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©Ferrata Storti Foundation Original Article MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells Jose L.R. Brito,1 Brian Walker,1 Matthew Jenner,1 Nicholas J. Dickens,1 Nicola J.M. Brown,2 Fiona M. Ross,3 Athanasia Avramidou,1 Julie A.E. Irving,4 David Gonzalez,1 Faith E. Davies,1 and Gareth J. Morgan1 1Institute for Cancer Research, Section of Haemato-Oncology, London; 2Department of Medical and Molecular Genetics, Division of Genetics and Molecular Medicine, King’s College London, School of Medicine; 3Leukaemia Research Fund UK Myeloma Forum Cytogenetics Group, Wessex Regional Genetics Laboratory, Salisbury; 4Northern Institute for Cancer Research, Newcastle University, UK ABSTRACT Acknowledgments: we would like to thank Bud Flanagan and Background Kay Kendall leukemia funds for The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple funding this work. myeloma patients and is associated with poor prognosis, through an unknown mecha- Manuscript received May 30, nism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple 2008. Revised version arrived myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis September 2, 2008. of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upreg- Manuscript accepted ulation are still unclear. September 23, 2008. Design and Methods Correspondence: Gareth Morgan, Brookes Lawley The expression of MMSET was analyzed using a novel antibodyFoundation. The involvement of Building, Institute of Cancer MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated Research, 15 Cotswold Road, knockdown combined with several biological assays. In addition, the differential gene Sutton, Surrey, SM2 5NG, UK. expression of MMSET-induced knockdown was analyzed with expression microarrays. E-mail: MMSET gene targets in primary patient material was analyzed by expression microarrays. [email protected] Results Storti We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14)©Ferrata multiple myeloma patient samples. Conclusions In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival. Key words: MMSET, WHSC1, myeloma. Citation: Brito JLR, Walker B, Jenner M, Dickens NJ, Brown NJM, Ross FM, Avramidou A, Irving JAE, Gonzalez D, Davies FE, and Morgan GJ. Multiple myeloma SET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells. Haematologica 2009; 94:78-86. doi: 10.3324/haematol.13426 ©2009 Ferrata Storti Foundation. This is an open-access paper. | 78 | haematologica | 2009; 94(1) MMSET involvement in t(4;14) MM Introduction Design and Methods Multiple myeloma (MM) is characterized by the Patients and sample preparation clonal expansion of plasma cells in the bone marrow. This study was performed on 231 bone marrow (BM) Recent advances in autologous stem cell transplanta- samples with informed consent obtained in accordance tion and chemotherapeutic treatments have improved with the Declaration of Helsinki received by the the long-term survival of patients with MM. However, Leukaemia Research Fund UK Myeloma Forum despite such advances, MM remains an incurable dis- Cytogenetic Database between March 2001 and ease for which there is a need to identify novel thera- December 2005. Plasma cells were isolated from newly peutic targets. There is a well recognized range of clin- diagnosed myeloma patients to a purity of more than ical outcomes in MM reflecting the heterogeneous 90% using CD138 microbeads and magnetic assisted nature of the disease which is characterized by multi- cell sorting (Miltenyi Biotech, Bergisch Gladbach, ple genetic abnormalities. In the past two decades our Germany). Selected cells were used for FISH and RNA understanding of the molecular pathology of MM has extraction as described.13, 14 increased significantly. This increase in knowledge has led to the identification of a wealth of chromosomal Genome-wide expression arrays and genetic abnormalities, amongst which recurrent For expression arrays, 100 ng of total tumor RNA was translocations targeting the immunoglobulin heavy amplified using the 2-cycle target labeling kit chain locus (IGH) at the 14q32 locus are the best char- (Affymetrix) and as specified by the manufacturer’s acterized. These translocations occur in up to 60% of instructions. Amplified cRNA (15µg) was hybridized to MM patients and deregulate a number of oncogenes the Human genome U133 Plus 2.0 arrays as described.13,14 including cyclins D1 (11q13) and D3 (6p21), C-MAF All samples were analyzed with dCHIP 2006 (16q23) together with FGFR3 and MMSET (4p16).1 (www.dchip.org).15 In addition, functional clustering These molecular events are thought to occur early in analysis was performed using DAVID Bioinformatics the natural history of the disease promoting myeloma- Resource 2007 (http://david.abcc.ncifcrf.gov).16 genesis and defining the clinical outcome. The most important of these translocations clinically is the Comparison of expression array samples t(4;14), which is present in 15-20% of MM cases and MMSET knockdown samples were compared to con- is associated with poor prognosis.1 trol Foundationsamples using dCHIP, for each cell line. In addition, The t(4;14) translocation leads to the simultaneous patient samples (n=231) were used to compare those overexpression of two genes, FGFR3 and MMSET.2,3 with and without a t(4;14) translocation (Online While the role and mechanism by which FGFR3, a Supplementary Appendix). receptor tyrosine kinase promotes myelomagenesis has been shown in various in vitro and in vivoStortistudies, Multiple myeloma cell lines and cell culture the involvement of MMSET upregulation in t(4;14) All cell lines were acquired from either ATCC or myeloma is still poorly explored.4-10 Moreover, about DSZM, with the exception of KMS-11, which was 30% of MM tumors with the t(4;14) translocation kindly provided by Dr. Otsuki (Kawasaki Medical have recently been reported to lack expression of School, Japan) (Online Supplementary Appendix). FGFR3 due to the loss of der(14).11 Interestingly, the poor prognosis associated with the t(4;14) in such Nuclear-cytosol extraction and Western blotting analysis patients lacking FGFR3 expression remains unchanged, The cytosolic and nuclear fractions of MM cell lines lending further support to a potential role for were isolated and Western blotting analysis performed MMSET.5, 11 MMSET has recently been shown to have (Online Supplementary Appendix). histone methyltransferase©Ferrata activity and knockdown studies have demonstrated that MMSET upregulation MMSET knockdown and REIIBP cloning contributes to cellular adhesion, clonogenic growth MM cell lines were permeabilized to siRNAs using and tumorigenicity.12 Despite these studies, the genes streptolysin-O (SLO) as described previously17-19 (Online that are directly or indirectly regulated by MMSET and Supplementary Appendix). the mechanisms by which MMSET promotes t(4;14) MM remain unknown. Cell proliferation assay In this study, using RNAi mediated knockdown and Cell proliferation was assessed using the CellTiter 96® overexpression of the MMSET isoform, REIIBP com- AQueous Non-Radioactive Cell Proliferation Assay (Promega, bined with global expression arrays on myeloma cell UK) (Online Supplementary Appendix). lines and patient samples, we have identified genes involved in cell cycle progression, cell adhesion and Statistical analysis regulation of apoptosis that are differentially Statistical significance was determined by the two- expressed by MMSET in t(4;14) MM. This study sig- way ANOVA test and Pearson’s correlation using nificantly sheds light on our better understanding of GraphPad Prism version 4.00 for Windows (GraphPad the molecular mechanisms by which MMSET pro- Software, California, US). The minimal level of signifi- motes t(4;14) myelomagenesis. cance was p<0.05. haematologica | 2009; 94(1) | 79 | J.L.R. Brito et al. MMSET knockdown affects cell proliferation, Results cell cycle and apoptosis In order to demonstrate the involvement of MMSET MMSET isoforms are up-regulated at the protein upregulation in the pathogenesis of t(4;14) MM we level in multiple myeloma cell lines with the t(4;14) knocked down MMSET expression using specific translocation siRNAs. MMSET knockdown was initially optimized MMSET isoforms up-regulated in MM cell lines and in three myeloma cell lines, RPMI8226, JIM-3 and H929 patient samples with the t(4;14) have been studied using the streptolysin-O method.19 We found that two using quantitative real time PCR (qRT-PCR).5,20 siRNAs, 273 and 490, designed within exons 23 and 24 However, due to the complex nature of the alternative of MMSET respectively reduced the expression of splicing of MMSET, the use of any region of MMSET MB4-2II and MB4-3II isoforms by greater than 90% in mRNA amplifies or detects multiple transcript iso- the t(4;14)-positive cell lines JIM-3 and H929 in relation forms, making the data difficult to interpret accurately to the negative control siRNA (Figure 2A). The expres- (Figure 1A). More recently, antibodies have been used sion of REIIBP was not affected by the knockdown, this to determine the expression of MMSET isoforms in finding was expected as REIIBP has an extended half- cell lines.12,21 However, these studies have used a limit- life of 65 hours compared to four hours for MMSET II, ed number of cell lines carrying the t(4;14) transloca- MB4-2II and MB4-3II isoforms (data not shown).
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