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.