Proteomic Evaluation of Pathways Associated with Dexamethasone-Induced Apoptosis and Resistance in Multiple Myeloma.

Glucocorticoids are the mainstay of treatment for patients with myeloma (MM). Unfortunately many patients become resistant to therapy and the median survival is approximately 4.5 years. This study has used a global protein-expression approach to further characterise the pathways of dexamethasone (dex)-induced apoptosis and resistance in the sensitive and resistant MM.1 sub-line’s (MM.1S and MM.1R respectively). Following dex treatment, protein from MM.1S was separated by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using a pH range of 4–7 in the first dimension. Changes in expression pattern were identified between dex-treated and untreated cells with 24 spots downregulated and 3 spots upregulated. Identification by mass spectrometry (4700 Proteomics analyser: MALDI TOF-TOF, Applied Biosystems) demonstrated that 10/24 of the downregulated proteins were involved in cell survival and proliferation whereas the three upregulated proteins were involved in post translational modification, protein folding and trafficking. A comparison with gene expression studies identified a number of corresponding genes, as well as a number of proteins/genes highlighted in one study but not the other. Interestingly the upregulation of FKBP51 a key regulatory component of the Hsp90 steroid receptor complex was observed in both gene and protein analysis. Importantly 2D-PAGE of the dex-resistant line MM.1R demonstrated no increase in FKBP51, confirming its role in mediating dex-induced apoptosis. The Hsp90 receptor complex is important in maintaining the glucocorticoid receptor (GR) in a state receptive to dex and comprises of a number of molecules including the GR, Hsp90, and the immunophilin’s FKBP51 and FKBP52. Western and immunoblotting analysis of the complex in the MM.1S cell line identified FKBP51/52 and GR expression to increase in a time dependent manner whilst Hsp90 expression remained constant. No changes were observed in the resistant line post drug exposure. Overexpression of FKBP51 at resting state or the inability to induce expression following treatment with dex has been associated with glucocorticoid resistance in other diseases. Gene array analysis of 29 myeloma cases showed no statistical difference in FKBP51 expression between presenting myeloma cases sensitive to dex and relapsed refractory cases resistant to dex, suggesting in myeloma resistance is not mediated by a resting state upregulation of FKBP51. RQ-PCR of MM.1S and a number of other cell lines sensitive to dex showed between 5 and 24-fold change in FKBP51 expression post drug exposure, whereas cell lines resistant to dex showed <2 -fold change. Similar results were observed in primary pateint cells, suggesting that an increase in FKBP51 following dex exposure may be a surrogate marker for dex sensitivity. In conclusion these protein profiling studies have identified a number of novel proteins involved in dex-induced apoptosis and resistance, many of which warrant further investigation.