Kawasaki Hideyoshi, Saotome Takuya, Usui Tatsuya, Ohama Takashi, Sato Koichi
Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
Laboratory of Veterinary Toxicology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
Oncol Rep. 2017 May;37(5):3128-3136. doi: 10.3892/or.2017.5520. Epub 2017 Mar 22.
In colorectal cancer, gain-of-function mutations in KRas play a critical role in malignant transformation. Tumor growth in colorectal cancer is known to be promoted by the intestinal myofibroblasts (IMFs) that localize adjacent to the cancer cells, but the mechanisms of interaction between KRas-mutated cancer cells and the myofibroblasts remain unclear. Here, we investigated the effects of KRas-mutated cells on the behavior of myofibroblasts by using mouse primary IMFs and cells of an IMF cell line (LmcMF) and a mouse colon epithelial cell line (aMoC1). Conditioned medium (CM) was collected from aMoC1 cells overexpressing a control vector or KRasV12 vector (KRasV12-CM), and the effects of KRasV12-CM on IMFs were analyzed by performing proliferation assays, wound-healing assays, Boyden chamber assays, and western blotting. Whereas KRasV12-CM exerted little effect on the differentiation and proliferation of primary IMFs, the CM promoted migration of both primary IMFs and LmcMF cells. In KRasV12-overexpressing aMoC1 cells, mRNA expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was higher than in mock-transfected aMoC1 cells, and HB-EGF promoted the migration of primary IMFs and LmcMF cells. Moreover, KRasV12-CM-induced IMF migration was suppressed by dacomitinib, an inhibitor of HB-EGF receptors. Notably, in LmcMF cells, both KRasV12-CM and HB-EGF activated extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK), whereas KRasV12-CM-induced migration of IMFs was suppressed following treatment with either an ERK inhibitor (FR180204) or a JNK inhibitor (SP600125). These results suggest that HB-EGF secreted from KRas-mutated colorectal cancer cells promotes IMF migration through ERK and JNK activation, which, in turn, could support cancer progression.
在结直肠癌中，KRas功能获得性突变在恶性转化中起关键作用。已知结直肠癌中的肿瘤生长是由定位于癌细胞附近的肠道肌成纤维细胞（IMF）促进的，但KRas突变癌细胞与肌成纤维细胞之间的相互作用机制仍不清楚。在这里，我们使用小鼠原代IMF以及IMF细胞系（LmcMF）和小鼠结肠上皮细胞系（aMoC1）的细胞，研究了KRas突变细胞对肌成纤维细胞行为的影响。从过表达对照载体或KRasV12载体的aMoC1细胞中收集条件培养基（CM），并通过进行增殖测定、伤口愈合测定、博伊登室测定和蛋白质印迹分析来分析KRasV12-CM对IMF的影响。虽然KRasV12-CM对原代IMF的分化和增殖影响很小，但该CM促进了原代IMF和LmcMF细胞的迁移。在过表达KRasV12的aMoC1细胞中，肝素结合表皮生长因子样生长因子（HB-EGF）的mRNA表达高于mock转染的aMoC1细胞，并且HB-EGF促进了原代IMF和LmcMF细胞的迁移。此外，达可替尼（一种HB-EGF受体抑制剂）抑制了KRasV12-CM诱导的IMF迁移。值得注意的是，在LmcMF细胞中，KRasV12-CM和HB-EGF均激活了细胞外信号调节激酶（ERK）和c-Jun氨基末端激酶（JNK），而在用ERK抑制剂（FR180204）或JNK抑制剂（SP600125）处理后，KRasV12-CM诱导的IMF迁移受到抑制。这些结果表明，KRas突变的结直肠癌细胞分泌的HB-EGF通过ERK和JNK激活促进IMF迁移，这反过来可能支持癌症进展。
