The Nuclear Orphan Receptors NR4A1 and NR4A3 as Putative Tumour Suppressor Genes in Aggressive Non-Hodgkin's Lymphomas.

Abstract 2967

Poster Board II-943

NR4A1 (Nur77, TR3, or NGFI-B), NR4A2 (Nurr1 or RNR-1) and NR4A3 (Nor-1 or Minor) are three members of the orphan nuclear hormone receptor (NR) family referred to as Nur77 family. These orphan NRs have been implicated in cell cycle regulation, apoptosis, inflammation, and more recently in carcinogenesis. The most convincing evidence that nuclear orphan receptors function as critical tumour suppressors is the observation that the NR4A1 and NR4A3 double knock out mouse develop rapidly acute myeloid leukemia (AML) involving abnormal expansion of hematopoietic stem cells and myeloid progenitors, decreased expression of the AP-1 transcription factors JunB and c-Jun and defective extrinsic apoptotic (Fas-L and TRAIL) signalling.

Because the NR4A1, NR4A2 and NR4A3 expression and their function in mature B-cell neoplasmas and ALL are unknown, we performed a comprehensive expression analyses on frozen tissue samples of the most common B-cell lymphomas and lymphoma cell lines on mRNA and protein levels using semi-quantitative real-time PCR and Western Blot analyses. Samples of peripheral CD19+ B-cells, hyperplastic lymph nodes (lymphadenopathy, LA) and CD77+ germinal center B-cells (GC B-cells), and mantle cells isolated from tonsils of healthy donors served as controls. Samples of AML with known reductions in NR4A levels were included as negative control. Comparing the NR4As expression on mRNA and protein level of each hematologic malignancy to peripheral CD19+ cells, LAs and to their cell of origin, significantly reduced expression levels for NR4A1 and NR4A3 were found in all lymphoma cell lines analysed (Ly 1, 3, 4, 7 and 8), in two of eight “low grade lymphoma” entities (B-CLL and follicular lymphomas grade II (=FLII)) and in three of five “high grade lymphoma” entities (diffuse large B-cell lymphomas (=DLBCL), FL III, mantle cell lymphomas (=MCL)) as well as in acute lymphatic leukemia (=ALL) (p<0.01). The magnitude of reduction in NR4A1 and NR4A3 expression in high grade lymphomas equalled those previously described for AML suggesting a similar biologic effect. To gain knowledge whether the reduced NR4A1 and NR4A3 expression has any impact on NR4A target genes, we analyzed the mRNA expression of two members of the extrinsic apoptotic pathway, namely Fas-L and TRAIL. TRAIL was significantly lower expressed in the “low grade lymphoma” entities (B-CLL and FLII) and in the “high grade lymphoma” entities (BL, DLBCL, FL III and MCL) compared to normal controls (p<0.01). Bim and Puma, two members of the BH3 only proteins inducing apoptosis via the intrinsic pathway during B cell development, were also significantly lower expressed in the “high grade lymphoma” entities (DLBCL and FL III) and Bim additionally in FLII (p<0.01). When correlating the NR4A1 and NR4A3 expression levels to Fas-L, TRAIL, Bim and Puma expression, a significant positive correlation was observed suggesting that reduced NR4A1 and NR4A3 expression may result in a reduction of extrinsic apoptotic signals. Our data suggests that NR4A1 and NR4A3 act as putative tumour suppressor in a substantial number of lymphoma entities and that their abrogation leads to a defective extrinsic and intrinsic apoptotic signalling.