Although calcineurin inhibitors are excellent immunosuppressive agents to inhibit allograft rejection, they may promote the growth of different tumors (7
). In this study, we define a mechanism in human cancer cells by which CsA can promote tumor growth through VEGF overexpression and angiogenesis, having direct relevance for the development of post-transplantation cancer. Although not shown, we have found that FK506, another calcineurin inhibitor (25
), also induces VEGF overexpression in these cells.
Some previous studies have suggested that CsA may have both pro-angiogenic and anti-angiogenic effects. CsA may inhibit VEGF-induced angiogenesis either alone or in presence of some angiogenesis inhibitors (22
). In contrast, Shihab et al (42
) demonstrated that during CsA-induced nephrotoxicity, VEGF and its receptors are overexpressed. Gottsch et al (43
) observed that CsA can promote angiogenesis in corneal ulcers. However, CsA may mediate completely opposite effects on the same signaling pathway in two different cell types (44
We suggest some possible mechanisms for the effect of CsA and other calcineurin inhibitors on VEGF overexpression. One possibility is that while CsA treatment blocks the calcineurin/NFAT-signaling pathway, it may also suppress negative regulators of VEGF expression and angiogenesis as proposed by others (20
). Thus, in the absence of any negative regulator, VEGF may be overexpressed in transplant patients, and it may induce the growth of micro tumors under immune suppressed conditions. Another possibility is that CsA-induced VEGF expression may be an indirect effect through TGF-β. CsA is a potent inducer of TGF-β (7
), and it has been reported that TGF-β can stimulate VEGF transcription (45
). However, CsA can also directly promote VEGF transcriptional activation as demonstrated in this study.
Apart from inhibiting NFAT, the calcineurin inhibitors may also regulate other signaling molecules involved in VEGF expression. Pan et al (46
) recently showed that CsA inhibits carabin, a novel endogenous inhibitor of calcineurin. Carabin may also inhibit the Ras signaling pathway, suggesting CsA can activate Ras (a known inducer of VEGF) through the inhibition of carabin. Cho et al (47
) reported that while CsA or FK506 suppress calcineurin, they may also unleash the PKC signaling pathways to promote the expression of the linker for activation of T cells (LAT). In this study, we have shown for the first time that CsA activates PKCζ and PKCδ isoforms in human renal cancer cells. Our observations show that blockade of the PKCζ and PKCδ pathways inhibits CsA-induced VEGF
transcriptional activation. We have also found that CsA can promote the association of PKCζ and PKCδ with the transcription factor Sp1, and can induce Sp1 DNA-binding activity. We have previously reported that PKCζ can phosphorylate Sp1 (27
), and thus we suggest that CsA may activate Sp1 through its association with PKCζ.
It is established that HIF-α and pVHL play major roles in the regulation of VEGF
in renal cancer. The 786-0 cells lack VHL
); and in the absence of pVHL, HIF-2α is stabilized in these cells (36
). Our findings suggest that HIF-2α is not involved in CsA-mediated VEGF
transcription. However, others have demonstrated that CsA may regulate gene expression through either degradation of HIF-1α or prevention of HIF-1α protein accumulation (48
). In this study, we have found that CsA-induced VEGF
transcription is mediated primarily through the PKCζ-Sp1 and PKCδ-Sp1 pathways. We have also observed that CsA-mediated VEGF
transcription is partially inhibited by wt-pVHL, which prevents the association between PKCζ and Sp1. It has been shown that pVHL can also directly bind to Sp1, and may prevent Sp1-mediated gene transcription (26
). We and others have previously reported that pVHL can bind to atypical PKC isoforms and either prevent their membrane translocation (34
) or promote their degradation (50
), supporting our present findings. The effect of CsA on VEGF
expression was smaller in pVHL-intact cells compared with pVHL-deficient cells. Thus, loss of pVHL, as occurs early in the course of renal cell carcinoma development, may sensitize a cell to CsA-induced growth promoting effects. However, although pVHL appears to be a critical regulator of CsA-mediated VEGF
expression, we cannot rule out a pVHL-independent pathway that may involve other PKC isoforms, like PKCδ.
In summary, the mechanism(s) underlying the development of post-transplantation cancer should be thoroughly evaluated such that select agents can be used to target cancer development. Our in vitro and in vivo studies in this report clearly demonstrate the role of overexpressed VEGF in the development of CsA-induced post-transplantation cancer. Thus, targeting the pathways that promote VEGF overexpression in response to calcineurin inhibitors might serve as novel therapeutics for the prevention and treatment of post-transplantation cancer.