2530 - Stiff Matrix Promotes Radioresistance in Lung Cancer via the Rho/ROCK-YAP/TEAD4 Signaling Axis

Purpose/Objective(s): Previous studies on the mechanisms of anti-cancer treatment resistance have primarily focused on the interactions between tumor cells and their microenvironment from cellular biological and immunological perspectives. Emerging evidence highlights the critical role of physical interactions between tumor cells and the microenvironment in tumor progression and treatment response, revealing novel drivers of treatment resistance. However, the impact of physical factors in the tumor microenvironment on tumor cell radiosensitivity remains poorly understood. This study investigates how extracellular matrix (ECM) stiffness influences lung cancer radiosensitivity, aiming to identify biophysical targets for precision radiotherapy strategies.

Materials/Methods: ECM models with varying stiffness were constructed for in vitro and in vivo experiments. Radiosensitivity of lung cancer cells (A549 and H460) cultured on matrices of different stiffness was evaluated using flow cytometry, comet assay and ?-H2A.X detection assay. RNA sequencing and proteomic analysis were performed to explore mechanotransduction-related molecular mechanisms. Techniques including fluorescence resonance energy transfer (FRET), transmission electron microscopy (TEM), immunofluorescence, CoIP, WB, ChIP and inhibitor assays were employed to validate signaling pathways. Subcutaneous xenograft models in mice were established to assess tumor radiosensitivity.

Results: Lung cancer cells on stiff matrices exhibited significantly reduced apoptosis, enhanced proliferation, and less DNA damage following irradiation (10 Gy or 8 Gy). Upregulation of homologous recombination repair (HRR) associated proteins (BRCA1, BRCA2, MRE11, RAD51) was observed in stiff matrix groups. Transcriptomic and proteomic analyses revealed enrichment of HRR, Rho GTPase activation, Rho GTPase effectors, Hippo pathway and cell cycle regulation pathways in stiff matrix-cultured cells. Mechanistically, in stiff matrix groups, the Rho/ROCK signaling pathway was activated, which enhanced F-actin polymerization to promote YAP nuclear translocation and TEAD4 transcriptional activity. CoIP and ChIP results confirmed YAP/TEAD4 interaction and the transcription of HRR genes. In vivo, subcutaneous tumors in the stiff matrix groups displayed significant radioresistance, with larger tumor volumes compared to the soft matrix groups at the observational endpoint. IHC results of tumor tissues further validated the aforementioned molecular mechanisms.

Conclusion: This study demonstrates that the stiff matrix can promote lung cancer radioresistance through the Rho/ROCK-F-actin-YAP/TEAD4 signaling pathway, which enhances HRR gene expression. These findings elucidate the role of physical factors in lung cancer radiosensitivity and identify potential biophysical targets, advancing the theoretical foundation for precision radiotherapy.