Smac Mimetic-Induced Upregulation of CCL2/MCP-1 Triggers Migration and Invasion of Glioblastoma Cells and Influences the Tumor Microenvironment in a Paracrine Manner

Abstract

Second mitochondria-derived activator of caspase (Smac) mimetics are considered as promising anticancer therapeutics that are currently under investigation in early clinical trials. They induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are frequently overexpressed in cancer. We previously reported that Smac mimetics, such as BV6, additionally exert non-apoptotic functions in glioblastoma (GBM) cells by stimulating migration and invasion in a nuclear factor kappa B (NF-κB)-dependent manner. Because NF-κB target genes mediating these effects are largely unknown, we performed whole-genome expression analyses. Here, we identify chemokine (C-C motif) ligand 2 (CCL2) as the top-listed NF-κB-regulated gene being upregulated upon BV6 treatment in GBM cells. BV6-induced upregulation and secretion of CCL2 are required for migration and invasion of GBM cells because knockdown of CCL2 in GBM cells abolishes these effects. Co-culture experiments of GBM cells with non-malignant astroglial cells reveal that BV6-stimulated secretion of CCL2 by GBM cells into the supernatant triggers migration of astroglial cells toward GBM cells because CCL2 knockdown in BV6-treated GBM cells impedes BV6-stimulated migration of astroglial cells. In conclusion, we identify CCL2 as a BV6-induced NF-κB target gene that triggers migration and invasion of GBM cells and exerts paracrine effects on the GBM's microenvironment by stimulating migration of astroglial cells. These findings provide novel insights into the biological functions of Smac mimetics with important implications for the development of Smac mimetics as cancer therapeutics.

Figure 1

BV6-induced, NF-κB-mediated upregulation and secretion of CCL2 in GBM cells. (A) A172 cells expressing SR or EV were treated with 2 μM BV6 for 9 hours. mRNA expression of CCL2 was analyzed by quantitative reverse transcriptase (qRT)-PCR. Fold increase in mRNA levels is shown with mean and SEM of three independent experiments performed at least in duplicate. (B) T98G and U87MG cells were treated for indicated times with 2.5 μM BV6. mRNA expression of CCL2 was analyzed by qRT-PCR. Fold increase in mRNA levels is shown with mean and SEM of three to six independent experiments performed at least in duplicate. * P < .05, ** P < .01, and *** P < .001. (C) T98G and U87MG cells were treated with 2.5 μM BV6 (12 hours for T98G; 9 hours for U87MG). CCL2 protein expression in supernatants was determined by FACS analysis. Mean and SD of three to four independent experiments are shown. * P < .05 and ** P < .01.

Figure 2

BV6-induced upregulation of CCL2 triggers migration and invasion in GBM cells. (A) Cells were transiently transfected with siRNA against CCL2 or siCtrl. CCL2 mRNA expression in GBM cells was analyzed by qRT-PCR. Fold change in mRNA is shown with mean and SEM of three independent experiments performed at least in duplicate. * P < .05 and *** P < .001. (B) CCL2 protein expression in supernatants was assessed by FACS analysis. Mean and SD of four independent experiments performed in duplicate are shown. * P < .05 and *** P < .001. (C and D) GBM cells were transiently transfected with siRNA against CCL2 or control siRNA and treated with 2.5 μM BV6 or DMSO for 24 hours. Migration was assessed by transwell migration assay (C); and invasion, by matrigel-precoated Transwell migration chamber (D). Fold increase in migration or invasion relative to untreated cells transfected with control siRNA with mean and SD of four independent experiments performed at least in duplicate is shown. * P < .05 and *** P < .001. (E) GBM cells were preincubated for 10 minutes with 1 ng/ml recombinant CCL2 and then CCL2 was added to the upper and lower migration chamber at same concentration. Migration was assessed after 24 hours by transwell migration assay. Fold increase in migration relative to untreated cells with mean and SD of three independent experiments performed at least in duplicate is shown. * P < .05.

Figure 3

BV6-stimulated GBM cells induce astroglial cell migration in a co-culture model. (A and B) SVG or NHA-E6/E7/hTERT cells were treated for indicated times with 1 μM (SVG) or 2.5 μM (NHA-E6/E7/hTERT) BV6. Expression levels of cIAP1, cIAP2, and XIAP (A) and expression and/or phosphorylation of p100, p52, p65, and IκBα (B) were analyzed by Western blotting; β-actin served as loading control. Representative blots of four independent experiments are shown. (C) SVG or NHA-E6/E7/hTERT cells were treated for 24 hours with 1 μM (SVG) or 2.5 μM (NHA-E6/E7/hTERT) BV6. Migration was assessed after 24 hours by transwell migration assay. Fold increase in migration relative to untreated cells with mean and SD of three to four independent experiments performed at least in duplicate is shown; ns, not significant. (D) SVG or NHA-E6/E7/hTERT cells in the upper chamber of the transwell plate were co-cultured with T98G or U87MG cells in the bottom chamber that were pretreated with 2.5 μM BV6 for 4 hours to stimulate cytokine expression. Migration was assessed after 24 hours by transwell migration assay. Fold increase in migration relative to untreated cells with mean and SD of three independent experiments performed at least in duplicate is shown. * P < .05 and ** P < .01.

Figure 4

BV6-stimulated CCL2 release by GBM cells induces astroglial cell migration in a co-culture model. (A and B) SVG cells in the upper chamber of the transwell plate were co-cultured with T98G or U87MG cells in the bottom chamber that were transiently transfected with siRNA against CCL2 or siCtrl and pretreated with 2.5 μM BV6 for 4 hours to stimulate cytokine expression. Migration was assessed after 24 hours by transwell migration assay. Fold increase in migration relative to untreated cells with mean and SD of three to four independent experiments performed at least in duplicate is shown. * P < .05, ** P < .01, and *** P < .001. (C) Scheme of BV6-induced upregulation of CCL2 and its influence on GBM cells and the tumor microenvironment. Smac mimetic BV6 activates NF-κB signaling pathway in GBM and astroglial cells. BV6-induced NF-κB activation in GBM cells induces CCL2 upregulation and triggers migration and invasion of GBM cells in an autocrine/paracrine manner. CCL2 secretion of BV6-pretreated GBM cells increases migration of cells of astroglial cells in a paracrine manner.