Mitophagy in breast cancer: Regulatory mechanisms and therapeutic potential.

Introduction
Breast cancer (BC) is the most prevalent malignancy among women, with 2.3 million new diagnoses and 665,700 deaths reported in 2022, and an estimated 3 million new diagnoses are projected by 2040.[1,2] At present, the primary treatments for BC are surgery, chemotherapy, radiation therapy, endocrine therapy, immunotherapy, and targeted therapy.[3] In addition to standard chemotherapy, targeted radiotherapy at the sites of metastasis has been shown to have the potential to improve patients’ prognosis.[4] Recent studies have shown that the targeting of osteoclast production in BC patients with bone metastases is promising.[5] However, drug resistance and relapse continue to pose significant challenges.[6] Triple-negative breast cancer (TNBC) accounts for 10–15% of BC cases and is associated with increased invasiveness and a poor prognosis.[7,8] Despite advances in immunotherapy and targeted therapy, reliable biomarkers to predict the patient response are lacking, making conventional chemotherapy the standard treatment for TNBC.[9,10] Therefore, an urgent need is to develop novel treatment strategies to improve the prognosis of patients with BC, particularly those with TNBC.
Autophagy is a highly conserved process of self-degradation and dynamic energy recycling during the proliferation, differentiation, and maturation of eukaryotic cells through which damaged organelles, misfolded cytoplasmic proteins, and macromolecules are degraded, thereby providing energy and essential substances for cellular homeostasis and survival.[11,12] In mammalian cells, autophagy encompasses macroautophagy (MA), microautophagy (MI), and chaperone-mediated autophagy.[13] In 2005, Lemasters identified mitophagy as a subtype of MA that selectively targets and degrades impaired mitochondria, thereby ensuring mitochondrial quality control and cellular stability.[14,15] The proper function of mitophagy is crucial for maintaining both mitochondrial and cellular homeostasis. This process involves the recognition and encapsulation of damaged mitochondria by autophagosomes, followed by fusion with lysosomes for subsequent degradation.[16,17] We have summarized the general process of mitophagy in Figure 1.
Mitochondria-targeted therapies have been extensively explored in preclinical studies, highlighting the critical role of mitochondrial homeostasis in disease management. Strategies, including the use of mitochondrial antioxidants, mitochondrial decoupling agents, gene therapies targeting mitochondrial dysfunction, and mitochondrial modulators, aim to restore mitochondrial function, reduce oxidative stress, and sustain energy production.[18–21] These approaches have shown promise in the treatment of neurodegenerative diseases, metabolic disorders, and ischemia–reperfusion injury.[22] Mitophagy has garnered particular attention in cancer research because of its role in promoting cancer cell development through aberrant mitophagy. Currently, preclinical studies targeting mitophagy pathways have emerged, identifying novel therapeutic pathways that disrupt tumor metabolism and promote cancer cell death.[23,24] Because of the high degree of molecular heterogeneity in BC, studying mitophagy can aid in the development of personalized treatment strategies tailored to the specific characteristics of individual patients’ tumors. Therefore, in-depth research into the regulatory mechanisms of mitophagy and its role in BC progression and drug resistance is crucial for discovering new biomarkers and therapeutic targets, which is highly important for the development of novel BC treatment strategies.
This review aims to summarize current research findings on the significance of mitophagy in BC, assess its potential impact on BC development and progression, and explore the potential of regulating mitophagy as a novel therapeutic and diagnostic approach for BC. Finally, we discuss the mitophagy modulators that may enter clinical trials in the future and the unanswered questions in current research, with the aim of providing a scientific basis for the clinical management and treatment of BC and offering guidance for future research directions.

Pathway and Mechanism of Mitophagy
The mechanism of mitophagy was summarized by Wang et al.[22] In general, mitophagy can be divided into Parkin-dependent and Parkin-independent pathways. The Parkin-dependent pathway is particularly crucial in neurodegenerative diseases as it facilitates the clearance of damaged mitochondria, thereby helping to maintain neuronal health.[25] Pathways mediated by receptors, including B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2)/Adenovirus E1B 19 kDa interacting protein 3 (BNIP3), BNIP3-like (BNIP3L)/NIX, and FUN14 domain-containing 1 (FUNDC1), are considered Parkin-independent mitophagy pathways.[26] These pathways collectively regulate the quality and quantity of mitochondria and are closely associated with physiological and pathological conditions, including cancer, aging, and neurodegeneration.[26]

Parkin-dependent mitophagy
Over the past decade, numerous studies have focused on PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy, which is considered the most common and significant mitophagy pathway.[27] PINK1 is a serine/threonine kinase encoded by the PARK6 gene and contains an N-terminal mitochondrial targeting sequence.[28] Encoded by the PARK2 gene, Parkin is an E3 ubiquitin ligase consisting of several domains, including ubiquitin-like, in-between-RING (IBR), repressor element of Parkin, really interesting new gene 0 (RING0), RING1, and RING2 domains.[28,29] Mutations in PINK1 and Parkin lead to autosomal recessive Parkinson’s disease (PD), suggesting that impaired PINK1/Parkin-mediated mitophagy plays a pathogenic role in the disease etiology.[30] RNA interference-mediated inhibition of PINK1 expression reduces mitophagy levels. However, the overexpression of Parkin can reverse these effects, whereas PINK1 activity cannot compensate for the damage caused by Parkin deficiency.[31] Park et al[28] found that PINK1 mutation results in a phenotype similar to the Parkin mutation phenotype and that Parkin expression significantly ameliorates the PINK1 mutation phenotype in Drosophila, further indicating that Parkin plays a role downstream of PINK1.
PINK1 functions as a mitochondrial damage sensor. In healthy mitochondria, PINK1 expression is too low to be detected, as PINK1 is gradually translocated from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM), where it is cleaved by mitochondrial processing peptidase and presenilin-associated rhomboid-like (PARL), followed by degradation through the N-terminal regulatory pathway.[32–34] When the mitochondrial membrane potential (MMP, ΔΨm) is disrupted, PINK1 transport to the IMM is inhibited, causing its stable accumulation in the OMM.[35] Once stabilized in the OMM, PINK1 can phosphorylate ubiquitin at Ser65 (pSer65-Ub) and activate Parkin, an E3 Ub ligase, by phosphorylating Parkin at Ser65.[36,37] As a result, Parkin is phosphorylated and activated by PINK1, and together, they enhance the initial signal by modifying mitochondria with ubiquitin chains, which PINK1 further phosphorylates, establishing a positive feedback loop.
Parkin functions as a signal amplifier and ubiquitinates numerous proteins on the mitochondrial surface, including mitofusin 1 (MFN1), MFN2, mitochondrial Rho-GTPase 1 (Miro1), subunits of the TIM/TOM complex, voltage-dependent anion channels 1/2/3 (VDAC1/2/3), hexokinase 1 (HK1), and autophagy-related factors.[38–42] Furthermore, Parkin recruits autophagic adaptors to damaged mitochondria, promoting the formation of microtubule-associated protein 1 light chain 3 (LC3)-positive phagophores, which results in mitochondrial degradation by lysosomes.[43,44] Ubiquitination is a reversible process, as deubiquitinating enzymes (DUBs) can detach ubiquitin from modified substrates. The deubiquitinases identified to date include USP8, USP15, and USP30, which regulate PINK1/Parkin-mediated mitophagy.[45–48]
LC3 autophagy adaptor proteins also play important roles in mitophagy. All these adaptors contain two key domains, the ubiquitin-binding domain and the LC3-interacting region (LIR), which are essential for their classification as autophagy adaptors.[49,50] Sequestosome-1 (p62/SQSTM1), one of the first identified selective autophagy receptors, preferentially localizes between adjacent mitochondria and promotes the accumulation of damaged mitochondria through its polybromo 1 (PB1) oligomeric domain, which specifically facilitates the transport of damaged mitochondria into autophagosomes.[50,51] The NF-E2-related factor 2 (Nrf2) upregulates p62 transcription and continuously modulates its levels through a positive feedback loop.[52] Optineurin (OPTN) and nuclear dot protein 52 (NDP52/CALCOCO2) interact directly with LC3 through their respective LIRs to anchor ubiquitin-tagged mitochondria for autophagic degradation.[53,54] Human T-cell leukemia virus type I binding protein 1 (TAX1BP1) and nuclear dot protein 52 (NDP52) share highly similar functional domains, and autophagosomes can also be recruited via an LC3-independent pathway.[55] Studies have shown that the clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated nuclease 9 (CRISPR/Cas9) is used to “eliminate” these five autophagy receptors, reducing the primary prerequisites for mitophagy to OPTN and NDP52.[56] This process may occur because among the five autophagic adaptors, only NDP52 and OPTN can generate a separator film via an autophagy-related 8 (ATG8)-dependent positive feedback loop.[56]
TANK-binding kinase 1 (TBK1) directly or indirectly mediates the phosphorylation of all known autophagy adaptors, increasing their binding affinity for the ubiquitin chain and the ATG8 protein, thereby promoting mitophagy.[57,58] However, OPTN and NDP52 are considered its primary substrates. OPTN is phosphorylated at Ser177, which increases its binding affinity for LC3, and at Ser473 and Ser513, which further increases its ubiquitin chain-binding activity.[59–62]

Parkin-independent mitophagy

BNIP3 and BNIP3L/NIX
BNIP3 is an OMM protein and a homolog of the BCL-2 family; it contains a BH3 domain and is expressed in various cell types.[63] BNIP3 features a distinctive C-terminal transmembrane (C-TM) domain and a broad composite N-terminal region.[64] Under stress conditions such as hypoxia, BNIP3 forms stable homodimers through its C-TM domain and anchors to the OMM, a key step for effective mitochondrial clearance.[65] The interaction between BNIP3 and LC3 is enhanced when Ser17 and Ser24, located near the LIR of BNIP3, are phosphorylated.[66] Studies have shown that the UNC-51-like kinases 1 (ULK1) mediates the phosphorylation of Ser17 in BNIP3, whereas TBK1 may be involved in the phosphorylation of Ser24 in BNIP3.[67] In addition, under hypoxic conditions, C-Jun N-terminal kinase 1/2 (JNK1/2) and protein phosphatase 1/2a (PP1/2 A) can phosphorylate and dephosphorylate BNIP3 at the Ser 60/Thr 66 sites.[68]
NIX, also known as BNIP3L, shares 56% homology with BNIP3.[69,70] NIX possesses a C-terminal TM domain and an LIR motif.[71] NIX plays a key role in the selective degradation of mitochondria during reticulocyte development. The loss of NIX impairs mitochondrial clearance in reticulocytes, causing a compensatory expansion of erythroid precursors, anemia, and erythroid–myeloid hyperplasia.[72] NIX also mediates mitophagy following cerebral ischemia–reperfusion injury.[73] NIX can directly bind to LC3 and induce mitophagy.[74] However, NIX-mediated mitophagy can also occur via LC3-independent pathways.[75] The phosphorylation of Ser34 and Ser35 near the LIR of NIX enhances the NIX–LC3 interaction, increasing the recruitment of mitochondria by autophagosomes.[76,77] Studies have shown that ULK1 mediates the phosphorylation of Ser35 in NIX, whereas TBK1 may phosphorylate Ser34 in NIX.[67] Furthermore, the production of reactive oxygen species (ROS) promotes the transfer of Rheb, a small GTPase, to mitochondria, where its interaction with NIX triggers mitophagy.[78,79] Similar to BNIP3, the function of NIX depends on phosphorylation of its C-terminal region, a critical process for NIX to successfully recruit mitochondria and promote mitophagy.[80] Furthermore, NIX-mediated mitophagy is required for retinal ganglion cell differentiation and somatic reprogramming to induce the formation of pluripotent stem cells.[81,82]
Because of the high degree of sequence similarity between BNIP3 and NIX, the expression of BNIP3 enhances mitochondrial clearance efficiency in reticulocytes lacking NIX.[83] Knockout of the BNIP3 gene leads to increased expression of the NIX protein; however, this increase is insufficient to compensate for the reduction in mitophagy caused by BNIP3 deletion.[69] Furthermore, mitophagy is significantly inhibited in the absence of BNIP3, further exacerbating apoptosis and kidney tissue damage.[84] Notably, PINK1 and Parkin are involved in NIX/BNIP3L and BNIP3-mediated mitophagy. NIX is ubiquitinated by Parkin and recruits the mitochondrial phagocytic receptor NBR1 for mitochondrial degradation.[85]

FUN14 domain containing 1 (FUNDC1)
FUNDC1 is a mitochondrial outer membrane protein consisting of 155 amino acids that encodes three highly hydrophobic α-helical segments in humans. This protein possesses a C-terminal region, an N-terminal region exposed to the cytosol, and a region within the intermembrane space.[86] FUNDC1 initiates mitophagy by interacting with LC3 through its LC3-interacting region (LIR) motif, which is located in the N-terminal region, under hypoxic conditions.[87] Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy sensing. AMPK facilitates the recruitment of the autophagy initiator ULK1 to mitochondria during energy expenditure.[88,89] FUNDC1 is phosphorylated at Ser17 by ULK1, which promotes the interaction between LC3B and FUNDC1, thereby increasing mitophagy.[90] In addition, the mitochondrial E3 ligase MARCH5 (also known as MITOL) modulates Parkin-independent mitophagy under hypoxic conditions by ubiquitylating and degrading FUNDC1.[91] PGAM family member 5 (PGAM5) positively regulates mitophagy by enhancing the interaction between FUNDC1 and LC3 through dephosphorylation at Ser13.[92,93] In conclusion, FUNDC1-induced mitophagy is regulated by both ubiquitin-mediated degradation and phosphorylation. In addition, FUNDC1 is closely related to mitochondrial fission, mitochondrial autophagy, and mitochondrial fusion. Dysfunctional mitochondria must be separated from the mitochondrial network through mitochondrial fission or other unknown mechanisms before being cleared by mitochondrial autophagy. The phosphorylation of FUNDC1 at the Ser13 site promotes its interaction with Opa1 and reduces its interaction with Drp1, thereby inhibiting mitochondrial fission.[94] Recent studies have shown that FUNDC1 mRNA expression is significantly increased in various tumor tissues, including breast, prostate, pancreatic, ovarian, lung, colorectal, and cervical cancer tissues. Furthermore, the expression of FUNDC1 is correlated with the prognosis, suggesting that FUNDC1 may serve as an independent prognostic biomarker.[86,95]

Other mitophagy receptors: BCL2L13, FKBP8, AMBRA1, MCL-1, PHB2, and cardiolipin
BCL2L13 is a single-pass membrane protein anchored to the OMM and is characterized by two LIR motifs.[96] Under Parkin-independent conditions, BCL2L13 interacts with LC3B through conserved LIR motifs and with ULK1 to localize the autophagy initiation complex to mitochondria, thereby promoting mitophagy.[97] Recent studies have shown that the way in which ULK1 binds to BCL2L13 depends on the kinase activity of ULK1, which forms a trimolecular complex with LC3B, and that ULK1 is required for BCL2L13-dependent mitophagy.[97] BCL2L13 additionally mediates mitophagy by regulating mitochondrial fission.[98] BCL2L13 phosphorylation also appears to help regulate BCL2L13–LC3 interactions, as mutations in Ser272 near the second LIR motif reduce mitophagy.[98] FK506-binding protein 8 (FKBP8) is a recently identified mitophagy receptor in the OMM that features a C-terminal TM domain and an N-terminal LIR motif.[99] The LIR motif of FKBP8 strongly binds to LC3A, recruiting it to facilitate mitophagy in a Parkin-independent manner.[99] Furthermore, FKBP8 can escape from easily degradable mitochondria and localize to the endoplasmic reticulum through unknown mechanisms.[99] The autophagy and beclin 1 regulator 1 (AMBRA1) is swiftly recruited to the OMM following mitochondrial depolarization, where it binds LC3 directly to trigger mitophagy and interacts with the ATPase family AAA domain containing 3A (ATAD3A) complex to promote Parkin-dependent mitophagy.[100,101] Moreover, AMBRA1 induces Parkin-independent mitophagy by recruiting the E3 ubiquitin ligase HUWE1 (also known as URE-B1, MULE, and LASU1).[102] Myeloid cell leukemia-1 (MCL-1) is a mitophagy receptor that directly binds LC3A and contains three classical LIR motifs at its C-terminus.[103] Prohibitin 2 (PHB2) enhances PINK1–PRKN-mediated mitophagy by stabilizing PINK1 through the PARL–PGAM5 pathway and augmenting the mitochondrial recruitment of parkin RBR E3 ubiquitin protein ligase (PRKN).[104] During mitochondrial depolarization, cardiolipin (CL) is transferred from the IMM to the OMM, where it binds LC3 to induce mitophagy.[105]

Other E3 ubiquitin ligases that mediate mitophagy
Recently, additional E3 ubiquitin ligases, including ariadne RBR E3 ubiquitin protein ligase 1 (ARIH1), mitochondrial ubiquitin ligase 1 (MUL1), Gp78, seven in absentia homolog (SIAH)-1, and HUWE1, have been identified as participants in the ubiquitination of mitochondrial proteins and the induction of mitophagy.[106] When PINK1/Parkin function is impaired, these E3 ubiquitin ligases can compensate for PINK1/Parkin-mediated mitophagy. Parkin is highly expressed in neurons, but its expression is often reduced in cancer cells. In contrast, ARIH1 is overexpressed in cancer cells, particularly in breast and lung adenocarcinomas, and promotes mitophagy in a PINK1-dependent manner.[107] PINK1 recruits SynPhilin-1 (α-synuclein-interacting protein) to the mitochondria, and the direct interaction between SIAH1 and SynPhilin-1 facilitates mitophagy.[108] MUL1 stabilizes PINK1 on the mitochondrial membrane, thereby promoting mitophagy, although it is not associated with mitochondrial depolarization.[109] The MUL1 pathway compensates for PINK1/Parkin deficiency in both Drosophila and mammalian models.[110] Furthermore, MUL1 is upregulated in cancerous tissues compared with normal tissues.[111] We succinctly summarize the molecular mechanisms of mitophagy in Figure 2.

Role of Mitophagy in the Pathogenesis of BC

Mitophagy plays a dual role in the development of BC
Currently, studies have described changes in mitochondrial autophagic activity across various human tumors, indicating a dual role in cancer.[96,112] During the early stages of tumorigenesis, mitophagy inhibits tumor development by eliminating dysfunctional mitochondria.[113] However, in advanced cancers, mitophagy can promote tumor growth and invasion by supplying energy and nutrients, particularly when aberrant mitophagy results in mitochondrial dysfunction and ROS production.[114] The accumulation of dysfunctional mitochondria or the elimination of normally functioning mitochondria is a critical factor determining whether mitophagy promotes or inhibits tumorigenesis. Further research is needed to elucidate these mechanisms and develop targeted therapies for cancer.
Mitophagy plays a pivotal role in shaping anti-tumor immunity by modulating the metabolic and functional states of immune cells within the tumor microenvironment.[17,115] In T cells, for instance, defective mitophagy leads to the accumulation of damaged mitochondria, a key driver of CD8+ T cell exhaustion. Conversely, enhanced mitophagic activity supports the development of long-lived memory T cells.[116,117] Similarly, BNIP3- and NIX-mediated mitophagy is crucial for fostering memory in natural killer (NK) cells.[118] Paradoxically, mitophagy can also promote tumor progression by inducing the polarization of macrophages towards an immunosuppressive M2 phenotype, which secretes factors that facilitate immune evasion.[119,120] This dual functionality establishes mitophagy as a critical regulatory node determining the success of anti-tumor immune responses, positioning it as a promising therapeutic target to improve cancer immunotherapy. Furthermore, the influence of mitophagy is intimately linked with mitochondrial dynamics (fusion and fission) and calcium homeostasis, together orchestrating ultimate cell fate decisions.[121,122]

Roles of mitophagy-related genes/proteins in the pathogenesis of BC
Mitochondrial dysfunction and the accumulation of mitochondrial DNA (mtDNA) mutations are commonly observed in human cancers. Recent studies have shown that the dysregulation of mitophagy regulators is closely associated with the development and progression of cancer. Various mitophagy-related molecules, including PINK1, Parkin, BNIP3, and FUNDC1, are implicated in tumorigenesis [Figures 1 and 3].

Parkin/PARK2 and PINK1/PARK6
Studies have shown that solid breast, ovarian, colon, and lung cancers exhibit PARK2/Parkin gene deletions or loss-of-function mutations, which inhibit mitophagy, leading to the accumulation of damaged mitochondria, increased ROS production, genomic instability, and cancer progression.[123,124] In certain BCs, the PARK2-containing FRA6E (6q26) fragile region is frequently lost.[125] Parkin downregulates the expression of CDK6 and increases the degradation of hypoxia-inducible factor-1 alpha (HIF-1α) through ubiquitination, thereby inhibiting cancer progression. Reintroducing the Parkin gene into Parkin expression-deficient MCF7 BC cells resulted in reduced proliferation rates both in vitro and in vivo, as well as a decrease in the migratory capacity. Parkin protein and mRNA expression are reduced in most patients with BC, and Parkin inhibits the metastasis of BC cells by interacting with HIF-1α, making Parkin a potential therapeutic target.[126]
The evidence suggests that PINK1 plays a dual role in cancer, although the precise mechanisms underlying this function remain to be elucidated.[127,128] Li et al[129] examined the expression of PINK1 in 150 BC patients with distinct BC subtypes using tissue microarray immunohistochemistry. Their analysis revealed a significant correlation between PINK1 staining and the histological grade of BC. Furthermore, the ablation of PINK1 in MDA-MB-231 cells via the CRISPR/Cas9 strategy resulted in a reduced proliferative capacity of these cells. A proteomic analysis revealed that PINK1 deletion caused substantial alterations in the expression of proteins involved in energy metabolism, particularly those related to glycolytic capacity.[129] These findings suggest that PINK1 may serve as a marker of BC and possess tumor-promoting characteristics, providing novel insights into the regulatory role of PINK1 in BC.

BNIP3
A substantial body of evidence has shown that BNIP3 expression is closely associated with BC. BNIP3 has been implicated in both the development and progression of BC, as well as in BC metastasis. A study revealed that BNIP3 gene expression was upregulated in breast cancer brain metastases (BCBMs), which was correlated with a poorer prognosis.[130] This finding is further supported by studies by Mustaf et al[131] who observed significantly higher BNIP3 protein expression in aggressive BC tissues than in adjacent normal tissues, suggesting that BNIP3 overexpression may promote tumorigenesis. Importantly, however, the deletion of BNIP3 can lead to mitochondrial dysfunction and increased ROS production, which can activate HIF-1α, thereby promoting BC cell proliferation and metastasis.[132] Experimental mouse models have corroborated these findings, demonstrating that reduced BNIP3 expression is associated with enhanced tumor metastasis and that BNIP3 knockout can increase the tumor size and promote multiorgan metastasis.[133] These contradictory findings may result from variations in experimental models and subject selection, highlighting the complexity of the role of BNIP3 in BC.
BNIP3 is also associated with various histological types of BC and may serve as a valuable prognostic marker. The BNIP3 mRNA is highly expressed in ductal carcinoma in situ (DCIS) and is associated with high-grade phenotypes and invasive disease.[134] Notably, BNIP3 protein expression is significantly increased in the cytoplasm of DCIS and invasive carcinoma tissues compared with normal breast tissue. BNIP3 expression is associated with better survival outcomes of patients with invasive carcinoma but with shorter disease-free survival of patients with DCIS.[135] Another study indicated that low BNIP3 mRNA and protein expression in invasive carcinoma are associated with poor prognostic features, although not with hypoxic responses, further reinforcing the hypothesis that BNIP3 expression may serve as a prognostic indicator for BC.[136]
BNIP3 may represent a promising therapeutic target for cancer treatment. A study employing both in vitro and in vivo experiments using TNBC cells showed that the combination of CW1 and ionizing radiation significantly enhanced autophagy and cytotoxicity by downregulating the BNIP3 protein, emphasizing its potential in targeted therapies.[137]

NIX
The role of NIX in BC remains underexplored. NIX mRNA levels were found to be elevated in most BC samples compared with normal tissues; however, in mouse tumor xenograft studies, NIX knockdown promoted tumor growth.[138,139] In addition, NIX mRNA expression was upregulated in DCIS and correlated with hypoxia.[134]

FUNDC1
The FUNDC1 mRNA and protein are frequently overexpressed in BC and are linked to disease progression, tumor size, metastasis, and poor survival outcomes.[86,95] Thus, FUNDC1 may serve as a potential biomarker for BC. Moreover, FUNDC1 has been reported to facilitate cancer progression via the Ca2+–NFATC1–BMI1 pathway.[140] Other studies have shown that melatonin inhibits TNBC progression via the lnc049808-FUNDC1 pathway.[141] Therefore, FUNDC1 may represent a promising biomarker and therapeutic target in BC treatment. Future studies are needed to investigate how FUNDC1 triggers the mitophagy process in BC.

ULK1
ULK1 is a critical autophagy-related gene located on chromosome 12q24.3, which encodes a serine/threonine kinase that functions as the mammalian counterpart to the yeast ATG1 gene.[142] Hypoxia induces the translocation of ULK1 to mitochondria, where it phosphorylates the mitophagy receptor FUNDC1, thereby initiating mitophagy.[143] Metastasis is the leading cause of BC-related mortality, with bone metastasis observed in 65–80% of patients with advanced-stage tumors.[144,145] Previous studies have shown that ULK1 expression is significantly downregulated in most BC tissues, which correlates with BC progression and serves as a novel prognostic biomarker for patients with BC.[146] Researchers found that under hypoxic conditions, the loss of ULK1 impairs mitophagy, resulting in the accumulation of ROS and the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, which ultimately induces an aggressive phenotype in BC cells and promotes bone metastasis.[147] Moreover, the ULK1 activator LYN-1604 has been shown to induce TNBC cell death by activating ULK1.[148] The activation of ULK1-mediated autophagy may represent a promising therapeutic approach for BC treatment.

Other mitophagy receptors

PHB2
The role of PHB2 in BC has been largely elucidated. PHB2 protein expression differs significantly between luminal A and other molecular subtypes of BC, suggesting that PHB2 may serve as a potential biomarker for distinguishing luminal A subtype of BC. In addition, the expression level of PHB2 on the membrane of BC cells may correlate with the treatment response, suggesting a new therapeutic target for the personalized treatment of BC.[149] Furthermore, PHB2 has been identified as a tumor suppressor in BC, particularly in relation to ERα.[150] The overexpression of Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) and the dephosphorylation of PHB2 at Ser39 in ERα-positive patients lead to the loss of PHB2 function, which correlates with a poor prognosis.[151] A peptide inhibitor, endoplasmic reticulum aminopeptidase (ERAP), has been identified as a regulator of the estrogen/ERα pathway by inhibiting the BIG3–PHB2 interaction, potentially overcoming tamoxifen resistance in BC cells.[152] These findings suggest that targeting the BIG3–PHB2 interaction could serve as a therapeutic strategy for luminal BC.

AMBRA1
Studies have shown that the AMBRA1 mRNA and protein are significantly upregulated in BC tissues and are associated with the tumorigenesis and progression of BC, potentially through the mitogen-activated protein kinase (MAPK) signaling pathway, chronic myeloid leukemia pathway, and the vascular endothelial growth factor (VEGF) signaling pathway.[153] In terms of therapeutic efficacy, AMBRA1 regulates autophagy via ATG12 and modulates the sensitivity of BC cells to epirubicin (EPI); AMBRA1 expression is inversely correlated with EPI sensitivity in BC cells. Studies have also confirmed the role of AMBRA1 in modulating EPI sensitivity in vivo.[154] Another study proposed a novel pathway that inhibits paclitaxel-induced apoptosis in BC cells through the AMBRA1/Akt/FoxO1/Bim/mitochondrial pathway, which modulates chemosensitivity.[155] Moreover, miR-3653 regulates BC metastasis by targeting ATG12 and AMBRA1 to inhibit the autophagy process, thereby suppressing epithelial–mesenchymal transition (EMT).[156] These findings suggest that AMBRA1 could serve as a therapeutic target for patients with BC.

FKBP8
Sylvia Fong and colleagues[157] reported that FKBP8 is downregulated in aggressive tumors and that FKBP8 expression reduces metastatic progression to 4T1 tumors in tumor-bearing mice. Therefore, FKBP8 expression may play a crucial role in tumor metastasis. Another study revealed that signal peptide peptidase (SPP), which promotes tumor progression by mediating the degradation of FKBP8, is significantly upregulated in BC.[158] Therefore, targeting the interaction between SPP and FKBP8 may represent a promising strategy for BC therapy.

BCL2L13
A study conducted by Petry et al[159] revealed that BCL2L13 mRNA expression is inversely correlated with Erb-b2 receptor tyrosine kinase 2 (ERBB2, also known as HER2) expression and tumor growth in individuals with node-negative BC. Therefore, high expression of BCL2L13 may be associated with a better prognosis for patients with BC.

Mitophagy adaptors of LC3: p62/SQSTM1 and OPTN

p62/SQSTM1
Abnormal overexpression of the p62 protein is frequently observed in BC, and the p62 mRNA is ubiquitously expressed in both normal and cancer tissues.[160,161] In TNBC, p62 overexpression is strongly associated with advanced clinical stages, a greater proportion of positive lymph nodes, and lymphovascular invasion.[162] Furthermore, accumulating evidence indicates that breast cancer stem cells (BCSCs), a subpopulation pivotal for BC recurrence and drug resistance, exhibit an indefinite capacity for self-renewal, thereby driving tumorigenesis.[163,164] P62 expression is increased in BCSCs, and high p62 levels are associated with poor clinical outcomes. In vitro experiments have shown that p62 is crucial for the self-renewal capacity of BC cells and enhances BC stem cell properties by stabilizing the MYC mRNA through the repression of let-7a and let-7b expression.[165] Therefore, targeting p62 could represent a promising therapeutic strategy to overcome drug resistance mediated by BC stem cells and prevent tumor recurrence.

OPTN
One study revealed that high expression of OPTN, an autophagy receptor, was associated with prolonged survival of patients with TNBC. Furthermore, OPTN may act as a repressor of TNBC metastasis by inhibiting the TGFβ signaling pathway, which promotes metastasis.[166] Another study reported that OPTN mRNA levels were positively correlated with the recurrence-free survival (RFS) and distant metastasis-free survival of patients with TNBC, suggesting that higher OPTN expression is associated with a better prognosis. OPTN is a negative regulator of TGFβ receptor/SMAD signaling and inhibits TNBC metastasis.[167] Therefore, OPTN expression may serve as a potential predictor of the metastasis risk in patients with TNBC.

Other E3 ubiquitin ligases: MUL and ARIH1
MUL1 exerts an oncogenic effect on BC. Therapies targeting MUL1, either through gene therapy or the development of specific drug inhibitors, may represent new therapeutic strategies for BC treatment.[168,169]
Villa et al[107] found that ARIH1 is widely expressed in BC and protects against chemotherapy-induced cell death. This discovery challenges the traditional notion that the primary regulator of mitophagy is a tumor suppressor, suggesting that ARIH1-mediated mitophagy may increase cancer cell resistance to chemotherapy. In addition, ARIH1 plays a role in the EMT and cancer progression. In BC cells, silencing ARIH1 significantly attenuates cancer stem cell properties and tumor formation, whereas ARIH1 overexpression promotes the EMT and cell invasion, suggesting that ARIH1 is a potential target for BC therapy.[170] In immunotherapy studies, ARIH1 overexpression promoted cytotoxic T-cell infiltration, inhibited tumor growth, and enhanced the efficacy of programmed death ligand 1 (PD-L1) blockade. A mechanism of immune checkpoint blockade resistance mediated by the loss of ARIH1 has been identified in invasive breast cancer (IBC)-insensitive tumor models (4T1-derived mouse TNBC models), demonstrating that the activation of ARIH1 can increase the efficacy of cancer immunotherapy.[171]
In summary, this section delineates the relationships between mitophagy-related genes/proteins and the development and progression of BC. More importantly, it highlights their roles in diagnosis and treatment, as illustrated in Table 1.

Other mitophagy-related regulators

BRCA1
Breast cancer susceptibility gene 1 (BRCA1) is a critical tumor suppressor in BC, with over 75% of tumors in women with BRCA1 mutations being TNBC.[172] Chen et al[173] discovered that BRCA1 deficiency impairs mitophagy by blocking ATM–AMPK–DRP1-mediated mitochondrial fission and activating the NLRP3 inflammasome, creating a tumor-associated microenvironment that promotes BC proliferation and metastasis. Another study showed that BRCA1 and PINK1/Parkin expression are negatively correlated in the cancerous breast tissue of patients with BC, with BRCA1 knockdown inhibiting cancer cell growth and high BRCA1 expression correlating with shorter RFS of patients with BC.[174] These findings suggest that the PINK1–Parkin–BRCA1 axis may represent a novel mechanism for BC treatment.

MUC1
Mucin 1 (MUC1) is a heterodimeric transmembrane protein that is aberrantly overexpressed in many human cancers.[175] Previous studies have shown that MUC1 downregulates ATAD3A expression and protects PINK1 from ATAD3A-mediated cleavage, revealing that mitophagy through the MUC1/ATAD3A/PINK1 axis is a novel mechanism supporting the malignancy of BC cells.[176]

Uncoupling protein 1
Uncoupling protein 1 (UCP1), a member of the mitochondrial anion carrier protein family, regulates the MMP and exhibits binding capabilities for long-chain fatty acids, thereby contributing to the maintenance of the lipid metabolic balance.[177,178] Studies have demonstrated that UCP1 can inhibit the metastasis and invasion of TNBC by activating both mitophagy and pyroptosis. The inhibition of mitophagy by cyclosporine A (CsA) diminished the regulatory effects of UCP1 on TNBC metastasis and invasion.[178] These findings underscore the therapeutic potential of mitochondrial proteins in the treatment of TNBC.
Researchers have inferred that several genes or proteins are altered in BC and influence tumor cell growth and metastasis through the regulation of mitophagy; however, the precise mechanisms, as well as the potential involvement of additional unidentified genes, remain unclear. Investigating targets associated with mitophagy may provide a novel approach to BC treatment.

Effect of mitophagy on the tumor immune microenvironment in BC
Mitophagy affects the tumor immune microenvironment through a variety of mechanisms, especially the regulation of immune cell activity. First, mitophagy is able to regulate the metabolic state of tumor cells, making them more inclined toward aerobic glycolysis (Warburg effect), thereby changing the metabolite composition in the tumor microenvironment. This metabolic reprogramming not only provides energy support to tumor cells but also inhibits immune cell activity by producing immunosuppressive metabolites such as lactate.[179,180] In addition, mitophagy may promote immune evasion by influencing direct interactions between tumor cells and immune cells. For example, one study revealed that cancer cells can pass mitochondria carrying mtDNA mutations to tumor-infiltrating lymphocytes through mitochondrial metastasis, leading to T-cell dysfunction and thus a decrease in T-cell antitumor immunity. Patients with mtDNA mutations receiving PD-1 blockade therapy have a poor prognosis.[181] In addition, Liu et al[182] analyzed the transcriptome and clinical information of patients with TNBC in the TCGA and GEO databases and screened five prognosis-related mitophagy-related genes (BSG, JMJD6, DNAJA3, DISC1, and SQSTM1) to construct a risk prediction model; they observed significant differences in the tumor immune microenvironment between the high-risk and low-risk groups. Another study used single-cell RNA sequencing data to screen for genes associated with mitophagy (MRPS5, C20orf27, PSMB5, PYCR1, HEBP1, CBR1, PTMS, LSM2, and NDUFS3) and divided patients with TNBC into high- and low-risk groups.[183] These studies revealed that the expression patterns of mitochondria-related genes (MRGs) can predict the prognosis and immunotherapy response of patients with TNBC and provide a reference for personalized treatment.

Design of Biosensors to Detect the Role of Mitophagy in BC
Breast cancer lung metastasis significantly contributes to patient mortality, yet the absence of targeted therapies and efficient drug screening tools for this condition presents a critical clinical challenge.[184,185] Identifying potential targets for BC metastasis, which are critical components for the effective treatment of malignant tumors, is essential for early detection and prevention. Early intervention can significantly improve the patient’s prognosis, reduce the likelihood of advanced disease progression, and improve overall survival rates. Research has shown that, compared with normal cells, cancer cells typically exhibit increased mitochondrial viscosity.[186] Yuan et al[187] developed a mitochondrion-targeted fluorescent biosensor, PMV-12, with low cytotoxicity. After one hour of PMV-12 staining, the fluorescence intensity in the two types of BC cells was significantly higher than that in normal cells, indicating that PMV-12 could differentiate cancer cells from normal cells based on differences in mitochondrial viscosity, thereby establishing a foundation for research on BC lung metastasis. After treating 4T1 cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), PMV-12 exhibited considerable overlap with MitoTracker Deep Red (MTDR), with a colocalization coefficient of 0.94. This finding demonstrated that PMV-12 is firmly anchored to the mitochondrial membrane, independent of changes in the MMP, enabling mitochondrial imaging for up to 24 hours. Furthermore, PMV-12 can be used to track the gradual fusion of mitochondria-containing autophagic vesicles with lysosomes and to monitor BC lung metastasis in vivo.[187]
In addition, Liao et al[188] developed the G-cleave LC3B biosensor, which integrates split-luciferase technology with an LC3B cleavage sequence to enable rapid assessment of autophagic activity in BC cells. The specificity of the biosensor was validated through ATG4B gene knockout experiments. Results demonstrated that resveratrol potently induces autophagy and acts synergistically with doxorubicin (DXR) to significantly promote apoptosis and reduce the survival rate of MDA-MB-231 cells. This finding not only proposes a potential new therapeutic strategy for BC but also underscores the utility of the G-cleave LC3B biosensor in screening autophagy-modulating compounds.[188]
The development of such novel biosensors provides promising avenues for elucidating the role of mitophagy in BC and advancing its clinical translation. PMV-12, the first fluorescent sensor designed to explore the connection between mitophagy and cancer metastasis, allows real-time monitoring of mitochondrial status and visualization of lung metastatic lesions, highlighting its diagnostic value. Meanwhile, the G-cleave LC3B biosensor offers a rapid and specific means to evaluate autophagic flux, greatly streamlining drug screening efforts targeting autophagy. Future research will focus on developing dynamic imaging tools with higher temporal resolution to overcome existing limitations in tracking rapid processes such as mitophagy. These advances will deepen our understanding of the underlying biological mechanisms and accelerate the clinical application of combinatory therapies targeting mitophagy.

Mitophagy-related Genes as Biomarkers and Prognostic Factors for BC
The early diagnosis of BC is crucial for improving patient survival, and abnormal expression of mitophagy-related proteins may be closely linked to the progression and metastasis of BC. The role of mitophagy as both a biomarker and a prognostic factor for BC has gained increasing recognition in recent medical research. Zhou et al[189] identified significant differences in the expression patterns of mitophagy-related genes such as PINK1, ULK1, FUNDC1, and p62 in various BC subtypes, which are closely related to the tumor microenvironment and prognosis. In this study, BC samples were classified into two subtypes based on the expression of 29 mitophagy-related genes using bioinformatics analysis. These subtypes were significantly associated with ethnicity, intrinsic subtype, and BC pathological classification. In addition, gene set variant analysis and immune microenvironment analysis revealed significant differences in cancer-related and immune-related features between the two subtypes, suggesting that mitophagy activity could serve as a novel prognostic indicator for BC.[189]

Mitophagy as a Potential Therapeutic Target
As previously discussed, the modulation of mitophagy may emerge as a key strategy for BC treatment, as impaired mitophagy is linked to the development and metastasis of BC and plays a dual role in its progression. Consequently, both mitophagy inducers and mitophagy inhibitors have potential as effective therapeutic agents in BC treatment. Moreover, the identification of novel mitophagy regulators represents a promising pathway for the development of mitophagy-based cancer therapies.

Inhibition of mitophagy improves BC treatment resistance
With the ongoing advancements in BC treatments, the survival rate of patients with BC has significantly improved. However, drug resistance remains a significant challenge in BC treatment. When BC cells develop resistance to anticancer drugs, treatment efficacy is significantly diminished, severely impacting patients’ prognosis. Therefore, investigating the mechanisms of BC drug resistance and identifying effective therapeutic strategies are crucial for enhancing treatment efficacy and improving patients’ prognosis. The chemical drug cisplatin induces mitophagy in liver cancer cells, and the use of the dynamin-related protein 1 (DRP1) inhibitor Mdivi-1 blocks this process, enhancing cell apoptosis and thereby improving chemotherapy efficacy.[152] Similarly, inhibiting mitophagy can restore chemotherapy sensitivity in patients with esophageal squamous cell carcinoma.[190] Therefore, targeting mitophagy may significantly increase the efficacy of anticancer therapies, as has also been observed in BC-related studies.

Chemotherapy sensitivity
Mitophagy may contribute to the development of drug resistance, and its inhibition may increase the sensitivity of BC cells to chemotherapy. Chemotherapy remains the primary treatment for BC.[191] Most chemotherapy drugs exert cytotoxic effects by inducing mitochondrial dysfunction and oxidative stress, but their effectiveness can be compromised by mitophagy-dependent drug resistance.[192] Previous studies have shown that the 70-kDa heat shock proteins (Hsp70) expression is increased in BC and other cancers and that its overexpression is associated with tumor progression.[193] Vincristine has been shown to reduce the proinvasive and promigratory effects of Hsp70 by acetylating Hsp70 at K126, potentially implicating mitophagy in drug resistance.[194] However, the specific molecular mechanisms by which mitophagy contributes to drug resistance remain insufficiently explored in this study. A better understanding of these mechanisms could facilitate the development of novel strategies to combat or prevent drug resistance in cancer therapy. In addition, DXR, a key chemotherapy drug for TNBC, induces mitochondrial damage and activates the PINK1/Parkin mitophagy pathway in cancer cells. The exogenously expressed miRNA miR-218-5p can target and inhibit the Parkin mRNA, blocking DXR-induced mitophagy and increasing cancer cell sensitivity to DXR, potentially reducing drug resistance.[195,196] The combination of miR-218-5p with DXR could enhance the anticancer effects of DXR and may represent a novel therapeutic strategy to combat TNBC resistance. This study also highlights the potential of codelivering miRNA and DXR using nanocarriers, paving the way for novel drug delivery systems. However, these findings are based primarily on in vitro studies, and further in vivo validation is needed before their clinical application.
Clinical research has revealed that patients with BC experience muscle atrophy following a single cycle of epirubicin–cyclophosphamide (EC) chemotherapy, accompanied by a significant impairment of mitophagy. Specifically, the protein levels of the key mitophagy markers, PINK1, Parkin, and Mul1, were markedly reduced after EC chemotherapy. Mitophagy is a crucial process for the clearance of damaged mitochondria; its impairment can lead to the accumulation of fragmented and dysfunctional mitochondria, subsequently increasing ROS production and promoting cell apoptosis. These findings suggest that EC chemotherapy may contribute to mitochondrial dysfunction and cell apoptosis by inhibiting mitophagy, which could be a significant factor in the muscle atrophy and functional decline observed in BC patients.[197]

Radiotherapy sensitivity
In addition, radiation therapy plays a critical role in BC treatment; however, tumor cells deprived of oxygen often exhibit radioresistance. p53 exerts a significant radiosensitizing effect on hypoxic BC cells by inhibiting Parkin-mediated mitophagy.[198] A relatively new treatment, sonodynamic therapy (SDT), uses the synergistic effect of low-intensity ultrasound and sonosensitizers and holds potential as a noninvasive cancer treatment. Song et al[199] found that ROS produced by 5-aminolevulinic acid (ALA)-SDT can trigger PINK1/Parkin-mediated mitophagy, which may confer a protective effect on 5-ALA-SDT-induced MCF-7 cell death. Therefore, inhibiting mitophagy may increase the sensitivity of BC cells to SDT.

New designs of mitochondria-targeted nanomedicines
Drug-loaded nanoparticles show significant promise in therapeutics because of their ability to precisely target cancer cells. Li et al[200] developed a mitochondria-targeting nanomedicine containing artemisinin (ART) and liensinine (Lien) for BC treatment. Although ART has anticancer properties, its efficacy is limited, but it is significantly enhanced by the creation of ART- Poly (D,L-lactide-co-glycolide) (PLGA)/ C18-PEG2000-TPP (CPT)/DLPE-S-S-mPEG4000 (DSSP) nanostructures. However, cancer cells may develop resistance to this treatment at relatively high drug concentrations. This study further revealed that Lien inhibits ART-induced mitophagy, preventing the clearance of damaged mitochondria and thereby enhancing the anticancer effect of ART. These findings suggest that blocking mitophagy may increase the effectiveness of BC treatments.

Interaction with apoptosis
Inhibiting mitophagy may increase the sensitivity of BC cells to treatment by promoting apoptosis. Apoptosis and autophagy are known to be interdependent processes.[201] Under certain conditions, mitophagy is considered a self-protective mechanism that allows cancer cells to resist apoptosis or necrosis.[202] Mitophagy inhibits the release of proapoptotic factors and the production of ROS by degrading damaged mitochondria, thereby counteracting the initiation of apoptosis.[203] Studies have shown that warangalone, a natural compound, reduces BC cell viability by increasing ROS production, causing mitochondrial damage, decreasing the membrane potential, and thus inducing mitochondrial-related apoptosis. In addition, warangalone activates PINK1/Parkin-mediated mitophagy. When combined with a PINK1 small interfering RNA (siRNA), warangalone-induced apoptosis is enhanced.[204] These findings suggest that warangalone may reduce treatment sensitivity by stimulating mitophagy through mitochondrial damage, while mitophagy inhibition enhances its anticancer effect. Therefore, combining warangalone with mitophagy inhibitors may be a promising strategy for BC treatment. Mitophagy and apoptosis are complex processes with mutual interactions. Although this study provides insights into how warangalone affects these processes, it does not fully elucidate their relationship. Similarly, silibinin-induced mitochondrial fission leads to mitophagy via the PINK1/Parkin signaling pathway, which alleviates silibinin-induced BC cell apoptosis. The inhibition of mitophagy synergistically promotes silibinin-induced apoptosis through PINK1 or Parkin knockdown.[205] In addition, chloroquine (CQ) significantly enhances isobacterin (IH)-induced mitochondrial division and apoptosis in TNBC cells by inhibiting autophagy/mitophagy without affecting estrogen-dependent BC cells. These findings suggest that IH could be further developed as a new chemotherapeutic drug and that the combination of CQ and IH may become a new strategy for TNBC treatment.[206] In addition, in the study of ER-positive BC, researchers synthesized novel mitochondria-targeted derivatives of mito-fisetin (mF3 and mF7) and tested their anticancer activity. mF3 exerts promising anticancer effects by disrupting the mitophagy response and inducing apoptosis.[207]

RNA interference
RNA interference is a biological process in which double-stranded RNA (dsRNA) molecules trigger the silencing of specific genes, a phenomenon initially discovered in Caenorhabditis elegans.[208] This discovery has been progressively integrated into cancer research, particularly in treatment strategies. The silencing of mitophagy-related genes increases BC sensitivity to treatment. Under hypoxic conditions, glycerophosphocholine phosphodiesterase 1 (GPCPD1) undergoes depalmitoylation by lysophospholipase 1 (LYPLA1), leading to its migration to the OMM and binding to VDAC1. This process disrupts VDAC1 oligomerization and promotes the polyubiquitination of VDAC1 by PRKN, thereby activating mitophagy. The overexpression of GPCPD1 confers resistance to doxorubicin (DOX) in TNBC cell lines, whereas RNA interference-mediated silencing of GPCPD1 increases their sensitivity to DOX.[209] Moreover, ubiquinone oxidoreductase complex assembly factor 6 (NDUFAF6) is significantly overexpressed in BC, which is correlated with poorer patient outcomes. Silencing NDUFAF6 diminishes the proliferation and migratory capacities of BC cells.[210] Other overexpressed mitophagy-related genes may also be targeted using similar RNA interference strategies, suggesting a promising therapeutic approach.

Activating mitophagy promotes BC cell death
Mitophagy can promote cancer cell survival by adapting to stress conditions; however, excessive mitochondrial clearance may lead to cell death. In contrast to mitophagy inhibition, mitophagy activation can increase the sensitivity of BC cells to anticancer drugs. Excessive or prolonged mitophagy can impair mitochondrial health during tumorigenesis and metastasis, potentially resulting in autophagic cell death.[211,212]

Excessive mitochondrial clearance induces death
Flubendazole, initially developed as an antiparasitic agent, has recently been shown to effectively induce autophagy and apoptosis in BC cells, demonstrating its antitumor potential.[213] Studies have shown that flubendazole increases DRP1 protein expression by disrupting the permeability and mitochondrial function of the OMM, triggering the mitochondrial translocation of the PINK1 and Parkin proteins, and thereby promoting excessive mitophagy and inhibiting BC cell proliferation and migration. In addition, flubendazole upregulates the expression of Eva-1 homolog A (EVA1A), and EVA1A knockdown partially blocks the expression and phosphorylation of DRP1, thereby affecting flubendazole-induced mitophagy.[214] Thus, flubendazole may exert its anticancer effects on BC through EVA1A–DRP1-mediated mitophagy.

Activation of mitophagy enhances the effectiveness of immunotherapy
Harnessing the properties of mitophagy to improve the effectiveness of immunotherapy for BC has become a hot topic of current research, especially in TNBC. By activating mitophagy, tumor cells can become more sensitive to immune checkpoint inhibitors. Xie et al[215] found that mitochondria are involved in the localization and regulation of the PD-L1 protein on cell membranes and mitochondria and that the excessive accumulation of PD-L1 on cell membranes is closely related to resistance to immunotherapy drugs. The activation of mitophagy through the ATAD3A–PINK1 axis can alter the distribution and degradation of PD-L1, thereby increasing the efficacy of immunotherapy. Therefore, the inhibition of ATAD3A may restore good antitumor immunity and improve the therapeutic efficacy of chemoimmunotherapy for TNBC. In addition, one previous study reported that the codelivery of CAR-T cells and a mitophagy agonist (BC1618) through a hydrogel could improve the metabolic status of CAR-T cells and increase their persistence and antitumor activity in the tumor microenvironment, thereby improving the efficacy of TNBC immunotherapy.[216] In BC, raloxifene inhibits the growth of BC cells by inducing mitophagy and activating the aryl hydrocarbon receptor(AhR)/Nrf2/HO-1 axis, reducing ROS levels and inhibiting NLRP3 inflammasome activation. These results suggest that the mitophagy and AhR signaling pathways play key roles in tumor therapy and may increase the efficacy of immunotherapy by regulating the immune microenvironment.[217]

Research on other mitophagy-related drugs in BC
Based on the aforementioned findings, mitophagy has emerged as a pivotal factor in the management of BC, suggesting that its modulation could serve as a viable therapeutic strategy. Nevertheless, given the relatively recent focus on mitophagy, research on mitophagy modulators is still in its early stages, with the majority of evidence stemming from preclinical studies.

Natural compounds
In recent years, numerous natural compounds have been reported to exert antitumor effects. These compounds offer advantages such as ease of extraction and minimal side effects. However, their limited specificity and poorly understood anticancer mechanisms have hindered their widespread application.[218] Urolithin A (UA), a metabolite derived from the ellagitannins found in pomegranates, walnuts, and berries, has antitumor effects by inhibiting cancer cell proliferation and improving drug resistance.[219,220] UA is widely recognized for its pivotal role in facilitating mitophagy.[221] Researchers have shown that UA activates the transcription factor EB (TFEB)-mediated mitophagy in tumor-associated macrophages, promotes TFEB nuclear translocation, and inhibits the secretion of pro-proliferative inflammatory factors such as interleukin-6 (IL-6), thereby improving the tumor microenvironment. UA primarily influences the fusion of autophagosomes with lysosomes. The safety of UA treatment in mice was validated through in vivo tumor models, which also showed a significant reduction in tumor size.[222] These findings suggest that UA could be a promising therapeutic agent for BC, and further studies are needed to confirm the impact of the TFEB C212S mutation on tumorigenesis.
Ionophore natural products are a class of hydrophobic molecules that disrupt ion gradients, thereby eliciting various biological effects.[223] Gramicidin A, a 15-residue ion-channel-forming peptide with a molecular weight of 1882 Da produced by Bacillus brevis, was studied in the human BC cell line MCF-7 to investigate its cytostatic effects.[224] Gramicidin A disrupts the ion gradient across the cell membrane and localizes to mitochondria, causing mitochondrial dysfunction and inducing mitophagy, which ultimately leads to the significant inhibition of cancer cell growth.[225] Despite research revealing that gramicidin A can induce mitophagy, the molecular mechanisms by which it specifically localizes to mitochondria and alters their function require further elucidation.
Traditional Chinese medicine, known for its efficacy and minimal side effects, is used to treat BC.[226,227] Arsenic sulfide (As4S4), commonly referred to as realgar, is a traditional Chinese mineral medicine recognized for its potent antineoplastic properties.[228] However, its clinical use is limited by high toxicity, low solubility, and poor bioavailability.[229] Fang et al[230] developed an acid-ground nano-realgar processed product (NRPP), which exhibited enhanced antitumor efficacy compared with conventional realgar. MTG and LTR staining revealed that NRPP promoted mitochondrial and lysosomal colocalization in a concentration-dependent manner, suggesting the occurrence of mitophagy, which was further confirmed by the increase in LC3–mitochondrial colocalization. The activation of the p53/BNIP3/NIX pathway by NRPP likely contributed to its antitumor effects.[230] Thus, NRPP has potential as a therapeutic agent for BC. Cyclovirobuxine D (CVB-D) is a natural alkaloid extracted from the traditional Chinese herb Buxus sinica that has cardioprotective and anti-ischemic effects, and recent studies have shown that it has significant anticancer properties.[231,232] A recent study revealed that CVB-D can bind directly to YAP targets and then activate the FOXO3a/PINK1–Parkin axis to trigger excessive mitophagy, thereby promoting apoptosis and inhibiting TNBC cell proliferation. These findings illustrate the potential of CVB-D as a promising candidate for TNBC therapy.[233]

Novel-targeted drugs
Previous studies have shown that mitophagy can be induced by iron deficiency.[234] Cristian Sandoval-Acuña et al[235] modified the traditional iron chelator deferoxamine (DFO) by attaching a triphenylphosphonium (TPP+) group, resulting in mitochondrially targeted deferoxamine (mitoDFO). This modification converts the iron overload therapeutic agent into an anticancer agent by selectively targeting mitochondrial iron metabolism in cancer cells. The resulting reduction in intracellular iron levels within tumor cells causes mitochondrial dysfunction and mitophagy, along with increased expression of PINK1 and BNIP3.[235] These effects collectively suppress tumor growth and metastasis, suggesting a novel and clinically significant strategy for targeting cancer cells.
Interestingly, Dong et al[236] introduced the mitophagy modulator characterization system (MMCS), which facilitates the elucidation of causal relationships between mitophagy regulation and pharmacological effects. The MMCS assists in identifying mitophagy modulators and provides valuable insights for the therapeutic development of these modulators.

Combination therapy
In BC treatment, a multidisciplinary team (MDT) is a key strategy to improve the survival rate and quality of life of patients by integrating the expertise of specialists in surgery, internal medicine, radiotherapy, pathology, imaging, and other disciplines to provide patients with individualized treatment plans, which can more effectively target mitophagy to enhance the treatment effect. As mentioned earlier, approaches such as drug therapy, immunotherapy, and gene therapy can achieve synergistic effects through MDT. For example, the combination of chemotherapy drugs and immunotherapy drugs can simultaneously induce mitophagy and enhance immune responses. Dankó et al[237] investigated the combined effects of rapamycin and doxycycline on BC cell growth. Their study revealed that this combination strategy induces mitophagy, leading to mitochondrial dysfunction and cell death. Specifically, the combination treatment significantly reduced tumor cell proliferation and growth by inhibiting the mTOR signaling pathway and activating autophagy.[237] In addition, gene therapy can be combined with drug therapy to increase the efficacy of drugs by regulating gene expression.[209,238] Cepharanthine is a natural anti-inflammatory and antineoplastic drug and a novel autophagy inhibitor.[239] The results showed that cepharanthine-mediated inhibition of autophagy/mitophagy effectively enhanced the anti-TNBC cell activity of the chemotherapy drug epirubicin.[240] In addition, Li et al[241] reported that fasting enhances the direct anticancer effect of sorafenib through the ROS–p53 pathway, which is mediated by mitophagy, and further improves the effect of immunotherapy by regulating the immune microenvironment, especially by increasing the activity of T cells and NK cells. Therefore, triple therapy involving fasting combined with sorafenib and immunotherapy (such as anti-PD-1 antibodies) provides a new strategy and a theoretical basis for the comprehensive treatment of BC.
Within the framework of multidisciplinary collaboration, mitophagy can be targeted at multiple levels. This synergistic strategy not only improves the precision of treatment but also provides a more personalized treatment plan for patients with BC. An evaluation of the synergistic effect of mitophagy modulators in combination with existing BC treatments may result in improved treatment outcomes.

Current clinical research progress on mitophagy and targeted therapeutic strategies
Currently, research on mitophagy in BC primarily consists of preclinical studies. Several mitophagy regulators have entered clinical trials, mainly focusing on the treatment of neurodegenerative diseases and certain metabolic disorders. Mitophagy regulators that are currently undergoing or have completed clinical trials include terazosin, idebenone, nicotinamide riboside, Ganoderma, urolithin A, curcumin, resveratrol, and spermidine, with the compound Ganoderma currently in phase 3 clinical trials.[242–245] In addition, the diabetes drug metformin is beginning to be used by an increasing number of people because of its proposed autophagy-stimulating properties.[246] Metformin induces mitophagy in tumor cells and normal cells through the AMPK pathway.[247]
At present, the strategies for targeting mitophagy mainly include regulating the mitophagy pathway, regulating mitochondrial metabolism, and combining therapeutic strategies. In summary, mitochondrial autophagy modulators select mainly the PINK1–Parkin-mediated pathway for BC drug targets. Other drug targets include BNIP3-, NIX-, and TFEB-mediated pathways, among which the pathways that are effective against TNBC target the ATAD3A–PINK1 axis to overcome chemotherapy and immunotherapy resistance. These important drug targets can provide valuable references for the development of drugs for clinical application. In addition, mitochondrial function is closely related to cellular metabolism, and regulating mitochondrial metabolism can also indirectly affect mitophagy. For example, alterations in mitochondrial metabolism can affect the level of oxidative stress in the tumor microenvironment, which in turn affects mitophagy and tumor cell aggressiveness.[248] Mitophagy modulators can be used in combination with other antitumor drugs to enhance the therapeutic effects.
In summary, the role of mitophagy in BC is multifaceted, and its modulation could provide new therapeutic avenues. Further research is needed to fully elucidate the mechanisms involved and translate these findings into clinical applications. The main findings are summarized in Table 2.

Research Progress on Mitophagy in Other Diseases
In addition to the close relationship between mitophagy and BC, its role in a variety of diseases has also received widespread attention, especially in neurodegenerative diseases, cardiovascular diseases, and certain cancers. Studies have shown that the dysregulation of mitophagy is closely related to the occurrence and progression of these diseases and that the regulation of mitophagy may become a potential therapeutic strategy.
Mitophagy plays a crucial role in maintaining cellular homeostasis and neuronal health. Dysregulation of this process is closely associated with the onset and progression of neurodegenerative diseases such as Alzheimer’s disease (AD), PD, and Huntington’s disease (HD).[249] Previous studies have shown that mutations in the PINK1 and PRKN genes are common in patients with PD and that OPTN and TBK1 mutations are common in patients with amyotrophic lateral sclerosis.[29,30,250] Zhang et al[251] reported that PHB2 is involved in the development of PD by interacting with endoplasmic reticulum stress and Parkin. One study revealed that abnormalities in mitophagy are also associated with the abnormal accumulation of the tau protein. In the early stages, tau protein accumulation promotes mitophagy, which helps to clear damaged mitochondria. However, with increasing accumulation, the tau protein prevents Parkin from translocating to mitochondria, inhibits mitophagy, leads to mitochondrial dysfunction and neurotoxicity, and further exacerbates the pathological process of AD.[252] In patients with PD, mutations in key proteins, such as α-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2), promote neuronal death by affecting Parkin-mediated mitophagy.[22,253,254]
The role of mitophagy in cardiovascular disease has also been extensively studied. Mitophagy is closely related to the occurrence and treatment of aberrant cardiac development, aging, cardiac hypertrophy, heart failure, myocardial infarction, ischemia–reperfusion injury, metabolic cardiomyopathy, and vascular atherosclerosis.[255,256] For example, in ischemia–reperfusion injury, mitophagy can be modulated by the ULK1/Rab9/Rip1 signaling pathway or the Parkin-dependent pathway to affect cardiomyocyte survival.[248–250] The inhibition of excessive mitophagy and inadequate promotion of mitophagy can alleviate myocardial ischemia/reperfusion injury.[257–260] Recent studies have shown that PINK1-mediated mitophagy plays a protective role in stress overload-induced cardiac hypertrophy by inhibiting the mtDNA–cGAS–STING pathway.[261] Therapeutically, melatonin has been shown to improve mitophagy, activate the AMPK–OPA1 signaling pathway, promote cardiomyocyte survival, and ameliorate myocardial ischemia–reperfusion injury.[262] In addition, triptolide inhibits excessive Parkin-mediated mitophagy and improves myocardial remodeling by restoring FoxP3 activity.[263]
In some other cancers, the role of mitophagy is complex. At present, many studies on lung cancer, glioblastoma, pancreatic cancer, gastric cancer, colorectal cancer, and hepatocellular carcinoma exist.[264] We summarize the most recent advances in mitophagy and related treatments for these diseases in Table 3.

Conclusions and Future Perspectives
This review provides an overview of the current research progress on the roles of mitophagy in BC development, the treatment response, and disease prognosis. Studies have shown that mitophagy is closely associated with the proliferation, invasion, metastasis, and therapeutic response of BC. However, current research has several limitations, including an incomplete understanding of the mechanisms underlying mitophagy in different BC subtypes and the absence of specific drugs targeting mitophagy. The efficacy of activating versus inhibiting mitophagy as a therapeutic strategy remains uncertain. Significant prospects exist for the future development of new small-molecule drugs or nanoparticle-based therapeutics to precisely modulate mitophagy, as well as the integration of technologies such as CRISPR gene editing and single-cell analysis to further investigate the role of mitophagy in BC. In addition, future studies should focus on elucidating the specific signaling pathways involved in mitophagy across different BC subtypes, including luminal A, luminal B, HER2-enriched, and TNBC subtypes. The identification of biomarkers to predict the response to mitophagy-targeted therapies is another critical area of research. Furthermore, exploring the potential of combination strategies that integrate mitophagy modulation with existing treatments, such as chemotherapy, immunotherapy, or targeted therapy, may provide valuable insights into overcoming treatment resistance and improving patient outcomes. We remain optimistic that, with continued research, mitophagy modulation may emerge as a promising therapeutic strategy for BC, facilitating the selective elimination of tumor cells.

Conflicts of interest
None.