Pyrvinium Targets CD 133 in Human Glioblastoma Brain Tumor – Initiating Cells

Purpose: Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133þ treatment-refractory population. Experimental Design:Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved. Results:We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition ofWnt/b-catenin signaling andother essential stemcell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133þ stem cells that drive glioblastoma relapse andmortality. Conclusions: Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers. Clin Cancer Res; 21(23); 5324–37. 2015 AACR. Introduction Despite improvements in cancer treatment, many patients experience disease progression, relapse, and reduced overall survival. Prior research has focused on molecular mechanisms or genetic alterations implicated in drug resistance. However, the contribution of intratumoral heterogeneity to therapy failure and relapse must be acknowledged (1). Individual tumor cells can display variable proliferation, apoptosis, metabolism, and other "hallmarks of cancer" (2). Therefore, mechanisms driving intratumoral cellular variability present appealing therapeutic targets. Intratumoral cancer cell heterogeneity was identified by prospective isolation via marker-directed cell sorting of malignant stem-like cell subpopulations, with corresponding phenotypic diversity. The repertoires of cell surface markers that identify tumor-initiating cells (TICs) across multiple solid tumors include CD133 (3–6), Stem cell antigen 1 (Sca1; refs. 7–10), CD44 (11, 12), CD24 (13, 14), and epithelial-specific antigen (ESA; refs. 13, 15). However, unlike clinical biomarkers (like EGFR, HER2, and KRAS), the functional significance of these proteins in tumor progression remains poorly understood. Different cell surface markers may indicate heterogeneous TIC subpopulations with different stem cell characteristics, degrees of differentiation, and malignant biologic behaviors. Moreover, using surface markers to dissect intratumoral heterogeneity is complicated by cancer cells alternating between TIC and non-TIC states (16). Determinants of stemness have been shown to contribute to treatment failure, irrespective of whether the tumor cell population exists in dynamic equilibrium. Self-renewal, the cardinal property of stemness, is defined by the ability of a cell, at each cell division, to generate an identical copy of itself and a cell of the same or different phenotype (17). Cancer may thus be thought of as a disease of unregulated self-renewal (18). CD133 is a marker of self-renewing hematopoietic (19) and neural (20) stem cells that also identifies TIC populations in multiple human cancers (3–6). CD133 expression correlates with McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario, Canada. McMaster Centre for Functional Genomics, McMaster University, Hamilton, Ontario, Canada. Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada. Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada. Department of Pathology and Molecular Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada. Department of Surgery, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Sheila K. Singh, McMaster University, 1200 Main Street West, MDCL 5027, Hamilton, ON L8S 4K1, Canada. Phone: 905-521-2100, ext. 75237; Fax: 905-521-992; E-mail: ssingh@mcmaster.ca doi: 10.1158/1078-0432.CCR-14-3147 2015 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 21(23) December 1, 2015 5324 on November 11, 2017. © 2015 American Association for Cancer Research. clincancerres.aacrjournals. org Downloaded from Published OnlineFirst July 7, 2015; DOI: 10.1158/1078-0432.CCR-14-3147