Abstract
Inhibition of the protein neddylation process by the small-molecule inhibitor MLN4924 has been recently indicated as a promising direction for cancer treatment. However, the knowledge of all biological consequences of MLN4924 for cancer cells is still incomplete. Here, we report that MLN4924 inhibits tumor necrosis factor-alpha (TNF-α)-induced matrix metalloproteinase 9 (MMP9)-driven cell migration. Using real-time polymerase chain reaction (PCR) and gelatin zymography, we found that MLN4924 inhibited expression and activity of MMP9 at the messenger RNA (mRNA) and protein levels in both resting cells and cells stimulated with TNF-α, and this inhibition was closely related to impaired cell migration. We also revealed that MLN4924, similar to TNF-α, induced phosphorylation of inhibitor of nuclear factor kappa B-alpha (IκB-α). However, contrary to TNF-α, MLN4924 did not induce IκB-α degradation in treated cells. In coimmunoprecipitation experiments, nuclear IκB-α which formed complexes with nuclear factor kappa B p65 subunit (NFκB/p65) was found to be highly phosphorylated at Ser32 in the cells treated with MLN4924, but not in the cells treated with TNF-α alone. Moreover, in the presence of MLN4924, nuclear NFκB/p65 complexes were found to be enriched in c-Jun and cyclin dependent kinase inhibitor 1 A (CDKN1A/p21) proteins. In these cells, NFκB/p65 was unable to bind to the MMP9 gene promoter, which was confirmed by the chromatin immunoprecipitation (ChIP) assay. Taken together, our findings identified MLN4924 as a suppressor of TNF-α-induced MMP9-driven cell migration in esophageal squamous cell carcinoma (ESCC), likely acting by affecting the nuclear ubiquitin–proteasome system that governs NFκB/p65 complex formation and its DNA binding activity in regard to the MMP9 promoter, suggesting that inhibition of neddylation might be a new therapeutic strategy to prevent invasion/metastasis in ESCC patients.
Highlights
Esophageal squamous cell carcinoma (ESCC) is a predominant histological type accounting globally for about 87% of all esophageal cancers ranked as the sixth leading cause of cancer death worldwide [1]
We observed by gelatin zymography or Western blotting that the treatment of esophageal squamous cell carcinoma (ESCC) cells with TNF-α significantly increased secretion of matrix metalloproteinase 9 (MMP9), but not of MMP2 or MMP14, in a dose-dependent manner (Figure 1A and Figure S1)
Upregulation of MMP9 gene expression induced by TNFα was confirmed by the quantitative real-time polymerase chain reaction assay and showed nearly 4-fold and 14-fold increase in the MMP9 messenger RNA (mRNA) level in KYSE70 and KYSE150 cells, respectively, under TNF-α (Figure 1B)
Summary
Esophageal squamous cell carcinoma (ESCC) is a predominant histological type accounting globally for about 87% of all esophageal cancers ranked as the sixth leading cause of cancer death worldwide [1]. Despite recent improvements in diagnostics and therapy with neoadjuvant chemotherapy or radiochemotherapy, the 5-year survival rate for ESCC patients is low and does not exceed 20% [2]. Recurrent long-term exposure of esophageal epithelium to harmful factors is known to cause tissue damage and chronic inflammation which leads to constitutive activation of signaling pathways, promoting cell survival and proliferation [5,6]. Chronic inflammation has been indicated as an additional contributor to ESCC pathogenesis [7]. Several pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), secreted mostly by cells of the immune system, have been shown to be upregulated in patients with ESCC, indicating their potential effect on the tumor development [7,8]
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