Published OnlineFirst February 6, 2018; DOI: 10.1158/1078-0432.CCR-17-2923 Cancer Therapy: Preclinical Clinical Cancer Research Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma Renata E. Gordon1,2, Li Zhang3, Suraj Peri4, Yin-Ming Kuo5, Fang Du1, Brian L. Egleston4, Jessica M. Y. Ng6, Andrew J. Andrews5, Igor Astsaturov2,7, Tom Curran6, and Zeng-Jie Yang1,3 Abstract Purpose: The role of cholesterol biosynthesis in hedgehog approved Smoothened antagonist), were utilized to inhibit pathway activity and progression of hedgehog pathway medul- medulloblastoma growth in vivo. loblastoma (Hh-MB) were examined in vivo. Statins, commonly Results: Cholesterol biosynthesis was markedly enhanced in used cholesterol-lowering agents, were utilized to validate cho- Hh-MB from both humans and mice. Inhibition of cholesterol lesterol biosynthesis as a therapeutic target for Hh-MB. biosynthesis dramatically decreased Hh pathway activity and Experimental Design: Bioinformatic analysis was performed reduced proliferation of medulloblastoma cells. Statins effectively to evaluate the association between cholesterol biosynthesis with inhibited medulloblastoma growth in vivo and functioned syn- hedgehog group medulloblastoma in human biospecimens. ergistically in combination with vismodegib. Alterations in hedgehog signaling were evaluated in medulloblas- Conclusions: Cholesterol biosynthesis is required for toma cells after inhibition of cholesterol biosynthesis. The pro- Smoothened activity in the hedgehog pathway, and it is indis- gression of endogenous medulloblastoma in mice was examined pensable for the growth of Hh-MB. Targeting cholesterol bio- after genetic blockage of cholesterol biosynthesis in tumor cells. synthesis represents a promising strategy for treatment of Statins alone, or in combination with vismodegib (an FDA- Hh-MB. Clin Cancer Res; 24(6); 1375–88. Ó2018 AACR. Introduction reductase (HMGCR) is rate limiting for cholesterol biosynthesis. HMGCR converts HMG-CoA to mevalonate and is the target Cholesterol is a key component of cellular membranes and a of statins (2). NAD(P) steroid dehydrogenase-like (NSDHL), a precursor for steroid hormones and bile acids. The brain is the 3b-hydroxysterol dehydrogenase, is involved in the removal of most cholesterol-rich organ, containing approximately 20% of two C-4 methyl groups in one of the later steps of cholesterol total body cholesterol. In the brain, cholesterol is primarily biosynthesis. Deficiency in NSDHL dramatically impairs choles- generated by de novo synthesis, as the blood–brain barrier in terol biosynthesis in both humans and mice (3). In particular, vertebrates prevents its uptake from the bloodstream (1). Almost conditional deletion of Nsdhl gene in cerebellar granule neuronal all cholesterol in the brain is present in an unesterified, free form. precursors inhibits cholesterol synthesis and dramatically Cholesterol is synthesized through a series of approximately represses proliferation (4). The final step in cholesterol synthesis 30 enzymatic reactions, starting with the substrate acetyl CoA is catalyzed by the enzyme 24-dehydrocholesterol reductase (Fig. 1A). The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (DHCR24), which converts desmosterol to cholesterol by satu- rating the C-24,25 double-bond in the side chain. Triparanol, the first synthetic cholesterol-lowering drug, works by antagonizing 1 Cancer Biology Program, Fox Chase Cancer Center, Temple University Health DHCR24 (5). System, Philadelphia, Pennsylvania. 2Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia. 3Jiangsu Key Laboratory of In addition to its function as a component of cell membranes, Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Soochow cholesterol regulates many signaling pathways, including the University, Suzhou, Jiangsu, China. 4Biostatistics and Bioinformatics Research hedgehog (Hh) pathway. The Hh pathway plays a critical role in Facility, Fox Chase Cancer Center, Temple University Health System, Philadel- brain development and function. In the absence of Hh ligand, the 5 phia, Pennsylvania. Cancer Epigenetics Program, Fox Chase Cancer Center, antagonizing receptor Patched1 (Ptch1) suppresses the seven- 6 Temple University Health System, Philadelphia, Pennsylvania. Children's transmembrane protein, Smoothened (Smo), thus precluding Research Institute, Children's Mercy Kansas City, Missouri. 7Molecular Thera- peutics Program, Fox Chase Cancer Center, Temple University Health System, downstream signaling. The interaction between Hh and Ptch1 Philadelphia, Pennsylvania. relieves inhibition of Smo, which then triggers a cascade of events culminating in activation of the glioma-associated oncogenes 1 Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). and 2 (Gli1 and Gli2). Gli proteins translocate into cell nuclei and promote transcription of Hh pathway target genes, including Cancer Therapy: Preclinical Ptch1 and Gli1 (6, 7). Extensive evidence suggests that some Corresponding Author: Zeng-Jie Yang, Fox Chase Cancer Center, 333 Cottman exogenous oxysterols, such as 20 (S)-hydroxycholesterol and Avenue, Philadelphia, PA 19111. Phone: 215-214-1545; Fax: 215-728-2741; E-mail: 25-hydroxycholesterol, generated by cholesterol oxidation, can [email protected] activate Smo by binding to a site located in the extracellular doi: 10.1158/1078-0432.CCR-17-2923 cysteine-rich domain (CRD). This indicates that cholesterol deri- Ó2018 American Association for Cancer Research. vatives are capable of triggering Hh pathway activation in www.aacrjournals.org 1375 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst February 6, 2018; DOI: 10.1158/1078-0432.CCR-17-2923 Gordon et al. in vitro as well as tumor growth in vivo. Furthermore, we show that Translational Relevance cholesterol is necessary for maintaining Smo activity in the Medulloblastoma is the most common malignant brain absence of Ptch1. Statins, antagonists of cholesterol biosynthesis, tumor in children. Approximately 30% of human medullo- exhibited promising therapeutic effects in mouse medulloblas- blastoma arise from aberrant activation of hedgehog (Hh) toma models. Moreover, statins synergized with the clinically pathway. Vismodegib and sonidegib are two FDA-approved approved Smo antagonist, vismodegib, to prevent medulloblas- Hh pathway inhibitors that have demonstrated efficacy in toma progression in vivo. These findings elucidate an important clinical trials of Hh pathway–associated medulloblastoma role for endogenous cholesterol in medulloblastoma tumorigen- (Hh-MB). Despite a dramatic response to these inhibitors in esis and demonstrate that cholesterol biosynthesis represents a initial clinical trials, drug resistance and developmental toxi- novel therapeutic target for the treatment of medulloblastoma cities arise in humans as predicted by mouse models. Here, we and, potentially, other Hh pathway–associated malignancies. demonstrate that cholesterol is required for Hh pathway signal transduction in tumor cells. Statins, inhibitors of cholesterol Materials and Methods biosynthesis, block hedgehog pathway activity in medullo- blastoma and synergize with vismodegib to inhibit tumor Animals growth. Given the excellent safety profile, low cost and ready All mice were maintained under barrier conditions in the availability of cholesterol inhibitors, targeting cholesterol Laboratory Animal Facility at Fox Chase Cancer Center (Phila- biosynthesis represents a promising strategy for treatment of delphia, PA), and all experiments were performed in accordance with procedures approved by the Fox Chase Cancer Center Hh-MB and potentially other malignancies caused by Hh fl/fl pathway activation. Animal Care and Use Committee. Nsdhl mice were kindly provided by Dr. Gail Herman (The Ohio State University, T2 Columbus, OH). Math1-Cre mice, Math1-CreER mice, T2 fl/fl þ/À GFAP-CreER mice, Ptch1 mice, Ptch1 mice, R26R-eYFP mice, and SmoM2 (R26SmoM2)micewerepurchasedfromThe recipient cells (8–11). Two recent studies revealed that cholesterol Jackson Laboratory. CB17/SCID mice were generated by the itself synergizes with Hh ligand to activate Smo through binding animal facility laboratory at Fox Chase Cancer Center. to the CRD (12, 13). The physical interaction between Smo and Tamoxifen (Sigma-Aldrich) was dissolved in corn oil and cholesterol was further confirmed in studies of the crystal structure administrated by oral gavage at a dosage of 200 mg/kg daily of Smo (14). These findings suggest that cholesterol functions as a during 3 subsequent days. Animals were sacrificed for further native ligand for Smo activation and that Hh signaling may be analyses after 7 days from initial tamoxifen treatment. influenced by the local availability of cholesterol. Insufficient Hh signaling can lead to birth defects, whereas Compounds excessive activity of the Hh pathway is associated with the for- The following compounds were used for our in vitro and in vivo mation of human malignancies, including medulloblastoma, the studies: simvastatin (Cayman Chemicals), atorvastatin (Cayman most common malignant brain tumor in children. The Hh Chemicals), triparanol (Sigma-Aldrich), vismodegib (Chemlea- pathway is activated in approximately 30% of human medullo- der Biochemical), and water-soluble cholesterol (cholesterol- blastoma.