Calcineurin inhibitor-induced and Ras-mediated overexpression of VEGF in renal cancer cells involves mTOR through the regulation of PRAS40.
Calcineurin inhibitor-induced and Ras-mediated overexpression of VEGF in renal cancer cells involves mTOR through the regulation of PRAS40.
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肾癌细胞中钙调磷酸酶抑制剂诱导和 Ras 介导的 VEGF 过度表达涉及 mTOR 通过 PRAS40 的调节。
DOI：
10.1371/journal.pone.0023919
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Pal S
中科院分区：
文献类型：
--
作者：
Basu A;Banerjee P;Contreras AG;Flynn E;Pal S
恶性肿瘤是免疫抑制剂治疗患者的主要问题。我们已经证明，使用钙调磷酸酶抑制剂（CNIs）治疗可以诱导原致癌Ras的激活，并可能通过血管内皮生长因子（VEGF）的过度表达促进人类肾癌的快速进展。有趣的是，我们发现cni诱导的VEGF过表达和癌细胞增殖被雷帕霉素治疗抑制，这表明哺乳动物雷帕霉素靶蛋白（mTOR）通路可能参与了这一致瘤过程。在这里，我们研究了mTOR通路在介导CNI和ras诱导的人肾癌细胞中VEGF过表达中的作用（786-0和Caki-1）。通过启动子荧光素酶检测，我们发现（使用siRNA）敲除raptor显著降低了cni诱导的VEGF启动子活性，提示mTOR复合物1 （mTORC1）在cni诱导的VEGF转录中的作用。已知mTOR在其负调节因子PRAS40磷酸化后被激活，PRAS40是mTORC1的一部分。我们观察到，CNI处理和H-Ras的激活（通过转染活性H-Ras质粒）显著增加了PRAS40的磷酸化，而使用Ras的显性阴性质粒转染细胞，显著降低了PRAS40的磷酸化。蛋白激酶C (PKC)-ζ和PKC-δ是cni诱导肿瘤通路的关键中间信号分子，与PRAS40形成复合物；我们发现CNI处理增加了PRAS40和PKC之间的复合物形成，特别是(PKC)-ζ。使用药理学抑制剂抑制PKC活性可显著降低h - ras诱导的PRAS40磷酸化。在肾癌细胞中，PRAS40过表达可显著下调CNI-和h - ras诱导的VEGF转录激活。最后，我们观察到CNI处理增加了体内肾肿瘤组织中phoho - pras40的表达。总之，在cni诱导的VEGF过表达和肾癌进展中，PRAS40的磷酸化对于mTOR的激活至关重要。
Malignancy is a major problem in patients treated with immunosuppressive agents. We have demonstrated that treatment with calcineurin inhibitors (CNIs) can induce the activation of proto-oncogenic Ras, and may promote a rapid progression of human renal cancer through the overexpression of vascular endothelial growth factor (VEGF). Interestingly, we found that CNI-induced VEGF overexpression and cancer cell proliferation was inhibited by rapamycin treatment, indicating potential involvement of the mammalian target of rapamycin (mTOR) pathway in this tumorigenic process. Here, we examined the role of mTOR pathway in mediating CNI- and Ras-induced overexpression of VEGF in human renal cancer cells (786-0 and Caki-1). We found that the knockdown of raptor (using siRNA) significantly decreased CNI-induced VEGF promoter activity as observed by promoter-luciferase assay, suggesting the role of mTOR complex1 (mTORC1) in CNI-induced VEGF transcription. It is known that mTOR becomes activated following phosphorylation of its negative regulator PRAS40, which is a part of mTORC1. We observed that CNI treatment and activation of H-Ras (through transfection of an active H-Ras plasmid) markedly increased the phosphorylation of PRAS40, and the transfection of cells using a dominant-negative plasmid of Ras, significantly decreased PRAS40 phosphorylation. Protein kinase C (PKC)-ζ and PKC-δ, which are critical intermediary signaling molecules for CNI-induced tumorigenic pathway, formed complex with PRAS40; and we found that the CNI treatment increased the complex formation between PRAS40 and PKC, particularly (PKC)-ζ. Inhibition of PKC activity using pharmacological inhibitor markedly decreased H-Ras-induced phosphorylation of PRAS40. The overexpression of PRAS40 in renal cancer cells significantly down-regulated CNI- and H-Ras-induced VEGF transcriptional activation. Finally, it was observed that CNI treatment increased the expression of phosho-PRAS40 in renal tumor tissues in vivo. Together, the phosphorylation of PRAS40 is critical for the activation of mTOR in CNI-induced VEGF overexpression and renal cancer progression.
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