MMP-2 regulates Src activation via repression of the CHK/MATK tumor suppressor in osteosarcoma

Abstract

Background: Doxorubicin, a first-line anticancer drug for osteosarcoma treatment, has been the subject of recent research exploring the mechanisms behind its chemoresistance and its ability to enhance cell migration at sublethal concentrations. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase and zinc-dependent endopeptidase, is well-known for degrading the extracellular matrix and promoting cancer metastasis. Our previous work demonstrated that nuclear MMP-2 regulates ribosomal RNA transcription via histone clipping, thereby controlling gene expression. Additionally, MMP-2 activity is regulated by the non-receptor tyrosine kinase and oncogene, Src, which plays a crucial role in cell adhesion, invasion, and metastasis. Src kinase is primarily regulated by two endogenous inhibitors: C-terminal Src kinase (Csk) and Csk homologous kinase (CHK/MATK).

Aim: In this study, we reveal that the MMP-2 gene acts as an upstream regulator of Src kinase activity by suppressing its endogenous inhibitor, CHK/MATK, in osteosarcoma cells.

Methods and results: We show that enhanced osteosarcoma cell migration which is induced by sublethal concentrations of doxorubicin can be overcome by inactivating the MMP-2 gene or overexpressing CHK/MATK. Our findings highlight the MMP-2 gene as a promising additional target for combating cancer cell migration and metastasis. This is due to its role in suppressing on the gene and protein expression of the tumor suppressor CHK/MATK in osteosarcoma.

Conclusion: By targeting the MMP-2 gene, we can potentially enhance the effectiveness of doxorubicin treatment and reduce chemoresistance in osteosarcoma.

Keywords: Csk homologous kinase (CHK/MATK); Src; Src family kinases (SFK); doxorubicin; matrix metalloproteinase-2 (MMP-2); osteosarcoma.

FIGURE 1

Effect of knocking out MMP‐2 gene on osteosarcoma cell migration. (A). Fold change MMP‐2 gene expression for U2OS WT and MMP‐2 KO cells (p < .0001, t‐test). (B). Above; MMP‐2 protein levels for WT and MMP‐2 KO cells. Below; quantification of MMP‐2 protein levels to α‐tubulin in WT and KO cells (p < .0001, t‐test). (C). MMP‐2 activity levels in WT and KO cell lysates and excreted MMP‐2 in cultured OPTI‐MEM medium, validating our MMP‐2 KO cells. (D). Above; transwell migration assay of U2OS WT, MMP‐2 KO, MMP‐2 KO cells with exogenous MMP‐2, and MMP‐2 KO cells with WT cultured media containing secreted MMP‐2 at 10× magnification. Black scale bar in right bottom corner of each image represents 200 μm. Below; quantification of percentage cells migrated after 24 h after normalizing to WT (p < .0001, one‐way ANOVA). (E) Left; wound closure assays of U2OS WT and MMP‐2 KO (n = 3) at 10× magnification. Black scale bar in right bottom corner of each image represents 200 μm. Right; quantification of % wound closure at 24 and 48‐h time‐points (p < .001 between WT and KO, one‐way ANOVA). Inactivating the MMP‐2 gene significantly impeded the migration of U2OS cells, and the addition of exogenous or secreted active MMP‐2 did not recover their migratory phenotype.

FIGURE 2

Role of MMP‐2 in enhancement of cell migration by sublethal concentrations of doxorubicin. (A) Left; percent cytotoxicity of doxorubicin at various concentrations after 24 h (0, 0.2, 0.4, 0.6, and 1 μM). Right; percent cytotoxicity of doxorubicin at 48 h, showing 0.4 μM doxorubicin as a sublethal concentration of doxorubicin for U2OS cells, as it is statistically non‐significant from the vehicle (p > .05, one‐way ANOVA). (B) Left; wound closure assays for U2OS WT −/+ 0.4 μM doxorubicin at 24 and 48‐h at 10× magnification. Black scale bar in right bottom corner of each image represents 200 μm. Right; quantification of % wound closure at 24 h (upper) and 48 h (lower) time‐points (n = 3). (C) Left; wound closure assays for U2OS MMP‐2 KO −/+ 0.4 μM doxorubicin at 24 and 48 h at 10× magnification. Black scale bar in right bottom corner of each image represents 200 μm. Right; quantification of % wound closure at 24 h (upper) and 48 h (lower) time‐points for WT and KO (n = 3). Although the sublethal concentration of 0.4 μM doxorubicin enhances U2OS cell migration, this enhancement in cell migration is lost when MMP‐2 gene is inactivated.

FIGURE 3

MMP‐2 regulates Src phosphorylation at Tyr‐416. (A). Top; levels of Src phosphorylation at Tyr‐416 and Tyr‐527 after 0 and 0.4 μM treatment at 24 h for U2OS WT (p < .01 and p > .05, t‐test respectively). Bottom; quantification of pSrc at Tyr‐416 (left) and pSrc at Tyr‐527 (right) for U2OS WT. (B). Top; levels of Src phosphorylation at Tyr‐416 and Tyr‐527 after 0 and 0.4 μM treatment at 24 h for U2OS MMP‐2 KO. Bottom; quantification of pSrc at Tyr‐416 (left) and pSrc at Tyr‐527 (right) for U2OS MMP‐2 KO (n = 3) (p > .05, t‐test). In the presence of a sublethal concentration of doxorubicin, Src activation and phosphorylation at Tyr‐416 occurs in an MMP‐2‐dependent manner.

FIGURE 4

MMP‐2 knockout upregulates expression of Src Family Kinase inhibitors. (A) Fold gene expression of endogenous Src kinase inhibitors (CHK/MATK, Csk, CDC2), showing significant upregulation of CHK/MATK in U2OS MMP‐2 KO, compared to WT cells (p < .01, one‐way ANOVA). (B) Top; protein levels of CSK and CHK/MATK in U2OS WT and MMP‐2 KO cells. Bottom; quantification of CSK and CHK/MATK protein levels to 𝛃‐Actin levels in WT and MMP‐2 KO cells (n = 3). (C) Top; protein levels of CSK and CHK/MATK in 0.4 μM doxorubicin treated U2OS WT and MMP‐2 KO cells. Bottom; quantification of CSK and CHK/MATK protein levels to 𝛃‐Actin levels in 0.4 μM doxorubicin treated U2OS WT and MMP‐2 KO cells. ns p > .5, **p < .01, ***p < .001 (t‐test). Loss of MMP‐2 gene caused significant re‐expression of the endogenous SFK inhibitor, CHK/MATK.

FIGURE 5

Overexpression of CHK/MATK impedes osteosarcoma cell migration in the presence of a sublethal concentration of doxorubicin. (A) GFP‐expression of U2OS WT + GFP‐MATK stables, in comparison to U2OS WT, in both GFP and bright field (White scale bar at bottom right corner = 200 μm). (B) gene fold expression for CHK/MATK in WT + GFP‐MATK compared to WT (p < .05, t‐test). (C) Left; protein expression for CHK/MATK in WT and WT + GFP‐MATK, validating expression in WT + MATK stables. Right; Quantification of CHK/MATK protein levels to GAPDH in WT and MATK stable cell lines (p < .0001, t‐test). (D) Top; wound closure assays comparing untreated U2OS WT and U2OS WT + MATK stables, showing complete closure at 48 h at 10× magnification. Black scale bar in right bottom corner of each image represents 200 μm. Bottom; quantification of % of wound closure of WT and WT + MATK stables at 24 and 48 h (n = 3) (p > .05, one‐way ANOVA). (E) Top; wound closure assays for 0.4 μM doxorubicin treated U2OS WT and U2OS WT + MATK stables at 10× magnification. Black scale bar in right bottom corner of each image represents 200 μm. Bottom; quantification of % wound closure of U2OS WT and U2OS WT + MATK stables at 24 and 48‐h time points (p < .05, one‐way ANOVA). (F) Top; levels of Src phosphorylation at Tyr‐416 and Tyr‐527 after 0 μM and 0.4 μM treatment at 24 h for U2OS WT + MATK Stable cells. Bottom; quantification of pSrc at Tyr‐416 (left) and pSrc at Tyr‐527 (right) for U2OS WT + MATK Stable cells for both treatments (p > .05, t‐test). Results show significant inhibition of cell migration in the WT + MATK stables when treated with 0.4 μM doxorubicin.

FIGURE 6

Graphical abstract. Left; MMP‐2 gene represses CHK/MATK expression. This suppression allows for activation of Src by sublethal concentrations of doxorubicin. Right; inactivation of MMP‐2 gene allows for re‐expression of CHK/MATK by its turn inhibit doxorubicin‐induced Src activation and cell migration.