Abstract
Mantle cell lymphoma (MCL) remains incurable with current therapeutic approaches. Proteins expressed aberrantly at the MCL plasma membrane are candidate molecules for the development of targeted therapy. In the current study, we characterized globally the expression of proteins in the MCL plasma membrane and in purified lipid raft fractions in comparison with normal B cells using proteomic and bioinformatic analyses. Plasma membrane fractions were prepared from MCL patients in leukemic phase of disease and were then separated on 1D SDS-PAGE gels; sequential gel slices were digested with trypsin and the extracted peptides identified by LC/MS-MS. This approach overcomes the limitations of 2D gels for membrane proteins and also provides information on protein localization and post-translational modifications. Bioinformatics identified 111 intrinsic transmembrane proteins, from which we profiled selected proteins across primary MCL cases, MCL derived cell lines and normal B cells by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Several transmembrane proteins, including CD27/CD70 and CD31 (PECAM) were abnormally expressed when compared to normal B cells. CD70 was significantly up-regulated (> 10 fold) in MCL patients along with its cognate receptor, CD27 which was also up-strongly up-regulated (4–9 fold), suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and PKC-βII were also detected in plasma membranes, suggesting constitutive activation of these proteins in MCL. Since activated PKC-βII has been reported to be recruited to lipid rafts during BCR signaling and to control IêB kinase lipid raft recruitment and activation, we undertook shotgun proteomics and protein profiling of MCL lipid rafts, purified using sucrose gradient centrifugation. This analysis revealed an abnormal composition of lipid rafts in MCL. Raftlin a myristoylated lipid raft B-cell specific protein, required for the integrity of lipid rafts and BCR signal transduction, was markedly down-regulated in MCL, as was the lymphoid transmembrane adaptor protein, Cbp/PAG (PAG1), which controls lymphocyte activation. These data were confirmed by RT-PCR which showed significant down-regulation of both genes in MCL. In comparison, other constitutive lipid raft proteins, such as Csk, Blk, Fyn and Lyn kinases and flotillin 1 were expressed within lipid rafts at levels similar to normal B lymphocytes. However, PKC-bII was not localized within lipid rafts indicating aberrant signaling via this molecule in MCL. Conversely, 5-lipoxygenase (5-LO/ALOX5) a key enzyme in leukotriene biosynthesis, which is normally expressed in either the nucleus or cytoplasm, was unexpectedly associated with lipid rafts isolated from MCL cells and was up-regulated ~7-fold in MCL as compared to normal B cells. Comparable results were obtained in chronic lymphocytic leukemia (CLL). Aberrant expression of 5-lipoxygenase has been associated with increased proliferation and suppression of apoptosis in other malignancies. To assess the possible functional activity of this pathway in malignant B-cells, the effects of inhibitors of 5-LO activity (AA861) and FLAP (MK886) its activating enzyme, were assessed on MCL cell lines and primary CLL cells. Both inhibitors induced high levels of apoptosis in a time-dependent and concentration-dependent manner in MCL cell lines and CLL cells, indicating an important role for this enzyme and the leukotriene biosynthetic pathway in MCL and other B-cell malignancies. Thus, using shotgun proteomics and protein expression profiling we have identified a subset of transmembrane proteins with aberrant expression and aberrant subcellular localization in MCL plasma membranes that may contribute to the pathology of the disease and are potential therapeutic targets in treating MCL.
Plasma membrane organization and function: moving past lipid rafts
