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.