Molecular Mechanisms of the Antiangiogenic and Antitumor Effects of Mycophenolic Acid
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1656 Molecular mechanisms of the antiangiogenic and antitumor effects of mycophenolic acid Sophie Domhan,1,2,3 Stefan Muschal,1 endothelial and fibroblast cells at 6 and 12 h after MPA Christian Schwager,1 Christian Morath,2 treatment. Network analysis revealed a critical role for Ute Wirkner,1 Wilhelm Ansorge,1 MYC signaling in endothelial cells treated with MPA. Christian Maercker,1 Martin Zeier,2 Moreover, we found that the antiangiogenic effects of MPA Peter E. Huber,1,3 and Amir Abdollahi1,3,4 were organized by coordinated communications between MYC and NDRG1, YYI, HIF1A, HDAC2, CDC2, GSK3B, and 1Department of Radiation Oncology, German Cancer Research PRKACB signaling. The regulation of these ‘‘hub nodes’’ Center, and 2Department of Nephrology, University of Heidelberg was confirmed by real-time quantitative reverse transcrip- 3 Medical School, Heidelberg, Germany and Center of Cancer tion-PCR and protein analysis. The critical involvement of Systems Biology, Department of Medicine, Caritas St. Elizabeth’s Medical Center, Tufts University School of Medicine; 4Children’s MYC in the antiangiogenic signaling of MPA was further Hospital Boston, Vascular Biology Program & Harvard Medical shown by gene knockdown experiments. Together, these School, Department of Surgery, Karp Family Research data provide a molecular basis for the antiangiogenic and Laboratories, Boston, Massachusetts antifibrotic effects of MPA, which warrants further clinical investigations. [Mol Cancer Ther 2008;7(6):1656–68] Abstract The relative risk for the development of malignancies Introduction following solid organ transplantation seems to be de- Mycophenolic acid (MPA) is a potent uncompetitive creased in patients treated with the immunosuppressive inhibitor of inosine monophosphate dehydrogenase agent mycophenolic acid (MPA). However, the molecular (IMPDH), the rate-limiting enzyme in the de novo synthesis mechanisms of the antineoplastic effects of MPA are not of guanosine nucleotides (1, 2). Guanine nucleotides are completely understood. Here, we report that human crucial prerequisites for cell proliferation and many cellular endothelial cells and fibroblasts are highly sensitive to functions including transmembrane and intracellular sig- MPA treatment. We found that U87 glioblastoma cells naling, DNA replication, and RNA and protein synthesis were resistant to MPA treatment in vitro. However, U87 (1, 2). Mycophenolate mofetil (MMF; CellCeptR), the tumor growth was markedly inhibited in vivo in BALB/c morpholinoethyl ester prodrug of MPA, is approved for nude mice, suggesting that MPA exerted its antitumor the prevention of acute graft rejection in kidney, heart, and effects via modulation of the tumor microenvironment. liver transplantation (3). Accordingly, microvascular density and pericyte coverage As newer immunosuppressive regimens have steadily were markedly reduced in MPA-treated tumors in vivo. reduced the incidence of acute rejection and have extended Using functional in vitro assays, we showed that MPA the life expectancy of allograft recipients, post-transplant potently inhibited endothelial cell and fibroblast prolifera- malignancy has become an important cause of mortality (4). tion, invasion/migration, and endothelial cell tube forma- Unlike other immunosuppressants such as calcineurin tion. To identify the genetic participants governing the inhibitors and azathioprine, the relative risk for the antiangiogenic and antifibrotic effects of MPA, we development of post-transplant malignancies seems to be performed genome-wide transcriptional analysis in U87, decreased in MPA-treated patients (4, 5). In contrast to other immunosuppressive agents, MMF appears to convey a dose-dependent protective effect against malignant transformation, with patients maintained on 3 g/d MMF Received 9/20/07; revised 2/27/08; accepted 4/8/08. manifesting a lower relative risk for skin cancer than those Grant support: Deutsche Krebshilfe 106997 (U. Wirkner, P.E. Huber, and on 2 g/d (5). These clinical observations have led to a A. Abdollahi), DFG National Priority Research Program the Tumor-Vessel renaissance in investigating nonimmunologic and more Interface SPP1190 (S. Muschal, C. Schwager, P.E. Huber, and A. Abdollahi), National Aeronautics and Space Administration Specialized specifically the antitumor activities of MPA (4, 6–8). Center of Research NNJ04HJ12G (P.E. Huber and A. Abdollahi), Tumor- The mechanisms discussed for the potential anticancer zentrum Heidelberg-Mannheim, and Medical Faculty of University of Heidelberg Medical School (A. Abdollahi and P.E. Huber). effects of MPA include inhibition of DNA synthesis and cell The costs of publication of this article were defrayed in part by the cycle arrest at the G1-S boundary, induction of differenti- payment of page charges. This article must therefore be hereby marked ation in a variety of human tumor cell lines, suppression advertisement in accordance with 18 U.S.C. Section 1734 solely to of glycosylation and expression of several adhesion mole- indicate this fact. cules relevant in tumor metastasis process (4, 6–8). Requests for reprints: Sophie Domhan, DKFZ, INF 280, 69120 Heidelberg, Germany. Phone: 49-6221-422515; Fax: 49-6221-422514. Until recently, the testing of potential anticancer agents E-mail: [email protected] was almost solely based on investigating direct effects of a Copyright C 2008 American Association for Cancer Research. compound on tumor cells. However, tumor microenviron- doi:10.1158/1535-7163.MCT-08-0193 ment consisting of tumor interstitial cells (e.g., fibroblasts MolCancerTher2008;7(6).June2008 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2008 American Association for Cancer Research. Molecular Cancer Therapeutics 1657 and extracellular matrix) and tumor vasculature (e.g., (Becton Dickinson), cells were plated, and after endothelial cells recruited by tumors) are increasingly 12-h incubation, cells were fixed and stained with Diff- recognized as critical targets of conventional as well as Quick II reagents (Dade Behring). For invasion/migration novel cancer therapeutics (9, 10). assay, Matrigel-coated (0.78 mg/mL) Transwells with 8 Am Here, we aimed to uncover the cellular and molecular pore size (Becton Dickinson) were used. The HDMVEC or effects of MPA particularly on the tumor microenviron- fibroblasts were added to the Transwells (upper compart- ment compartment. First, we did a comparative analysis to ment). Chemoattractant medium containing 2 ng/mL detect the sensitivity of tumor versus microenvironmental vascular endothelial growth factor and 4 ng/mL basic cells to MPA-mediated antimitotic effects. Human endo- fibroblast growth factor (500 AL) was added to 24-well thelial cells and fibroblasts were among the most MPA plates (lower wells). The Transwells were transferred to the sensitive cells in vitro. Interestingly, we found a large 24-well plates, and after 18 h of incubation, cells that had discrepancy between in vitro (resistant) and in vivo invaded the underside of the membrane were fixed and (sensitive) response of human brain tumor cells (U87) to stained with Diff-Quick II solution (Dade Behring), sealed MPA, suggesting the tumor microenvironment as a on slides, and counted by microscopy (number of migrated potential target for MPA. Accordingly, we found here that cells per eight optical fields at Â40 objective and Â10 microvascular density and pericyte coverage were mark- oculars). Experiments were done at least in quadruplicates. edly reduced in MPA-treated U87 tumors in vivo. Next, we Animal Studies and Immunohistology investigated the antiangiogenic activity of MPA in func- The animal experiments were conducted according to the tional angiogenesis assays such as endothelial cell tube guidelines of the German Animal Protection Law and formation and endothelial cell migration/invasion. We approved by the state agency supervising animal experi- analyzed the transcriptional response to MPA in microvas- mentation (Regierungspraesidium). For tumor growth cular endothelial cells as the effector cells of tumor experiments, athymic 8-week-old, 20 g BALB/c nu/nu mice angiogenesis and interstitial fibroblasts as the effector cells were obtained from Charles River Laboratories. Human of tumor fibrogenesis. A comprehensive analysis of tran- U87 glioblastoma cells (5  106 in 100 AL PBS) were injected scriptome data and functional confirmation of critical s.c. into the right hind limb of the mice. Tumor volume was signaling patterns and pathway components was done to determined by caliper measurements using the formula: identify the molecular mechanisms of MPA-induced anti- volume V = length  width  width  0.5. Animals were angiogenic effects. Our data suggest a novel strategy to treated with 120 mg/kg b.i.d. oral gavage MMF identify and classify compounds with potential antiangio- (CellCeptR; Roche), the morpholinoethyl ester prodrug of genic property based on combined functional and integra- MPA. Treatment started 45 h after s.c. tumor cell injection. tive transcriptome analysis. For histologic analysis, tumors were excised and snap frozen in isopentane, cooled by liquid nitrogen, and kept at À80jC. Frozen tissues were sectioned (6 Am), mounted on Materials and Methods silan-coated slides, and fixed in ice-cold methanol (1min) Reagents and Cell Culture and acetone (2 min). After washing with 1 PBS (pH 7.2), Primary isolated human dermal microvascular endothe- the sections were incubated with Image-iT FX signal lial cells