3094 - Reversing Radioresistance in Hepatocellular Carcinoma by Targeting Autophagy and Cancer Stem Cells via the CD44/mTOR Signaling Axis

Purpose/Objective(s): Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, and while stereotactic body radiation therapy (SBRT) is a crucial treatment modality, radioresistance limits its therapeutic efficacy. Cancer stem cells (CSCs) and dysregulated autophagy facilitate tumor adaptation and survival under radiotherapeutic stress. CD44, a key CSC marker, interacts with the mTOR pathway to regulate autophagy and cell survival, contributing to radioresistance. This study investigates the CD44/mTOR axis as a therapeutic target to overcome HCC radioresistance and enhance SBRT outcomes.

Materials/Methods: HCC cell lines (Huh7, Hep3B, SK-Hep-1) and their radioresistant derivatives (Huh7-IR) were utilized to examine the role of CD44 and mTOR in autophagy and CSC-mediated resistance. Gene and protein expression of autophagy regulators (mTOR, ULK1, SQSTM1, LC3B-II) and CSC markers (CD44, CD133, ABCB1) were analyzed by qPCR and Western blotting. Functional assays, including ROS measurement, apoptosis, cell cycle analysis, and sphere formation, assessed the impact of mTOR inhibition (rapamycin) and activation (MHY1485) on CSC properties. shRNA-mediated CD44 knockdown was performed to evaluate its effect on mTOR-driven autophagy and survival signaling. The Cancer Genome Atlas (TCGA) database was analyzed to determine the clinical relevance of these pathways in HCC progression and treatment resistance.

Results: TCGA analysis demonstrated significant upregulation of CD44, mTOR, ULK1, and SQSTM1 in advanced and metastatic HCC, correlating with tumor aggressiveness. In radioresistant Huh7-IR cells, mTOR inhibition led to a marked reduction in CSC markers, with CD133 and CD44 expression decreasing by more than 50%, and autophagic flux being suppressed as evidenced by a 60% reduction in LC3B-II and SQSTM1 levels. Knockdown of CD44 further suppressed mTOR activation, leading to decreased CSC survival and a significant increase in radiation-induced apoptosis. Functional assays confirmed that sphere formation and ROS-driven survival were significantly impaired, demonstrating the role of CD44/mTOR in maintaining CSC-mediated resistance. Conversely, mTOR activation preserved CSC phenotypes and autophagic survival, reinforcing its role in therapy resistance. These findings collectively indicate that targeting the CD44/mTOR axis effectively disrupts the autophagy-CSC crosstalk, enhancing radiosensitivity in resistant HCC models.

Conclusion: This study establishes the CD44/mTOR signaling axis as a key driver of radioresistance in HCC. Targeting this pathway disrupts autophagy-mediated CSC survival and enhances radiosensitivity, providing a strong rationale for integrating mTOR inhibitors into SBRT strategies for treatment-refractory HCC. Further validation in preclinical models will facilitate the translation of these findings into future therapeutic approaches.