Elucidation of Proteins Involved in the Bortezomib Resistance of Multiple Myelomas though iTRAQ Analysis
J Micallef, J Chen, M Dharsee, S Ackloo, K Evans, H Chang. University Health Network, University of Toronto, Toronto, Canada; Ontario Cancer Biomarker Network, Toronto, Canada
Background: Multiple myeloma (MM) is a neoplasm of terminally differentiated plasma cells in the bone marrow. Despite advances in clinical care, MM remains an almost universally fatal disease. Bortezomib, a novel proteasome inhibitor represents a promising new clinical strategy for relapsed and refractory MM. However, only 30-40% of MM patients respond to this treatment. The mechanism of resistance is not understood.
Design: In order to gain insights into the mechanism of bortezomib resistance, we examined the differentially expressed proteins between 8226-S (bortezomib sensitive) and 8226-R5 (bortezomib resistant) cell lines using iTRAQ mass spectrometry (MS). The MS based multiple-reaction-monitoring technique (MRM) was then used to independently verify the quantitative differences of the protein expression levels between the two cell lines. RNA interference strategies are being utilized in order to determine the differentially expressed proteins that maybe involved in drug resistance and the associated signaling pathways.
Results: We identified 176 proteins to be differentially expressed between 8226/S and 8226/R5 by iTRAQ mass spectrometry. Among these proteins, 115 (65%) were up-regulated and 61 (35%) were down-regulated. Using a biological systems analysis approach we then identified 36 cancer relevant proteins. From this list, we chose two proteins that may contribute to bortezomib resistance. One such protein is Ubiquilin-4, an ubiquitin-like protein involved in the unfolded protein response which has been shown to protect against cell death. The second protein we chose was MARCKS, a PKC substrate that has been implicated in conferring drug resistance to ovarian carcinoma cells. Differential expression of these proteins between resistant and sensitive cells was verified by MRM and then confirmed by RT-PCR and western blot analysis. Knockdown of these proteins through siRNA techniques is being used to determine their role in MM bortezomib resistant cell lines. Similar strategies will also be used to identify additional proteins that may be involved in drug resistance.
Conclusions: The use of MS based techniques is a powerful tool that will allow us to identify relevant signaling pathways involved in MM drug resistance. Such knowledge will guide the design of additional therapeutic targets and help tailor patient specific treatment.
Category: Pan-genomic/Pan-proteomic Approaches to Diseases
Monday, March 22, 2010 1:00 PM
Poster Session II # 224, Monday Afternoon