Annexin-A1 Regulates Apoptosis-Autophagy Switch in a 5-FU-Resistant Colorectal Cancer Stem-Like Model Through the...

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths globally, primarily due to treatment failure associated with metastasis and recurrence driven by chemoresistant cancer stem-like cells (CSCs). Annexin A1 (Anx-A1) is implicated in regulating apoptosis and autophagy, two crucial mechanisms contributing to chemoresistance. However, the precise role of Anx-A1 in modulating these mechanisms in CRC CSCs, specifically via the PI3K/AKT/mTOR pathway, is not well understood. This study aimed to explore the role of Anx-A1 in modulating apoptosis-autophagy interplay in a 5-FU-resistant colorectal CSC model using HCT 116, particularly through its regulatory effects on the PI3K/AKT/mTOR signaling pathway. A 5-FU-resistant CRC CSC model was developed by sequentially exposing HCT 116 cells to 5-fluorouracil (5-FU). The model was characterized by assessing CSC markers (CD133, ALDH1, ABCG2, Oct-3/4) via flow cytometry and Simple Western analysis. Anx-A1 knockdown and overexpression were performed using siRNA and expression plasmids, respectively. Chemosensitivity was evaluated through cell viability assays, while apoptosis and autophagy markers were assessed by caspase assays, proteome profiler arrays, Western blotting, and autophagy flux analysis. The expression levels of PI3K, p-PI3K, AKT, p-AKT, and mTOR were quantified using Simple Western analysis. The 5-FU-resistant HCT 116 demonstrated significantly elevated IC₅₀ values, heightened expression of CSC markers, and increased Anx-A1 expression. Knockdown of Anx-A1 markedly enhanced chemosensitivity, significantly reduced cell viability, and modulated CSC marker expression. Anx-A1 knockdown led to mild induction of apoptosis characterized by increased caspase activity, concurrent downregulation of multiple apoptotic proteins, and substantial autophagy induction demonstrated by increased LC3-II and p62 expression, and enhanced autophagosome degradation. Analysis of the PI3K/AKT/mTOR pathway revealed that Anx-A1 knockdown significantly increased phosphorylated PI3K p85α and AKT1/2/3, and substantially downregulated mTOR, illustrating the dynamic regulation of apoptosis-autophagy interplay. Anx-A1 modulates 5-FU induced-chemoresistance in colorectal CSCs by orchestrating the apoptosis-autophagy balance through the PI3K/AKT/mTOR pathway. Targeting Anx-A1 provides a promising therapeutic strategy to enhance chemosensitivity and overcome resistance in CRC treatment.