Introduction
Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent endopeptidases overexpressed in several cancer types (1,2). In particular, matrix metalloproteinase-2 (MMP-2) gained considerable attention for its ability to degrade the extracellular matrix, facilitating detachment from primary tumors and migration to secondary tumor sites, thereby underscoring its critical role in cancer metastasis (3). Currently MMP inhibitors employed for cancer treatment predominately target extracellular MMPs, but the lack of specificity (broad targeting of multiple MMPs) renders them less effective in impeding cancer metastasis due to dose-limiting toxicity (4,5). Recent findings reveal that MMP-2 also localizes to different subcellular compartments, including the nuclei of osteosarcoma U2OS cells. However, the contribution of intracellular MMP-2 in osteosarcoma cell migration remains largely unexplored (6,7).
Previous research demonstrated that MMP-2 expression is regulated downstream of Src activity, a non-receptor tyrosine kinase and oncogene, through the extracellular signal-regulated kinases (ERK) pathway (8–11). Src kinase, a member of Src Family Kinases (SFKs), is overexpressed in cancer, and serves a critical role in cell adhesion, invasion and cancer metastasis (9). Src’s catalytic activity is regulated via phosphorylation at Tyr-416 for full activation, and Tyr-527 for inhibition (12). Although phosphorylation at Tyr-527 is indicative of Src inhibition, SFKs’ receptor protein-tyrosine kinases may non-catalytically bind to the SH2 and SH3 domains to inhibit the Src kinase (13,14). The overactivation of SFKs, such as Src, can be attributed in part to the reduced expression of their endogenous inhibitors (13).
The most common endogenous inhibitors of SFKs include C-terminal Src kinase (Csk) and the Csk homologous kinase (CHK/MATK) (14). While Csk is ubiquitously expressed in mammalian cells, CHK/MATK is predominantly found in hematopoietic cells and neurons (14–16). Csk and CHK/MATK share a similar structural composition with Src, possessing a SH2, SH3 and kinase domain; however, they lack the C-terminal tail phosphorylation site and N-terminal myristoyl group (14). Despite their structural resemblance, the binding domains of Csk and CHK/MATK exhibit differences, as their SH2 domains engage with distinct phosphoproteins and target Csk and CHK/MATK to various cellular compartments (17). Both inhibitors were previously reported to catalyze the phosphorylation of the C-terminal tail tyrosine of Src at Tyr-527, but recent studies have shown CHK/MATK to be ineffective at phosphorylating Src C-terminal regulatory Tyr-527 (13). Unlike Csk, CHK/MATK has also been shown to directly bind to Src via a non-catalytic mechanism, thereby preventing autophosphorylation at Tyr-416 and inhibiting Src activation without affecting Tyr-527 phosphorylation, and subsequently, inhibit cellular processes such as cell migration (18,19).
Doxorubicin, an anthracycline antibiotic, is commonly used to treat various cancer types, including osteosarcoma, breast cancer and leukemia (20). Specifically, in osteosarcoma, it serves as a first line drug treatment; however, low concentrations result in drug resistance, while at high concentrations cause significant toxicity (21,22). Due to doxorubicin’s toxic effects on the heart, brain, liver and kidneys, doxorubicin doses need to be lowered in various clinical settings and research has increasingly focused on the cellular mechanisms influenced by different concentrations of doxorubicin (20). For instance, a study by Mohammed et al. (2021) investigated the impact of a sublethal concentration of doxorubicin on several cancer cell lines, revealing that sublethal concentrations enhances cell migration and invasion through SFK activation in both non-invasive and invasive cancer cell lines, including U2OS (23). Furthermore, doxorubicin has been reported to increase the expression of MMP-2 and MMP-9 in cardiac myocytes (24,25). These findings align with the study by Mohammed et al. (2021), as a sublethal concentration of doxorubicin activates SFKs, augmenting the expression of MMP-2, and consequently, enhancing cell migration (23).
The role of intracellular MMP-2 is increasingly being implicated not only in cancer cell invasion, but also in cell migration (6). In our previous studies, we reported that nuclear MMP-2 regulates ribosomal RNA transcription through histone clipping, thereby modulating gene expression and cell proliferation (7). This discovery has opened up a new avenue of research on the role of intracellular/nuclear MMP-2 in regulating gene expression, as cleavage of histones will lead to modified chromatin structure and epigenetic alterations regulating gene expression. In the current study, we examined the impact of sublethal concentrations of doxorubicin on enhancing the invasiveness and migration of U2OS cells in the absence of MMP-2 gene. We reported that knocking out of MMP-2 gene considerably hinders osteosarcoma cell migration and inhibits doxorubicin-induced cell migration. Additionally, we found that the MMP-2 gene plays a role in regulating Src activation, and consequently, cell migration. We also report that inactivation of MMP-2 inhibits Src activation through upregulating the endogenous Src inhibitor, CHK/MATK. Lastly, although a sublethal concentration of doxorubicin promotes osteosarcoma cell migration, combining this treatment with CHK/MATK overexpression in osteosarcoma cells hinders, or at least partially attenuates, cell migration. We conclude that a deeper understanding of the role of intracellular/nuclear MMP-2 in cell migration may pave the way for new strategies to effectively target cancer migration and metastasis.