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Inhibition of matrix stiffness relating integrin β1 signaling pathway inhibits tumor growth in vitro and in hepatocellular cancer xenografts.

ABSTRACT:

Background

Cancer development is strictly correlated to composition and physical properties of the extracellular matrix. Particularly, a higher matrix stiffness has been demonstrated to promote tumor sustained growth. Our purpose was to explore the role of matrix stiffness in liver cancer development.

Methods

The matrix stiffness of tumor tissues was determined by atomic force microscopy (AFM) analysis. In vitro, we used a tunable Polyacrylamide (PA) hydrogels culture system for liver cancer cells culture. The expression level of integrin β1, phosphorylated FAK, ERK1/2, and NF-κB in SMMC-7721 cells was measured by western blotting analysis. We performed MTT, colony formation and transwell assay to examine the tumorigenic and metastatic potential of SMMC-7721 cells cultured on the tunable PA hydrogels. SMMC-7721 cancer xenografts were established to explore the anticancer effects of integrin inhibitors.

Results

Our study provided evidence that liver tumor tissues from metastatic patients possessed a higher matrix stiffness, when compared to the non-metastatic group. Liver cancer cells cultured on high stiffness PA hydrogels displayed enhanced tumorigenic potential and migrative properties. Mechanistically, activation of integrin β1/FAK/ ERK1/2/NF-κB signaling pathway was observed in SMMC-7721 cells cultured on high stiffness PA hydrogels. Inhibition of ERK1/2, FAK, and NF-κB signaling suppressed the pro-tumor effects induced by matrix stiffness. Combination of chemotherapy and integrin β1 inhibitor suppressed the tumor growth and prolonged survival time in hepatocellular cancer xenografts.

Conclusion

A higher matrix stiffness equipped tumor cells with enhanced stemness and proliferative characteristics, which was dependent on the activation of integrin β1/FAK/ERK1/2/NF-κB signaling pathway. Blockade of integrin signals efficiently improved the outcome of chemotherapy, which described an innovative approach for liver cancer treatment.

SUBMITTER: Wang C

PROVIDER: S-EPMC8620230 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin ?5/?1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS:hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin ?5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3?, p-GSK-3?, and ?-catenin) were evaluated by western blot analysis. RESULTS:Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin ?5/?1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3?, p-GSK-3?, and ?-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin ?5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin ?5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and ?-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3?, and p-GSK-3? were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin ?5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin ?5 antibody groups. At the same time, expression of GSK-3? and p-GSK-3? was upregulated and ?-catenin levels showed no difference between the groups with or without anti-integrin ?5 antibody. Since Akt, p-Akt, GSK-3?, and p-GSK-3? displayed the same changes between the groups with or without anti-integrin ?5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3? was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3?, and ?-catenin were unchanged. These results suggested that expression of p-GSK-3? was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS:Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin ?5.