Metformin Reverses Progesterone Resistance in Endometrial Cancer by Targeting the AMPK-FOXO1-CALB2 Pathway

INTRODUCTION: Progesterone therapy is standard for fertility-preserving endometrial cancer (ECC) patients, but primary and secondary resistance limit its efficacy. Metformin (MET) combined with progesterone shows promise, yet the synergistic mechanism remains unclear. MATERIALS AND METHODS: In vitro cellular studies and nude mouse xenograft models were used. Cell proliferation, migration, and apoptosis were assessed by CCK8, flow cytometry, Western blot, and immunofluorescence. RNA sequencing and cell transfection verified that Forkhead box protein O1(FOXO1) inhibits ECC cell proliferation by downregulating Calbindin2 (CALB2). Xenograft models confirmed FOXO1's inhibitory role. RESULTS: MET combined with Megestrol acetate (MPA) inhibited the proliferation of ECC cells and induced cell apoptosis, and its effect was stronger than that of a single drug. MET promoted FOXO1 nuclear localization. FOXO1 suppressed ECC cell proliferation and migration in vitro and in vivo. MET inhibited proliferation via FOXO1-dependent CALB2 downregulation. Both MET treatment and CALB2 knockdown caused mitochondrial Ca2+ overload, membrane potential depolarization, and apoptosis, effects that were reversed by FOXO1 knockdown or CALB2 overexpression. DISCUSSION: MET with MPA inhibits proliferation and induces apoptosis in MPA-sensitive and resistant ECC cells. MET facilitates FOXO1 nuclear localization via adenosine monophosphate-activated protein kinase (AMPK) activation and AKT serine/threonine kinase 1 (Akt) inhibition. CALB2 is a key downstream target of nuclear FOXO1. The AMPK-FOXO1-CALB2 axis may represent a novel pathway activated by MET, inhibiting ECC proliferation and MPA resistance. CONCLUSION: The AMPK-FOXO1-CALB2 axis mediates MET-induced mitochondrial dysfunction and apoptosis, providing a mechanistic basis for MET to overcome progesterone resistance in ECC.