Abstract
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.
Highlights
Recent improvements in immunosuppressive therapies have significantly reduced the incidence of acute rejection of allografts, and increased the survival of transplant patients [1,2]
We have found that calcineurin inhibitors (CNIs) can activate the proto-oncogenic H-Ras in human renal cancer cells [24]; and we have shown that CNI-induced vascular endothelial growth factor (VEGF) overexpression is mediated through the activation of protein kinase C (PKC)-f and Protein kinase C (PKC)-d [22,23], which are potential downstream targets of Ras [25]
We show that CNI-induced and Ras-PKCmediated VEGF overexpression can be channeled through the mTOR complex1 (mTORC1) signaling pathway, and this is mediated through the regulation of prolinerich Akt substrate of 40 kDa (PRAS40)
Summary
Recent improvements in immunosuppressive therapies have significantly reduced the incidence of acute rejection of allografts, and increased the survival of transplant patients [1,2]. These agents may contribute to higher rates of mortality due to an increased risk of cancer [3,4,5,6]. Calcineurin inhibitors (CNIs) are excellent immunosuppressive agents to inhibit allograft rejection; they may promote the growth of different tumors [9,10,11,12]. Guba et al [19] suggested that CsA treatment can induce the expression of angiogenic cytokines
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