Preto A, Figueiredo J, Velho S, Ribeiro A S, Soares P, Oliveira C, Seruca R
Institute of Molecular Pathology and Immunology, University of Porto, 4200-465 Porto, Portugal.
J Pathol. 2008 Feb;214(3):320-7. doi: 10.1002/path.2295.
BRAF kinase is a downstream target of KRAS and activates the MAPK pathway. These two molecules are prone to mutations in sporadic microsatellite unstable (MSI) colorectal carcinomas (CRC) and BRAF V600E mutations are inversely associated with oncogenic KRAS mutations. The biological significance of BRAF V600E oncogenic activation is not well established in this type of tumour. We aimed to study proliferation and survival effects induced by BRAF inhibition in MSI CRC cell lines harbouring distinct genetic backgrounds (BRAF V600E or KRAS G13D). Suppression of BRAF in BRAF V600E MSI CRC cell lines by RNA interference significantly inhibited proliferation and induced apoptosis, as demonstrated by BrdU incorporation and TUNEL assay, respectively. No significant differences were seen in proliferation and apoptosis, in cell lines harbouring KRAS G13D, after BRAF inhibition. We further analysed proliferation-associated molecules (pERK1/2, cyclin D1, p27 Kip1) and apoptosis-associated molecules (Bcl-2, Bax, pAkt, pBad, XIAP) in all cell lines. After BRAF down-regulation, we found a more pronounced decrease in ERK1/2 phosphorylation and cyclin D1 expression levels in BRAF-mutated cell lines in comparison to KRAS mutated cells. Upon BRAF inhibition, we also found an increase in p27(Kip1) levels and a more pronounced decrease in the levels of anti-apoptotic protein Bcl-2, specifically in cell lines with BRAF V600E. In conclusion, we have shown that MSI KRAS and BRAF mutant CRC cell lines respond differently to BRAF knockdown. This report provides evidence supporting BRAF as a good target for therapeutic intervention in patients with sporadic MSI CRC harbouring activating mutations in BRAF but not in KRAS.
BRAF激酶是KRAS的下游靶点，可激活丝裂原活化蛋白激酶（MAPK）通路。这两种分子在散发性微卫星不稳定（MSI）结直肠癌（CRC）中容易发生突变，且BRAF V600E突变与致癌性KRAS突变呈负相关。BRAF V600E致癌激活在这类肿瘤中的生物学意义尚未完全明确。我们旨在研究BRAF抑制对具有不同遗传背景（BRAF V600E或KRAS G13D）的MSI CRC细胞系增殖和存活的影响。通过RNA干扰抑制BRAF V600E MSI CRC细胞系中的BRAF，分别通过BrdU掺入和TUNEL检测表明，显著抑制了增殖并诱导了凋亡。BRAF抑制后，在携带KRAS G13D的细胞系中，增殖和凋亡未见明显差异。我们进一步分析了所有细胞系中与增殖相关的分子（pERK1/2、细胞周期蛋白D1、p27 Kip1）和与凋亡相关的分子（Bcl-2、Bax、pAkt、pBad、XIAP）。BRAF下调后，我们发现与KRAS突变细胞相比，BRAF突变细胞系中ERK1/2磷酸化和细胞周期蛋白D1表达水平下降更为明显。BRAF抑制后，我们还发现p27（Kip1）水平升高，抗凋亡蛋白Bcl-2水平下降更为明显，特别是在具有BRAF V600E的细胞系中。总之，我们已经表明，MSI KRAS和BRAF突变的CRC细胞系对BRAF基因敲低的反应不同。本报告提供了证据，支持BRAF作为散发性MSI CRC患者治疗干预的良好靶点，这些患者的BRAF而非KRAS存在激活突变。
