Constitutive NF-kappaB activation confers interleukin 6 (IL6) independence and resistance to dexamethasone and Janus kinase inhibitor INCB018424 in murine plasmacytoma cells - PubMed (original) (raw)

Constitutive NF-kappaB activation confers interleukin 6 (IL6) independence and resistance to dexamethasone and Janus kinase inhibitor INCB018424 in murine plasmacytoma cells

Yanqiang Yang et al. J Biol Chem. 2011.

Abstract

Myeloma cells are dependent on IL6 for their survival and proliferation during the early stages of disease, and independence from IL6 is associated with disease progression. The role of the NF-κB pathway in the IL6-independent growth of myeloma cells has not been studied. Because human herpesvirus 8-encoded K13 selectively activates the NF-κB pathway, we have used it as a molecular tool to examine the ability of the NF-κB pathway to confer IL6 independence on murine plasmacytomas. We demonstrated that ectopic expression of K13, but not its NF-κB-defective mutant or a structural homolog, protected plasmacytomas against IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas with visceral involvement when injected intraperitoneally into syngeneic mice. These IL6-independent clones were dependent on NF-κB activity for their survival and proliferation but were resistant to dexamethasone and INCB018424, a selective Janus kinase 1/2 inhibitor. Ectopic expression of human T cell leukemia virus 1-encoded Tax protein, which resembles K13 in inducing constitutive NF-κB activation, similarly protected plasmacytoma cells against IL6 withdrawal-induced apoptosis. Although K13 is known to up-regulate IL6 gene expression, its protective effect was not due to induction of endogenous IL6 production but instead was associated with sustained expression of several antiapoptotic members of the Bcl2 family upon IL6 withdrawal. Collectively, these results demonstrate that NF-κB activation cannot only promote the emergence of IL6 independence during myeloma progression but can also confer resistance to dexamethasone and INCB018424.

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Figures

FIGURE 1.

K13 protects the T1165 murine plasmacytoma cell line against IL6 withdrawal-induced apoptosis. A, expression of FLAG-K13 in T1165 cells as revealed by Western blotting (IB) with a FLAG antibody. B and C, T1165 cells expressing an empty vector or K13 were grown in triplicate in a 96-well plate in the presence or absence of IL6, and cell viability was measured 48 h later using an MTS assay. The values shown are mean ± S.D. of two independent experiments performed in triplicate. *, p ≤ 0.05 as compared with vector cells (B). Cells were stained with SYTOX Green, a cell-impermeable nuclear dye that stains the nuclei of dead cells, and were examined under a fluorescence microscope or under phase-contrast microscope and photographed (C). D, DNA content analysis shows significant increase in sub-G0/G1 fraction in T1165-vector cells upon withdrawal of IL6, which was absent in K13-expressing cells.

FIGURE 2.

Role of NF-κB activation in K13-induced protection against IL6 withdrawal-induced apoptosis in T1165 cells. A, status of the NF-κB pathway as measured by an EMSA in T1165-vector and T1165-K13−IL6 cells. The position of the induced NF-κB complexes is marked by the arrow, whereas the asterisk marks the position of the constitutive complexes. The difference in the size of the constitutive and the induced NF-κB complexes is probably due to their different subunit composition. B, increase in phosphorylated IκBα and decrease in total IκBα in T1165-K13−IL6 cells. Tubulin serves as a loading control. C, lack of increase in phosphorylation of JNK and AKT in T1165-K13−IL6 cells. Data shown are representative of two independent experiments. D, wild-type K13 protects T1165 cells against IL6 withdrawal-induced apoptosis, whereas its NF-κB-defective mutant 58AAA and vFLIP E8 fail to do so. Cell viability was measured using a MTS-based assay. *, p ≤ 0.05.

FIGURE 3.

Protective effect of K13 against IL6 withdrawal-induced apoptosis is reversed by NF-κB inhibitors. A–C, T1165-vector and K13−IL6 cells were treated in triplicate with the indicated concentrations (μ

) of Bay-11-7082, arsenic trioxide (As2O3), and dexamethasone, and cell viability was measured after ∼72 h using an MTS assay. The values shown are mean ± S.D. of a representative of two independent experiments performed in triplicate. *, p ≤ 0.05 compared with vector cells.

FIGURE 4.

Role of NF-κB activation in Tax-induced protection against IL6 withdrawal-induced apoptosis. A, immunoblot (I.B.) showing equivalent expression of wild-type Tax and its mutant constructs in T1165 cells. B, wild-type Tax and its M47 mutant activate NF-κB in T1165 cells, whereas the M22 fails to do so. The status of the NF-κB pathway was measured in nuclear extracts by an ELISA-based NF-κB (p65/RelA)-DNA binding assay kit (Transfector, Clontech). *, p ≤ 0.05 compared with vector cells upon IL6 withdrawal. C, wild-type Tax and its M47 mutant protect against IL6 withdrawal-induced apoptosis, whereas the M22 mutant fails to do so. Cell viability was measured using a MTS-based assay. The values shown are mean ± S.D. of a representative experiment performed in triplicate. *, p ≤ 0.05.

FIGURE 5.

Mechanism of protection against IL6 withdrawal conferred by K13. A, ELISA showing lack of murine IL6 secretion in the conditioned medium of T1165-vector cells grown in the presence of human IL6 (Hu-IL6) and T1165-K13 cells grown in the presence or absence of hu-IL6 for 72 h. B, ELISA showing lack of murine IL6 secretion in the conditioned medium of T1165-vector cells treated with TNF-α. Conditioned medium (C.M.) from SP2 cells was used as a positive control for murine IL6. C, conditioned medium collected from T1165-K13 or T1165-vector cells fail to protect a fresh batch of T1165 from IL6 withdrawal-induced apoptosis, indicating a lack of IL6 secretion. T1165 cells were grown in triplicate in the presence and absence of mIL6 (10 ng/ml) or in the presence of 10% C.M. collected from T1165-vector, T1165-K13, or murine IL6-secreting SP2/mIL6 cells, and cell survival was measured using an MTS-based assay as described for Fig. 1_B. D_, immunoblot analysis showing lack of phosphorylation of STAT1 and STAT3 in T1165-K13 cells when grown in the absence of IL6 for the indicated time points. Phosphorylation of STAT1 and STAT3 at residues Tyr-701 and Tyr-705 were measured using the indicated phospho-specific antibodies. E, T1165-vector and K13−IL6 cells were treated in triplicate with the indicated concentrations (μ

) of JAK1/2 inhibitor INCB018424, and cell viability was measured after ∼72 h using an MTS assay. The values shown are mean ± S.D. of a representative of two independent experiments performed in triplicate. F, immunoblot analysis showing lack of caspase activation and up-regulated expression of Bcl2 family members in T1165-K13 cells upon withdrawal from IL6 for the indicated time points. Unlike T1165-vector cells, T1165-K13 cells did not show cleavage of caspase 3 and PARP and maintain the expression of Mcl-1, Bcl-2, and Bcl-xL upon IL6 withdrawal. G, immunoblot analyses showing ectopic expression of Bcl-2, Bcl-xL, and Mcl-1 in T1165 cells as revealed by Western blotting with indicated antibodies. Tubulin served as a loading control. H, T1165 cells overexpressing an empty vector or indicated Bcl2 family members or K13 were grown in triplicate in a 96-well plate in the presence or absence of IL6, and cell viability was measured 48 h later using an MTS assay. The values shown are mean ± S.D. of two independent experiments performed in triplicate. *, p ≤ 0.05 compared with vector cells upon IL6 withdrawal.

FIGURE 6.

K13 protects the B9 murine plasmacytoma cell line against IL6 withdrawal-induced apoptosis via NF-κB activation. A, expression of FLAG-K13 in B9 cells as revealed by Western blotting with a FLAG antibody. B, B9 cells expressing an empty vector or K13 were grown in triplicate in a 96-well plate in the presence or absence of IL6, and cell viability was measured 48 h later using an MTS assay. The values shown are mean ± S.D. of two independent experiments performed in triplicate. *, p ≤ 0.05 versus vector cells. C, B9-vector and B9-K13 cells were treated in triplicate with the indicated concentrations (μ

) of Bay-11-7082, and cell viability was measured after ∼72 h using an MTS assay. B9-K13 cells were grown in the absence of IL6. *, p ≤ 0.05. D, immunoblot showing lack of phosphorylation of STAT1 and STAT3 in B9-K13 cells when grown in the absence of IL6.

FIGURE 7.

T1165-K13−IL6 cells establish peritoneal plasmacytomas without pristane preconditioning and lead to disseminated disease involving visceral organs. A and B, BALB/cAnNCr mice were injected intraperitoneally with the indicated cells, and tumor growth was monitored by physical examination (A) or bioluminescence imagine (B) as described under “Materials and Methods.” C, plasmacytomas isolated at autopsy from mice injected with the T1165-Luc-K13−IL6 cells. D, splenomegaly in mice injected with T1165-Luc-K13−IL6 cells (right panel) as compared with a normal spleen in those injected with the T1165-Luc-vector cells (left panel). E, immunoblot analysis showing the presence of FLAG-tagged K13 in the parental T1165-Luc-K13−IL6 cells and in cells isolated from the abdominal plasmacytoma and spleen of mice injected with the T1165-Luc-K13−IL6 cells.

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Constitutive NF-kappaB activation confers interleukin 6 (IL6) independence and resistance to dexamethasone and Janus kinase inhibitor INCB018424 in murine plasmacytoma cells - PubMed (original) (raw)