Hill A, McFarlane S, Mulligan K, Gillespie H, Draffin J E, Trimble A, Ouhtit A, Johnston P G, Harkin D P, McCormick D, Waugh D J J
Centre for Cancer Research and Cell Biology, Queens University Belfast, Belfast City Hospital, Belfast, Northern Ireland.
Oncogene. 2006 Oct 5;25(45):6079-91. doi: 10.1038/sj.onc.1209628. Epub 2006 May 1.
Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).
利用经过验证的四环素（tet）调控的MCF7原始细胞（MCF7F）表达系统来调节CD44标准型（CD44s）的表达，我们报告了CD44s以及透明质酸（HA）/CD44s信号传导的一个新转录靶点EMS1/皮层肌动蛋白在支持乳腺癌转移中的功能重要性。在功能实验中，tet调控的CD44s诱导增强了MCF7F细胞通过补充HA的基质胶的迁移和侵袭能力。通过表达谱分析确定EMS1/皮层肌动蛋白是HA/CD44信号传导的一个新转录靶点，在一系列乳腺癌细胞系中的定量PCR和免疫印迹实验验证了这种关联。结果表明，CD44促进EMS1/皮层肌动蛋白转录的机制基础依赖于NFκB机制，因为对IκB激酶-2的药理抑制或p65 Rel A表达的抑制减弱了CD44诱导的皮层肌动蛋白mRNA转录水平的增加。在低侵袭性、缺乏CD44的MCF7F和T47D细胞中过表达c-myc标记的小鼠皮层肌动蛋白构建体增强了它们的侵袭能力。此外，在表达CD44的MCF7F-B5和MDA-MB-231乳腺癌细胞中使用RNAi选择性抑制皮层肌动蛋白，证明了皮层肌动蛋白对CD44s促进转移的功能重要性，结果显示这导致CD44促进的侵袭以及CD44促进的与骨髓内皮细胞（BMECs）的黏附减弱。
