While the impact of increased matrix stiffness on promoting aggressive behaviors in hepatocellular carcinoma (HCC) cells is well established, its influence on chemoradiotherapy resistance and the precise mechanisms underlying this effect remain unclear. To investigate the contribution of matrix stiffness to HCC development, we developed innovative in vivo animal models featuring precisely controlled liver stiffness levels, along with a matching adjustable hydrogel-based culture platform. This combined strategy enabled detailed analysis of how mechanical signals affect HCC cell growth and DNA repair processes in both laboratory and living-organism settings. Elevated stiffness markedly boosted cell proliferation and protected cells against DNA damage by altering the expression and localization of the metabolic enzyme PFKFB3. In particular, high stiffness reduced PFKFB3 ubiquitination by decreasing E3 ubiquitin ligase NEDD4 levels, thereby stabilizing PFKFB3 protein levels. This led to enhanced glycolysis and ultimately supported HCC expansion. Additionally, stiff matrix conditions improved the DNA damage repair capacity of irradiated HCC cells, with PFKFB3 nuclear translocation playing a key role in stiffness-dependent repair through its interaction with Ku70. These findings reveal a PFKFB3-dependent mechanism by which heightened matrix stiffness drives HCC progression and reduces radiotherapy effectiveness. The study underscores the importance of matrix stiffness in tumor expansion and DNA repair in HCC, while uncovering a novel non-metabolic role for PFKFB3.
