MMP-2 Knockout Impairs Cell Migration in Osteosarcoma
To validate our U2OS MMP-2 knockout cell line, we analyzed the mRNA levels and examined the fold change of MMP-2 gene expression between the wildtype (WT) and MMP-2 knockout (KO). qPCR analysis of MMP-2 mRNA showed a significant difference between the KO and WT, with the KO displaying an 833-fold decrease in MMP-2 transcripts compared to the WT cells (Figure 1a). The MMP-2 gene inactivation in KO was also confirmed at the protein level by performing western blot and analyzing the activity of MMP-2 in our WT and KO cells by gelatin zymography (Figure 1a).
In order to assess the impact of MMP-2 KO on cancer cell migration, transwell migration assays were conducted on U2OS WT and KO cell lines. Our findings demonstrated that while the WT cells displayed a high level of migration ability, the MMP-2 KO cells exhibited complete inhibition of cell migration (Figure 1b). Interestingly, adding active MMP-2 enzymes exogenously to the serum-free media of KO cells did not restore cell migration. Likewise, adding the culture media from WT cells, that contains active secreted MMP-2, to the KO cells did not enhance cell migration. It is noteworthy that although prior research had established that MMP-2 inhibitors results in inhibition of tumor invasion and angiogenesis attributable to the activity of extracellular MMP-2 (26), our study is the first to demonstrate a significant inhibition in osteosarcoma cell migration as a result of inactivating MMP-2 gene in the presence of active extracellular MMP-2.
To further examine the effect of MMP-2 KO on the migratory ability of osteosarcoma cells, we conducted wound closure assays on both the WT and MMP-2 KO cells for the duration of 48 hours until complete wound closure was observed in the WT (Figure 1c). Minimal wound closure (approximately 35% closure vs. 100% closure in WT) was observed in the MMP-2 KO at 48 hours time-point (Figure 1c). Thus, knocking out the MMP-2 gene in osteosarcoma cells, in the presence of active extracellular MMP-2, results in a significant inhibition of cancer cell migration, suggesting that the MMP-2 gene, rather than extracellular protein, plays a major role in cancer cell migration pathways.