Ras/TGF-beta pathway downstream in liver metastasis of colorectal cancer

在结直肠癌（CRC）中，在近40%的患者中发现K-ras突变，并且其具有预后意义（Onozato W等人，J Surg Oncol，2010）。此外，其在具有突变的K-ras基因的CRC细胞系中的敲低导致细胞增殖和锚定非依赖性生长能力的强烈降低（Shirasawa S，Science，1993），这两者都反映了转移能力。另一方面，在CRC促进步骤中已经报道了聚糖结构的稳健改变，伴随着显著的表型变化，但是没有报道提到K-ras突变和聚糖变化的关系。（材料和方法）我们通过包含41种凝集素的凝集素阵列检测了体细胞敲除K-ras基因的CRC细胞系（DLD 1和HCT 116）的聚糖变化，以阐明K-ras突变诱导的聚糖变化是否在CRC促进中起关键作用。（结果）（1）在DLD 1中，K-ras基因敲除细胞（DKO-3、DKS-8）和野生型细胞（DLD 1、DKS-5）的MAL、MPA、UEA-Ⅰ和TJA-Ⅱ信号均明显减弱。(2)在HCT 116中，我们将HCT 116野生型基因型细胞（HCT 116，Hk 2 -10）与敲除突变K-ras基因的细胞（Hke-3）进行比较，MAL和MPA均显著下降。(3)在这两种细胞系中，可以与MAL和MPA结合的聚糖的表达变化与凝集素印迹的结果一致，并且证实通过去除突变的K-ras基因通常改变两种凝集素信号。结论K-ras基因突变可能参与了大肠癌的重要表型改变，并伴有唾液酸识别异常。

(Background) In colorectal cancer(CRC), K-ras mutation is found in nearly 40% of the patients, and it is of prognostic significance(Onozato W et al, J Surg Oncol, 2010). Moreover, its knockdown in CRC cell lines with mutated K-ras gene results in robust reduction of ability of cell proliferation and anchorage independent growth(Shirasawa S, Science, 1993), both of which reflect metastatic ability. On the other hand, robust alterations of glycan structures have been reported in CRC promotion steps, accompanied by remarkable phenotypic changes, however there is no report that mentions the relationship of K-ras mutation and glycan change.(Materials and Methods) We examined glycan change of CRC cell lines(DLD1 and HCT116) somatically knocked out for K-ras gene by lectin array including 41 lectins to elucidate whether K-ras mutation-induced glycan changes play a critical role in CRC promotion.(Result)(1) In DLD1, we compared DLD1wild type genotype cells(DLD1, DKS-5) with those that were knocked out for mutated K-ras gene(DKO-3, DKS-8), and the signals of MAL, MPA, UEA-I, and TJA-II were remarkably decreased in the knockdown cells.(2) In HCT116, we compared HCT116 wild type genotypic cells(HCT116, Hk2-10) with those that were knocked out for the mutated K-ras gene(Hke-3), and both MAL and MPA were remarkably declined.(3) In both cell lines, expression changes of glycans which can bind with MAL and MPA were consistent with the results of the lectin blotting, and both lectin signals were confirmed to be commonly altered by removing the mutated K-ras gene.(Conclusion) Mutated K-ras may be involved in critical phenotype change of CRC, accompanied by abnormal sialic acid recognition.