2656 - Inhibition of miR-96-5p Overcomes Radioresistance in Rectal Cancer by Unleashing p66Shc-Driven Ferroptosis and Inflammatory Signaling
Presenter(s)
F. P. Wu1, and C. Song2; 1The Fourth Hospital of Hebei Medical University, Shijiazhuang, China, 2Radiotherapy Department,the Fourth Hospital of Hebei Medical University, Shijiazhuang, hebei, China
Purpose/Objective(s): Radiotherapy resistance in locally advanced rectal cancer (LARC) presents a major clinical challenge and is closely intertwined with an immunosuppressive, inflammation-rich tumor microenvironment (TME). Ferroptosis, a form of regulated cell death driven by lipid peroxidation, has emerged as a key mediator of radiation response and is intrinsically linked to inflammatory signaling. This study investigates the novel miR-96-5p/p66Shc axis, exploring its role in regulating radiosensitivity through the crosstalk between ferroptosis and inflammation in the TME
Materials/Methods: Utilizing the radioresistant rectal cancer cell line HR-8348, we employed bioinformatic prediction, dual-luciferase reporter assays, and gain/loss-of-function approaches (mimics, inhibitors, siRNA) to validate molecular targeting. Radiosensitivity was assessed in vitro via clonogenic survival, cell viability (CCK-8), and measurements of lipid peroxidation, mitochondrial reactive oxygen species (ROS), and ultrastructure. Expression of key ferroptosis and inflammatory markers (ACSL4, GPX4, COX2/PTGS2) was analyzed by western blot and immunofluorescence. An in vivo xenograft model in nude mice was used to confirm the findings.
Results: We identified p66Shc as a direct target of miR-96-5p. Inhibition of miR-96-5p upregulated p66Shc, leading to increased mitochondrial ROS, elevated lipid peroxidation, and a marked shift towards a ferroptosis-prone state, characterized by upregulation of ACSL4 and COX2 and downregulation of GPX4. This significantly enhanced cellular radiosensitivity in vitro. The effect was reversed by either p66Shc knockdown or the ferroptosis inhibitor Liproxstatin-1, confirming the axis's mechanistic role. In vivo, miR-96-5p knockdown synergized with irradiation to induce pronounced tumor regression, accompanied by congruent molecular changes in the tumors (increased COX2/ACSL4, decreased GPX4).
Conclusion: Our findings reveal that the miR-96-5p/p66Shc axis promotes radioresistance in rectal cancer by repressing radiation-induced ferroptosis. Inhibiting miR-96-5p activates a p66Shc/ROS/ferroptosis pathway, thereby overcoming radioresistance. Crucially, this process concurrently triggers a robust pro-inflammatory response, as evidenced by significant COX2 upregulation. Therefore, targeting the miR-96-5p/p66Shc axis represents a promising dual-pronged therapeutic strategy to not only sensitize tumors to radiotherapy but also to potentially remodel the associated immunosuppressive and inflammatory tumor microenvironment.