Abstract
Abstract
Gene expression profiling studies sub-classified diffuse large B-cell lymphomas (DLBCL) into two clinically distinct types: germinal center B cell (GCB)-like and activated B-cell (ABC)-like tumors, characterized by long and short survival, respectively. At least two markers of the GCB-phenotype - BCL6 and HGAL - are IL-4 target genes whose high expression independently predicts better overall survival. Gene expression analysis of DLBCL also demonstrated higher levels of mRNA expression of components of the IL-4 signaling pathway (IL-4Rα, IRS, p110 subunit of PI-3 kinase, and PKC delta) in the GCB-like DLBCL (Alizadeh et al Nature. 2000;403:503). Identification of IL-4 inducible genes in normal B-lymphocytes revealed additional IL-4 target genes that are expressed at higher levels in GCB-like DLBCL compared to ABC-like DLBCL. Together, these observations support the distinct activity of the IL-4 signaling pathway in DLBCL subtypes. Accordingly, IL-4 stimulation of GCB-like (SUDHL6, SUDHL4 and OCILY19) and ABC-like (OCILY10 and OCILY3) DLBCL cell lines increased expression of its known target genes only in GCB-like, but not in ABC-like DLBCL. Further, IL-4 stimulation led to AKT phosphorylation in the ABC-like but not in the GCB-like cells. Conversely, IL-4 induced STAT6 phosphorylation (pSTAT6) in all the tested GCB-like and in the OCILY10 cell lines but not in the OCILY3 ABC-like cell-line. IL-4 induced progressive accumulation of large quantities of pSTAT6 in both the cytoplasm and in the nucleus in the GCB-like DLBCL. In contrast, in IL-4 treated ABC-like OCILY10 cells, pSTAT6 did not accumulate in either cytoplasm or nucleus, and much smaller amounts of pSTAT6 were detected in the nuclear extracts from stimulated cells. The latter observation was at least partially attributed to different extent of pSTAT6 nuclear deposphorylation and proteasomal degradation in the GCB-like and ABC-like DLBCL, as determined by exposure of these cell lines to STAT6 nuclear export inhibitor (leptomycin B) and phosphatase and proteasome inhibitors. Nuclear protein tyrosine phosphatase assays revealed significantly higher phosphatase activity in the ABC-like compared to the GCB-like DLBCL cell-lines. Evaluation of mRNA expression of 51 known tyrosine phosphatases in the GCB-like and ABC-like DLBCL tumors based on array data revealed that mRNA expression of 13 protein tyrosine phosphatases was significantly higher (p<0.001) in the ABC-like compared to GCB-like DLBCL. In vitro pSTAT6 dephosphorylation studies suggested that PTPN2, expressed in ABC-like but not in the GCB-like cell-lines, is a putative STAT6 phosphatase. Taken together with the independent prognostic significance of IL-4 target genes (e.g., BCL-6 and HGAL) and distinct effects of IL-4 on proliferation and immuno-chemosensitivity of GCB-like and ABC-like cells (Abstract by Nechushtan et al), the observed differences in the intracellular IL-4 signaling might contribute to different clinical outcome of patients with DLBCL.