CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27Kip1 mRNA translation Silvia Galardi, Massimo Petretich, Guillaume Pinna, Silvia D’amico, Fabrizio Loreni, Alessandro Michienzi, Irina Groisman, Silvia Anna Ciafrè To cite this version: Silvia Galardi, Massimo Petretich, Guillaume Pinna, Silvia D’amico, Fabrizio Loreni, et al.. CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27Kip1 mRNA translation. Scientific Reports, Nature Publishing Group, 2016, 6, 10.1038/srep25219. hal-01412513 HAL Id: hal-01412513 https://hal.archives-ouvertes.fr/hal-01412513 Submitted on 8 Dec 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. www.nature.com/scientificreports OPEN CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27Kip1 mRNA Received: 25 November 2015 Accepted: 08 April 2016 translation Published: 04 May 2016 Silvia Galardi1, Massimo Petretich2,†, Guillaume Pinna2, Silvia D’Amico3, Fabrizio Loreni3, Alessandro Michienzi1, Irina Groisman2,* & Silvia Anna Ciafrè1,* The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via′ 3 untranslated regions (3′UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27Kip1 by specifically targeting its ′3 UTR, and competes with miR-221/222 binding at an overlapping site in the 3′UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27Kip1 3′UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27Kip1 mRNA. This leads to higher levels of p27Kip1 in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma. The cytoplasmic element binding protein 1 (CPEB1) is the founding member of a family of four conserved sequence-specific RNA-binding proteins (CPEB1–4) that regulate gene expression at the post-transcriptional level.1 CPEBs specifically bind to the cytoplasmic polyadenylation element (CPE; consensus sequence UUUUUAU) in 3′ UTRs of messenger RNAs and control, along with other cellular factors, both translational repression and activation through regulation of poly(A) tail length2. Initially CPEBs were identified as maternal mRNA regulators, responsible of cytoplasmic polyadenylation of dormant mRNAs with short poly(A) tails and consequent induction of their translation during early embryonic development3,4. In the last years, the data about the biochemistry and biology of CPEBs has grown rather substantially and it is now clear that these proteins are key mediators of several important biological processes ranging from cell cycle control and cancer to learning and memory5,6. Some evidences suggest a potential tumor suppressive role for CPEB1. The expression level of CPEB1 mRNA were found decreased in ovarian, melanoma and gastric cancer as well as in cell lines derived from breast, mye- loma and colorectal cancer7–11. This reduction has been associated with the capacity of malignant cells to promote invasion, angiogenesis, to increase resistance to nutritional stress and to induce epithelial-to-mesenchymal tran- sition12,13. Moreover, CPEB1-knockout human foreskin fibroblasts proliferate more rapidly, bypassing senescence and are subject to the “Warburg effect”, an aerobic glycolysis considered to be a hallmark of cancer cells14. GBM is the most common and aggressive primary brain tumor. Despite interventional therapy, the overall prognosis for GBM patients remains poor15; thus, it is essential to understand its molecular pathogenesis, in order to provide new insight into modern therapy. Among factors whose mis-regulation has been linked to GBM progression, the cyclin-dependent kinase inhibitor p27Kip1 is an established prognostic marker and its expres- sion has been inversely related to tumor grade and positively related to favorable outcome of GBM patients16,17. p27Kip1 plays a pivotal role in the control of cell proliferation, differentiation, and apoptosis17–19; it is considered 1Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, 00133 Rome, Italy. 2Institute for Integrative Biology of the Cell, IBITECS, CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198, Gif-sur- Yvette cedex, France. 3Department of Biology, University of Rome “Tor Vergata”, 00133 Rome, Italy. †Present address: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.G. (email: [email protected]) SCIENTIFIC REPORTS | 6:25219 | DOI: 10.1038/srep25219 1 www.nature.com/scientificreports/ Figure 1. CPEB1 is downregulated in glioblastoma and affects proliferation of glioma cell lines. (A) Microarray data for CPEB1 gene expression from TCGA dataset consisting of normal (n = 10) and GBM (n = 542) samples; or from “Sun brain” dataset consisting of normal (n = 23) and GBM (n = 81) samples, are shown as a box-plots. A non-parametric t test was performed to calculate p values as indicated in the text. The horizontal line in each plot represents the mean value. (B) Total RNA was extracted from A172, LN18, U87 and T98G glioblastoma cell lines, or from cultured human astrocytes and endogenous CPEB1 mRNA expression was analysed by RT-qPCR. CPEB1 mRNA expression in all samples is reported as compared to that in “normal human brain” (Ambion-Thermofisher #AM7962), set as = 1. ANOVA test in combination with Dunnett’s test were performed to calculate p values. (C) Cells were transfected with the empty plasmid (C) or pCPEB1 plasmid7 and, after 48 h, viable cells were counted using a hemocytometer. Student’s t-test was performed. D, T98G cells transfected with siRNAs against CPEB17 or a non-targeting control siRNA (siC) were analysed as in (C). In this and all the subsequent figures, *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were performed three times (biologic replicates). All histograms in all figures refer to mean ± S.D. an unusual tumor-suppressor protein, as its deregulated expression in cancer is almost exclusively due to mis- regulation of its gene expression at the mRNA or the protein level, rather than due to mutations in the p27Kip1 gene. Different mechanisms of misregulation of p27Kip1 expression were proposed in human gliomas. Particularly, specific microRNAs (miRNAs), a class of post-transcriptional negative regulators that control gene expression by binding to 3′ UTRs, were shown to control p27Kip1 expression in several tumors20–23. Among miRNAs regulating p27Kip1, miR-221/222 were shown to negatively affect the translation of p27Kip1 mRNA in glioblastoma20,24 where the increased expression level of miR-221/222 clearly correlates with malignancy, as opposed to that of p27Kip1. In the present study, we found that CPEB1 is significantly downregulated in human gliomas and that the restoration of its expression impairs GBM cell lines growth. We demonstrated that CPEB1 is directly involved in p27Kip1 expression regulation, by specifically targeting the 3′ UTR of p27Kip1 and counteracting miR-221/222 action. Results and Discussion CPEB1 ectopic modulation affects proliferation of glioma cells. To evaluate the possible role of CPEB1 in glioma malignancy, we searched the Oncomine database25 for CPEB1 expression in large glioblastoma datasets. Among them, in the Cancer Genome Atlas (TCGA) showing the distribution of CPEB1 mRNA expres- sion in 542 GBM tissues versus 10 normal brain tissues, we found that the CPEB1 mRNA levels in the GBM group were significantly lower than those in the normal brain group (Log2FC = − 5.607, p = 5.68E-15, Fig. 1A). This observation was confirmed in another independent dataset, the “Sun brain”, comparing 81 GBM samples to 23 healthy controls (Log2FCGBMvsCTRL = − 5.360, p = 5.10E-21, Fig. 1A). We then applied real-time PCR to SCIENTIFIC REPORTS | 6:25219 | DOI: 10.1038/srep25219 2 www.nature.com/scientificreports/ quantitatively analyse endogenous CPEB1 expression in a panel of human GBM cell lines and we determined that CPEB1 was not expressed in LN-18, A172, and U87MG cell lines and it was 20-fold less expressed in T98G cell line as compared with normal human brain and astrocytes (Fig. 1B). To elucidate the function of CPEB1 in GBM, we ectopically altered its expression and found that the prolifer- ation rate of glioma cells was strongly modified. Fig. 1C shows the results of a proliferation assay where the via- bility of T98G and A172 cells transfected with the empty vector, was compared with that of cells transfected with a plasmid expressing FLAG-tagged CPEB1 (pCPEB1, Fig. S1A): the expression of CPEB1 induced on average a 2-fold decrease