Inhibition of VCP modulates NF-κB signaling pathway to suppress multiple myeloma cell proliferation and osteoclast differentiation

Abstract

Multiple myeloma (MM) is the second most common hematological malignancy, in which the dysfunction of the ubiquitin-proteasome pathway is associated with the pathogenesis. The valosin containing protein (VCP)/p97, a member of the AAA+ ATPase family, possesses multiple functions to regulate the protein quality control including ubiquitin-proteasome system and molecular chaperone. VCP is involved in the occurrence and development of various tumors while still elusive in MM. VCP inhibitors have gradually shown great potential for cancer treatment. This study aims to identify if VCP is a therapeutic target in MM and confirm the effect of a novel inhibitor of VCP (VCP20) on MM. We found that VCP was elevated in MM patients and correlated with shorter survival in clinical TT2 cohort. Silencing VCP using siRNA resulted in decreased MM cell proliferation via NF-κB signaling pathway. VCP20 evidently inhibited MM cell proliferation and osteoclast differentiation. Moreover, exosomes containing VCP derived from MM cells partially alleviated the inhibitory effect of VCP20 on cell proliferation and osteoclast differentiation. Mechanism study revealed that VCP20 inactivated the NF-κB signaling pathway by inhibiting ubiquitination degradation of IκBα. Furthermore, VCP20 suppressed MM cell proliferation, prolonged the survival of MM model mice and improved bone destruction in vivo. Collectively, our findings suggest that VCP is a novel target in MM progression. Targeting VCP with VCP20 suppresses malignancy progression of MM via inhibition of NF-κB signaling pathway.

Keywords: NF-κB; VCP; multiple myeloma; osteoclast differentiation; ubiquitination.

Figure 1

Elevated VCP is associated with poor survival in MM patients and siVCP inhibits MM cell proliferation in vitro. (A) VCP mRNA levels were significantly increased in MM samples (n = 351) compared with NP (n = 22) and MGUS (n = 44) samples. (B) Higher VCP level was associated with a shorter OS in TT2 cohort (n = 351, High: n = 75, Low: n = 276). (C) Box plot representing VCP expression in eight MM subgroups from TT2 cohort. (D) WB analysis of VCP expression in siVCP cells. (E) MTT assay examined the effect of siVCP on MM cell proliferation. (F) WB analysis showed that siVCP inhibited CDK4/6 expression in MM cells. The MM patient survival data were plotted by Kaplan-Meier curve, and the survival of patients with low and high expression of VCP was compared through a log-rank test. Data are presented as the mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

VCP20 inhibits MM cell proliferation and induces cell apoptosis. (A) The structure of VCP20. (B) Predicted binding modes of VCP20 targeting VCP. (C) Inhibition ratio of VCP20 in MM cells. (D) Flow cytometry analysis indicated that VCP20 induced MM cell apoptosis. (E) WB analysis showed that VCP20 increased the expressions of apoptotic protein. (F) WB analysis showed that VCP20 decreased the CDK4/6 expression. The concentration of VCP20 was as follow: ARP1, 575.6 nM; H929, 661 nM. Data are presented as the mean ± SD; **p < 0.01; ***p < 0.001.

Figure 3

VCP regulates MM cell proliferation via modulating NF-κB signaling pathway. (A) Volcano plots showed the upregulated (red) and downregulated (blue) genes upon silencing VCP in MM cells. (B) KEGG pathway analysis of RNA-seq data indicated that VCP was associated with NF-κB signaling pathway. (C) WB analysis of P-P65 and P65 expressions in siVCP cells compared with NC cells. (D) WB analysis of P-P65 and P65 expressions in the cells treated with VCP20 compared with WT cells. (E) VCP20 increased IκBα expression. (F) The ubiquitination level of IKBα in MM cells with the treatment of VCP20 or not.

Figure 4

VCP20 inhibits osteoclast differentiation via downregulating NF-κB P65. (A) TRAP staining showed that VCP20 inhibited osteoclast differentiation in RAW 264.7 cells treated with RANKL and M-CSF. Scale bar: 250 μm. (B) Quantitative analysis of multinucleated osteoclasts. (C) The biological characteristics of exosomes were detected by transmission electron microscopy. Scale bar: 100 nm. (D) Detection of VCP, Alix and β-actin in ARP1 WT cells and exosomes from ARP1 WT cells by WB analysis. (E) Exosomes from ARP1 WT cells rescued cell proliferation inhibited by VCP20 in MM cells (1×10⁶ MM cells were treated with 20 μg exosomes or not for 48 h). (F) TRAP staining revealed that exosomes from ARP1 WT cells induced osteoclast differentiation. Scale bar: 250 μm. (G) Quantitative analysis of multinucleated osteoclasts. (H) WB assay confirmed that exosomes from ARP1 WT cells increased the expressions of P-P65 and NFATC1 that were downregulated by VCP20 in RAW264.7 cells. (I) RT-PCR assay showed that VCP20 decreased the levels of osteoclast differentiation related markers, and this effect was partially compensated by exosomes from ARP1 WT cells. VCP20: 100 nM; exosomes: 2 μg/mL each well. Data are presented as the mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

VCP20 impedes MM cell proliferation and prolongs MM mice survival in vivo. (A) Photographic images of xenograft mice at Day 31 (n = 4, VCP20: i.p. 20 mg/kg, twice a week). (B) Images of the harvested xenograft tumors. (C) Mean tumor weight of NOD/SCID mice. (D) Time course of tumor growth. V = 0.52 × larger diameter × (smaller diameter)². (E) Representative micro-CT images of the bones. (F) BMD of 5TMM3VT mice in Control and VCP20 groups. (G) BV/TV of 5TMM3VT mice in Control and VCP20 groups. (H) VCP20 treatment started from the day after injection (VCP20: i.p. 20 mg/kg, twice a week) and continued until the mice were dead. (I) VCP20 extended the survival period of MM mice (n = 8). Data are presented as the mean ± SD; *p < 0.05; **p < 0.01.

Figure 6

Schematic depiction illustrates that VCP20 targeting VCP is a promising agent for inhibiting cellular proliferation and improving bone marrow microenvironment in MM.