Matrix stiffness-mediated effects on stemness characteristics occurring in HCC cells

Abstract

Matrix stiffness as an important physical attribute of extracellular matrix exerts significant impacts on biological behaviors of cancer cells such as growth, proliferation, motility, metabolism and invasion. However, its influence on cancer stemness still remains elusive. Here, we explore whether matrix stiffness-mediated effects on stemness characteristics occur in HCC cells. As the substrate stiffness increased, HCC cells exhibited high proportion of cells with CD133(+)/EpCAM(+), high expression levels of CD133, EpCAM, Nanog and SOX2, greater self-renewing ability and oxaliplatin resistance. Simultaneously, their phosphorylation levels of Akt and mTOR, as well as p-4E-BP and SOX2 expressions were also obviously upregulated. Conversely, knockdown of integrin β1 partially attenuated higher stiffness-mediated stemness characteristics in HCC cells, and reversed the phosphorylation levels of Akt and mTOR, and expressions of p-4E-BP and SOX2, suggesting that integrin β1 may deliver higher stiffness signal into HCC cells and activate mTOR signaling pathway. Additionally, mTOR inhibitor suppressed the mTOR phosphorylation level and expression levels of p-4E-BP and SOX2 in HCC cells grown on higher stiffness substrate, as well as depressed their stemness properties significantly, favoring a regulating role of mTOR signaling pathway in matrix stiffness-mediated effects on stemness. In summary, matrix stiffness may be involved in the process of stemness regulation via activating integrin β1/Akt/mTOR/SOX2 signaling pathway. To the best of our knowledge, this study first reveals a novel regulating pathway to direct the stemness characteristics in HCC cells.

Keywords: hepatocellular carcinoma; integrin β1; mTOR; matrix stiffness; stemness.

Figure 1. Higher matrix stiffness induces and enhances the stemness characteristics of HCC cells

A. Increasing matrix stiffness enhances the proportion of cells with CD133(+)/ EpCAM(+) in two HCC cells; B. Higher stiffness remarkably upregulates the expression of CD133, EpCAM, and stemness-related transcription factor Nanog and SOX2 in two HCC cells. Error bars indicate SEM, ***, P value < 0.001; **, P value< 0.01; *, P value< 0.05. C. and D. Higher stiffness-induced HCC cells exhibit greater ability in morphology and quantity of spheres compared with the controls. Error bars indicate SEM, ***, P value < 0.001; **, P value< 0.01; *, P value< 0.05. E. Oxaliplatin resistance analysis shows that as the stiffness increases, the early apoptosis percentage and the late apoptosis percentage in the treated HCC cells are all descended. F. Low expression of PARP indicates the existence of higher stiffness-mediated oxaliplatin resistance in HCC cells.

Figure 2. Integrin β1 knockdown and mTOR inhibitor reverse higher matrix stiffness-mediated HCC stemness characteristics

A. The proportion of cells with CD133(+) or EpCAM(+) all decrease in the transfected HCC cells with LV-ITGB1-RNAi under higher stiffness stimulation. B. Oxaliplatin resistance analysis shows that higher stiffness-induced HCC cells with LV-ITGB1-RNAi present higher proportion of early and late apoptosis as compared with that of the controls. C. and D. The number of spheres derived from higher stiffness-induced HCC cells treated with LV-ITGB1-RNAi and Rapamycin also significantly diminish. Error bars indicate SEM, ***, P value < 0.001; **, P value< 0.01; *, P value< 0.05.

Figure 3. Integrin β1 and mTOR signaling pathway are involved in the regulation of matrix stiffness-mediated HCC stemness characteristics

A. Increasing matrix stiffness upregulates the phosphorylation levels of Akt and mTOR, as well as the expressions of mTOR downstream molecules p-4E-BP and SOX2 in two HCC cells. B. Integrin β1 knockdown suppresses the phosphorylation level of Akt and mTOR, attenuates p-4E-BP and SOX2 expression in two HCC cells, while rapamycin, an mTOR-inhibitor, not only results in an obvious decrease of mTOR phosphorylation level and its downstream molecules expression, but also produces a slight increase in AKT phosphorylation level.

Figure 4. Schematic diagram of the proposed mechanism by which matrix stiffness drives integrin β1/Akt/mTOR/SOX2 signaling pathway to regulate the stemness characteristics of HCC