Yonsei Med J.  2018 Jan;59(1):43-50. 10.3349/ymj.2018.59.1.43.

TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling

Affiliations
  • 1Department of Hematology, The First Affiliated Hospital of Xi'an Jiao Tong University, Xi'an 710061, China. zhangmeizmst@163.com

Abstract

PURPOSE
Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis.
MATERIALS AND METHODS
TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway.
RESULTS
TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells.
CONCLUSION
TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.

Keyword

TRIM56; multiple myeloma; proliferation; apoptosis; inflammatory cytokine; TLR3/TRIF

MeSH Terms

Adaptor Proteins, Vesicular Transport/*metabolism
Apoptosis/drug effects
Cell Line, Tumor
Cell Proliferation/drug effects
Cytokines/secretion
*Disease Progression
Down-Regulation/drug effects
Gene Knockdown Techniques
Humans
Multiple Myeloma/*metabolism/*pathology
Poly I-C/pharmacology
*Signal Transduction/drug effects
Toll-Like Receptor 3/*metabolism
Tripartite Motif Proteins/deficiency/*metabolism
Ubiquitin-Protein Ligases/deficiency/*metabolism
Adaptor Proteins, Vesicular Transport
Cytokines
Toll-Like Receptor 3
Tripartite Motif Proteins
Ubiquitin-Protein Ligases

Figure

  • Fig. 1 Down-regulation of TRIM56 in MM cells. (A and B) qRT-PCR and western blot was performed to examine the expression level of TRIM56 mRNA and protein in normal PCs and different MM cell lines (NCI-H929, RPMI8226, LP-1, OPM2, and U266 cells). *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; PC, plasma cell.

  • Fig. 2 TRIM56 inhibited MM cell proliferation and induced apoptosis. Western blot analysis of TRIM56 level in U266 cells (A) transfected with poly (dA:dT) and in RPMI8226 cells (E) introduced with si-control, T56si-1, or T56si-2. Cell viability was evaluated by MTT assay in U266 cells (B) with poly (dA:dT) stimulation and in RPMI8226 cells (F) transfected with si-control or T56si-2. Apoptosis detection was performed by flow cytometry analysis in U266 cells (C) with poly (dA:dT) transfection and in 2 µM ATO-treated RPMI8226 cells (G) with si-control or T56si-2 transfection. The relative caspase3 activity was measured by the Caspase3 Colorimetric Assay Kit in U266 cells (D) with poly (dA:dT) transfection and in 2 µM ATO-treated RPMI8226 cells (H) introduced with si-control or T56si-2. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; ATO, arsenic trioxide.

  • Fig. 3 TRIM56 enhanced the secretion of cytokines in MM cells. ELISA was conducted to measure the concentrations of IFN-β (A and D), IL-6 (B and E), and TNF-α (C and F) in poly (dA:dT)-treated U266 cells and T56si-2-transfected RPMI8229 cells. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

  • Fig. 4 TRIM56 knockdown inactivated TLR3/TRIF signaling pathway in MM cells. (A) Western blot analysis of TLR3, TRIF, and RIP1 protein in RPMI8226 cells transfected with T56si-2 or si-control. (B) Quantitative analysis of TLR3, TRIF, and RIP1 protein levels. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; TLR3, toll-like receptor3; TRIF, toll-IL-1 receptor domain-containing adaptor inducing interferon-β; RIP1, receptor-interacting protein 1.

  • Fig. 5 TRIM56 deficiency promoted the progression of MM via interrupting TLR3/TRIF signaling pathway. RPMI8226 cells were transfected with T56si-2 or along with poly (I:C) for further analysis at 48 h post-transfection. (A) Cell viability of transfected RPMI8226 cells was assessed by MTT assay. (B) Apoptotic rate of transfected RPMI8226 cells was detected by flow cytometry. (C) The relative caspase3 activity of transfected RPMI8226 cells was measured by the Caspase3 Colorimetric Assay Kit. The concentrations of secreted cytokines IFN-β (D), IL-6 (E), and TNF-α (F) in transfected RPMI8226 cells were measured by ELISA. (G) Western blot analysis of TLR3, TRIF, and RIP1 protein expression in transfected RPMI8226 cells. *p<0.05. TRIM56, tripartite-mortif-containing protein 56; MM, multiple myeloma; TLR3, toll-like receptor3; TRIF, toll-IL-1 receptor domain-containing adaptor inducing IFN-β; RIP1, receptor-interacting protein 1; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; ATO, arsenic trioxide.


Reference

1. Munshi NC, Anderson KC, Bergsagel PL, Shaughnessy J, Palumbo A, Durie B, et al. Consensus recommendations for risk stratification in multiple myeloma: report of the International Myeloma Workshop Consensus Panel 2. Blood. 2011; 117:4696–4700.
Article
2. Palumbo A, Anderson K. Multiple myeloma. N Engl J Med. 2011; 364:1046–1060.
Article
3. Manier S, Sacco A, Leleu X, Ghobrial IM, Roccaro AM. Bone marrow microenvironment in multiple myeloma progression. J Biomed Biotechnol. 2012; 2012:157496.
Article
4. Wang F, Zhang W, Guo L, Bao W, Jin N, Liu R, et al. Gambogic acid suppresses hypoxia-induced hypoxia-inducible factor-1α/vascular endothelial growth factor expression via inhibiting phosphatidylinositol 3-kinase/Akt/mammalian target protein of rapamycin pathway in multiple myeloma cells. Cancer Sci. 2014; 105:1063–1070.
Article
5. Pratt G, Goodyear O, Moss P. Immunodeficiency and immunotherapy in multiple myeloma. Br J Haematol. 2007; 138:563–579.
Article
6. Chiron D, Jego G, Pellat-Deuceunynck C. Toll-like receptors: expression and involvement in multiple myeloma. Leuk Res. 2010; 34:1545–1550.
Article
7. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010; 11:373–384.
Article
8. Lester SN, Li K. Toll-like receptors in antiviral innate immunity. J Mol Biol. 2014; 426:1246–1264.
Article
9. Matsumoto M, Seya T. TLR3: interferon induction by double-stranded RNA including poly(I:C). Adv Drug Deliv Rev. 2008; 60:805–812.
Article
10. Chiron D, Pellat-Deceunynck C, Amiot M, Bataille R, Jego G. TLR3 ligand induces NF-κB activation and various fates of multiple myeloma cells depending on IFN-α production. J Immunol. 2009; 182:4471–4478.
Article
11. Abdi J, Engels F, Garssen J, Redegeld F. The role of toll-like receptor mediated signalling in the pathogenesis of multiple myeloma. Crit Rev Oncol Hematol. 2011; 80:225–240.
Article
12. Munir M. TRIM proteins: another class of viral victims. Sci Signal. 2010; 3:jc2.
Article
13. Ozato K, Shin DM, Chang TH, Morse HC 3rd. TRIM family proteins and their emerging roles in innate immunity. Nat Rev Immunol. 2008; 8:849–860.
Article
14. Meroni G, Diez-Roux G. TRIM/RBCC, a novel class of ‘single protein RING finger’ E3 ubiquitin ligases. Bioessays. 2005; 27:1147–1157.
Article
15. Hatakeyama S. TRIM family proteins: roles in autophagy, immunity, and carcinogenesis. Trends Biochem Sci. 2017; 42:297–311.
Article
16. Wang J, Liu B, Wang N, Lee YM, Liu C, Li K. TRIM56 is a virus- and interferon-inducible E3 ubiquitin ligase that restricts pestivirus infection. J Virol. 2011; 85:3733–3745.
Article
17. Shen Y, Li NL, Wang J, Liu B, Lester S, Li K. TRIM56 is an essential component of the TLR3 antiviral signaling pathway. J Biol Chem. 2012; 287:36404–36413.
Article
18. Saha MN, Jiang H, Yang Y, Reece D, Chang H. PRIMA-1Met/APR-246 displays high antitumor activity in multiple myeloma by induction of p73 and Noxa. Mol Cancer Ther. 2013; 12:2331–2341.
Article
19. Park WH, Seol JG, Kim ES, Hyun JM, Jung CW, Lee CC, et al. Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. Cancer Res. 2000; 60:3065–3071.
20. Tsuchida T, Zou J, Saitoh T, Kumar H, Abe T, Matsuura Y, et al. The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular double-stranded DNA. Immunity. 2010; 33:765–776.
Article
21. Watanabe M, Hatakeyama S. TRIM proteins and diseases. J Biochem. 2017; 161:135–144.
Article
22. Qin Y, Cui H, Zhang H. Overexpression of TRIM25 in lung cancer regulates tumor cell progression. Technol Cancer Res Treat. 2016; 15:707–715.
Article
23. Yamada Y, Takayama KI, Fujimura T, Ashikari D, Obinata D, Takahashi S, et al. A novel prognostic factor TRIM44 promotes cell proliferation and migration, and inhibits apoptosis in testicular germ cell tumor. Cancer Sci. 2017; 108:32–41.
Article
24. Chen Y, Guo Y, Yang H, Shi G, Xu G, Shi J, et al. TRIM66 overexpresssion contributes to osteosarcoma carcinogenesis and indicates poor survival outcome. Oncotarget. 2015; 6:23708–23719.
Article
25. Yi J, Huang D, Li X, Jiang G, Dong J, Liu Y. TRIM26 acts as a tumor suppressor in non-small-cell lung cancer. Int J Clin Exp Pathol. 2016; 9:6385–6390.
26. Sutton SK, Koach J, Tan O, Liu B, Carter DR, Wilmott JS, et al. TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells. Oncotarget. 2014; 5:10127–10139.
Article
27. Lee OH, Lee J, Lee KH, Woo YM, Kang JH, Yoon HG, et al. Role of the focal adhesion protein TRIM15 in colon cancer development. Biochim Biophys Acta. 2015; 1853:409–421.
Article
28. Ai L, Kim WJ, Alpay M, Tang M, Pardo CE, Hatakeyama S, et al. TRIM29 suppresses TWIST1 and invasive breast cancer behavior. Cancer Res. 2014; 74:4875–4887.
Article
29. Harashima N, Inao T, Imamura R, Okano S, Suda T, Harada M. Roles of the PI3K/Akt pathway and autophagy in TLR3 signalinginduced apoptosis and growth arrest of human prostate cancer cells. Cancer Immunol Immunother. 2012; 61:667–676.
Article
30. Salaun B, Coste I, Rissoan MC, Lebecque SJ, Renno T. TLR3 can directly trigger apoptosis in human cancer cells. J Immunol. 2006; 176:4894–4901.
Article
31. Khvalevsky E, Rivkin L, Rachmilewitz J, Galun E, Giladi H. TLR3 signaling in a hepatoma cell line is skewed towards apoptosis. J Cell Biochem. 2007; 100:1301–1312.
Article
32. Morikawa T, Sugiyama A, Kume H, Ota S, Kashima T, Tomita K, et al. Identification of Toll-like receptor 3 as a potential therapeutic target in clear cell renal cell carcinoma. Clin Cancer Res. 2007; 13:5703–5709.
Article
33. Jiang Q, Wei H, Tian Z. Poly I:C enhances cycloheximide-induced apoptosis of tumor cells through TLR3 pathway. BMC Cancer. 2008; 8:12.
Article
34. Bohnhorst J, Rasmussen T, Moen SH, Fløttum M, Knudsen L, Børset M, et al. Toll-like receptors mediate proliferation and survival of multiple myeloma cells. Leukemia. 2006; 20:1138–1144.
Article
Full Text Links
  • YMJ
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr