Subramaniam Venkateswaran, Vincent Isabella R, Gardner Helena, Chan Emily, Dhamko Helena, Jothy Serge
Department of Laboratory Medicine, St. Michael's Hospital, 30 Bond Street, Toronto, ON, Canada.
Exp Mol Pathol. 2007 Oct;83(2):207-15. doi: 10.1016/j.yexmp.2007.04.008. Epub 2007 May 13.
Colon cancer is among the leading causes of cancer death in North America. CD44, an adhesion and antiapoptotic molecule is overexpressed in colon cancer. Cofilin is involved in the directional motility of cells. In the present study, we looked at how CD44 might modulate cell migration in human colon cancer via cofilin. We used a human colon cancer cell line, HT29, which expresses CD44, HT29 where CD44 expression was knocked down by siRNA, SW620, a human colon cancer cell line which does not express CD44, stably transfected exons of CD44 in SW620 cells and the colon from CD44 knockout and wild-type mouse. Western blot analysis of siRNA CD44 lysates showed increased level of AKT phosphorylation and decreased level of cofilin expression. Similar results were also observed with SW620 cells and CD44 knockout mouse colon lysates. Experiments using the AKT phosphorylation inhibitor LY294002 indicate that AKT phosphorylation downregulates cofilin. Immunoprecipitation studies showed CD44 complex formation with Lyn, providing an essential link between CD44 and AKT phosphorylation. LY294002 also stabilized Lyn from phosphorylated AKT, suggesting an interaction between Lyn and AKT phosphorylation. Immunocytochemistry showed that cofilin and Lyn expression were downregulated in siRNA CD44 cells and CD44 knockout mouse colon. siRNA CD44 cells had significantly less migration compared to HT29 vector. Given the well-defined roles of CD44, phosphorylated AKT in apoptosis and cancer, these results indicate that CD44-induced cell migration is dependent on its complex formation with Lyn and its consequent regulation of AKT phosphorylation and cofilin expression.
结肠癌是北美癌症死亡的主要原因之一。CD44是一种黏附及抗凋亡分子，在结肠癌中过度表达。丝切蛋白参与细胞的定向运动。在本研究中，我们探究了CD44如何通过丝切蛋白调节人结肠癌中的细胞迁移。我们使用了表达CD44的人结肠癌细胞系HT29、用小干扰RNA（siRNA）敲低CD44表达的HT29、不表达CD44的人结肠癌细胞系SW620、在SW620细胞中稳定转染CD44外显子以及来自CD44基因敲除小鼠和野生型小鼠的结肠。对siRNA CD44裂解物的蛋白质免疫印迹分析显示，AKT磷酸化水平升高，丝切蛋白表达水平降低。在SW620细胞和CD44基因敲除小鼠结肠裂解物中也观察到了类似结果。使用AKT磷酸化抑制剂LY294002的实验表明，AKT磷酸化会下调丝切蛋白。免疫沉淀研究显示CD44与Lyn形成复合物，这为CD44与AKT磷酸化之间提供了关键联系。LY294002还使Lyn免受磷酸化AKT的影响，表明Lyn与AKT磷酸化之间存在相互作用。免疫细胞化学显示，在siRNA CD44细胞和CD44基因敲除小鼠结肠中，丝切蛋白和Lyn的表达下调。与HT29载体相比，siRNA CD44细胞的迁移明显减少。鉴于CD44、磷酸化AKT在细胞凋亡和癌症中的明确作用，这些结果表明，CD44诱导的细胞迁移依赖于其与Lyn形成复合物以及随之对AKT磷酸化和丝切蛋白表达的调节。
