Tumor and Stem Cell Biology microRNA-148a Is a Prognostic oncomiR That Targets MIG6 and BIM to Regulate EGFR and Apoptosis in Glioblastoma

Great interest persists in useful prognostic and therapeutic targets in glioblastoma. In this study, we report the definition of miRNA (miR)-148a as a novel prognostic oncomiR in glioblastoma. miR-148a expression was elevated in human glioblastoma specimens, cell lines, and stem cells (GSC) compared with normal human brain and astrocytes. High levels were a risk indicator for glioblastoma patient survival. Functionally, miR-148a expression increased cell growth, survival, migration, and invasion in glioblastoma cells and GSCs and promoted GSC neurosphere formation. Two direct targets of miR-148a were identified, the EGF receptor (EGFR) regulator MIG6 and the apoptosis regulator BIM, which rescue experiments showed were essential to mediate the oncogenic activity of miR-148a. By inhibiting MIG6 expression, miR-148a reduced EGFR trafficking to Rab7expressing compartments, which includes late endosomes and lysosomes. This process coincided with reduced degradation and elevated expression and activation of EGFR. Finally, inhibition of miR-148a strongly suppressed GSC and glioblastoma xenograft growth in vivo. Taken together, our findings provide a comprehensive analysis of the prognostic value and oncogenic function of miR-148a in glioblastoma, further defining it as a potential target for glioblastoma therapy. Cancer Res; 74(5); 1541–53. 2014 AACR. Introduction Glioblastoma is an extremely aggressive tumor that accounts for the majority of deaths due to primary brain neoplasms (1). Despite the most advanced treatment with combinations of surgery, radiotherapy, and chemotherapy, glioblastoma is associated with a median survival of only 14 months (2). Factors responsible for glioblastoma malignancy and poor prognosis include rapid cell growth, resistance against apoptosis, and distant invasion of the surrounding brain (1, 3). Receptor tyrosine kinase (RTK) pathways are deregulated in the vast majority of glioblastomas (4, 5). Among RTKs, the EGF receptor (EGFR) is themost commonly altered (6). It ismutated and/or amplified in 40% and overexpressed in >60% of tumors (7, 8). Activation of EGFR induces tumor cell growth, migration, and invasion, as well as resistance to chemotherapy and radiation (6, 9). EGFR signaling and protein half-life are tightly regulated (10). Mitogen-inducible gene 6 (MIG6) regulates EGFR signaling and turnover by binding EGFR and directly inhibiting tyrosine kinase activity, increasing clathrin-dependent EGFR endocytosis and trafficking into the lysosome, and promoting EGFR degradation (11–13). Ablation of MIG6 induces tumor formation, supporting a tumor suppressor function of MIG6 (11, 14). The MIG6 gene is located on chromosome 1p36, which is subject to focal deletions in glioblastoma. The Cancer Genome Atlas (TCGA) data analysis showed that 15 out of 430 glioblastoma samples contain homozygous deletions in 1p36 (14) but that MIG6 expression is downregulated in approximately 50% of primary tumor samples and glioblastoma cell lines (11). Therefore MIG6 deletions only account for a small fraction of the glioblastoma tumors with reduced MIG6 expression. Resistance to apoptosis is a big obstacle in glioblastoma therapy (15, 16). Apoptosis in the intrinsic pathway is regulated by the balance between proapoptotic (Bax, Bak, BIM, and Bad) and antiapoptotic (Bcl-2 and Bcl-xL) members of the Bcl-2 family (17). Proapoptotic BIM (BCL2L11) is localized to the mitochondria where it initiates the mitochondrial cell death pathway by directly activating Bax/Bak-dependent apoptosis. BIM has been shown to be an important mediator of targeted therapy-induced apoptosis in solid tumors. BIM is downregulated in 29% of glioblastoma cases based on TCGA analysis (18, 19). However, the causes of BIM downregulation in glioblastoma are not known. microRNAs (miRNA) are short noncoding RNA molecules that regulate gene expression by binding to the 30 untranslated region (30UTR) of target mRNA and inducing mRNA Authors' Affiliations: Departments of Microbiology, Immunology and Cancer Biology, Neurology, and Cancer Center, University of Virginia, Charlottesville, Virginia; Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom; and Department of Neurosurgery, Brigham and Women's Hospital, Boston, Massachusetts Deceased. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Roger Abounader, University of Virginia, PO Box 800168, Charlottesville, VA 22908. Phone: 434-982-6634; Fax: 434-2436843; E-mail: [email protected]; and Sean Lawler, Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, 4 Blackfan Circle, HIM 930A, Boston, MA 02115. Phone: 617-525-5066; Fax: 617726-5079; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-1449 2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org 1541 on January 28, 2018. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 14, 2014; DOI: 10.1158/0008-5472.CAN-13-1449