Classical and/or alternative NF-kappaB pathway activation in multiple myeloma

Abstract

Mutations involving the nuclear factor-kappaB (NF-kappaB) pathway are present in at least 17% of multiple myeloma (MM) tumors and 40% of MM cell lines (MMCLs). These mutations, which are apparent progression events, enable MM tumors to become less dependent on bone marrow signals that activate NF-kappaB. Studies on a panel of 51 MMCLs provide some clarification of the mechanisms through which these mutations act and the significance of classical versus alternative activation of NF-kappaB. First, only one mutation (NFKB2) selectively activates the alternative pathway, whereas several mutations (CYLD, NFKB1, and TACI) selectively activate the classical pathway. However, most mutations affecting NF-kappaB-inducing kinase (NIK) levels (NIK, TRAF2, TRAF3, cIAP1&2, and CD40) activate the alternative but often both pathways. Second, we confirm the critical role of TRAF2 in regulating NIK degradation, whereas TRAF3 enhances but is not essential for cIAP1/2-mediated proteasomal degradation of NIK in MM. Third, using transfection to selectively activate the classical or alternative NF-kappaB pathways, we show virtually identical changes in gene expression in one MMCL, whereas the changes are similar albeit nonidentical in a second MMCL. Our results suggest that MM tumors can achieve increased autonomy from the bone marrow microenvironment by mutations that activate either NF-kappaB pathway.

Figure 1

Steady-state levels of components of the NF-κB pathway in MM cells. (A) Steady-state levels of NF-κB subunits in nuclear-enriched protein fraction from MMCLs. Nuclear extracts were prepared, and expression of the proteins indicated at right was analyzed on immunoblots: known and unknown (?). NF-κB mutations for MMCLs with high NF-κB index are indicated in parentheses. (B) Immunoblot for CYLD in MMCLs.

Figure 2

Effect of Smac-mimetic on NF-κB signaling in MMCLs with different genetic abnormalities of NF-κB pathway components. (A) Immunoblot of of MMCLs cultured for 16 hours in medium alone or in the presence of 50nM Smac-mimetic. Nuclear and cytosol extracts were prepared, and expression of the proteins indicated at the right was analyzed. NF-κB mutations in different MMCLs are indicated. (B) Activation of NF-κB target gene expression after incubation of cells with Smac-mimetic. The average expression of 3 target genes (TNFAIP3, NFKB2, and IL2RG) was detected by quantitative PCR in control cells and cells cultured for 16 hours in medium with 50nM Smac-mimetic. Four groups were compared: (1) 3 MMCLs with biallelic deletions of cIAP1/2, (2) 5 MMCLs with mutations in TRAF3, (3) one cell line with biallelic deletions of TRAF2, and (4) 3 MMCLs with low NF-κB index and no known mutations in NF-κB pathway. The ΔNF-κB(3.1) index was determined as the differences between treated and control samples. Data are mean ± SE (“Genes comprising the NF-κB index in myeloma”). (C) Immunoblot for TRAF2 coimmunoprecipitated with NIK in MMCLs. In some cases, the cells were incubated with MG132 for 3 hours to stabilize endogenous NIK. NIK was immunoprecipitated and loaded on the gel (IP:NIK), or cytosol extracts were directly loaded. Samples were analyzed with TRAF2 antibody. (D) Immunoblot for NIK in MMCLs cultured for 16 hours in medium alone or in the presence of 50nM Smac-mimetic or Smac-mimetic plus MG132. NF-κB mutations in different MMCLs are indicated. (E) Proposed mechanism of activation of the NF-κB pathways through NIK-protein in MM cells with different genetic abnormalities (top to bottom): WT, ΔcIAP1/2, ΔTRAF2, ΔTRAF3, and NIK lacking TRAF3-binding site.

Figure 3

Knockdown of NIK expression by RNA interference inhibits both NF-κB pathways. (A) Immunoblot of NIK protein levels after infection of NIK shRNA in MMCLs that have cIAP1/2 deletion. (B) Knockdown of NIK affects NF-κB signaling via classical and alternative pathways. Nuclear extracts were prepared, and expression of the proteins indicated at right was analyzed on Western blots. NF-κB mutations in different MMCLs are indicated. (C) Knockdown of NIK inhibits nuclear NF-κB DNA binding. DNA-binding activity of the indicated NF-κB subunits was quantified by enzyme-linked immunosorbent assay. (D) Inhibition of NF-κB target gene expression after knockdown of NIK in MM cells. The −ΔNF-κB(3.1) = decreased expression for average of 3 target genes (TNFAIP3, NFKB2, and IL2RG) in MM cells treated with NIK shRNA versus empty vector. Data are mean ± SE.

Figure 4

Effect of overexpression of NIK in cell lines with low NF-κB index. (A) Immunoblot of MMCLs after infection with empty vector or NIK gene. Nuclear and cytosol extracts were prepared, and expression of the proteins indicated at right was analyzed on Western blots. (B) Overexpression of NIK and IKK-β activates nuclear NF-κB DNA binding. DNA-binding activity of the indicated NF-κB subunits was quantified by enzyme-linked immunosorbent assay. (C) Activation of NF-κB target gene expression after infection of cells with NIK gene. The Δ NF-κB(3) = increased expression for average of 3 target genes (TNFAIP3, BIRC3, and IL2RG) in MM cells infected with NIK gene versus empty vector. Data are mean ± SE.

Figure 5

Effect of overexpression of NIK and IKK-β in cell lines with low NF-κB index. (A) Overexpression of IKK-β activates classical NF-κB pathway. Immunoblot of MMCLs after infection with empty vector or constitutively active IKK-β gene. Nuclear and cytosol extracts were prepared, and expression of the proteins indicated at right was analyzed. (B) Activation of NF-κB target gene expression after infection of cells with NIK or IKK-β. Average of 11 target gene expression in cells infected with NIK or IKK-β corrected by subtraction of expression in cells infected with empty vector. Data are mean ± SE.

Figure 6

Correlation in expression changes of genes after infection of cells with NIK or IKK-β. (A) Correlation in expression changes of 569 total genes in SACHI cells. (B) Correlation in expression changes of 458 total genes in H929 cells.