Breast cancer (BC) is a prevalent malignancy that significantly impacts women's health. Triple-negative BC (TNBC) accounts for approximately 20 % of all BCs and is defined by aggressive clinical behavior and a poor prognosis (Liu et al., 2024a). Because of the absence of estrogen (ER) and progesterone receptors (PR), as well as human epidermal growth factor receptor 2 (HER2), TNBC is refractory to endocrine and targeted therapies (Choupani et al., 2023). Consequently, chemotherapy remains the primary treatment option. While most TNBC patients initially show a response, over 50 % of them develop chemoresistance, leading to recurrence, metastasis, and increased mortality (Inayatullah et al., 2024). The complexity of chemotherapy resistance mechanisms has sparked considerable research interest, yet many underlying factors remain poorly understood.

AMP-activated protein kinase (AMPK) plays a central role in the regulation of the metabolisms of cellular energy and nutrients (Malik and Shaw, 2025). AMPK comprises a catalytic α-subunit and a regulatory β-subunit and a γ-subunit, forming a heterotrimeric complex (Huynh et al., 2023). The protein kinase AMP-activated catalytic subunit alpha 1 (AMPKα1) and alpha 2 (AMPKα2) are two α-subunits of AMPK, which are encoded by PRKAA1 and PRKAA2, sharing approximate 75 % amino acid sequence (Steinberg and Hardie, 2023). Previous studies have described tumor suppressive functions of AMPK signaling through different mechanisms including inactivating the mTOR signaling pathway, downregulation of the glycolytic pathway, or suppression of the epithelial-mesenchymal transition (EMT) to inhibit cancer metastasis (Shackelford and Shaw, 2009, Chen et al., 2022). However, recent studies demonstrate that AMPK mitigates metabolic stress and cancer cell death to promotes cancer cell survival. Energy stress-mediated AMPK activation inhibits ferroptosis (Lee et al., 2020). AMPKα2 phosphorylates ULK1, a critical autophagy initiator, leading to autophagy and subsequent promotion of cancer cell survival and chemoresistance under stress conditions (Fang et al., 2022, Rao et al., 2017). Activation of AMPK signaling is essential for TNBC recovery from metabolic stress (Lo et al., 2023), and AMPKα2 is indispensable for TNBC cell survival during chemotherapy and lung metastasis (Cheng et al., 2023). Notably, most studies only focus on AMPKα and did not distinguish whether the observed biological functions originated from AMPKα1 or AMPKα2. Since there were reports on the controversial roles of AMPK signaling in TNBC, accurate identification of AMPKα1 or AMPKα2 and corresponding underlying mechanisms in chemo-resistance are needed.

Ferroptosis is a type of programed cell death driven by iron-dependent phospholipid peroxidation (Liang et al., 2022). The characteristics of ferroptosis are increased level of free ferrous ions (Fe2 +), and accumulation of lipid peroxides in the cell membrane (Liang et al., 2022). Previous studies have revealed that chemotherapeutic agents, such as doxorubicin (DOX), induce cancer cell death mainly through cell apoptosis and ferroptosis (Shen et al., 2024). DOX can provoke ferroptosis by dysregulation of labile iron pool (LIP) and increased level of Fe2+ (Qiu et al., 2024). Ferritin is the predominant iron storage protein which prevents ferroptosis by sequestering Fe2+ to reduce LIP (Deng et al., 2023). The protein level of ferritin is regulated by ferritinophagy, a selective autophagic degradation of ferritin mediated by nuclear receptor coactivator 4 (NCOA4) (Galy et al., 2024). Activation of ferritinophagy release free Fe2+ from ferritin to increase LIP, thereby promoting ferroptosis. Thus, blocking ferritinophagy or high level of ferritin confers chemoresistance to cancer cells. Indeed, ferroptosis pathways are usually inactivated, or iron metabolism is disturbed in chemo-resistant cancers (Zhu et al., 2023, Mao et al., 2024, Song et al., 2024). However, as a key regulator of cellular metabolisms, the roles of AMPK, or specifically AMPKα2 or AMPKα2, in iron metabolism and ferroptotic resistance in chemo-resistant cancers remain obscure.

In this study, we revealed a high expression of AMPKα2 in DOX resistant TNBC. Elevated level of AMPKα2 phosphorylated NCOA4 at S151, which led to degradation of NCOA4 protein through chaperone-mediated autophagy (CMA) and subsequent defective ferritinophagy that accumulates FTH1 in TNBC cells. Higher levels of FTH1 reduce LIP and inhibit DOX induced ferroptosis. Depletion of AMPKα2 re-sensitized DOX resistant TNBC cells to DOX treatment, implying that targeting AMPKα2 could be a therapeutic strategy for managing chemoresistance of TNBC.