Discussion
Osteosarcoma, a prevalent bone cancer in adolescents, continues to exhibit high mortality rates of 30-40%, with metastasis occurring in at least 25% of patients at diagnosis (31). Although MMP-2 is known to degrade the extracellular matrix and facilitate cancer cell invasion and metastasis, targeting extracellular MMPs clinically has proven insufficient in inhibiting metastasis (3). Recent findings indicate that MMP-2 is also present in various subcellular compartments, including the nuclei of various cells such as osteosarcoma U2OS cells (7,32). We previously reported that nuclear MMP-2 can regulate gene expression of ribosomal RNA (7). Thus, examining the role of intracellular/nuclear MMP-2 in cancer cell migration pathways is crucial for developing effective osteosarcoma treatments targeting metastasis.
We here investigated the consequences of MMP-2 knockout on U2OS cell migration. We observed that wild-type (WT) cells exhibited complete wound closure within 48 hours, whereas MMP-2 knockout (KO) cells showed only 35% closure. Moreover, KO cells were unable to migrate in the presence of externally supplemented active MMP-2 within the culture media. These findings imply that the MMP-2 gene significantly hinders osteosarcoma cell migration, with extracellular MMP-2 having a minimal impact. We propose that the observed effect is attributed to the presence of intracellular/nuclear MMP-2. This protease, typically involved in the degradation of extracellular matrix components, has been found to play a role in various subcellular compartments including the nucleus (6,7). The presence of intracellular/nuclear MMP-2 in this context could potentially contribute to the enhanced cell migration observed in osteosarcoma cells treated with sublethal concentrations of doxorubicin. Further investigation is necessary to fully understand the underlying mechanisms and the role of MMP-2 in modulating osteosarcoma cell migration. Our observations align with a previous study on retinoblastoma (RB) that investigated the effects of inactivating MMP-2 or MMP-9 in cell migration, invasion, and angiogenesis (33). These studies, collectively, indicate that targeting the MMP-2 gene is crucial for impeding cancer cell migration.
At present, doxorubicin is a frequently utilized anticancer chemotherapy for treating osteosarcoma (21). Although low doxorubicin doses cause reduced cardiotoxic effects, numerous studies have reported that sublethal concentrations of doxorubicin may enhance cancer cell migration and lead to chemoresistance (23,25). Accordingly, exploring methods to boost the efficacy of this low-dose treatment in osteosarcoma cases is necessary. Huang et al. (2012) investigated a mechanism linked to chemoresistance, which involves the DNA-binding protein HMGB1 (21). As one of the three anticancer drugs, doxorubicin triggers the upregulation of HMGB1 in osteosarcoma cells. This action leads to the formation of a complex with the autophagy regulator Beclin1, thereby increasing chemoresistance. Furthermore, suppressing HMGB1 effectively restored chemosensitivity in osteosarcoma cells to lower concentrations of doxorubicin (21). In a related study, Tian et al. (2020) observed a similar chemoresistance effect of doxorubicin on osteosarcoma cells. They discovered that doxorubicin treatment promoted stem cell-like characteristics in osteosarcoma, such as enhanced cell migration and proliferation, which ultimately resulted in resistance to the drug (34). Additionally, Tian et al . found that apatinib effectively deactivated the STAT3/Sox2 pathway and reduced doxorubicin-induced cell migration and chemoresistance (34). We investigated the effects of sublethal doxorubicin concentrations on osteosarcoma cells lacking the MMP-2 gene to identify an additional target for enhancing doxorubicin effectiveness. Our results show that MMP-2 knockout prevents increased cell migration in response to sublethal doxorubicin concentrations. Additionally, we demonstrated that MMP-2 gene is upstream mediator of Src kinase in the migration pathway, thus we suggest that MMP-2 expression is not only downstream of Src activation as previously reported (23).
We also found that MMP-2 regulates Src kinase activity via suppression of the endogenous Src inhibitor, CHK/MATK, in osteosarcoma cells. Cheuhet al. (2021) identified that CHK/MATK is suppressed and epigenetically silenced in human colorectal cancer cells, suggesting its potential role as a tumor suppressor (18). To investigate MMP-2’s role in regulating the expression of endogenous Src inhibitors Csk and CHK/MATK in osteosarcoma, we analyzed gene fold expression and protein levels in U2OS WT and MMP-2 KO cells. While Csk gene fold expression was slightly elevated in MMP-2 KO cells, CHK/MATK expression was dramatically increased. Protein level analysis showed a substantial upregulation of CHK/MATK in MMP-2 KO cells, with Csk levels remaining similar between WT and KO cells. Under conditions where CHK/MATK level is elevated, achieved through MMP-2 knockout or CHK/MATK overexpression, sublethal concentrations of doxorubicin fail to activate Src kinase activity. These findings suggest that intracellular/nuclear MMP-2 is responsible for downregulating and suppressing the potential tumor suppressor CHK/MATK in osteosarcoma. Furthermore, the enhanced osteosarcoma cell migration induced by sublethal doxorubicin concentrations can be overcome by overexpressing CHK/MATK in WT cells. Thus, we propose that the MMP-2 gene is an additional target to consider, as it influences the gene and protein expression of the tumor suppressor CHK/MATK in osteosarcoma. Clinically, we anticipate that inhibiting intracellular/nuclear MMP-2 and allowing CHK/MATK re-expression will enhance the effectiveness of sublethal doxorubicin treatments in osteosarcoma patients.
In addition, we analyzed the effects of overexpressing CHK/MATK in U2OS WT cells to unveil the impact on osteosarcoma migration. Surprisingly, simply overexpressing CHK/MATK in U2OS WT cells did not reduce their migration. However, CHK/MATK overexpression was able to inhibit the doxorubicin-induced enhancement of cell migration. Previous research shows in normal untreated cells, SFKs remain in the stable inactive conformation until activated in some cellular events, including doxorubicin treatment and cell migration (17,23). We then activated the Src system through sublethal concentrations of doxorubicin, as shown in our earlier experiments, and therefore, examined Src phosphorylation at Tyr-416 and Tyr-527 in our U2OS WT cells overexpressing CHK/MATK cells. In parallel with our data showing that CHK/MATK overexpression inhibited doxorubicin-induced cell migration, there was no effect on Src activation/phosphorylation when these cells were treated with sublethal concentration of doxorubicin. Additionally, unlike Csk, CHK/MATK is also known to suppress multiple active forms of SFKs by a non-catalytic mechanism that directly binds to the C-terminal tail of SFKs. This inhibiting mechanism is independent of any Src phosphorylation that occurs at Tyr-527. Despite the lack of phosphorylation at Tyr-527, we also observed non-significant change in phosphorylation at Tyr-416 when cells overexpressing CHK/MATK were treated with doxorubicin. As a result, overexpressing CHK/MATK inhibits doxorubicin-induced Src activation and phosphorylation at Tyr-416 as well as the enhancement of cell migration in osteosarcoma.
Correspondingly, Pichot et al. (2009) investigated the effects of targeting SFKs with dasatinib in combination with doxorubicin treatments to inhibit the migration and invasion of breast cancer cells. A synergistic effect between dasatinib and doxorubicin treatments were observed, resulting in inhibiting cell migration, proliferation, thus significantly reducing IC50 of doxorubicin (35). We also report similar effects of overexpressing CHK/MATK to inhibit doxorubicin-induced Src activation and U2OS cell migration.