This work shows for the first time that myeloma cells from MM patients express BCL3
and that high expression of BCL3
at the time of diagnosis is associated with reduced survival. Furthermore, in a comparison of molecularly defined subgroups, BCL3
expression was significantly increased in a high risk subgroup characterized by overexpression of cell cycle-and proliferation-related genes (28
). Our data are consistent with the concept that high BCL3
expression is an event associated with poor outcome.
We have not shown that BCL3
is an independent adverse prognostic factor and the exact relevance of elevated expression of BCL3
in myeloma high-risk disease is currently unclear. Interestingly, Annunziata et al.
has shown that the high risk subgroup with high expression of BCL3
has a very low average expression of NFκB signature genes (39
). NFκB signature genes are genes that are activated by the classical and/or alternative NFκB pathway (39
). It is therefore tempting to speculate that the nuclear presence of Bcl-3 as after IL-6 stimulation activates a different set of genes than activation of the classical and/or alternative NFκB pathway in myeloma cells.
The array data was confirmed with qPCR in a material with 10 patients. In another randomly selected material with eight patients with MM, Bcl-3 protein was detected in seven of eight samples by Western blot. We also examined the expression of Bcl-3 at protein level by immunohistochemical staining of biopsies from MM patients and patients with plasmacytoma, and found that 11% (two of 18) of the biopsies stained positive for Bcl-3. This proportion of Bcl-3+
plasma cell malignancies is similar to the proportion of B-cell lymphomas expressing Bcl-3 (6%) (9
). The discrepancy between the proportion of patient samples positive for BCL3
at mRNA level and biopsies positive for Bcl-3 at protein level may be caused by lower sensitivity of immunostaining of biopsies, a well-known problem when using immunohistochemistry as detection method. However, the number of patient samples tested was small, and we cannot rule out that there is reduced translation or post-translational modifications.
Our data showed that stimulation of myeloma cells with various cytokines well-known to promote proliferation increased Bcl-3 expression. These data indicate that the gene encoding Bcl-3 is indeed a common target gene for growth promoting cytokines in myeloma cells. As growth-promoting cytokines are frequently present in the bone marrow of MM patients, cytokine signaling is a highly possible mechanism for upregulation of Bcl-3 in myeloma cells in vivo
. Importantly, and in concordance with the patient data, we found that the induction of Bcl-3 by cytokines was associated with increased proliferation of myeloma cells. However, in line with the results from Brocke-Heidrich et al.
), we could not show any effect on thymidine incorporation or apoptosis when down regulating Bcl-3 with siRNA in the INA-6 cell line (data not shown). These results are from only one cell line and further studies are needed to clarify the exact role of Bcl-3.
Additional potential mechanisms for upregulation of Bcl-3 in MM patients are gains/amplification of the BCL3
gene, as described in patients with anaplastic large cell lymphoma, or translocations involving chromosome 19, as found in patients with CLL (40
). Our studies substantiate that both these mechanism may be active in freshly isolated MM patients cells. The BCL3
gene is located at chromosome 19, a chromosome with frequent trisomy in MM patients having a hyperdiploid tumor (42
In our study, intracellular localization of Bcl-3 was dependent of type of stimulus given to the cell. Nuclear accumulation of Bcl-3, as after IL-6 stimulation, has been shown to be associated with p50/Bcl-3- or p52/Bcl-3 dependent gene activation and subsequent proliferation in CYLD
-lacking keratinocytes (26
). It is possible that similar mechanisms are active in myeloma cells, and may explain why we observed activation of p50 in IH-1 after IL-6 stimulation.
The starting point of this study was our observation that IL-6, IL-21 and TNF-α induced expression of BCL3 in the myeloma cell lines OH-2 and IH-1. Our study demonstrates that also the protein, Bcl-3, is produced in cell lines by cytokine stimulation and is associated with increased proliferation of the cells. We show for the first time that Bcl-3 is present in a subset of MM patients, and that the high gene expression at the time of diagnosis is associated with inferior prognosis as demonstrated in a large cohort of newly diagnosed patients. Our results indicate a potential role for Bcl-3 in the development of multiple myeloma, and further studies are needed to clarify this.