Essential Role of PDGFRα-p70S6K Signaling in Mesenchymal Cells During Therapeutic and Tumor Angiogenesis In Vivo
Role of PDGFRα During Angiogenesis
Discovery of the common and ubiquitous molecular targets for the disruption of angiogenesis, that are independent of the characteristics of malignant tumors, is desired to develop the more effective antitumor drugs. In this study, we propose that the platelet-derived growth factor receptor-α (PDGFRα)-p70S6K signal transduction pathway in mesenchymal cells, which is required for functional angiogenesis induced by fibroblast growth factor-2, is the potent candidate. Using murine limb ischemia as a tumor-free assay system, we demonstrated that p70S6K inhibitor rapamycin (RAPA) targets mesenchymal cells to shut down the sustained expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), via silencing of the PDGFRα-p70S6K pathway. Irrespective of the varied expression profiles of angiogenic factors in each tumor tested, RAPA constantly led the tumors to dormancy and severe ischemia in the time course, even associated with upregulated expression of VEGF from tumors. Because RAPA showed only a minimal effect to hypoxia-related expression of VEGF in culture, these results suggest that RAPA targets the host-vasculature rather than tumor itself in vivo. Thus, our current study indicates that the PDGFRα-p70S6K pathway is an essential regulator for FGF-2–mediated therapeutic neovascularization, as well as for the host-derived vasculature but not tumors during tumor angiogenesis, via controlling continuity of expression of multiple angiogenic growth factors.
Angiogenesis is required for tumor progression, as supported by a number of animal studies demonstrating a reduction in tumor growth by antiangiogenic agents.1–3 Vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis, and inhibition of VEGF activity by the overexpression of a soluble high-affinity receptor, fms-like tyrosine kinase-1 (FLT-1), induced tumor dormancy.4,5 These studies thus suggest that VEGF-related signaling could be a target for tumor angiogenesis; however, an independent study indicated that the antitumor effect of FLT-1 was highly dependent on the expression level of VEGF in each tumor type studied,6 suggesting a possible limitation of the anti-VEGF strategy. Therefore, the discovery of common molecular targets of tumor angiogenesis that are independent of the profile of angiogenic growth factor expression in each tumor type would be desirable for the development of broad-acting antitumor agents.
A recent elegant study explored the potential of an immunosuppressive drug, rapamycin (RAPA), which is antiangiogenic; in that study, tumor regression was successfully demonstrated.7 Although immunosuppressive therapy for patients after organ transplantation promotes the risks of cancer development and recurrence, the use of RAPA or related derivatives was thought to possibly reduce the chance of the development of malignancies. The antiangiogenic effect of RAPA was suggested to involve the reduced expression of VEGF by tumor according to the data from cultured cells7; however, its exact mechanism of action in vivo remains unknown.
Independently, we recently demonstrated the critical role of mesenchymal, but not endothelial cell (EC), expression of angiogenic polypeptides during therapeutic angiogenesis using fibroblast growth factor-2 (FGF-2) to treat critical limb ischemia.8,9 FGF-2 stimulated the local expression of VEGF and hepatocyte growth factor/scatter factor (HGF/SF), an alternative angiogenic factor, in mesenchymal cells (MCs; including pericytes, vascular smooth muscle cells and adventitial fibroblasts) in the vasculature.9 Interestingly, the time course of FGF-2–mediated HGF/SF expression was biphasic; early upregulation did not require new protein synthesis, whereas later upregulation was mediated and maintained by endogenous platelet–derived growth factor receptor-α (PDGFRα)- p70S6K pathways.9
Because not only VEGF but also host-derived FGF-2 activity is likely to be involved in tumor progression,10 and also because RAPA is a specific inhibitor of p70S6K via reducing the activity of target of rapamycin (TOR), we hypothesized that the antitumor effect of RAPA might involve the PDGFRα-p70S6K signaling pathway in host-originated stromal MCs, which should be independent of the various angiogenic signals from each tumor. In this study, we provide evidence indicating that RAPA targets stromal MCs rather than tumor cells, thereby turning off the PDGFRα-p70S6K system, and in turn silencing angiogenic signals.
Materials and Methods
Cells and Reagents
All cells used in this study were purchased from American Type Culture Correction. The following intracellular signal inhibitors were used at each of the following concentrations on human smooth muscle cells (HSMCs) and MRC5 cells, as previously described9: Ras, Ras-inhibitory peptide (50 μmol/L, Alexis Japan); p70S6K, rapamycin (100 ng/mL, Sigma-Aldrich Japan); PKC, bisindolylmaleimide (100 nmol/L, Sigma); PI3K, wortmannin (120 nmol/L, Sigma); MEK, U0126 (10 μmol/L, Promega K.K.); PKA, PKA-inhibitory peptide (1 μmol/L, Calbiochem); and NF-κB, ALLN (5 μmol/L, Roche Diagnostics). Neutralizing antibodies (anti-PDGF-AA and anti-PDGFRα from goat), and control goat IgG were from R&D systems. Stocks of recombinant SeVs (SeV-FGF2 and SeV-luciferase) were prepared as previously described.8,9 Recombinant SeV-expressing extracellular domain of human PDGFRα was constructed as follows: cDNA fragment (amplicon size=1575 bp) was amplified using synthetic primers with restriction enzyme tag (forward BglII: 5′-aaAGATCTatggggacttccc-atccggc-3′; reverse NheI: 5′-ttGCTAGCtcacttgtcatcgtcgtccttgtagtcttcagaacgcagggt-3′), and whole sequence was determined as completely matched to reported sequence (GenBank No. NM006206) by capillary sequencer. The clone was inserted into a template plasmid encoding SeV18+, and SeV-hsPDGFα was recovered as described.8,9,11 The secretion of soluble human PDGFα via SeV-hsPDGFα was determined by Western blotting (data not shown).
Male C57BL/6 (6 weeks old) and balb/c nu/nu mice (5 weeks old) were from KBT Oriental Co, Ltd (Charles River Grade, Tosu, Saga, Japan). All animal experiments were performed according to approved protocols and in accordance with recommendations for the proper care and use of laboratory animals by the Committee for Animals, Recombinant DNA, and Experiments Using Infectious Pathogen at Kyushu University, and according to The Law (No. 105) and Notification (No. 6) of the Japanese Government.
Limb Ischemia Models
The details of the surgical treatment and evaluation of limb prognosis were described previously.8,9 For the gene transfer, 25 μL of vector solutions were injected into two portions of the thigh muscle soon after the completion of the surgical procedures. In vivo suppression of endogenous PDGF-AA activity was performed as previously described using PDGF-AA-specific neutralizing goat polyclonal IgG (cross-reactive to human and murine proteins) via a disposable micro-osmotic pump (Model 1007D, ALZA Co) as previously described.8,9
For tumor implantation, 106 SAS or MH134 cells were intradermally implanted into the abdominal wall, and the tumor volume was evaluated every other day. Seven days after implantation, RAPA (1.5 mg/kg per day) was intraperitoneally administered daily. On day 7 or 28, mice were euthanized, and the tumors were subjected to enzyme-linked immunosorbent assay (ELISA).
Enzyme-Linked Immunosorbent Assay
Protein contents in murine limb muscles, tumors, and culture medium were determined using Quantikine Immunoassay systems for murine (recognizes both 164 and 120 amino acid residue forms) and human VEGF-A, human FGF-2 (available to both human and murine), human HGF (R&D Systems Inc), and rat HGF (available to murine HGF, Institute of Immunology Inc) according to the manufacturer’s instructions, as previously described.8,9
Northern Blot Analysis
Total cellular RNA, isolated using the ISOGEN system (Wako Pure Chemicals), was electrophoresed and transferred onto a nylon membrane. The filters were hybridized overnight at 60°C with random [α-32P] dCTP-labeled probes. The bands were then visualized and subjected to densitometry using a photoimager.
Total RNA was extracted from ischemic limb muscles with the ISOGEN system followed by RNase-free DNase I (Boehringer). Aliquots of total RNA(25 ng) were reverse-transcribed and amplified in triplicate with the TaqMan EZ RT-PCR kit and a Sequence Detection System, model 7000 (PE Biosystems). The nucleotide sequences of PCR primers and TaqMan probes are listed in the Table. The murine GAPDH control reagents were used as the internal standard. The target quantity was determined from the relative standard curves constructed with serial dilutions of the control total RNA. The expression level of the target gene was normalized by the GAPDH level in each sample.
Laser Doppler Perfusion Images
Intratumor blood perfusion was evaluated using a laser Doppler perfusion image (LDPI) analyzer (Moor Instruments), as previously described.8,9 To eliminate background noise due to intestinal blood flow, a blue sheet was inserted into the peritoneal cavity just before the measurement. To minimize the data variables due to ambient light and temperature, the LDPI index was expressed as the ratio of intratumor pixels to those in the scrotum.
All data were expressed as mean±SEM and were analyzed by one-way ANOVA with Fisher’s adjustment. For the survival analysis, the survival rate, expressed by limb salvage score,12 was analyzed using Kaplan-Meyer’s method. The statistical significance of the survival experiments was determined using the log-rank test, and P<0.05 was considered to be statistically significant.
FGF-2 and PDGF-AA Cooperatively Enhance VEGF and HGF/SF Expression via FGF-2–Mediated Upregulation of PDGFRα
To assess the role of PDGF-AA signaling in the angiogenic response of the host vasculature, we first examined the induction of VEGF and HGF via FGF-2 using cultured human MCs (MRC5 and HSMCs) under serum-free conditions. As shown in Figure 1A, FGF-2, but not PDGF-AA stimulated VEGF in the culture medium of MRC5 cells (Figure 1A, left), and inversely, PDGF-AA, but not FGF-2, upregulated VEGF in the medium of HSMCs (Figure 1A, right). On the other hand, costimulation using FGF-2 and PDGF-AA demonstrated a synergistic effect as regards both VEGF expression (Figure 1A) and HGF/SF (data not shown), in both types of cell. Similar to MRC-5 cells, a cell line of murine fibroblasts, NIH3T3, also showed synergistic effect of FGF-2 and PDGF-AA on expression of VEGF and HGF (data not shown), indicating that such effect might be a common system in mesenchymal cells irrespective of animal species. Northern blot analyses showed FGF-2–mediated upregulation of PDGFRα transcription in both types of cell (Figure 1B), whereas PDGF-AA did not alter FGFR1 expression (data not shown); these findings suggest that FGF-2 modulates the response to PDGF-AA regarding VEGF and HGF/SF expression in mesenchymal cells via the transcriptional regulation of PDGFRα.
FGF-2–Dependent, Mesenchymal Expression of VEGF and HGF/SF Expression Is Mediated by PDGFRα, Which Can Be Shut by RAPA
In addition to the cooperative effect of FGF-2 and PDGF-AA on VEGF and HGF/SF expression in MCs, we previously found that FGF-2 also enhanced the endogenous expression of PDGF-AA via Ras and p70S6K signaling, which contributed to the sustained expression of HGF/SF in HSMCs.9 We thus considered the possibility that a similar system might also be seen in the context of VEGF and HGF/SF expression in fibroblasts (MRC5). As also observed in previous studies, it was found that FGF-2 representatively upregulated both the VEGF and HGF/SF proteins, and these effects were abolished by an MEK inhibitor, Ras-inhibitory peptide, and the p70S6K inhibitor (RAPA) (Figure 2A). Repetitive Northern blot analysis of the time course of FGF-2–mediated VEGF expression exhibited the biphasic (at 3 hours and later) upregulation of VEGF (Figure 2B), as previously seen in the case of HGF/SF expression using HSMCs.9 Early VEGF expression was not affected, but the later/sustained expression was completely diminished by RAPA treatment (Figure 2B). Furthermore, FGF-2–mediated upregulation of VEGF protein was completely abolished by anti-PDGFRα antibody (Figure 2C); a similar finding observed with RAPA (Figure 2A). As the same results were obtained regarding HGF/SF expression (data not shown), the PDGFRα system was suggested to play a critical role in FGF-2–mediated enhancement and continuity of VEGF and HGF/SF expression in MCs.
PDGFRα Plays a Critical Role in the Therapeutic Effect of FGF-2 in Murine Critical Limb Ischemia
To examine the possible cascade-like link of FGF-2-PDGFRα-VEGF/HGF in vivo, we assessed two independent murine models of limb ischemia, namely a “limb salvage model” in C57 BL/6 mice and an “autoamputation model” in balb/c nu/nu mice,8 using SeV-FGF2 in vivo.8,9,10–15 Overexpression of FGF-2, which was assessed by ELISA (data not shown), resulted in the upregulation of both PDGF-A and PDGFRα mRNA quantitatively assessed by real-time PCR in a limb salvage model (Figure 3A). Similar enhancement of VEGF and HGF/SF expression by FGF-2 was also observed in the same tissue samples; this effect was diminished by anti–PDGF-AA neutralizing antibody, as well as by RAPA treatment (Figure 3B). This effect of RAPA was also confirmed on the protein level (Figure 3C); furthermore, the therapeutic effect of FGF-2 in the autoamputation model was abolished by anti–PDGF-AA antibody and RAPA (Figure 3D), indicating that the PDGFRα system plays a critical role in FGF-2-mediated therapeutic angiogenesis.
RAPA Induces Tumor Dormancy Irrespective of the Variety in the Expression of Angiogenic Factors in Each Type of Tumor
These results from the tumor-free systems suggest that the PDGFRα-p70S6K signal pathway in MCs is essential, and that RAPA imitates the effects of anti–PDGF-AA antibody in FGF-2–mediated angiogenesis. However, an issue arose from this line of questioning regarding whether or not RAPA might be able to affect ubiquitous angiogenic responses, irrespective of angiogenic stimuli. To clarify this issue, we examined tumor angiogenesis using two independent tumor cell lines, ie, SAS-human oral squamous cell carcinoma, which expresses VEGF, FGF-2, and PDGF-AA at high levels; and MH134-murine hepatocellular carcinoma,16 which secretes far less VEGF and FGF-2 than the former, and shows null expression of PDGF-AA.
RAPA reduced the growth of both tumor types (Figure 4A), indicating that the antitumor effect of RAPA was likely to be independent of the respective expression patterns of angiogenic growth factors in each type of tumor. To obtain further and direct evidence of tumor-independent antitumor effect of PDGFRα-p70S6K pathway, we conducted additional experiments assessing tumor growth via intratumor injection of SeV-hsPDGFRα, which expressed ectodomain of human PDGFRα. As expected, SeV-hsPDGFRα significantly inhibited both tumor types (Figure 4B). Tumor weight was also assessed at the end of experiments, and significantly reduced in both tumors treated with hsPDGFRα compared with luciferase (SAS-luciferase: 415.1±104.9 mg versus SAS-hsPDGFRα=54.3±9.6 mg, MH134-luciferase: 3930.4±304.4 mg versus MH134-hsPDGFRα=2654.4±296.5 mg, P=0.0027 and P=0.0106, respectively).
To confirm an interpretation that antitumor effect of RAPA might be independent of the expression pattern of angiogenic factors, we assessed the in vitro and in vivo expression of VEGF with or without RAPA. In culture, 100 ng/mL of RAPA significantly reduced endogenous VEGF secretion approximately 30% to 50% from the SAS baseline, and also from the baseline of the other tumors assessed (squamous cell carcinomas: QG56, TF, KN, EBC-1; and adenocarcinoma: PC9) under normoxia condition; similar findings obtained in the other laboratory.7 In addition, no effect of RAPA on PDGF-AA and FGF-2 expression was observed in each tumor type (data not shown). In vivo, however, intratumor expressions of VEGF treated with RAPA for 3 or 7 days were significantly upregulated in MH134 tumor compared with those treated with buffer (Figure 5A), and further, Doppler flow image analyses revealed that RAPA reduced blood flow in both tumors on 7 days after the start of RAPA injection (Figure 5B).
These paradoxical results might be explained as follows: RAPA treatment might have induced hypoxia and, as a result, VEGF was upregulated via a hypoxia-dependent mechanism, thereby overcoming the RAPA-mediated downregulation. This hypothesis was also confirmed as follows; RAPA showed significant, but only minimal effect on hypoxia (2.5% O2)-induced VEGF expression in cultured MH134 (Figure 5C), and similar findings were also obtained in all other cell lines tested (data not shown).
Thus, we next evaluated the source of VEGF in SAS, a xenograft model, using human- and murine-specific ELISA systems. RAPA significantly upregulated human VEGF without effect to murine VEGF content in solid tumors (Figure 5D), indicating that the upregulation of tumor cell–derived VEGF was mediated by hypoxia due to host vasculature-targeted antiangiogenesis independent to the variation of expression of angiogenic stimuli by each tumor type.
In this study, we demonstrated that the PDGFRα signal transduction pathway plays an essential role in MCs, not only in the context of therapeutic treatments, but also as regards tumor angiogenesis, irrespective of the variety in the expression patterns of angiogenic substances in each type of tumor. Thus, the PDGFRα-p70S6K signal transduction pathway in host-derived vasculature was concluded to be a ubiquitous molecular target for inducing tumor dormancy (a scheme representing potential mechanisms is summarized in an online Movie available in the online data supplement at http://circres.ahajournals.org).
The biological role of PDGFRα has been controversial for quite some time. PDGF-AA induces DNA synthesis as well as the proliferation of NIH3T3 cells, but in other cells, it inversely inhibits the chemotactic response induced by other agents.17 Whereas little evidence is available regarding endothelial expression of PDGF-receptors, PDGF ligands, not only PDGF-AA and -BB, but also -CC, a novel member of PDGF,18 apparently stimulate in vivo angiogenesis,19,20 suggesting that other angiogenic stimulators may mediate PDGF-dependent angiogenic processes. Our current study, as well as a previous study,9 strongly suggest that the PDGFRα system may be critical for the maintenance of angiogenic signals using VEGF and HGF/SF in MCs. However, one limitation of the current study was that it did not include a determination of the PDGF ligands that might be critical during angiogenesis, Because all such ligands can activate PDGFR-α, leading to different cellular responses. Our recent independent study revealed that the neutralization of PDGF-BB also disrupted FGF-2-mediated therapeutic effects in an autoamputation model (Y. Yonemitsu, M. Onimaru, and M. Tanii, unpublished data, 2003), and SeV-hsPDGFRα, which traps both PDGF-AA and -BB, efficiently reduced the growth of SAS in vivo (Figure 4B), suggesting that the cooperative activities of PDGF ligands may be required for the efficient promotion of angiogenesis.
A paradox was observed among the results of this study, namely, that RAPA reduced the FGF-2–mediated upregulation of both VEGF and HGF to those of control levels at hypoxia in limb ischemia (Figure 3C); however, RAPA had no effect on the level murine VEGF in SAS cells with null HGF expression (Figure 5D). A possible explanation for this finding may be as follows: the baseline host MC expression of VEGF and HGF required for tumor angiogenesis in SAS was too low to be detected by ELISA, as endogenous HGF expression was undetectable. These cells in tumor, however, were exposed to hypoxia and thus VEGF was upregulated in hypoxia-dependent manner, irrespective of RAPA treatment. This explanation is reasonable because ischemia-mediated upregulation of VEGF was not affected by RAPA (Figure 3C); furthermore, such an imbalance in the expression levels of VEGF and HGF could disturb the blood flow of neovessels, as shown previously.9
We here demonstrated that both RAPA and SeV-hsPDGFRα suppressed tumor growth irrespective of expression profiles of angiogenic growth factors; however, the effect of SeV-hsPDGFRα against MH135 was likely to be relatively modest compared with that obtained with RAPA (Figures 4A and 4B). The possible explanations were as follows: (1) the expression level of hsPDGFRα was not sufficient during experimental course, because transgene expression of SeV shows its peak at 2 days after gene transfer, and is reduced around 1/10 to 1/100 at 7 days even in nude mice8; (2) a possible PDGFRα-independent effect of RAPA, including direct growth inhibition of ECs,21,22 provided additional antitumor activity in RAPA-treated group; and (3) SAS tumors that have higher levels of PDGF-AA secretion than MH134 are more dependent on PDGF for angiogenesis. The 3rd explanation may be supported by our recent data, indicating that PDGF-AA is also an autocrine regulator for stimulating angiogenic growth factors including VEGF in SAS tumors (Y. Shikada and Y. Yonemitsu, unpublished data, 2004). Ideally, experiments using knockout of PDGF-A allele in mesenchymal cells in vivo might solve this issue; however, it is still difficult to establish such mice at present, because such mice showed lethal phenotype pre- and postnatally.23
What is the role of endogenous FGF-2, but not exogenous, in limb ischemia as well as tumor models for angiogenesis? At this time, we do not have any direct data regarding this question, however, some recent observations may be a clue to explain this point. As we previously demonstrated, limb ischemia–mediated upregulation of HGF was significantly and considerably reduced in FGF-2−/− mice compared with that seen in FGF-2+/+ mice,9 and similar findings were found in case of VEGF (Y. Yonemitsu et al, unpublished data, 2003). Furthermore, tumor growth of MH134 was significantly suppressed in FGF-2−/− mice (Y. Yonemitsu and S. Shibata, unpublished data, 2004). Together with these results and a published report by another laboratory, indicating that gene transfer of soluble FGFR1 could effectively reduce tumor growth,10 it is highly suspected that endogenous FGF-2 may essentially participate in therapeutic as well as tumor angiogenesis in vivo.
Although a number of studies have demonstrated that VEGF is an important angiogenic mediator of tumor growth, the anti-VEGF strategy is likely to depend on the expression levels of VEGF in each tumor type.6 Other angiogenic switches (eg, HGF/SF, angiopoietin-2, which can be induced by FGF-29,24) may possibly rescue tumor angiogenesis when the VEGF signal is intercepted, because tumor- and/or host-derived FGF-2, for example, is required for the maintenance of tumor vasculature. On the other hand, only the expression level of PDGF-AA in tumors was significantly correlated to the survival of patients with neuroblastomas, even when a high level of expression of various angiogenic factors was detected25 and further, an inhibitor of PDGFRs is likely to be effective in patients with malignant tumors.26 More recently, an important study has demonstrated an evidence indicating that a tyrosine kinase inhibitor SU6668 specific for PDGFRs blocked further growth of end-stage tumors in experimental animals, eliciting detachment of pericytes in tumor vasculature, whereas an inhibitor targeting VEGFRs (SU5416) was only effective against early-stage tumors,27 indicating the requirement of PDGFR signals for the maintenance of tumor growth as well as tumor vasculature. The apparent advance of the current study over these recent reports explored the discovery of an essential pathway downstream of the PDGFRα. Based on these findings, it is thus suggested that an anti-PDGFRα-p70S6K strategy, which can be mimicked by RAPA and abolishes host-derived VEGF and HGF/SF that are required for well-organized tumor vasculature, could be applicable for treatment of a broader range of tumor species than an anti-VEGF system.
No assessment of the contribution of bone marrow–derived stem cells (BM-SCs), which have been shown to contribute to tumor angiogenesis, is a limitation of the current study. Notably, such populations respond to angiogenic growth factors, including FGF-228 and VEGF,29 demonstrating potentials to promote tumor growth. Therefore, further studies are called for to clarify whether PDGFRα-p70S6K system may directly contribute to recruitment of BM-SCs to tumor vasculature.
In conclusion, our current study suggests the utility of a host vasculature–targeted antiangiogenic therapy mediated by the blockade of the PDGFRα-p70S6K signal transduction pathway. Because RAPA itself frequently evokes unfavorable symptoms in the clinical setting,30 related derivatives or novel compounds targeted the PDGFRα-p70S6K with reduced adverse effects could be unique antitumor agents at a future date.15,16
This work was supported in part by Grants-in-Aid (Y.Y. and K.S.) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (12557020, 13307009, and 13877028) and by a Grant of Promotion of Basic Science Research in Medical Frontier of the Organization for Pharmaceutical Safety and Research (Y.Y. and K.S., project No. MF-21). The authors thank Ryoko Nakamura-Hashimoto for her excellent help with the animal experiments.
Original received November 4, 2003; revision received March 11, 2004; accepted March 22, 2004.
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