿瘤细胞对西妥昔单抗产生耐药性。

BACKGROUND

In addition to the mutational status of KRAS, the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG) might function as bona fide biomarkers of cetuximab (Ctx) sensitivity for most EGFR-driven carcinomas.

METHODS

Lentivirus-delivered small hairpin RNAs were employed to specifically reduce AREG or EREG gene expression in wild-type KRAS A431 squamous cell carcinoma cells. Colony-forming assays were used to monitor the impact of AREG and EREG knockdown on Ctx efficacy. Amphiregulin and EREG protein expression levels were assessed by quantitative ELISA in parental A431 cells and in pooled populations of A431 cells adapted to grow in the presence of Ctx. A phosphoproteomic platform was used to measure the relative level of phosphorylation of 42 distinct receptor tyrosine kinases before and after the acquisition of resistance to Ctx.

RESULTS

Stable gene silencing of either ligand was found to notably reduce the expression of the other ligand. Parental A431 cells with normal expression levels of AREG/EREG exhibited significantly increased growth inhibition in response to Ctx, compared with derivatives that are engineered to produce minimal AREG/EREG. The parental A431 cells acutely treated with Ctx exhibited reduced basal expression levels of AREG/EREG. Pooled populations of Ctx-resistant A431 cells expressed significantly lower levels of AREG/EREG and were insensitive to the downregulatory effects of Ctx. Phosphoproteomic screen identified a remarkable hyperactivation of FGFR3 in Ctx-resistant A431 cells, which gained sensitivity to the cytotoxic and apoptotic effects of the FGFR3 TK inhibitor PD173074. The A431 parental cells acutely treated with Ctx rapidly activated FGFR3 and their concomitant exposure to Ctx and PD173074 resulted in synergistic apoptosis.

CONCLUSION

Cross-suppression of AREG/EREG expression may explain the tight co-expression of AREG and EREG, as well as their tendency to be more highly expressed than other EGFR ligands to determine Ctx efficacy. The positive selection for Ctx-resistant tumour cells exhibiting AREG/EREG cross-suppression may have an important role in the emergence of Ctx resistance. As de-repression of FGFR3 activity rapidly replaces the loss of EGFR-ligand signalling in terms of cell proliferation and survival, combinations of Ctx and FGFR3-targeted drugs may be a valuable strategy to enhance the efficacy of single Ctx while preventing or delaying acquired resistance to Ctx.

背景

除了 KRAS 的突变状态外，表皮生长因子受体 (EGFR) 配体 Amphiregulin (AREG) 和 Epiregulin (EREG) 可能作为西妥昔单抗 (Ctx) 敏感性的真正生物标志物，用于大多数 EGFR 驱动的癌。

方法

采用慢病毒介导的短发夹 RNA 特异性降低野生型 KRAS A431 鳞状细胞癌细胞中 AREG 或 EREG 基因的表达。集落形成试验用于监测 AREG 和 EREG 敲低对 Ctx 疗效的影响。通过定量 ELISA 评估亲本 A431 细胞和适应在 Ctx 存在下生长的 A431 细胞混合群体中 AREG 和 EREG 的蛋白表达水平。磷酸蛋白质组学平台用于测量获得 Ctx 耐药前后 42 种不同受体酪氨酸激酶的相对磷酸化水平。

结果

发现稳定沉默任一配体显著降低了另一个配体的表达。与工程设计产生最小 AREG/EREG 的衍生物相比，具有正常 AREG/EREG 表达水平的亲本 A431 细胞对 Ctx 的生长抑制作用显著增加。用 Ctx 急性处理的亲本 A431 细胞表现出 AREG/EREG 基础表达水平降低。对 Ctx 耐药的 A431 细胞混合群体表达的 AREG/EREG 水平明显较低，并且对 Ctx 的下调作用不敏感。磷酸蛋白质组学筛选发现 Ctx 耐药 A431 细胞中 FGFR3 的显著过度激活，该激活对 FGFR3 TK 抑制剂 PD173074 的细胞毒性和凋亡作用敏感。用 Ctx 急性处理的 A431 亲本细胞迅速激活 FGFR3，同时用 Ctx 和 PD173074 处理导致协同凋亡。

结论

AREG/EREG 表达的交叉抑制可能解释了 AREG 和 EREG 的紧密共表达，以及它们比其他 EGFR 配体更倾向于高度表达以决定 Ctx 疗效的原因。在出现 Ctx 耐药的情况下，对表现出 AREG/EREG 交叉抑制的 Ctx 耐药肿瘤细胞的正向选择可能具有重要作用。由于 FGFR3 活性的去抑制作用迅速取代了 EGFR 配体信号在细胞增殖和存活方面的丧失，因此 Ctx 和 FGFR3 靶向药物的组合可能是增强 Ctx 单一疗效的有价值策略，同时防止或延迟对 Ctx 的获得性耐药。