Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Gene Expression In Myeloma Cell Lines and Patients

Epigenetic aberrations play an important role in the development and progression of Multiple Myeloma (MM). 5-aza-2-deoxycytidine (5Aza-dC) and Trichostatin A (TSA) have been studied in reactivating the expression of epigenetically-silenced genes. IGFBP3 gene is a member of the insulin-like growth factor binding protein (IGFBP) family and can regulate cell growth and death by the ability to bind insulin-like growth factors (IGFs) as well as its IGF-independent effects involving binding to other molecules. Several in vitro, in vivo studies as well as clinical evidence point to IGFBP-3 as an anti-cancer molecule. In our study we analyzed global changes in gene expression profiles of MM cell lines, responding to 5Aza-dC and TSA and we evaluated the IGFBP3 expression in three myeloma cell lines and in samples from myeloma patients.

Human MM cell lines U266 and H929 were treated either with 0.5 micromol/L 5Aza-dC for 7 days or with 100 ng/mL TSA for 24 h or with the combination of 0.5 micromol/L 5Aza-dC for 7 days and 100 ng/mL TSA for additional 24 h. Control cells received no drug treatment. Applied Biosystems microarray platform ABI 1300 was used for carrying out microarray profiling and analysis. To classify up-regulated genes into functional categories the PANTHER Classification System was used (http://www.pantherdb.org). Stained or unstained bone marrow slides were collected from archival samples of 179 myeloma patients. For validation of microarray results, real-time reverse transcription-PCR (RT-PCR) was performed using Taqman Gene Expression Assays (Applied Biosystems).

After treatment with 5Aza-dC there was up regulation of gene expression in 698 genes in H929 cell line and 258 genes in U266 cell line. After treatment with TSA 719 genes were up regulated in H929 cell line and 742 genes in U266 cell line. The exposure to the combination of 5Aza-dC/TSA resulted in up-regulation of 921 genes in H929 cell line and 615 genes in U266 cell line. By using Panther classification system we classified up-regulated genes into functional categories and we identified several 5Aza-dC or TSA up-regulated genes that are involved in important cancer-related pathways including cell cycle, apoptosis, cell adhesion, oncogenesis and cell metabolism, including DNA repair and nucleosome assembly. Between these genes we particularly found interesting the expression changes of IGFBP-3 which was up regulated after treatment with 5-azacitidine in U266 cell line (level of induced expression was 5,16). The microarray data were validated in 3 MM cell lines and 179 patients samples by analysis of relative changes in IGFBP3 expression through ΔΔCt method. We found that the expression levels of IGFBP3 were significantly lower in U266 and RPMI myeloma cell lines, but not in H929 cell line and interestingly levels were lower in 54 percent of patients (Fig 1). It has been demonstrated decreased IGFBP-3 expression is associated with cancer progression and in our study we have shown that the down-regulation of this gene may be involved also in myeloma pathogenesis and mediate progression events, hence levels of IGFBP3 may be considered as a biomarker for disease staging. From a therapeutic point of view, up-regulation of IGFBP3 might be considered as target therapeutic strategy for monoclonal gammopathies/ smouldering Myelomas at high risk of progression to active myeloma.

Figure 1:

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Relative changes in IGFBP3 expression levels.

Figure 1:

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Relative changes in IGFBP3 expression levels.

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No relevant conflicts of interest to declare.