EMMPRIN-carrying T-MV increase tumor invasion through activation of p38/MAPK

Next, we were interested in the mechanism which mediates MV-induced tumor invasion.

Since pro-invasive T-MV we found to express HG-EMMPRIN at high levels and the role of MV-bound EMMPRIN for induction of MMP is well established (Muralidharan-Chari et al, 2010), we first investigated MMP levels in MCF-7 cells after stimulation with T-MVM. However, T-MVM did not augment the expression of selected MMP including MMP14, MMP15 or MMP9 (Fig. 16A). Moreover, also the expression of MMP-9 and MMP-2 remained unchanged in zymography assays of the supernatant of MCF-7 cells after T-MV stimulation (Fig. 16B). Only P-MV which were employed as negative control seemed to

induce a slight induction of MMP-9 on the mRNA and protein level, however, this did not reach statistical significance. Although T-MV have been previously described as carriers for MMP (Dolo et al, 1998; Ginestra et al, 1997), neither T-MVM nor T-MVS were found to express MMP-2 (data not shown) or MMP-9 in zymography assays (Fig. 16C). In contrast, benign MV from unstimulated human Mϕ (Mϕ-MV) or P-MV were positive for MMP-9, suggesting that the presence of MMP is not essential for the conveyance of a pro-invasive phenotype of MV. We then looked for the activation of other known pro-invasive EMMPRIN target genes in MCF-7 cells after stimulation with EMMPRIN-bearing T-MVM. However, neither VEGFA, CSF1 nor TNFα expression was increased in the cells in qRT-PCR screenings (Fig. 16D). Moreover, EMMPRIN is known to induce its own transcription via a positive feedback loop mechanism. Nonetheless, we also could not observe such autologous stimulation of EMMPRIN expression itself (Fig. 16D).

Fig. 16: T-MV have no effect on known EMMPRIN target genes in tumor cells

A, The expression of selected MMP was measured by qRT-PCR from total RNA samples of MCF-7 cells stimulated for 24 h with T-MV_M or P-MV (both 25 µg/ml) (means±SD, n=3). B, MCF-7 cells were stimulated for 24 h with increasing concentrations of T-MV_M or P-MV and MMP-2 and -9 levels were assessed in tumor cell supernatants by zymography assays. C, Comparative analysis of MMP-9 expression in whole cell lysates (=C) and their corresponding MV (zymography). MCF: MCF-7, SK: SK-BR-3, P: platelets. D, No changes in mRNA levels of known EMMPRIN target genes were detected in MCF-7 cells after 24 h of stimulation with T-MV_M or P-MV (both 25 µg/ml) by qRT-PCR from total RNA samples (means±SD, n=3). Modified from (Menck et al, 2014b).

For this reason, we decided to investigated signaling events upstream of target gene induction.

One signaling pathway that has been described to participate in EMMPRIN-mediated

signaling is the p38 (MAPK14) pathway (Lim et al, 1998). Indeed, in time course analyses of p38 pathway activation in MCF-7 cells stimulated with T-MV_M, we observed a phosphorylation, and thus activation, of p38 after 1 h of stimulation, while total p38 protein levels remained unchanged (Fig. 17A). To test if the activation of the p38 signaling cascade is indeed involved in MV-mediated tumor invasion, we pre-incubated MCF-7 cells for 2 h with the pyridinylimidazole compound SB-203580 which binds to the ATP-binding pocket of p38 and thereby prevents its catalytic activity (Kumar et al, 1999). After treatment of the cells with SB-203580 in concentrations which did not interefere with cellular viability (Fig. 17B), T-MV were added and found to induce significantly less tumor invasion in microinvasion assays (Fig. 17C). This indicates that p38 signaling is in fact important for T-MV to confer their autologous pro-invasive effect on tumor cell invasion.

Fig. 17: p38 signaling partially mediates the pro-invasive function of T-MV

A, MCF-7 cells were stimulated with T-MV_M (25 µg/ml) and expression of p38 and phosphorylated (P)-p38 was assessed by Western Blotting after different time points. B, MTT assay of MCF-7 cells treated for 96 h with 203580 (SB, 0,5 µM). n.s. = not significant C, Microinvasion assay of MCF-7 cells pre-treated for 2 h with SB-203580 before addition of 10 µg/ml T-MV_M (means±SD, n=3, *p<0,001). Modified from (Menck et al, 2014b).

Stimulation of MCF-7 cells with T-MV_M further induced an increase in phosphorylated c-jun which suggests an additional activation of the JNK-pathway (Fig. 18A). However, in parallel, we observed a concordant augmentation of total c-jun protein levels (Fig. 18A). This raised the question if the observed increase might be caused due to enhanced JUN transcription and thus synthesis of the c-jun protein. Therefore, we performed qRT-PCR analyses of MCF-7 cells which had been stimulated with T-MVM revealing that there was no increase, but, in contrast, a slight decrease in JUN mRNA levels (Fig. 18B). Since MV have often been demonstrated to function as vehicles for the transfer of proteins, we investigated whether this is also the case for the c-jun protein. Using Western Blots, we were indeed able to detect c-jun inside T-MVM (Fig. 18C) which suggests its transfer to the tumor cells via MV and might represent a possible explanation for the increased c-jun levels in MCF-7 after T-MV

stimulation. Nonetheless, inhibition of JNK signaling with a specific JNK inhibitor did not antagonize T-MV-induced tumor invasion (Fig. 18D) arguing against an involvement of JNK-signaling in the conveyance of the autologous pro-invasive effect of T-MV.

Fig. 18: JNK-signaling is not involved in the pro-invasive function of T-MV

A, Western Blot: MCF-7 were stimulated with T-MVM (25 µg/ml) and the cells harvested after the indicated time points to examine the expression of phosphorylated (P-) c-jun and total c-jun. B, Expression of JUN was slightly reduced in MCF-7 cells after 24 h of stimulation with T-MV_M (25 µg/ml) in qRT-PCR analyses from total RNA samples (means±SD, n=3, *p<0,01). C, The c-jun protein was detected inside T-MV_M by Western Blotting. Whole cell lysates (=C) were used as positive control. D, However, inhibition of c-jun signaling with a JNK-inhibitor (JNK-I) was not able to antagonize MV-induced tumor invasion of MCF-7 cells (microinvasion assays, means±SD, n=3). Modified from (Menck et al, 2014b).