Biology of Human Tumors Post-transcriptional Modifications Contribute to the Upregulation of Cyclin D2 in Multiple Myeloma Irena Misiewicz-Krzeminska 1,2,3 , María E. Sarasquete 1,2 , Carolina Vicente-Due ~ nas 2,4 , Patryk Krzeminski 1,2 , Katarzyna Wiktorska 3 , Luis Antonio Corchete 1,2 , Dalia Quwaider 1,2 , Elizabeta A. Rojas 1,2 , Rocío Corral 2,5 , Ana A. Martín 5 , Fernando Escalante 6 , Abelardo B arez 7 , Juan Luis García 1,2 , Isidro S anchez-García 2,4 , Ram on García-Sanz 1,2,5 , Jes  us F. San Miguel 8 , and Norma C. Guti  errez 1,2,5 Abstract Purpose: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3 0 UTR in CCND2 overexpression in multiple myeloma. Experimental Design: Eleven myeloma cell lines and 45 pri- mary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3 0 UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3 0 rapid amplification of cDNA ends (RACE)-PCR. Results: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3 0 RACE experiments revealed that changes in CCND2 3 0 UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3 0 UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Further- more, functional analysis showed significant CCND2 mRNA short- ening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggest- ing that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. Conclusions: Overall, these results highlight the impact of CCND2 3 0 UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma. Clin Cancer Res; 22(1); 207–17. Ó2015 AACR. Introduction Gene expression profiling has revealed that expression of CCND1, CCND2, or CCND3 is increased in virtually all multiple myeloma tumors, providing the hypothesis of a potential unify- ing event in multiple myeloma pathogenesis (1). In addition, D- cyclins have been proposed as molecular therapeutic targets in multiple myeloma (2). Cyclin D2 belongs to the group of D-type cyclin proteins that are cyclically expressed during the cell cycle. In physiologic conditions, cyclins D regulate the transition from G 1 –S phase by interaction with cyclin-dependent kinases 4 or 6, which further phosphorylate their substrates (3). One of them is Rb that promotes proliferation by release of E2F transcription factor. CCND1 is directly activated by t(11;14) and by biallelic dysregulation in patients with multiple myeloma with polys- omy 11, and CCND3 is overexpressed in patients with t(6;14). High levels of CCND2 are detected in multiple myeloma with t(4;14), t(14;16), and in a set of hyper and nonhyper- diploid multiple myeloma (4). The mechanisms by which CCND2 is upregulated in these cases are not completely deciphered. Apparently, CCND2 is not directly induced by IGH translocations, although the transcription factor Maf involved in t(14;16) transactivates the CCND2 promoter (5). Similarly, a pathway of CCND2 transactivation in multiple mye- loma by transcription factor ZKSCAN3 has been described (6). Nowadays, a large body of evidence indicates that miRNAs are key post-transcriptional regulators of gene expression. In fact, in silico algorithms have identified hundreds of predicted miRNAs targeting CCND2. Previously published data of our group showed a significant correlation between CCND2 upregulation and decreased expression of seven miRNAs in multiple myeloma: miR-15a, miR-19a, miR-19b, miR-20a, miR-135b, miR-196b, and miR-320 (7). In this regard, a downregulation of CCND2 has been demonstrated after transfection of myeloma cells with pre- miRNA-15a and -16, supporting the functional role of both 1 Centro de Investigacion del Cancer-IBMCC (USAL-CSIC), Salamanca, Spain. 2 Institute of Biomedical Research of Salamanca (IBSAL), Sal- amanca, Spain. 3 National Medicines Institute,Warsaw, Poland. 4 Exper- imental Therapeutics and Translational Oncology Program, Instituto de Biologia Molecular y Celular del Cancer, CSIC/Universidad de Salamanca, Salamanca, Spain. 5 Servicio de Hematología, Hospital Universitario, Salamanca, Spain. 6 Complejo Hospitalario de Le on, Le on, Spain. 7 Hospital Nuestra Se~ nora de Sonsoles,  Avila, Spain. 8 Clin- ica Universidad de Navarra, Centro de Investigaciones Medicas Apli- cadas (CIMA), Pamplona, Spain. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Norma C. Guti errez, Department of Hematology, Uni- versity Hospital of Salamanca, Paseo San Vicente, 58-182, Salamanca 37007, Spain. Phone: 34-923291384; Fax: 34-923294624; E-mail: normagu@usal.es doi: 10.1158/1078-0432.CCR-14-2796 Ó2015 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 207 on April 13, 2017. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst September 4, 2015; DOI: 10.1158/1078-0432.CCR-14-2796