The NF-κB subunit c-Rel regulates Bach2 tumour suppressor expression in B-cell lymphoma.

J E Hunter,H Sellier,J A Butterworth,K J Campbell,C M Bacon,B Zhao,N D Perkins,H D Thomas,B E Gewurz,S J Cockell

doi:10.1038/onc.2015.399

J E Hunter, H Sellier + Show 8 more

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https://doi.org/10.1038/onc.2015.399

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Abstract

The REL gene, encoding the NF-κB subunit c-Rel, is frequently amplified in B-cell lymphoma and functions as a tumour-promoting transcription factor. Here we report the surprising result that c-rel–/– mice display significantly earlier lymphomagenesis in the c-Myc driven, Eμ-Myc model of B-cell lymphoma. c-Rel loss also led to earlier onset of disease in a separate TCL1-Tg-driven lymphoma model. Tumour reimplantation experiments indicated that this is an effect intrinsic to the Eμ-Myc lymphoma cells but, counterintuitively, c-rel–/– Eμ-Myc lymphoma cells were more sensitive to apoptotic stimuli. To learn more about why loss of c-Rel led to earlier onset of disease, microarray gene expression analysis was performed on B cells from 4-week-old, wild-type and c-rel–/– Eμ-Myc mice. Extensive changes in gene expression were not seen at this age, but among those transcripts significantly downregulated by the loss of c-Rel was the B-cell tumour suppressor BTB and CNC homology 2 (Bach2). Quantitative PCR and western blot analysis confirmed loss of Bach2 in c-Rel mutant Eμ-Myc tumours at both 4 weeks and the terminal stages of disease. Moreover, Bach2 expression was also downregulated in c-rel–/– TCL1-Tg mice and RelA Thr505Ala mutant Eμ-Myc mice. Analysis of wild-type Eμ-Myc mice demonstrated that the population expressing low levels of Bach2 exhibited the earlier onset of lymphoma seen in c-rel–/– mice. Confirming the relevance of these findings to human disease, analysis of chromatin immunoprecipitation sequencing data revealed that Bach2 is a c-Rel and NF-κB target gene in transformed human B cells, whereas treatment of Burkitt's lymphoma cells with inhibitors of the NF-κB/IκB kinase pathway or deletion of c-Rel or RelA resulted in loss of Bach2 expression. These data reveal a surprising tumour suppressor role for c-Rel in lymphoma development explained by regulation of Bach2 expression, underlining the context-dependent complexity of NF-κB signalling in cancer.

Highlights

The tumour-promoting role of the NF-κB pathway is well established and results from its ability to regulate the expression of genes involved in multiple aspects of cancer cell biology.[1]
RelA and c-Rel function as effector subunits for the IκB kinase β-dependent, canonical NF-κB pathway.[7]. Of these NF-κB subunits, c-Rel is most closely associated with lymphoma and was first identified as the cellular homologue of the avian Rev-T retroviral oncogene v-Rel.[8–10] c-Rel is ubiquitously expressed in B cells regardless of developmental stage, the highest levels are observed in mature B cells.[11–13] c-rel knockout mice developed normally with no effects on B-cell maturation but do exhibit some immunological defects, including reduced B-cell proliferation and activation, abnormal germinal centres and reduced number of marginal zone B cells.[14–17] c-Rel is distinct from other NF-κB family members in its ability to transform chicken lymphoid cells in vitro.[8,18–20]
NF-κB is active in Eμ-Myc-derived lymphoma To determine if there are significant levels of NF-κB activity in Myc-driven B-cell lymphoma, with the potential to affect disease driven by this oncogene, we crossed 3 × κB-luc (NF-κB-Luc) reporter mice onto Eμ-Myc transgenic mice, allowing in vivo visualisation of NF-κB activity.[31]

Summary

Introduction

The tumour-promoting role of the NF-κB pathway is well established and results from its ability to regulate the expression of genes involved in multiple aspects of cancer cell biology.[1]. 3480 c-rel–/– B-cell lymphomas are more sensitive to apoptotic stimuli c-Rel and the other NF-κB subunits can contribute towards tumorigenesis by inducing the expression of antiapoptotic genes[34] and, consistent with this and the results, we found that when cultured ex vivo, tumour cell isolates from EμMyc/c-rel–/– mice showed increased sensitivity to the R-CHOP therapy components doxorubicin and vincristine (Figure 2e).

Results

Conclusion