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.