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Cancer is an increasing and major problem following solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors. We propose that the effect of calcineurin inhibitors on the expression of vascular endothelial growth factor (VEGF) leads to an angiogenic milieu that favors tumor growth. Here, we used 786-0 human renal cancer cells to investigate the effect of Cyclosporine (CsA) on VEGF expression. Utilizing a full-length VEGF promoter-luciferase construct, we found that CsA markedly induced VEGF transcriptional activation through the PKC signaling pathway, specifically involving PKCζ and PKCδ isoforms. Moreover, CsA promoted the association of PKCζ and PKCδ with the transcription factor Sp1 as observed by immunoprecipitation assays. Using promoter deletion constructs, we found that CsA-mediated VEGF transcription was primarily Sp1-dependent. Furthermore, CsA-induced and PKC-Sp1-mediated VEGF transcriptional activation was partially inhibited by pVHL. CsA also promoted the progression of human renal tumors in vivo, where VEGF is overexpressed. Finally, to evaluate the in vivo significance of CsA-induced VEGF overexpression in terms of post-transplantation tumor development, we injected CT26 murine carcinoma cells (known to form angiogenic tumors) into mice with fully MHC mismatched cardiac transplants. We observed that therapeutic doses of CsA increased tumor size, VEGF mRNA expression, and also enhanced tumor angiogenesis. However, co-administration of a blocking anti-VEGF antibody inhibited this CsA-mediated tumor growth. Collectively, these findings define PKC-mediated VEGF transcriptional activation as a key component in the progression of CsA-induced post-transplantation cancer.
Cancer is a common and major problem following organ transplantation (1-3). Malignant tumors develop in 15-20% of graft recipients within 10 years, and contribute to morbidity and mortality in these patients (4). There are three major ways by which malignant tumors may develop in transplant recipients: de-novo occurrence, recurrent malignancy, or transmission of malignancy from the donor (4). Some forms of cancer (e.g., cancers of kidney and skin, and lymphoma) increase markedly after kidney transplantation as compared to the general population or comparable patients on dialysis (1, 2). De-novo non-lymphoid cancers are a major cause of late death in liver transplant recipients (5). Thus, the prevention of cancer should be a major goal of future therapy following organ transplantation.
Immunosuppressive agents used in transplant recipients may play a critical role in the tumorigenic process. These agents are thought to compromise immune surveillance mechanism(s) for tumor cells (2, 6, 7), and/or interfere with normal DNA repair mechanisms (1). In particular, use of calcineurin inhibitors, including cyclosporine (CsA) and FK506, has significantly increased the incidence of post-transplantation cancer (7, 8). Hojo et al (7) demonstrated that CsA promotes cancer progression by direct cellular effect(s) through transforming growth factor-β (TGF-β) production that is independent of its effect on the host’s immune system. Similarly, FK506 has been shown to promote the proliferation of tumor cells through TGF-β (9). Koehl et al (10) reported that CsA augments the growth of tumor cells in vivo in doses that are sufficient to inhibit allograft rejection; and although they suggested a role of TGF-β in this process, they could not rule out the possible involvement of other angiogenic factors. Guba et al (11) suggested that CsA may induce the expression of an angiogenic cytokine, like vascular endothelial growth factor (VEGF), but did not demonstrate its function in the development of post-transplantation cancer.
VEGF is the most potent angiogenic factor described to date, playing important roles in tumor development (12, 13). It is expressed by tumor cells, endothelial cells, and a variety of cell types (13). VEGF is expressed in significant amounts in transplant recipients. Induced VEGF expression may mediate inflammatory cell trafficking into allografts (14, 15), and may promote both acute and chronic allograft rejection (16, 17). It is possible that high levels of VEGF expression in transplant recipients may provide an environment in which micro tumors can grow more efficiently due to enhanced VEGF-induced angiogenesis. The effect of CsA on VEGF expression may therefore be a risk factor for the development of post-transplantation cancer.
The calcineurin complex consists of three subunits, the catalytic-A, the regulatory-B, and calmodulin (18). Cellular Ca2+ binds to both calmodulin and the B subunit, displacing the inhibitory C-terminal peptide from the active site of the catalytic-A subunit (19). This process activates the catalytic subunit for its function as serine/threonine phosphatase, resulting in the activation of the nuclear factor of activated T cells (NFAT) family of transcription factors (20). While NFAT is functional in many cell types, it has been best studied in T cell activation responses, where it induces different cytokines, like IL-2 (21). It has also been suggested that the NFAT pathway may either induce or repress the expression of several angiogenic factors, including VEGF (20, 22-24). Thus, the calcineurin pathway may exert both positive and negative regulatory signals on different angiogenic molecules. The calcineurin inhibitor CsA binds to cyclophylin, a cytoplasmic protein, and the resultant complex binds to the regulatory-B subunit of calcineurin and prevents the activation of NFAT (25). This process may alter the regulatory switch for VEGF-induced angiogenesis, resulting in increased VEGF expression and tumor angiogenesis. However, the molecular mechanism by which CsA may mediate VEGF overexpression is completely unknown.
In the present study, we demonstrate that CsA can directly promote the transcriptional activation of VEGF in human renal cancer cells, involving the protein kinase C (PKC) signaling pathway, particularly the atypical and novel PKC isoforms. We also observe that CsA augments tumor growth following cardiac transplantation in vivo, and that this effect is in part dependent on increased VEGF expression.
Cyclosporine (CsA) (Novartis, East Hanover, NJ) and FK506 (Astellas, Deerfield, IL) were purchased from Children’s Hospital Boston pharmacy. The MEKK inhibitor PD98059, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, the generalized PKC inhibitor Calphostin C and the classical PKC inhibitor Go6976 were obtained from Calbiochem (La Jolla, CA). The small interfering RNA (siRNA) for HIF-2α and its control were purchased from Qiagen (Valencia, CA).
The development of our blocking anti-murine VEGF antibody (by DMB) was performed using reagents and techniques similar to those previously described (14). Briefly, noble rats were immunized with the N-terminal amino acid sequence of murine VEGF (CAPTTEGEQKSHEVIKFMDVYQRSY) coupled with keyhole limpet hemocyanin (KLH) using the maleimidobenzyl-N-hydroxylsuccinimide ester (MBS) crosslinker. Hybridoma fusion products were generated according to standard protocols, and were screened based on their anti-VEGF (peptide) optical density reading in ELISA. Secondary screens demonstrated two IgG producing clones, 2G11-2A05 and 1F07-1C02, which were subcloned and purified. In Western blots, anti-VEGF 2G11-2A05 bound murine VEGF with high affinity, producing a band of appropriate size similar to that using a commercially available polyclonal rabbit anti-human/mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA). We also found that anti-VEGF monoclonal antibody 2G11-A05 blocked murine VEGF-induced proliferation of endothelial cells in vitro using an assay previously described (14), and that 2G11-A05 neutralized VEGF function in vivo in our standardized VEGF-induced angiogenesis assay (14).
The human renal cancer cell lines (786-0 and Caki-1) and the murine colon adenocarcinoma cell line (CT26) were obtained from American Type Culture Collection, Manassas, VA. The cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT). 786-0 clonal cell lines stably transfected with either pFLAG-CMV2 (Neo cells, containing empty vector with neo cassette), or pFLAG-CMV2-VHL (VHL cells, containing wt-VHL) were grown in complete medium supplemented with G418 (0.5 mg/ml) (26). Human renal proximal tubular epithelial cells (TEC) were purchased from Clonetics (Walkersville, MD) and were cultured in complete epithelial medium (REGM BulletKit, MD).
A 2.6-kb VEGF promoter-luciferase construct in pGL2 basic vector (Promega, Madison, WI), containing the full-length VEGF promoter sequence (base pairs −2361 to +298 relative to the transcription start site), and the two deletion constructs (0.35- and 0.07-kb) of the 2.6-kb VEGF promoter were used in transient transfection assays (26, 27). The kinase inactive PKCζ plasmid (PKCζ KW) was a generous gift from Alex Toker (Beth Israel Deaconess Medical Center, Boston, MA) (27). The kinase inactive PKCδ plasmid (PKCδ KR) was a generous gift from Rakesh Dutta (Dana Farber Cancer Institute, Boston, MA) (27).
Cells were plated at 2 × 105 cells/well in 6-well plates, and were transfected with expression plasmids using the Effectene transfection reagent (Qiagen) (28). The total amount of transfected plasmid DNA was normalized using a control empty expression vector. Transfection efficiency was determined by co-transfection of the β-galactosidase gene and by measurement of β-galactosidase activity. For luciferase assays, cells were harvested 24 hours after transfection, and luciferase activity was measured using a standard assay kit (Promega) in a luminometer. The relative luciferase activity units were calculated as (light emission from experimental sample – light emission of lysis buffer alone)/micrograms of cellular protein in the sample (29).
Immunoprecipitations were performed with 0.5 mg of total protein at antibody excess using anti-Sp1 (Santa Cruz Biotechnology). Immunocomplexes were captured with protein A-sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ), and bead-bound proteins were subjected to Western blot analysis using either anti- PKCζ or anti- PKCδ (Santa Cruz Biotechnology).
Protein samples were run on SDS-polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane (NEN Life Sciences Product Inc, Boston, MA) (28). The membranes were incubated with anti-VEGF/anti-β-actin, anti-phospho PKC ζ/δ, anti-total PKC ζ/δ, anti-HIF-2α or anti-VHL (Santa Cruz Biotechnology), and subsequently incubated with peroxidase-linked secondary antibody. The reactive bands were detected by chemiluminescence (Pierce, Rockford, IL).
PKC activity was assayed in the presence of phosphatidylserine by measuring the incorporation of 32P into histone following standard methodology (27).
Nuclear extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA). EMSA was performed using a lightshift chemiluminescent EMSA kit (Pierce), utilizing the biotin-labeled Sp1 oligonucleotide duplex (Integrated DNA Technologies, Coralville, IA). The unlabeled Sp1 consensus oligonucleotide (for competition) and Sp1 antibody (for supershift) were purchased from Santa Cruz Biotechnology.
The concentrations of human and mouse VEGF in tissue culture supernatants were determined using Quantikine VEGF immunoassay kits (R & D Systems, Minneapolis, MN).
Human renal cancer cells (786-0) were injected subcutaneously in immunodeficient (nu/nu) mice. To evaluate the growth of tumors in allograft recipients, murine tumor cells (CT26) were injected subcutaneously into BALB/c mice one week before heart transplantation. Tumor volume was measured using a digital caliper at regular intervals. The volume was estimated by following a standard method (10), using the formula V = П / 6 × a2 × b, where a was the short and b the long tumor axis. Mice were sacrificed at designated times following injection or if complications occurred, which included signs of inactivity, cachexia, or decreased responsiveness.
BALB/c mice were used as recipients of fully MHC-mismatched C57BL/6 donor hearts. Vascularized intra-abdominal heterotopic heart transplantation was performed as described (14). Donor hearts were monitored daily (by measuring palpation) for the development of rejection.
Total RNA was prepared using the RNeasy isolation kit (Qiagen), and cDNA was synthesized using cloned AMV first-strand synthesis kit (Invitrogen, Carlsbad, CA). To analyze VEGF expression, real time PCR was performed using the Assays-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). As an internal control, GAPDH mRNA was amplified. Gene-specific primers were obtained from Applied Biosystems. Ct value (the cycle number at which emitted fluorescence exceeded an automatically determined threshold) for gene of interest (VEGF) was corrected by the Ct value for GAPDH and expressed as ΔCt. Data were reported as fold change in mRNA amount, which was calculated as follows: (fold change) = 2X (where X = ΔCt for control group - ΔCt for experimental group).
To stain the tumor vessels, tissue sections were incubated first with anti-mouse-CD31 (BD Pharmingen, San Diego, CA), and then with a species-specific horseradish peroxidase-conjugated secondary antibody using standard protocol (28). Specimens were developed in 3-amino ethylcarbazole and were counterstained in Gill’s hematoxylin. Vessel densities were quantified by grid-counting method at 400X magnification.
Statistical evaluation for data analysis was determined by Student’s t test. Differences with p<0.05 were considered statistically significant.
We first evaluated whether Cyclosporine (CsA) can induce VEGF expression in cancer cells. As renal cancer is common among transplant recipients (1-3), we made use of an established human renal cancer cell line (786-0) for our in vitro studies. The 786-0 cells were treated with either increasing concentrations of CsA or vehicle, and the expression of VEGF was examined by Western blot analysis. As shown in the Figure-1A, CsA significantly induced VEGF protein expression in these cells. We then evaluated whether CsA can regulate VEGF promoter activity in 786-0 and also in normal renal tubular epithelial cells (TEC). The cells were transiently transfected with a 2.6-kb full-length VEGF promoter-luciferase construct, and treated with either increasing concentrations of CsA or vehicle. The effect of CsA on VEGF promoter activity was assessed by luciferase assay. As shown in Figure-1B, CsA significantly induced VEGF transcriptional activation in a dose-dependent manner as compared to vehicle-treated controls in 786-0 cells and in TEC. Thus, CsA may regulate VEGF transcription in both renal cancer and renal epithelial cells.
We next determined the role of intermediary signaling molecules in CsA-mediated VEGF transcriptional activation. It has been demonstrated that CsA may regulate kinases, such as JNK, MAPK and PKC (30-32). To evaluate the roles of these kinases, we transfected 786-0 cells with the 2.6-kb VEGF promoter-luciferase construct, and treated them with CsA in absence/presence of pharmacological inhibitor; vehicle-treated cells served as controls. As shown in the Figure-1C, CsA significantly induced VEGF transcription as compared to vehicle-treated controls; although PD98059 (a MEKK inhibitor) and LY294002 (a PI3K inhibitor) had no significant effect, Calphostin C (a generalized PKC inhibitor) significantly inhibited CsA-mediated VEGF transcription. Furthermore, we observed that the effect of Calphostin C on CsA-mediated VEGF transcription was dose-dependent (Figure-1D). These results suggest that PKC is an important intermediary signaling molecule in CsA-mediated VEGF transcriptional activation.
Members of the PKC family can be subdivided into three major classes: classical, atypical and novel PKC isoforms (33). In next series of experiments we sought to determine which PKC isoforms may be important for CsA-mediated VEGF transcription. First, we used Go6976, a selective inhibitor of the classical PKC family (such as PKCα and PKCβ) (34). The 786-0 cells were transfected with the 2.6-kb VEGF promoter-luciferase construct, and treated with CsA in absence/presence of Go6976. Vehicle-treated cells served as control. We found that Go6976 failed to inhibit CsA-mediated VEGF transcriptional activation, and rather, to our surprise, somewhat increased VEGF transcription (Figure-2A). Thus, the classical PKC isoforms are not intermediaries in CsA-mediated VEGF transcriptional activation. In fact, they may facilitate a negative feed-back loop for VEGF expression.
Next, we wished to evaluate the roles of PKCζ and PKCδ, members of the atypical and novel PKC families respectively (33), in CsA-mediated VEGF transcription. The 786-0 cells were co-transfected with the 2.6-kb VEGF promoter-luciferase construct and either a specific dominant-negative mutant of PKCζ/PKCδ or the empty expression vector. Following transfection, the cells were treated with either CsA or vehicle. As shown in the Figure-2B, the dominant-negative mutants of both PKCζ and PKCδ dose-dependently inhibited CsA-mediated VEGF transcriptional activation, as compared to the empty vector-transfected and vehicle-treated controls. To next assess whether CsA increases the phosphorylation of PKCζ or PKCδ, 786-0 cells were treated with either different concentrations of CsA, or vehicle. Western blot analysis was performed using a phospho-specific antibody to either PKCζ or PKCδ. We found that CsA increased the phosphorylation of both PKCζ and PKCδ as compared to vehicle-treated controls (Figure-2C). However, there was no change in the amount of total PKCζ or PKCδ in these cells following CsA treatment. In separate experiments, we also found that CsA increased the kinase activity of both PKCζ (Figure-2D) and PKCδ (data not shown) as compared to vehicle-treated controls. Together, these findings suggest that PKCζ and PKCδ may act as critical intermediaries in CsA-induced VEGF transcription.
The transcription factor Sp1 is an established regulator of VEGF expression (26, 27). Moreover, we have previously observed that PKC can form complex with Sp1 to facilitate VEGF transcriptional activation (27). Since CsA increases PKCζ and PKCδ phosphorylation, we next evaluated whether CsA could promote the association between these two PKC isoforms and Sp1. By immunoprecipitation, we observed that CsA treatment indeed promoted the association of both PKCζ and PKCδ with Sp1 in 786-0 cells (Figure-3A). A similar level of association of both phospho-PKCζ and phospho-PKCδ with Sp1 was also found following CsA treatment (data not shown). By EMSA, we found that CsA treatment of 786-0 cells promoted the binding of Sp1 to a specific DNA probe containing an Sp1-binding site, and the induced binding of Sp1 to the probe was confirmed by both supershift and competition assays (Figure-3B). Thus, CsA-induced and PKC-Sp1-mediated pathways may play an important role in VEGF transcription.
We next determined if Sp1 facilitates CsA-mediated VEGF transcription. Besides Sp1, hypoxia inducible factor-α (HIF-α) is another well-established transcription factor for VEGF expression (26, 35). We utilized two 5’ deletion constructs of the 2.6-kb full-length VEGF promoter-luciferase plasmid (Supplementary Figure-1) (26); a 0.35-kb construct which has a deleted HIF-α binding site but intact Sp1 binding sites, and a 0.07-kb construct that has deleted binding sites for both HIF-α and Sp1. The 2.6-kb construct has binding sites for both HIF-α and Sp1. We transfected 786-0 cells with each of the VEGF promoter-luciferase constructs, and evaluated the effect of CsA on VEGF transcriptional activation by measuring luciferase activity. Vehicle-treated cells served as controls. We observed that there was a marked decrease in basal luciferase activity with the 0.35-kb construct compared with the 2.6-kb construct; however, the relative fold induction of each construct following CsA treatment was similar (~3-fold) (Figure-3C). These results suggest that HIF-α plays a major role in basal activation of the VEGF promoter in 786-0 cells, but it is unlikely to be involved in CsA-mediated VEGF transcription. In contrast, there was a marked reduction in basal luciferase activity with the 0.07-kb construct compared with the 0.35-kb construct; and there was no change in the activation of the 0.07-kb promoter following CsA treatment (Figure-3C). These results support a major role for Sp1 in both basal as well as CsA-mediated VEGF transcription.
Although we found that CsA may promote VEGF transcription in a HIF-α-independent manner, we next confirmed our findings using siRNA. It is established that in 786-0 cells HIF-1α is undetectable, but HIF-2α is active (36). Thus, we first knocked down HIF-2α in 786-0 cells using gene-specific siRNA, and then studied the effect of CsA on the induction of full-length 2.6-kb VEGF promoter in these cells as described above. As shown in Figure-3D, CsA promoted VEGF transcriptional activation in HIF-2α knock-down cells to a similar level as observed in control siRNA-transfected cells, although there was a decrease in basal promoter activity. The knock down of HIF-2α was confirmed by Western blot analysis (Figure-3D, right panel). Together, these results suggest that CsA can induce VEGF transcription in a HIF-α-independent manner; and that PKCζ- and PKCδ-mediated binding of Sp1 to the VEGF promoter may be one of the important regulatory factors in this induction process.
VHL has a critical role in the pathogenesis of renal cell carcinoma (37). It is known that 786-0 cells lack VHL (37, 38), while the Caki-1 renal cancer cell line retains the gene (38). Thus, we first tested whether CsA could promote the activation of the 2.6-kb VEGF promoter in Caki-1 cells as observed in 786-0 cells. We found that although there was induction in CsA-mediated VEGF promoter activity in VHL-containing Caki-1 cells, the effect was much lower compared with 786-0 cells lacking VHL (Figure-4A).
To dissect the specific role of VHL, we made use of 786-0 cells stably transfected with either empty vector (Neo) or wt-VHL (26), and studied the effect of CsA on VEGF promoter activity in these cells. As shown in Figure-4B, CsA promoted ~3-fold increase in the 2.6-kb VEGF promoter activity in Neo cells; however, the activation was blunted in wt-VHL cells. In addition, we observed that in contrast to Neo cells, CsA could not increase the association between PKCζ and Sp1 in wt-VHL cells (Figure-4C). These results suggest that the VHL protein (pVHL) may inhibit CsA-mediated VEGF transcriptional activation likely through the prevention of increased association between PKCζ and Sp1. However, we cannot rule out the presence of a pVHL-independent pathway that may also be involved in CsA-mediated VEGF transcription.
We next examined whether CsA can promote renal tumor growth in vivo. We used 786-0 human renal cancer cells, in which VEGF secretion was found to be increased following CsA treatment (Figure-5A). Tumor cells were injected subcutaneously in immunodeficient (nu/nu) mice. The mice (n=5 in each group) were then treated either with CsA (10 mg/kg/day) or with vehicle as control for 25 days. We observed that from day 5 following tumor injection, CsA treatment enhanced tumor growth as compared to vehicle-treated controls (Figure-5B). Tumors were harvested on day 25, and evaluated for VEGF expression. By real time PCR, we observed that CsA treatment significantly induced VEGF expression in the tumor as compared to vehicle-treated controls (Figure-5C). CsA treatment also markedly increased tumor vessel density as observed by CD31 staining (Figure-5D). Thus, CsA may promote tumor growth in vivo, and the overexpression of VEGF by the tumor cells as observed in our in vitro studies may be critical in mediating enhanced tumor angiogenesis. However, we cannot rule out a direct effect of CsA on endothelial cell proliferation.
To evaluate the in vivo significance of CsA-induced VEGF overexpression in terms of post-transplantation tumor development, we modified a previously reported mouse model of post-transplantation cancer (10). We made use of a syngenic tumor cell line (CT26), which can form VEGF-dependent angiogenic tumors. We found that these cells express HIF-1, as observed by others (39), while there was no expression of pVHL (data not shown). We first confirmed that treatment of CT26 cells with increasing doses of CsA markedly induced VEGF expression/secretion in these cells (Supplementary Figure-2). CT26 cells were then subcutaneously injected into Balb/C mice, and fully MHC mismatched cardiac transplants (C57BL/6) were performed in these mice 7 days later. This model may mimic a clinical situation in which few pre-existing tumor cells are present in patients undergoing transplantation. Following cardiac transplantation, the mice (n=5 in each group) were treated either with CsA (10 mg/kg/day) or with vehicle as control. Treatment was continued for 14 days (i.e., up to 22 days following tumor injection). We found that vehicle-treated mice rejected allografts within 10 days, whereas there was a significant prolongation of allograft survival in the CsA-treated group (data not shown). Tumor volume was monitored on alternate days. As shown in the Figure-6A, tumor size was significantly higher in the CsA-treated group as compared to the vehicle-treated control group on days 15 and 22 following tumor injection.
Tumors were harvested on day 22, and were initially evaluated for VEGF expression. We observed a significant increase in VEGF mRNA expression within tumors in the CsA-treated animals compared with vehicle-treated mice (Figure-6B). We also found that tumor vessel density was markedly increased in the CsA-treated group, as observed by CD31 staining (Figure-6C).
We next evaluated whether neutralization of VEGF can attenuate CsA-induced post-transplantation tumor growth. We used the same in vivo model of post-transplantation cancer as described above. Following CT26 tumor cell injection and cardiac transplantation, the mice (n=5 in each group) were treated with CsA (10 mg/kg/day) in absence or presence of a murine blocking anti-VEGF antibody; vehicle- and IgG isotype-treated mice served as controls. Treatments were continued until day 30 of tumor injection. We found that treatment with anti-VEGF alone resulted in some inhibition of tumor volume as compared to control (data not shown). As shown in the Figure-6D, CsA promoted tumor growth as compared to vehicle-treated controls, and blockade of VEGF substantially inhibited CsA-induced tumor development. However, the anti-VEGF treatment did not inhibit the tumor volume to the level of control, suggesting the possible roles of additional factors in CsA-mediated tumorigenic process, as proposed by others (7). Nevertheless, these findings suggest that CsA can promote the development of post-transplantation cancer through its effect on VEGF expression and VEGF-induced angiogenesis.
Although calcineurin inhibitors are excellent immunosuppressive agents to inhibit allograft rejection, they may promote the growth of different tumors (7, 9-11, 40). 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, 41). 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, 23). 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 (37, 38); 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, 49). 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.
This work was supported by a John-Merrill Grant (ASN-AST) and NIH grant DK64182 to S. Pal, ROTRF and Emerald Foundation grants to D. M. Briscoe, and NIH grant CA79830 to H. T. Cohen.