Wickenden J A, Jin H, Johnson M, Gillings A S, Newson C, Austin M, Chell S D, Balmanno K, Pritchard C A, Cook S J
Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Babraham, Cambridge, UK.
Oncogene. 2008 Dec 4;27(57):7150-61. doi: 10.1038/onc.2008.335. Epub 2008 Sep 22.
The RAF-mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase 1/2 (RAF-MEK1/2-ERK1/2) pathway is activated in many human tumours and can protect cells against growth factor deprivation; however, most such studies have relied upon overexpression of RAF or MEK constructs that are not found in tumours. Here we show that expression of the endogenous BRAF(V600E) allele in mouse embryonic fibroblasts from conditional knock-in transgenic mice activates ERK1/2, represses the BH3-only protein BIM and protects cells from growth factor withdrawal. Human colorectal cancer (CRC) cell lines harbouring BRAF(V600E) are growth factor independent for the activation of ERK1/2 and survival. However, treatment with the MEK1/2 inhibitors U0126, PD184352 or the novel clinical candidate AZD6244 (ARRY-142886) overcomes growth factor independence, causing CRC cell death. BIM is de-phosphorylated and upregulated following MEK1/2 inhibition in all CRC cell lines studied and knockdown of BIM reduces cell death, indicating that repression of BIM is a major part of the ability of BRAF(V600E) to confer growth factor-independent survival. We conclude that a single endogenous BRAF(V600E) allele is sufficient to repress BIM and prevent death arising from growth factor withdrawal, and CRC cells with BRAF(V600E) mutations are addicted to the ERK1/2 pathway for repression of BIM and growth factor-independent survival.
RAF-丝裂原活化蛋白激酶激酶1/2-细胞外信号调节激酶1/2（RAF-MEK1/2-ERK1/2）通路在许多人类肿瘤中被激活，并且可以保护细胞免受生长因子剥夺；然而，大多数此类研究依赖于肿瘤中未发现的RAF或MEK构建体的过表达。在这里，我们表明，来自条件性敲入转基因小鼠的小鼠胚胎成纤维细胞中内源性BRAF（V600E）等位基因的表达激活ERK1/2，抑制仅含BH3结构域的蛋白BIM，并保护细胞免受生长因子撤除的影响。携带BRAF（V600E）的人类结肠直肠癌（CRC）细胞系在激活ERK1/2和存活方面不依赖生长因子。然而，用MEK1/2抑制剂U0126、PD184352或新型临床候选药物AZD6244（ARRY-142886）治疗可克服生长因子独立性，导致CRC细胞死亡。在所有研究的CRC细胞系中，MEK1/2抑制后BIM去磷酸化并上调，敲低BIM可减少细胞死亡，这表明抑制BIM是BRAF（V600E）赋予生长因子非依赖性存活能力的主要部分。我们得出结论，单个内源性BRAF（V600E）等位基因足以抑制BIM并防止因生长因子撤除引起的死亡，并且具有BRAF（V600E）突变的CRC细胞对ERK1/2通路成瘾，以抑制BIM和实现生长因子非依赖性存活。
