Division of Nephrology and Transplantation Research Center, Children's Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 2010 Aug 13;285(33):25196-202. doi: 10.1074/jbc.M110.119446. Epub 2010 Jun 16.
The high incidence of cancer and its aggressive progression is a common and major problem in patients receiving immunosuppressive therapy. The calcineurin inhibitors (CNIs) may have protumorigenic effects and can promote the overexpression of several molecules inducing tumor growth. We have recently demonstrated that CNIs can mediate the transcriptional activation of the angiogenic cytokine vascular endothelial growth factor (VEGF) and promote a rapid progression of human renal cancer. Here, we investigated whether the CNI cyclosporine (CsA) and the mTOR inhibitor rapamycin (RAPA) could alter the mRNA stability of VEGF in 786-0 and Caki-1 renal cancer cells. Following actinomycin D treatment, we observed that CsA increased, whereas RAPA decreased the VEGF mRNA stability as observed by real time PCR. It is established that the mRNA-binding protein HuR may play a critical role in VEGF mRNA stability. By using HuR-siRNA, we found that the knockdown of HuR significantly decreased the CNI-induced VEGF mRNA stability. By Western blot analysis, it has been observed that CNI treatment induced the translocation of HuR from the nucleus to the cytoplasm; CNIs also induced the association between HuR and PKC-delta and promoted the phosphorylation of HuR. Finally, we found that the inhibition of PKC-delta using a dominant negative plasmid significantly decreased the CsA-induced cytoplasmic translocation of HuR and VEGF mRNA stability. Together, targeting the pathways that promote CNI-induced transcription as well as the mRNA stability of VEGF might serve as novel therapeutics for the prevention and treatment of cancer in immunosuppressed patients.
在接受免疫抑制治疗的患者中，癌症的高发率和侵袭性进展是一个常见且主要的问题。钙调神经磷酸酶抑制剂（CNIs）可能具有促肿瘤作用，并能促进诱导肿瘤生长的几种分子的过度表达。我们最近证实，CNIs 可以介导血管内皮生长因子（VEGF）等促血管生成细胞因子的转录激活，并促进人肾癌细胞的快速生长。在这里，我们研究了 CNI 环孢菌素（CsA）和 mTOR 抑制剂雷帕霉素（RAPA）是否可以改变 786-0 和 Caki-1 肾癌细胞中 VEGF 的 mRNA 稳定性。在用放线菌素 D 处理后，我们通过实时 PCR 观察到 CsA 增加，而 RAPA 减少了 VEGF mRNA 的稳定性。已确定 mRNA 结合蛋白 HuR 可能在 VEGF mRNA 稳定性中发挥关键作用。通过使用 HuR-siRNA，我们发现 HuR 的敲低显著降低了 CNI 诱导的 VEGF mRNA 稳定性。通过 Western blot 分析，观察到 CNI 处理诱导 HuR 从核转位到细胞质；CNIs 还诱导 HuR 与 PKC-delta 的结合，并促进 HuR 的磷酸化。最后，我们发现使用显性负性质粒抑制 PKC-delta 可显著减少 CsA 诱导的 HuR 细胞质易位和 VEGF mRNA 稳定性。总之，针对促进 CNI 诱导转录以及 VEGF mRNA 稳定性的途径，可能成为免疫抑制患者癌症预防和治疗的新疗法。
