USP15 inhibits multiple myeloma cell apoptosis through activating a feedback loop with the transcription factor NF-κBp65

Abstract

USP15 has been shown to stabilize transcription factors, to be amplified in many cancers and to mediate cancer cell survival. However, the underlying mechanism by which USP15 regulates multiple myeloma (MM) cell proliferation and apoptosis has not been established. Here, our results showed that USP15 mRNA expression was upregulated in MM patients. USP15 silencing induced MM cell proliferation inhibition, apoptosis, and the expression of nuclear and cytoplasmic NF-κBp65, while USP15 overexpression exhibited an inverse effect. Moreover, in vivo experiments indicated that USP15 silencing inhibited MM tumor growth and NF-κBp65 expression. PDTC treatment significantly inhibited USP15 overexpression-induced cell proliferation, apoptosis inhibition, and NF-κBp65 expression. USP15 overexpression promoted NF-κBp65 expression through inhibition of its ubiquitination, whereas NF-κBp65 promoted USP15 expression as a positive regulator. Taken together, the USP15-NF-κBp65 loop is involved in MM tumorigenesis and may be a potential therapeutic target for MM.

Fig. 1. USP15 expression in MM tissues and cell lines.

a The mRNA expression of USP15 in bone marrow samples from 80 patients with multiple myeloma (MM) and 15 patients with proliferative bone marrow (PBM) was measured by Real-time PCR. The expression of USP15 in five different MM cell lines and in control cells, non-cancerous bone marrow-derived plasma cells, was measured by Real-time PCR (b) and western blotting (c). **P < 0.01 compared with PBM or control

Fig. 2. Effects of USP15 RNA-interference and USP15 overexpression on MM cell proliferation.

After RPMI 8226 and U266 cells were transfected with siNC plus empty pLVX-Puro (vector), siUSP15#1, siUSP15#2, or pLVX-Puro-USP15, the expression of USP15 was measured by western blot (a–d) and cell proliferation was measured by the CCK-8 assay (e, f). ***P < 0.001 compared with siNC, vector or vector + siNC

Fig. 3. USP15 overexpression inhibits MM cell apoptosis.

a, b After RPMI 8226 and U266 cells were transfected with siNC plus empty pLVX-Puro (vector), siUSP15#1, siUSP15#2, or pLVX-Puro-USP15 for 48 h, apoptosis in the RPMI 8226 and U266 cells was measured by flow cytometry with Annexin V-FITC/PI staining. ***P < 0.001 compared with vector + siNC

Fig. 4. USP15 overexpression promotes the expression of Bcl-2, Bcl-xL, Survivin, and NF-κBp65.

After RPMI 8226 and U266 cells were transfected with siNC plus empty pLVX-Puro (vector), siUSP15#1, siUSP15#2, or pLVX-Puro-USP15, the expression of Bcl-2, Bcl-xL, Survivin, cleaved Caspase-3 (C-Caspase-3), cleaved PARP1 (C-PARP1), nuclear NF-κBp65, and cytoplasmic NF-κBp65 was measured by Real-time PCR (a, b) and/or western blot (c-g). (h) IκB-α and p-IκB-α expression in U266 cells was measured by western blot. IκB-α (i) or NF-κBp65 (j) was immunoprecipitated and immunoblotted in cells with empty pLVX-Puro (vector) or pLVX-Puro-USP15 transfection. ***P < 0.001 compared with vector + siNC

Fig. 5

USP15 knockdown inhibits tumor growth and related protein expression in MM in vivo. After RPMI 8226 cells were transfected with pLKO.1-USP15-shRNA or the negative control (shNC) and injected into the nude mouse xenograft model, the mice were sacrificed 48 days later, the tumor weight (a, b) and volume (c) were evaluated, and the expression of USP15, Bcl-2, Bcl-xL, Survivin, cleaved Caspase-3 (C-Caspase-3), cleaved PARP1 (C-PARP1), nuclear NF-κBp65, and cytoplasmic NF-κBp65 in xenograft tumors was measured by western blot (d, e). ***P < 0.001 compared with shNC

Fig. 6

PDTC treatment inhibits USP15 overexpression-induced cell proliferation and apoptosis inhibition in MM. After RPMI 8226 and U266 cells cultured in complete medium containing either PDTC (50 μM) or no inhibitors were transfected with pLVX-Puro-USP15, proliferation was measured with the CCK-8 assay (a, b) and apoptosis was measured by flow cytometry with Annexin V-FITC/PI staining (c, d). ***P < 0.001 compared with vector. ^###P < 0.001 compared with USP15

Fig. 7. PDTC treatment inhibits USP15 overexpression-induced expression of Bcl-2, Bcl-xL, Survivin and NF-κBp65 in MM cells.

After RPMI 8226 and U266 cells cultured in complete medium containing either PDTC (50 μM) or no inhibitor were transfected with pLVX-Puro-USP15, the expression of Bcl-2, Bcl-xL, Survivin, cleaved Caspase-3 (C-Caspase-3), cleaved PARP1 (C-PARP1), nuclear NF-κBp65, and cytoplasmic NF-κBp65 was measured by Real-time PCR (a, b) and/or western blot (c-h). *P < 0.05, ***P < 0.001 compared with vector. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 compared with USP15

Fig. 8. Regulatory feedback between USP15 and NF-κBp65.

a, b After RPMI 8226 and U266 cells were treated with PDTC (50 μM) or LPS (1 μg/ml), the expression of nuclear NF-κBp65 and cytoplasmic NF-κBp65 was measured by western blot. c, d Dual-luciferase assays in RPMI 8226 and U266 cells transfected with firefly luciferase constructs containing the 3′ UTR of USP15 with or without PDTC (50 μM) and LPS (1 μg/ml) treatment. After RPMI 8226 and U266 cells were treated with PDTC (50μM) or LPS (1 μg/ml), the expression of USP15 was measured by Real-time PCR (e, f) and western blot (g, h). i Schematic representation USP15-NF-κBp65 regulation of cell proliferation and apoptosis. ***P < 0.001 compared with untreated control