Capn4 is a marker of poor clinical outcomes and promotes nasopharyngeal carcinoma metastasis via nuclear factor-κB-induced matrix metalloproteinase 2 expression.
Bottom Line: The patients with NPC displaying higher Capn4 had a significantly shorter overall survival (P = 0.002) and progression-free survival (P = 0.003).These events resulted from Capn4 downregulation were associated with reduced expression of matrix metalloproteinase 2 (MMP2), Snail, and Vimentin.Together, these findings argue a novel function of Capn4 in invasion and metastasis of NPC, and thereby suggest that Capn4 may represent an independent prognostic factor and a potential therapeutic target in NPC.
Affiliation: Department of Anatomy, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.Show MeSH
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Mentions: To gain further insight into the mechanism by which Capn4 regulates migration and invasion of NPC cells, Western blot analysis was performed to examine expression of potential Capn4-targeting proteins that are involved in cell migration and invasion,23–27 as well as serve as proteolytic substrates of calpains, in which Capn4 acts as a key regulatory subunit.14,15 As shown in Fig.4a, downregulation of Capn4 by siRNA led to decreased expression of MMP2, Snail, and Vimentin, but increased expression of E-cadherin in NPC cells. In contrast, there was no change observed in expression of MMP9, N-cadherin, or β-catenin. Among the proteins that were downregulated in Capn4 siRNA cells, MMP2 plays an important role in multiple steps of cancer progression, including tumor invasion and metastasis,28–30 angiogenesis, as well as extracellular matrix remodeling in NPC.31 We then tested whether MMP2 contributes to Capn4-mediated regulation of NPC cell invasion. First, RT-PCR analysis confirmed a reduction in MMP2 mRNA after Capn4 was knocked down in NPC cells (Fig.4b). Second, MMP2 enzyme activity was determined using the gelatin zymography in media obtained from 5-8F cells and CNE2 cells transfected with Capn4 siRNA1 and siRNA2. The results showed lower enzyme activity in these cells than siRNA control cells, manifested by a band at 72 kDa (Fig.4c). Last, knockdown of MMP2 by siRNA significantly suppressed invasion capability of 5-8F cells and CNE2 cells in transwell assays. Conversely, overexpression of MMP2 in cells, in which Capn4 was silenced by siRNA, rescued the functional effect of Capn4 on cell invasion and subsequently restored the invasion activity of NPC cells (Fig.4c,d). Thus, these results suggest that Capn4 may promote metastasis of NPC via regulation of MMP2 expression.
Affiliation: Department of Anatomy, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.