Zuo Yufeng, Wu Yuexiu, Wehrli Bret, Chakrabarti Subrata, Chakraborty Chandan
Department of Pathology, University of Western Ontario, London, Ontario, Canada.
J Cell Biochem. 2015 Jan;116(1):124-32. doi: 10.1002/jcb.24950.
Members of Rho family GTPases including Cdc42 are known to play pivotal roles in cell migration. Cell migration is also known to be regulated by many protein kinases. Kinetworks KPSS 11.0 phospho-site screening of Cdc42-silenced Hs578T breast cancer cells revealed most dramatic change in ERK5 MAP kinase. In the present study, we set out to determine the relationship between Cdc42 and ERK5 and its significance in breast cancer cell migration and invasion. Specific siRNAs were used for knocking down Cdc42 or ERK5 in breast cancer cells. Increased ERK5 phosphorylation in breast cancer cells was achieved by infection of constitutively active MEK5 adenovirus. The cells were then subjected to cell migration or invasion assay without the presence of serum or any growth factor. We found that Cdc42 negatively regulated phosphorylation of ERK5, which in turn exhibited an inverse relationship with migration and invasiveness of breast cancer cells. To find out some in vivo relevance of the results of our in vitro experiments we also examined the expression of ERK5 in the breast cancer tissues and their adjacent normal control tissues by real-time RT-PCR and immunocytochemistry. ERK5 expression was found to be reduced in breast cancer tissues as compared with their adjacent uninvolved mammary tissues. Therefore, Cdc42 may promote breast cancer cell migration and invasion by inhibiting ERK5 phosphorylation and ERK5 expression may be inversely correlated with the progression of some breast tumors.
包括Cdc42在内的Rho家族GTP酶成员在细胞迁移中发挥关键作用。细胞迁移也受多种蛋白激酶调控。对Cdc42沉默的Hs578T乳腺癌细胞进行Kinetworks KPSS 11.0磷酸化位点筛选，结果显示ERK5丝裂原活化蛋白激酶变化最为显著。在本研究中，我们旨在确定Cdc42与ERK5之间的关系及其在乳腺癌细胞迁移和侵袭中的意义。使用特异性小干扰RNA（siRNA）敲低乳腺癌细胞中的Cdc42或ERK5。通过感染组成型活性MEK5腺病毒使乳腺癌细胞中ERK5磷酸化增加。然后在无血清或任何生长因子的情况下对细胞进行细胞迁移或侵袭试验。我们发现Cdc42负向调节ERK5的磷酸化，而ERK5磷酸化又与乳腺癌细胞的迁移和侵袭呈负相关。为了探究我们体外实验结果在体内的相关性，我们还通过实时逆转录聚合酶链反应（RT-PCR）和免疫细胞化学检测了乳腺癌组织及其相邻正常对照组织中ERK5的表达。结果发现，与相邻未受累乳腺组织相比，乳腺癌组织中ERK5表达降低。因此，Cdc42可能通过抑制ERK5磷酸化促进乳腺癌细胞迁移和侵袭，且ERK5表达可能与某些乳腺肿瘤的进展呈负相关。
