Programmed cell death in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a particularly aggressive and therapeutically challenging subtype of breast cancer, defined by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. This absence of actionable molecular targets contributes to its resistance to conventional treatments. This review provides an overview of the mechanistic functions, interrelated processes, and therapeutic implications of several programmed cell death (PCD) pathways—including apoptosis, pyroptosis, necroptosis, autophagy, and ferroptosis—in the context of TNBC pathogenesis and treatment. A conceptual framework is proposed for leveraging these interconnected cell death pathways as a basis for novel targeted interventions. Given the complex interplay among various PCD forms characterized by shared features such as inflammation, mitochondrial dysfunction, and overlapping molecular mediators, this integrated network offers promising opportunities for combinatorial therapeutic strategies. Modulation of one cell death pathway may influence others, potentially amplifying therapeutic efficacy. Furthermore, these PCD pathways are highly relevant to immunotherapy outcomes, offering a foundation for synergistic treatment modalities. This review provides an in-depth analysis of the crosstalk between immune-based therapies and PCD, along with a comprehensive discussion of derived therapeutic approaches. However, tumor diversity, resistance mechanisms, and discrepancies between preclinical models and human physiology pose major challenges in applying these findings clinically. The overarching goal is to present innovative insights and strategies to enhance the clinical management of TNBC and ultimately improve patient outcomes.

Graphical Abstract

Introduction

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype [1], characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression [2]. TNBC accounts for approximately 15–20% of all invasive breast cancer cases and is associated with heightened invasion, metastasis, recurrence, and poor survival [3]. It is marked by rapid tumor progression and early dissemination, contributing to significantly reduced overall survival. In the absence of specific therapeutic targets, conventional cytotoxic chemotherapy remains the mainstay of treatment for TNBC [4]. However, clinical outcomes remain suboptimal due to the frequent development of multidrug resistance.

Two primary challenges complicate the management of TNBC. First, the lack of hormone receptors and HER2 amplification renders hormone therapies and HER2-targeted treatments ineffective, leaving systemic chemotherapy as the only nonsurgical option [5]. Second, TNBC exhibits substantial heterogeneity at both intertumoral and intratumoral levels, manifesting in divergent clinical, pathological, histological, and molecular profiles [6]. Over time, the efficacy of chemotherapy diminishes as TNBC cells adapt and develop resistance mechanisms, underscoring the urgent need to identify novel therapeutic strategies and unique molecular targets to improve prognosis.

Cell death can be broadly categorized into accidental cell death (ACD) and programmed cell death (PCD) on the basis of morphological features, environmental triggers, and regulatory mechanisms [7]. ACD is an unregulated response to exogenous insults—such as mechanical injury, toxins, or oxidative stress—that overwhelm cellular defense mechanisms and result in rapid demise [7–9]. Conversely, PCD is a genetically regulated and biologically purposeful process, essential for tissue homeostasis, embryonic development, and immune surveillance [10]. Several PCD modalities have been identified, including apoptosis, pyroptosis, necroptosis, ferroptosis, autophagy-dependent cell death, lysosome-dependent cell death (LCD), parthanatos, and panoptosis [9, 11]. Among these, apoptosis, pyroptosis, necroptosis, autophagy, and ferroptosis are particularly relevant in cancer biology, each influencing tumor cell fate through distinct yet sometimes overlapping pathways. Understanding their regulatory networks and interactions is crucial for developing novel therapeutic paradigms for TNBC. Inducing PCD in cancer cells can suppress tumor growth and inhibit metastasis [12]. These cell death pathways also mediate antitumor effects by reshaping the tumor microenvironment, activating immune responses through the release of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [13, 14], and reversing drug resistance by inducing the release of bioactive compounds [11]. Importantly, recent studies have underscored the potential of integrating PCD mechanisms with immunotherapy. The emergence of immune checkpoint inhibitors, T cell therapies, and natural killer (NK) cell therapies has opened new avenues for combining immunotherapeutic strategies with cell death induction to enhance the overall effectiveness of TNBC treatment. This dual-pronged approach may not only facilitate tumor eradication but also help overcome immune evasion by modulating key components of the tumor microenvironment. Recent research has underscored the potential therapeutic benefits of modulating these pathways to enhance treatment outcomes. Despite the promising nature of preclinical findings, the translation of these discoveries into clinical practice poses significant challenges. This difficulty is largely attributed to the intricate interplay between various cell death pathways and the resistance mechanisms inherent in TNBC. Although research tools, including animal models and organoid models, facilitate the screening of potential therapies, their limited applicability to human physiology continues to be a substantial impediment to clinical translation.

Ongoing research aimed at elucidating the molecular underpinnings of cell death in TNBC is expected to reveal novel drug targets, potentially overcoming existing therapeutic limitations and driving the development of next-generation treatments. Overall, leveraging the interconnectivity of cell death pathways holds great promise for advancing both basic understanding and clinical management of TNBC. These topics are discussed at greater length within the body of this review.

The roles of different cell death pathways in TNBC

Multiple programmed cell death mechanisms play diverse and complex roles in TNBC progression and treatment. Apoptosis, the most well-characterized form of PCD, contributes significantly to TNBC cell clearance. However, TNBC frequently acquires resistance to apoptotic signals, diminishing the efficacy of therapies reliant on this mechanism [15]. Pyroptosis, a lytic and inflammatory form of cell death, exhibits dual roles. While it can eliminate tumor cells [16], it may also promote tumor growth under certain conditions [17]. Necroptosis has emerged as a pivotal compensatory mechanism in TNBC, particularly in cases where apoptotic pathways are impaired. Activation of necroptosis has been linked to improved responsiveness in resistant tumor phenotypes [18]. Autophagy, meanwhile, plays a paradoxical role in TNBC [19]. On one hand, it can promote cell survival by recycling intracellular components; on the other, excessive autophagic activity may trigger cell death by degrading essential cellular structures [20]. Ferroptosis, a recently characterized iron-dependent form of cell death, is increasingly recognized as a critical process in TNBC pathogenesis [14]. It is driven by lipid peroxidation and oxidative stress, which can directly kill tumor cells and modulate the tumor microenvironment to suppress progression [21, 22].

These five forms of cell death—apoptosis, pyroptosis, necroptosis, autophagy, and ferroptosis—have been the focus of intensive research, as each uniquely contributes to TNBC cell survival or death. The intricate interplay among these pathways provides a rich foundation for novel therapeutic interventions. Exploring their regulatory crosstalk and functional convergence is key to identifying new drug targets and formulating therapies. A deeper understanding of these cell death pathways and their integration into the tumor landscape can ultimately lead to more effective, personalized treatments for TNBC.

Apoptosis

Apoptosis is a fundamental form of PCD that plays a vital role in maintaining tissue homeostasis. It is a gene-regulated and orderly process involving nuclear fragmentation, cytoplasmic shrinkage, and cell disassembly into apoptotic bodies [23, 24]. A key distinction between apoptosis and necrosis is that apoptosis does not induce inflammation [25]. In cancer, where uncontrolled cell proliferation is a hallmark, inducing apoptosis has emerged as a central therapeutic strategy [26]. In TNBC, apoptosis is particularly crucial, as it promotes the elimination of malignant cells through tightly regulated signaling pathways. Dysregulation of apoptotic processes contributes to tumor progression and therapeutic resistance in TNBC [27].

Apoptosis is primarily triggered through two major signaling pathways: the extrinsic (death receptor-mediated) and intrinsic (mitochondrial) pathways [28]. The extrinsic pathway is initiated by the binding of extracellular death ligands—including Fas ligand (FasL), tumor necrosis factor (TNF)-α, and TNF-related apoptosis-inducing ligand (TRAIL)—to their corresponding death receptors such as Fas receptor (FasR), tumor necrosis factor receptor 1 (TNFR1), death receptor 4 (DR4), and death receptor 5 (DR5) [9, 29]. Ligand binding leads to the assembly of a death-inducing signaling complex (DISC) [30], which recruits and activates caspase-8. Activated caspase-8 then cleaves and activates caspase-3, a key executioner caspase responsible for the morphological and biochemical features of apoptosis [8, 31]. The intrinsic pathway, in contrast, is activated in response to intracellular stress signals such as DNA damage, oxidative stress, and endoplasmic reticulum dysfunction [32, 33]. Oxidative stress, characterized by the excessive accumulation of reactive oxygen species (ROS) [34], can compromise mitochondrial membrane integrity [14], leading to increased permeability and the release of pro-apoptotic factors such as cytochrome c (CytC) into the cytoplasm [35]. DNA damage triggers the activation of ATM and Rad3-related (ATR) proteins, which stabilize and activate the tumor suppressor p53 [36]. Activated p53 promotes the transcription of pro-apoptotic Bcl-2 family proteins, such as Bad and Bid, which antagonize anti-apoptotic proteins such as Bcl-2 [37, 38]. This interaction facilitates the oligomerization of Bax and Bak on the outer mitochondrial membrane, further enhancing membrane permeability [39]. The subsequent release of CytC into the cytoplasm leads to the formation of the apoptosome complex, composed of apoptotic protease activating factor 1 (Apaf-1), CytC, and procaspase-9 [40]. This complex activates caspase-9, which in turn activates caspase-3, thereby initiating the apoptotic cascade [41].

Apoptosis is essential for eliminating genetically unstable or damaged cells, thereby preventing malignant transformation and tumor development [33]. In TNBC, impairment of apoptotic signaling enables tumor cells to evade immune-mediated clearance, promoting sustained proliferation, metastasis, and resistance to therapy [42]. Tumor cells primarily circumvent destruction by upregulating anti-apoptotic proteins while downregulating pro-apoptotic proteins [43]. Differential expression of Bcl-2 family proteins plays a significant role in TNBC progression and drug resistance [44]. For instance, anti-apoptotic Bcl-2 and Bcl-xL overproduction inhibits the function of pro-apoptotic proteins, preventing the cytosolic release of CytC and thereby abrogating apoptotic signaling [45].

TNBC is additionally characterized by the overexpression of the mucin MUC1 [46], and its C-terminal subunit acts as an oncoprotein by interacting with various kinases and effectors [47]. Notably, MUC1 interacts with the nuclear transcription factor NF-κB p65 (RelA), a key regulator of cell survival and inflammation [9, 48]. This interaction enhances NF-κB activation and nuclear translocation, allowing it to bind the promoter of the gene encoding Bcl-2, leading to the upregulation of this anti-apoptotic protein and the consequent inhibition of CytC release and apoptosis induction [49, 50]. Additionally, MUC1 facilitates the activation of the epidermal growth factor receptor (EGFR)/PI3K/Akt signaling pathway [14], which further promotes the transcription of genes that inhibit apoptosis and enhance cell survival [51]. Given the central role of apoptotic dysregulation in TNBC pathogenesis, restoring apoptotic signaling is a promising therapeutic avenue. Current strategies include chemotherapeutic agents, targeted molecular therapies, and immunotherapeutics that aim to modulate key apoptotic regulators, including Bcl-2 family proteins, caspases, and noncoding RNAs involved in cell death regulation.

In summary, apoptosis is a critical cellular process that prevents the survival of aberrant cells and maintains tissue integrity. In TNBC, disruption of apoptotic signaling contributes significantly to tumor development, metastasis, and treatment resistance. Present understanding primarily centers on the extrinsic and intrinsic pathways and their molecular regulators. Continued investigation into the mechanisms governing apoptosis in TNBC is essential for the development of innovative, mechanism-based therapies aimed at improving clinical outcomes and patient survival (Fig. 1).

Fig. 1.

Apoptotic Activity and Regulation in TNBC. Apoptosis is predominantly activated via two distinct pathways: the extrinsic and intrinsic pathways. The extrinsic pathway is initiated by receptors of the TNF family, DISC, which subsequently activates caspase-8, leading to the activation of caspase-3. Conversely, the intrinsic pathway is activated by cellular stressors, including oxidative stress and DNA damage, which engage the mitochondrial pathway. Stress-induced alterations in mitochondrial membrane permeability result in the release of pro-apoptotic factors, such as CytC, which facilitates the activation of caspase-9, subsequently leading to caspase-3 activation. The convergence of both pathways at the activation of caspase-3 culminates in apoptosis. In TNBC cells, the overexpression of MUC1 directly interacts with the NF-κB p65 subunit. By activating NF-κB signaling and upregulating the expression of Bcl-2 family proteins, the release of CytC is inhibited. Additionally, MUC1 activates the EGFR/PI3K/Akt signaling pathway, collectively inhibiting apoptosis and promoting tumor cell survival and drug resistance

Pyroptosis

Pyroptosis is a highly inflammatory form of PCD, primarily initiated by the activation of inflammasomes [52]. This process initiates the activation of caspase family proteins, which subsequently cleave Gasdermin proteins [53], leading to pore formation in the cell membrane, ultimately causing cell swelling and lysis [14]. This process results in the release of intracellular contents, including DAMPs and pro-inflammatory cytokines, thereby triggering an acute inflammatory response in the surrounding tissue that resembles necrotic cell death [54, 55]. While both apoptosis and pyroptosis involve the activation of caspase family members, a key distinction is that pyroptosis culminates in lytic cell membrane rupture and the release of numerous inflammatory mediators, including interleukin-1β (IL-1β) and IL-18 [56]. Pyroptosis is characterized by membrane pore formation [14], as well as mitochondrial dysfunction, and robust cytokine release, which collectively contribute to inflammation and immune activation [57, 58]. In TNBC, pyroptosis plays a complex, dualistic role in tumor progression. On one hand, pro-inflammatory cytokines such as IL-1β can enhance tumor invasiveness and metastatic potential [59]. For instance, IL-1β has been shown to promote epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) traits in TNBC. Moreover, pyroptosis can induce the expression of chemokines such as C–C motif chemokine ligand 2 (CCL2) [60], which activates the Akt signaling pathway and elevates β-catenin levels, further driving EMT and CSC phenotypes [61]. On the other hand, pyroptosis can also initiate potent anti-tumor immune responses. IL-18, a cytokine released during pyroptosis, enhances the cytotoxic activity of NK cells and cytotoxic T lymphocytes (CTLs) against tumor cells [14, 62]. Additionally, the inflammatory milieu generated during pyroptosis facilitates the recruitment of immune effector cells into the tumor microenvironment [63], which can enhance immune-mediated tumor clearance [64]. Pyroptosis can be triggered by multiple upstream pathways, providing opportunities to selectively induce cell death in tumor cells [65].

In tumor cells, pyroptosis is regulated by multiple pathways and factors, which can be broadly classified into three major mechanisms: the classical inflammasome pathway (caspase-1-dependent), the nonclassical inflammasome pathway (dependent on caspase-4/-5 in humans and caspase-11 in mice), and a caspase-3-dependent pathway. The classical, or canonical, inflammasome pathway is primarily activated by the formation of multiprotein complexes known as inflammasomes [66]. These inflammasomes typically consist of three key components: sensor proteins—such as NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) or absent in melanoma 2 (AIM2); the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC); and the effector protein caspase-1 [67]. Sensor proteins detect PAMPs or DAMPs, leading to conformational changes that promote the assembly of the inflammasome complex. Specifically, NLRP3 utilizes its pyrin domain (PYD) to bind the PYD domain of ASC, initiating inflammasome assembly [68]. AIM2, on the other hand, detects intracellular double-stranded DNA through its hematopoietic interferon-inducible nuclear protein with a 200-amino acid repeat (HIN200) domain and similarly interacts with ASC [69]. ASC functions as a molecular bridge, linking sensor and effector proteins through its N-terminal PYD and C-terminal caspase recruitment domain (CARD) [70]. The PYD of ASC binds to the PYD of sensor proteins [71], while its CARD domain interacts with the CARD domain of caspase-1, thereby recruiting and activating the effector protease [72]. Once activated, caspase-1 processes pro-inflammatory cytokines, including pro-IL-1β and pro-IL-18, into their mature forms [73]. Caspase-1 also cleaves GSDMD, a critical executor of pyroptosis [74, 75]. Gasdermin family proteins generally contain an N-terminal domain, which has pore-forming capabilities, and a C-terminal domain, which acts as an autoinhibitory region that keeps the protein in an inactive state [76]. Upon cleavage, the inhibitory C-terminal domain is removed, liberating the active N-terminal domain. This fragment embeds into the plasma membrane to form transmembrane pores [77], allowing ions and water to enter the cell, ultimately causing cell swelling, lysis, and the release of inflammatory mediators, such as IL-1β and IL-18 [78, 79], triggering a strong inflammatory response. The nonclassical inflammasome pathway is initiated through direct activation of caspase-4/-5 in humans (or caspase-11 in mice) by intracellular lipopolysaccharide (LPS) [80]. Upon recognition of LPS, these caspases cleave GSDMD, leading to membrane pore formation and pyroptosis, similar to the classical pathway. Furthermore, caspase-4/5/11 activation can lead to secondary activation of caspase-1, thereby linking the nonclassical and classical pathways [81]. Leading to effects similar to the classical pathway. The caspase-3-dependent pathway is typically activated by external stimuli, such as chemotherapeutic agents (e.g., cisplatin and doxorubicin), oxidative stress, and DNA damage, all of which can activate caspase-3 [82, 83]. Once activated, caspase-3 cleaves Gasdermin E (GSDME), releasing its pore-forming N-terminal fragment. This process also leads to the activation of caspase-1 and triggers pyroptotic cell death [84].

There is a strong association between pyroptosis and the initiation, progression, and treatment of TNBC. Pyroptosis is characterized by the release of abundant pro-inflammatory cytokines such as IL-1β and IL-18. Within the tumor microenvironment, IL-1β has been implicated in promoting tumor growth, invasion, and metastasis and is correlated with unfavorable clinical outcomes [17]. In contrast, IL-18 has demonstrated the capacity to activate NK cells and CTLs [62, 85], thereby enhancing immune-mediated tumor cell destruction. GSDMD plays a central role in pyroptosis-mediated tumor suppression. Following cleavage by caspase-1 or caspase-4/5/11, the GSDMD N-terminal fragment forms pores in the tumor cell membrane, leading to osmotic imbalance, cell lysis, and the release of tumor-associated antigens [86]. These antigens are subsequently captured by DCs and presented to T cells, triggering a robust adaptive immune response that strengthens antitumor immunity [87]. Additionally, pyroptosis contributes to macrophage polarization toward the M1 phenotype, which is known for its pro-inflammatory and tumor-inhibitory effects. M1 macrophages secrete cytokines that suppress tumor proliferation and metastasis while promoting immune activation within the tumor microenvironment [88]. Pyroptosis can activate tumor suppressor genes such as p53, which not only promotes the apoptosis of tumor cells but also inhibits the growth of TNBC [89]. It has also been shown to induce mitochondrial dysfunction, thereby reducing ATP production and impairing energy metabolism, which limits tumor cell survival [90].

It serves as an important antitumor mechanism in TNBC by both directly eliminating cancer cells and enhancing the immune system’s ability to target tumors. A growing body of research is focusing on the molecular regulation of pyroptosis—particularly gene expression profiles and associated signaling pathways—in the context of TNBC. These insights are essential for guiding the development of innovative therapeutic strategies aimed at leveraging pyroptosis for more effective cancer treatment (Fig. 2).

Fig. 2.

The Mechanistic Activation of Pyroptosis. Pyroptosis is primarily regulated through three distinct pathways: the canonical pathway, which is dependent on caspase-1; the noncanonical pathway, reliant on caspase-4/5 in humans and caspase-11 in mice; and a pathway dependent on caspase-3. The canonical pathway is initiated when sensor proteins such as NLRP3 or AIM2 recognize PAMPs or DAMPs, resulting in the assembly of an inflammasome complex comprising ASC and caspase-1. This complex promotes the maturation of pro-inflammatory cytokines pro-IL-18 and pro-IL-1β and cleaves GSDMD, culminating in the disruption of the plasma membrane and the subsequent release of IL-18 and IL-1β, ultimately leading to cell death. The noncanonical pathway is triggered by LPS, which directly activates caspase-4/-5 or caspase-11, leading to the cleavage of GSDMD and the induction of pyroptosis. Additionally, chemotherapeutic agents can activate caspase-3, which subsequently cleaves GSDME and activates caspase-1, thereby inducing pyroptosis via a caspase-3-dependent mechanism

Necroptosis

Necroptosis is a regulated form of cell death that occurs independently of caspase activity. As a recently characterized form of programmed cell death, necroptosis is mechanistically distinct from both apoptosis and classical necrosis. It is predominantly governed by signaling pathways involving receptor-interacting protein kinases (RIPK1 and RIPK3) and the mixed lineage kinase domain-like protein (MLKL) [91]. Unlike apoptosis, which is a noninflammatory and orderly process that removes cells without disrupting tissue homeostasis, necroptosis is inherently pro-inflammatory [92]. This inflammatory nature arises from the release of intracellular contents following membrane rupture, a morphological hallmark that necroptosis shares with necrosis [75]. However, while necrosis is an uncontrolled, passive response to extreme cellular injury [31], necroptosis is an active, regulated process orchestrated by specific molecular mechanisms [93]. The release of cellular contents during necroptosis intensifies the surrounding inflammatory response [94]. The regulation of necroptosis is primarily mediated by the phosphorylation of MLKL, facilitated by apoptosis-regulating proteins such as RIPK1 and RIPK3 [95], and it interacts with ROS to modulate cell death and inflammatory signaling [96]. The incidence of necroptosis in TNBC provides a novel opportunity for patient treatment.

In contrast to apoptosis, necroptosis does not depend on caspases, particularly caspase-8. Instead, it is triggered through the activation of death receptors on the cell surface. When these receptors, such as FasR, TNFR, and DR4/DR5 bind their respective ligands, including FasL, TNF-α, and TRAIL, they typically initiate apoptosis through the formation of a DISC. This complex activates caspase-8, thereby promoting apoptotic cell death [93]. However, in cancer cells where caspase-8 expression is deficient or inhibited, the apoptotic cascade is disrupted. Under these conditions, RIPK1 becomes activated and undergoes deubiquitination, which facilitates its interaction with RIPK3 [97, 98]. This RIPK1–RIPK3 complex undergoes mutual phosphorylation and subsequently recruits MLKL. Phosphorylated MLKL oligomerizes and translocates to the plasma membrane, where it disrupts membrane integrity, culminating in necroptosis [99].

The activation of necroptosis holds particular significance in the context of TNBC, as it offers a potential strategy to overcome the resistance of tumor cells to apoptosis-based treatments [18]. Dysregulation of apoptotic pathways is a common feature in TNBC, often leading to chemoresistance and enabling tumor cells to evade cell death induced by conventional chemotherapeutic agents [92]. This evasion promotes uncontrolled tumor proliferation and contributes to aggressive disease progression [100]. Necroptosis addresses this therapeutic challenge by inducing irreversible cancer cell death via a mechanism distinct from apoptosis [101]. For instance, TNBC cells frequently exhibit elevated levels of the anti-apoptotic protein Bcl-2, which inhibits the activation of the caspase family and thereby blocks the apoptotic cascade [102]. In contrast, necroptosis proceeds independently of caspase activity and is instead mediated by a distinct set of signaling molecules, including receptor-interacting protein kinases RIPK1 and RIPK3, as well as MLKL. As a result, conventional anti-apoptotic resistance mechanisms in TNBC do not interfere with the necroptotic pathway. In its initial stages, necroptosis increases the permeability of tumor cell membranes, leading to the leakage of intracellular contents and ultimately resulting in cell death. This not only suppresses tumor proliferation and growth but also triggers immunological consequences that enhance anti-tumor responses [18]. Specifically, necroptosis promotes the release of high-mobility group box 1 protein (HMGB1) [14], which plays a central role in immune activation in TNBC. HMGB1 recruits immune cells and induces the release of cytokines such as IL-1α and IL-1β, both of which are involved in targeting tumor cells [103, 104]. HMGB1 is recognized by DCs, which are pivotal in orchestrating the adaptive immune response [105]. In TNBC patients, higher levels of DC infiltration are strongly correlated with improved clinical prognosis [106]. DCs engulf antigenic substances released from necrotic tumor cells, they undergo maturation and gain the ability to specifically recognize and target tumor cells presenting those antigens, thereby initiating a robust adaptive immune response [107]. Additionally, DCs secrete various cytokines, including IL-12 [108], which promotes the proliferation and activation of T cells and NK cells, further enhancing anti-tumor immunity [109].

As a caspase-independent programmed cell death pathway, necroptosis provides a promising alternative to trigger tumor cell death when apoptosis is suppressed in TNBC. Further exploration into the regulatory mechanisms of necroptosis and its interplay with immunotherapeutic strategies is expected to offer new theoretical insights for innovative treatments, potentially addressing the therapeutic limitations posed by apoptosis resistance in TNBC (Fig. 3).

Fig. 3.

The Activation and Anti-Tumor Mechanisms of Necroptosis. Necroptosis is initiated when death receptors on the cell surface, such as FasRs and TNFR1, bind to their respective ligands, leading to the formation of the DISC. In the absence or inhibition of caspase-8, RIPK1 is recruited and interacts with RIPK3, resulting in the phosphorylation of MLKL. The necrosome, a multiprotein complex, subsequently translocates to the plasma membrane, thereby initiating necroptosis. This pathway represents a caspase-independent mechanism of cell death with significant potential for anti-tumor applications

Autophagy

Autophagy is an evolutionarily conserved cellular degradation process that plays a fundamental role in maintaining homeostasis and cellular integrity by recycling cytoplasmic components through the formation of autophagosomes [110]. These vesicles encapsulate and degrade damaged proteins and organelles, contributing to intracellular stability and nutrient metabolism [111, 112]. In recent years, autophagy has emerged as a promising therapeutic target in TNBC, where it plays a paradoxical role by both promoting cancer cell survival and facilitating their death under certain conditions [113, 114]. In TNBC, autophagy is recognized as a double-edged sword [19]. While it promotes programmed cancer cell death by increasing autophagic activity, it also sustains tumor survival by providing essential nutrients through the recycling of cellular components, particularly under conditions of metabolic stress [20]. The regulation of autophagy significantly influences tumor proliferation, invasiveness, and therapeutic responsiveness.

The process of autophagy is tightly regulated by a series of autophagy-related genes (ATGs) and signaling pathways [115, 116]. ATG1, also known as UNC-51-like kinase 1 (ULK1), acts as a core regulator, responding to intracellular nutrient levels and upstream signaling cues [117]. Under nutrient-rich conditions, the mammalian target of rapamycin complex 1 (mTORC1) phosphorylates ULK1, thereby inhibiting autophagy [118]. In contrast, nutrient deprivation activates AMP-activated protein kinase (AMPK) [119], which suppresses mTORC1 activity and relieves its inhibition of the ULK1 complex composed of ULK1, ATG13, FIP200, and ATG101 [120, 121]. This ULK1 complex then translocates to the site of autophagosome initiation [122]. At this site, the ULK1 complex recruits the class III phosphatidylinositol 3-kinase (PI3K) complex, facilitating the formation of the phagophore, the precursor to the autophagosome [31]. The phagophore’s expansion requires two additional protein complexes: the ATG5-ATG12-ATG16L1 complex and the LC3 (microtubule-associated protein 1 light chain 3) protein [123]. This ATG5-ATG12-ATG16L1 complex localizes to the autophagosomal membrane, promoting the lipidation of LC3 family proteins [124]. Initially, LC3 is cleaved by ATG4 to form LC3-I, which is then conjugated with phosphatidylethanolamine (PE) to produce LC3-II, the membrane-bound form essential for autophagosome maturation [125]. The autophagosome then engages lysosomal fusion proteins while concurrently disassembling the ATGs on its outer membrane [111]. This process involves specific fusion proteins, such as Syntaxin 17 (STX17), which interacts with SNAP29 and VAMP8 to form the SNARE complex. The SNARE complex mediates membrane fusion, allowing lysosomal enzymes to degrade the autophagosomal contents into smaller molecules, which are then recycled for cellular use [115].

In TNBC, elevated expression of autophagy-related genes such as LC3 and Beclin-1 has been observed, leading to increased autophagic activity [126]. This heightened activity contributes to chemotherapy resistance and metastatic potential [127]. Clinically, downregulating the expression of LC3 and Beclin-1 has become a key strategy in TNBC therapy [128, 129]. While autophagy can suppress tumor initiation by eliminating defective organelles and proteins, it can also support tumor cell survival under stress by generating metabolic substrates through catabolic degradation [130]. In response to nutrient deprivation, TNBC cells activate autophagy to sustain growth and proliferation, converting macromolecules into reusable substrates [131]. Throughout tumor progression, cancer cells encounter various stressors such as chemotherapy, radiotherapy, and oxidative damage. Autophagy alleviates this stress by removing damaged cellular components, thereby enhancing tumor cell resilience [110]. Notably, autophagy in TNBC can suppress apoptosis by downregulating apoptosis-related proteins. For example, overexpression of Beclin 1 has been shown to reduce the levels of pro-apoptotic proteins such as BAX, caspase-3, and caspase-8, thereby impairing apoptosis [132]. Moreover, autophagy influences immune evasion mechanisms in TNBC by regulating immune cell function and differentiation [133]. This includes modulation of T cells and macrophages, allowing tumor cells to circumvent immune surveillance [134, 135]. However, excessive autophagy can be detrimental to both tumor and normal cells [136]. In conditions of extreme nutrient deprivation, for example, autophagy may lead to the degradation of essential cellular components, ultimately resulting in cell death [137, 138].

In summary, autophagy plays a dual and complex role in TNBC pathogenesis. Its intricate regulation underscores the necessity for further research aimed at converting its tumor-promoting effects into therapeutic advantages. A deeper understanding of autophagy’s molecular mechanisms and its dynamic interactions with tumor biology will be essential for designing safe and effective autophagy-targeted therapies (Fig. 4).

Fig. 4.

The Regulation and Function of Autophagy in TNBC. The regulation of autophagy is primarily mediated by ATGs and various signaling pathways. Under conditions of nutrient sufficiency, mTORC1 inhibits ULK1, thereby suppressing autophagy. Conversely, nutrient deprivation activates AMPK, which alleviates the inhibition of ULK1, facilitating the activation of the ULK1 complex, comprising ULK1, ATG13, FIP200, and ATG101. This activation leads to the recruitment of the PI3K complex and the subsequent formation of a phagophore. The elongation of the phagophore necessitates the involvement of the ATG5-ATG12-ATG16L1 complex and microtubule-associated proteins 1A/1B LC3 to develop an autophagosome. The autophagosome subsequently fuses with a lysosome to form an autolysosome, thereby initiating autophagy. In TNBC, the upregulation of Beclin 1 expression inhibits apoptosis, facilitating immune evasion by tumor cells and resulting in excessive autophagy

Ferroptosis

Ferroptosis is a distinct form of regulated cell death characterized by the excessive accumulation of intracellular iron and the subsequent peroxidation of lipids [125, 139]. It plays a critical role in the treatment of TNBC [21]. Unlike apoptosis and autophagy, ferroptosis is specifically driven by iron dependency and lipid peroxidation [140]. In TNBC, ferroptosis is intricately involved in tumor suppression through multiple mechanisms. First, ferroptosis inhibits TNBC cell proliferation by catalyzing lipid peroxidation through iron overload, ultimately disrupting cellular membrane integrity and function [141–143]. Second, ferroptosis can reshape the tumor microenvironment, thereby affecting the migration and invasiveness of TNBC cells [22]. Third, ferroptosis is closely tied to therapeutic responsiveness, with its induction enhancing the cytotoxic effects of anticancer treatments. Thus, modulating ferroptosis pathways offers a promising therapeutic avenue for TNBC management.

Ferroptosis is primarily regulated by two interconnected pathways: the classical iron metabolism pathway and the lipid metabolism pathway [144]. In the iron metabolism pathway, intracellular iron accumulation plays a central role. Transferrin, a major iron transport protein in the bloodstream, binds ferric ions and interacts with transferrin receptor 1 (TFR1) on the cell surface [145, 146]. During periods of high iron demand, such as during rapid tumor cell proliferation, TFR1 expression is upregulated to enhance iron uptake [147]. Upon cellular internalization via endocytosis, ferric ions are released in the acidic endosomal environment, while transferrin is recycled extracellularly [148]. The surplus iron is typically sequestered by ferritin, preventing iron-induced ROS generation [149, 150]. However, under proliferative or hypoxic conditions, TFR1 expression is further increased [151, 152], while pro-inflammatory cytokines, such as TNF-α and IL-6 [153], along with persistent iron demands, downregulate ferroportin (FPN1), the primary iron exporter, leading to iron overload [154]. Excess intracellular iron leads to the production of hydroxyl radicals through the Fenton reaction [155], thereby catalyzing ROS formation [156]. These ROS can target ferritin [157], releasing stored iron and expanding the labile iron pool (LIP). The tumor cell membrane, especially in TNBC, is enriched in polyunsaturated fatty acids (PUFAs), such as arachidonic acid and adrenic acid [158, 159], which are susceptible to ROS-induced lipid peroxidation [160]. This results in lipid peroxidation and the accumulation of lipid peroxides, a hallmark of ferroptosis [161]. The intracellular antioxidant defense system, primarily comprising glutathione peroxidase 4 (GPX4) and glutathione (GSH), plays a vital role in neutralizing lipid peroxides [162]. GPX4 utilizes GSH to reduce lipid peroxides into nontoxic lipid alcohols, thereby preventing ferroptosis [163, 164]. However, when GPX4 activity or GSH availability is diminished, lipid peroxide accumulation increases, facilitating ferroptosis initiation [21, 165].

Ferroptosis also significantly contributes to TNBC development and progression. Its induction requires both elevated free iron levels and excessive lipid peroxidation [166]. TNBC cells are particularly prone to ferroptosis due to their high iron and lipid content [167]. Iron overload in these cells promotes ROS generation and lipid peroxide accumulation, driving ferroptotic cell death. Concurrently, excessive lipid substrates enhance susceptibility to ROS-induced lipid peroxidation. Aberrations in iron regulatory proteins (IRPs) further exacerbate this process; overexpression of IRP1 and IRP2 in breast cancer disrupts iron homeostasis by increasing iron import and reducing iron storage, thereby promoting iron overload and ferroptosis [168]. GPX4 plays a pivotal antioxidant role in inhibiting ferroptosis by detoxifying lipid peroxides [169]. In TNBC, reduced GPX4 expression or activity impairs this defense mechanism, promoting ferroptotic vulnerability. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of oxidative stress responses, can limit ferroptosis by upregulating antioxidant genes. However, depletion of Nrf2 leads to ferritin accumulation within autophagosomes and enhances cellular sensitivity to ferroptosis [170, 171]. In TNBC cells, Nrf2 upregulation contributes to ferroptosis resistance, whereas its inhibition may enhance therapeutic efficacy [172]. Additionally, the tumor suppressor p53 can indirectly regulate ferroptosis by modulating Nrf2 signaling [173]. Notably, p53 is frequently inactivated in TNBC [174], suggesting that restoring p53 function may augment ferroptosis in TNBC cells through Nrf2 inhibition.

In summary, ferroptosis represents a promising anti-tumor mechanism in TNBC by inducing cancer cell death through iron accumulation, lipid peroxidation, and impairment of antioxidant defenses. Compared with conventional cell death pathways, ferroptosis exhibits higher specificity and therapeutic potential in targeting TNBC cells [175, 176]. Future research should focus on identifying novel regulatory factors of ferroptosis and exploring the synergistic effects of combining ferroptosis-inducing therapies with immunotherapy to improve clinical outcomes in TNBC (Fig. 5).

Fig. 5.

The Mechanistic Regulation of Ferroptosis. The initiation of ferroptosis can proceed through the classical iron metabolism pathway and the lipid metabolism pathway. In the iron metabolism pathway, transferrin interacts with the transferrin receptor TFR1 to facilitate the transport of iron ions. The intracellular accumulation of these iron ions, coupled with the reduced expression of the iron export protein FPN1, results in iron overload within the cell. This condition leads to the generation of ROS via the Fenton reaction, subsequently causing lipid peroxidation. In the lipid metabolism pathway, an imbalance in antioxidant system homeostasis, particularly involving GPX4 and GSH, leads to the accumulation of lipid peroxides, thereby inducing ferroptosis

Epigenetic regulation and potential pathways for treating TNBC

Epigenetic regulation governs gene expression without altering the underlying DNA sequence, primarily through mechanisms, such as DNA methylation, histone modifications, and the action of noncoding RNA [177]. DNA methylation typically represses gene expression by interfering with transcriptional activity [178], while histone modifications remodel chromatin architecture to either facilitate or restrict transcription factor access to DNA [179]. Non-coding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), regulate gene expression post-transcriptionally by either inhibiting mRNA translation or promoting mRNA degradation [180, 181]. These epigenetic processes play critical roles in TNBC, particularly in modulating cell death pathways through the regulation of key gene expression.

In the context of TNBC, aberrant DNA methylation has been shown to suppress the expression of apoptosis-related genes, such as caspase-8, thereby impairing apoptotic cell death [182]. Additionally, the tumor suppressor gene p53, which is central to genomic stability and the induction of apoptosis, can be transcriptionally silenced via hypermethylation of its promoter region. This downregulation of p53 contributes to genomic instability, enhanced tumorigenic potential, and diminished apoptotic responses [183, 184]. The methylation-mediated silencing of caspase-8 further endows tumor cells with anti-apoptotic properties, facilitating their survival and progression [185]. Conversely, in therapeutic settings, apoptosis in TNBC can be promoted through the assembly of the DISC with Fas-associated protein with death domain (FADD), caspase-8, and DR5 [186]. Epigenetic silencing is also implicated in the regulation of pyroptosis. For example, DNA methylation can suppress the transcription of GSDME thereby reducing pyroptotic activity in tumor cells and promoting immune evasion [84]. Similarly, zinc finger DHHC-type containing 1 (ZDHHC1), a putative tumor suppressor gene, is frequently inactivated through promoter methylation. Restoration of ZDHHC1 expression has been shown to enhance both apoptosis and pyroptosis in TNBC cells, contributing to its anti-tumor effects [187]. Moreover, promoter methylation of necroptosis-associated genes also plays a significant role in tumor development [188]. For example, in acute myeloid leukemia (AML) cells, DNA methylation silences RIPK3, reducing necroptosis and leading to drug resistance [189].

Additionally, histone modifications play a pivotal role in regulating cell death pathways, such as ferroptosis and autophagy, thereby influencing tumor cell proliferation and survival [190]. Specific histone modifications can modulate the expression of genes involved in iron metabolism and lipid peroxidation. For example, in pancreatic cancer, the upregulation of the histone acetyltransferase p300 enhances the acetylation of heat shock protein family A (Hsp70) member 5 (HSPA5), thereby promoting lipid peroxidation and sensitizing tumor cells to ferroptosis-induced cell death [191]. Similarly, CBP/p300, a well-characterized histone acetyltransferase, facilitates acetylation of histone H3 at lysine 27 (H3K27ac) [192]. In prostate cancer, CBP/p300-mediated histone acetylation activates autophagy pathways, which in turn suppress tumorigenic potential [193]. Furthermore, targeting histone methylation has emerged as a promising strategy to modulate ferroptosis. For instance, ORY-1001, a selective inhibitor of lysine-specific demethylase 1 (LSD1), inhibits the demethylation of histone marks H3K4me1/2 and H3K9me1/2. This leads to increased methylation at the promoter regions of glutathione synthase genes [194], thereby reducing glutathione synthesis, weakening the antioxidant defense of tumor cells, and enhancing their sensitivity to ferroptosis [195].

Non-coding RNAs also play a critical role in regulating programmed cell death pathways and modulating therapeutic responses in TNBC [196]. MicroRNAs (miRNAs), such as miR-15a and miR-16-1, have been shown to downregulate the anti-apoptotic protein Bcl-2, thereby promoting apoptotic cell death [197, 198]. Low expression levels of miR-30a have been associated with poor prognosis in TNBC, as miR-30a targets Beclin-1, a key regulator of autophagy. Downregulation of miR-30a leads to enhanced autophagic activity and tumor progression [199, 200]. Additionally, the long noncoding RNA (lncRNA) GAS5 promotes apoptosis by modulating the expression of pro-apoptotic proteins, thereby increasing the sensitivity of TNBC cells to chemotherapeutic agents [201, 202].

Ongoing research continues to explore the influence of epigenetic regulation on cell death pathways in TNBC, with the goal of developing more effective therapeutic strategies. Several epigenetically targeted drugs and interventions have been identified that can modulate key cell death mechanisms. These insights underscore the significance of epigenetic regulation in determining tumor progression and therapeutic response, providing new opportunities for improving clinical outcomes in TNBC (Table 1).

Table 1.

Applications of Programmed Cell Death Pathways in Epigenetic Regulation of TNBC

Cell death pathway involved	Types of epigenetic regulation	Drugs or molecules	Mechanism	References
Apoptosis	DNA methylation	Decitabine	TRAF6 triggers the breakdown of DNMT1, DNMT3A, and DNMT3B via the lysosome-dependent protein degradation pathway	[358]
	DNA methylation	Dazemetostat	The degradation of EZH2 leads to persistent disruption of endoplasmic reticulum function and excessive activation of the unfolded protein response, thereby initiating the apoptosis program of cancer cells	[359]
	Histone modification	Hydralazine	Induction of PPARγ up-regulation increases the expression of cyclin- dependent kinase inhibitors p21 and p27, thereby arresting the cell cycle at the G0/G1 phase	[360]
	Histone modification	YZ-836P	Induce TNBC cells to reduce the expression of CDK4, CDK6 and Cyclin D1, and upregulate p21 and p27, leading to cell arrest at the G1 phase and inducing apoptosis	[361]
	Histone modification	JQ1	Blocking Brd4 can interfere with cell cycle and apoptosis signaling pathways, leading to tumor cell apoptosis	[362]
	Non-coding RNA	miR-34a	Inducing the apoptosis of TNBC cells through the CD44 and NOTCH signaling pathways	[363]
Pyroptosis	DNA methylation	Decitabine	CP@Gel.CP@Gel facilitates the self-catalyzed production of reactive oxygen species, which triggers the activation of caspase-3. DAC prevents the methylation of GSDME, thereby elevating the protein level of GSDME and causing significant cell pyroptosis	[364]
	Histone modification	HDACi	Following HDACi treatment, the levels of GSDMA, GSDMB, and GSDME rise, which encourages immune cell infiltration and boosts the effectiveness of anti-cancer immunity	[365]
	Non-coding RNA	miR-1290	MiR-1290 can directly interact with the mRNA of NLRP3. It binds to a particular segment of the NLRP3 mRNA, hindering its translation and leading to reduced NLRP3 protein expression	[366]
		miR-155-5p	When used with Cetuximab, it enhances the pyroptosis of TNBC cells that overexpress the epidermal growth factor receptor by increasing GSDME-N and cleaved caspase-1	[367]
Necroptosis	Histone modification	ZMF-23	Inhibiting the activities of PAK1 and HDAC6 induces TNFα-regulated necroptosis, thereby further enhancing apoptosis	[368]
Autophagy	DNA methylation	DNMT1 inhibitor	Demethylate the CpG island in the promoter region of the Beclin-1 gene	[369]
	Histone modification	Pan-HDI	It interferes with the hsp90/histone deacetylase 6/HSF1/p97 complex, resulting in increased hsp levels. It also boosts autophagic flux, raises LC3B-II expression, and causes the breakdown of the autophagic substrate p62	[370]
	Histone modification	SAHA	When combined with olaparib, it enhances autophagy in TNBC cells and works together to inhibit the growth of TNBC cells with functional PTEN	[371]
	Non-coding RNA	circ-DNMT1	It can bind to both p53 and AUF1 simultaneously, and the nuclear translocation of p53 and AUF1 promotes autophagy	[372]
Ferroptosis	DNA methylation	SSZ	It enhances histone and DNA methylation at the MUC1 promoter, suppresses MUC1 gene transcription, reduces intracellular GSH levels, and heightens cell sensitivity to erastin-induced ferroptosis	[373]
	Non-coding RNA	LncFASA	LncFASA attaches directly to the Ahpc-TSA domain of PRDX1, hindering its peroxidase function by inducing liquid–liquid phase separation, which disrupts intracellular ROS balance, controls ferroptosis, and ultimately leads to the destruction of TNBC cells	[374]
	Non-coding RNA	POU2F2	It enhances the transcription of PTPRG-AS1, influences miR-376c-3p to increase SLC7A11 levels, thus preventing ferroptosis and aiding the progression of TNBC	[375]

Interactions among programmed cell death pathways

The crosstalk among various forms of PCD constitutes a complex and dynamic regulatory network in TNBC therapy [41]. Interactions among apoptosis, pyroptosis, necroptosis, ferroptosis, and autophagy can profoundly influence disease progression and therapeutic efficacy [97]. The boundaries between these pathways are often blurred, and their interplay complicates mechanistic distinctions. Among these, apoptosis serves as a central node with extensive cross-regulatory interactions [56, 203].

In TNBC, apoptosis and pyroptosis are interlinked and can be co-activated in response to certain stimuli [41]. Apoptotic processes can trigger pyroptosis via mitochondrial dysfunction and the release of ROS and ATP, which subsequently activate inflammasomes [204, 205]. In turn, pyroptosis induces the release of pro-inflammatory cytokines, such as IL-1β and TNF-α, which can promote apoptosis. IL-1β enhances apoptosis through NF-κB pathway activation [206], while TNF-α can activate caspase-8 [207], promoting apoptosis. Certain treatments can simultaneously induce both apoptosis and pyroptosis, thereby enhancing tumor cell death and improving therapeutic outcomes [92]. For example, caspase-3, a key executor of apoptosis, also cleaves GSDME, which activates caspase-1 and promotes pyroptosis [208], highlighting this as a promising target in TNBC. Autophagy and apoptosis also exhibit bidirectional regulatory interactions. Autophagy can facilitate apoptosis by degrading anti-apoptotic factors, whereas apoptosis may inhibit autophagy through caspase-mediated cleavage of autophagy-related proteins. In TNBC, prolonged chemotherapy often leads to multidrug resistance (MDR) [209]. While autophagy can support cancer cell survival under chemotherapeutic stress [210], it can also induce apoptosis in MDR cells, thereby counteracting resistance [211]. For instance, in TRAIL-resistant lung adenocarcinoma, metformin enhances TRAIL-induced apoptosis by downregulating c-FLIP and increasing autophagic flux [211, 212]. Furthermore, autophagy regulated by ATG1 has been linked to the activation of caspases, DNA fragmentation, and cytoskeletal disruption—hallmarks of apoptosis [213]. Overexpression of Bcl-2, an anti-apoptotic protein, inhibits autophagy by binding to Beclin 1 and reducing PI3K activity [214, 215], underscoring the therapeutic potential of targeting this axis in cancer treatment.

The balance between apoptosis and necroptosis is also essential for effective tumor cell elimination in TNBC. Resistance to traditional chemotherapeutic and targeted therapies often manifests as a decrease in apoptotic susceptibility. In such cases, redirecting cell death toward necroptosis offers an alternative strategy. Caspase-8 functions as a molecular switch between apoptosis and necroptosis [216]. When caspase-8 is inhibited, such as by the pan-caspase inhibitor Q-VD-OPh, RIPK1 is activated, initiating necroptosis via the RIPK1-RIPK3-MLKL axis [217]. AddFerroptosis is also intricately connected to apoptosis in TNBC. Compounds like erastin and its derivatives induce ferroptosis by inhibiting the cystine/glutamate antiporter System Xc− , thereby depleting intracellular cystine and suppressing GSH synthesis [218]. This leads to increased oxidative stress and lipid peroxidation, hallmarks of ferroptosis [219, 220]. The resulting ROS can damage mitochondria [221], triggering the release of CytC and activating downstream caspase cascades culminating in apoptosis [222]. These pathways present novel therapeutic targets and strategies for the treatment of TNBC.

In TNBC therapy, pyroptosis and necroptosis may act synergistically. Pyroptosis involves the release of inflammatory cytokines and intracellular contents, which can stimulate necroptotic pathways [223]. Doxorubicin, a commonly used chemotherapeutic agent, induces ROS accumulation, leading to caspase-3-mediated cleavage of GSDME and the onset of pyroptosis [224]. This is accompanied by the release of pro-inflammatory cytokines, such as IL-18, IL-1β, TNF-α, and IFN-γ [63], with TNF-α binding to TNFR1 and activating the RIPK1-RIPK3-MLKL signaling axis to induce necroptosis [225, 226]. Furthermore, pyroptosis regulators such as ATP can activate the NLRP3 inflammasome, initiating pyroptosis and potentially influencing other cell death pathways [212, 227]. Pyroptosis and autophagy are also interconnected in TNBC. Autophagy can suppress pyroptosis by degrading inflammasome components such as TRIM32 [228], which inhibits pyroptosis [229]. Conversely, pyroptosis-induced cytokines like IL-18 can activate NF-κB signaling, which upregulates autophagy-related genes and promotes autophagic activity [230, 231]. Interestingly, in hepatocellular carcinoma, ferroptosis and pyroptosis may exhibit antagonistic effects, with certain enzymes simultaneously promoting pyroptosis and inhibiting ferroptosis [232]. For example, 4-hydroxynonenal, a lipid peroxidation byproduct, can inhibit NLRP3 inflammasome activation and suppress pyroptosis in macrophages [233].

Autophagy also modulates necroptosis and ferroptosis. By degrading necroptosis-associated proteins such as RIPK1 and RIPK3, autophagy inhibits necroptotic signaling. This effect can be enhanced by agents, such as 3′-epi-12β-hydroxyfroside and HyFS, which suppress the Akt/mTOR pathway [234, 235]. Similarly, RUBCNL/PACER proteins downregulate RIPK1 activity, further inhibiting necroptosis [236]. In the context of ferroptosis, Anomanolide C (AC) induces autophagy-dependent ferroptosis by downregulating GPX4, leading to Fe²⁺ accumulation and suppression of TNBC cell growth [237]. Specific forms of autophagy, including ferritinophagy, mitophagy, and chaperone-mediated autophagy, can also promote ferroptosis by elevating intracellular free iron or lipid levels [238]. While oxidative stress and lipid peroxidation can trigger autophagy, excessive autophagy may, in turn, enhance ferroptosis [239]. Moreover, oxidative stress associated with ferroptosis can simultaneously activate apoptosis and autophagy, highlighting the interconnected nature of these pathways [204, 240].

A notable interplay exists between necroptosis and ferroptosis in TNBC, with RIPK1 and RIPK3 kinases modulating ferroptotic responses via regulation of iron metabolism-related proteins [241]. Evidence from ischemic stroke models shows that HSP90 can activate both necroptosis and ferroptosis by promoting RIPK1 phosphorylation and inhibiting GPX4 activity, suggesting that activation of necroptosis may also potentiate ferroptosis [242].

Investigating the interactions among different cell death mechanisms offers valuable insights for enhancing cancer treatment, particularly by overcoming tumor resistance and optimizing therapeutic strategies. Combining conventional treatments—such as chemotherapy, radiotherapy, targeted therapy, and immunotherapy—can induce multiple forms of cell death, potentially improving therapeutic efficacy and patient outcomes. This integrative approach is especially pertinent in TNBC, where elucidating the crosstalk among cell death pathways may shed light on tumor pathogenesis and therapeutic resistance, thereby guiding more effective interventions. Currently, TNBC treatment regimens include surgical resection, chemotherapy, radiation therapy, molecularly targeted agents, and immune-based approaches. The ongoing research aims to inform the development of innovative therapeutic strategies, establish comprehensive treatment frameworks, and promote precision medicine, ultimately expanding therapeutic options and improving the quality of life for patients with TNBC.

Immunotherapy and programmed cell death

Immunotherapy, as an emerging modality in TNBC treatment, plays a pivotal role in modulating PCD pathways [243, 244]. Immune checkpoint blockade, T cell-based therapies, and NK cell therapies interact with the regulation of cell death, the tumor microenvironment, and immune escape mechanisms [245].

Immune checkpoint inhibitors (ICIs) enhance antitumor immunity by blocking inhibitory signaling pathways between tumor cells and immune effector cells, thereby allowing cytotoxic T cells to exert apoptotic effects more effectively [246]. This is mediated through the activation of the caspase cascade [247]. Activated T cells can also produce ROS, elevating oxidative stress within tumor cells and inducing ferroptosis [248]. Moreover, ICIs may modulate the expression of iron metabolism-related genes, further promoting ferroptotic cell death [249]. Simultaneously, inflammatory cytokines such as IL-1β and IL-18, secreted by immune cells, can trigger pyroptosis in tumor cells [62, 250]. Blockade of the PD-1/PD-L1 axis has been shown to augment T cell activity and downregulate Beclin 1 expression, thereby suppressing autophagy in tumor cells [251]. In contrast, anti-CTLA-4 therapy enhances the infiltration of macrophages and dendritic cells (DCs) into the tumor microenvironment, whereupon they secrete cytokines that modulate autophagic activity [252]. In TNBC and other malignancies, high expression of the critical immunosuppressive checkpoint molecule T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) on tumor-associated macrophages (TAMs) is associated with immune evasion [253, 254]. Therapeutic targeting of TIM-3 with monoclonal antibodies can block its signaling, leading to macrophage reactivation [255], culminating in the induction of necroptosis in tumor cells, thereby enhancing their elimination [256].

T cell-based therapies, particularly chimeric antigen receptor T cell (CAR-T) and T cell receptor-engineered T cell (TCR-T) therapies, have demonstrated promise in TNBC management [257, 258]. CAR-T therapy utilizes genetically modified T cells that express receptors targeting specific surface antigens on TNBC cells [259], leading to their activation when they recognize these antigens on the surface of TNBC cells [260]. Upon antigen recognition, CAR-T cells release cytotoxic granules containing perforin and granzymes [261, 262]. Perforin forms pores in tumor cell membranes [263], facilitating the entry of granzyme B, which subsequently activates caspase-3 and caspase-8, initiating apoptosis [264, 265]. TCR-T therapy involves engineering T cells to express tumor-specific TCRs, enabling recognition of intracellular antigens presented by MHC molecules [266]. Ropporin-1, highly expressed in TNBC, has been identified as a target for TCR-T cells, allowing selective cytotoxicity against tumor cells [267]. This approach has already shown clinical utility in other solid tumors, such as melanoma, synovial sarcoma, nonsmall cell lung cancer, and hepatocellular carcinoma [268], underscoring its potential for TNBC therapy.

NK cells are innate immune effectors that exert potent cytotoxic effects against tumor cells [269, 270]. They eliminate cancer cells by releasing perforin and granzyme, promoting apoptosis, and by secreting TRAIL, which binds to death receptors on tumor cells [264, 271, 272]. Additionally, NK cells produce IFN-γ, which activates p53 signaling in tumor cells, upregulating pro-apoptotic proteins and downregulating anti-apoptotic ones, thereby enhancing apoptosis [38, 273, 274]. IFN-γ can also promote autophagy, aiding in macrophage-mediated phagocytosis and tumor cell clearance [275]. Furthermore, NK cells can trigger pyroptosis by secreting pro-inflammatory cytokines and activating DCs to enhance antigen presentation, leading to robust T cell responses [276]. These activated T cells, in turn, release various effector molecules and cytokines that induce additional forms of cell death, including necroptosis and ferroptosis, thereby amplifying tumor cell destruction [277].

The interplay between immunotherapy and cell death pathways is thus critical for the effective treatment of TNBC. Given the complex tumor microenvironment and myriad mechanisms that underlie immune evasion, such as PD-L1 overexpression, targeting immune checkpoints and enhancing immune cell function represent promising therapeutic avenues. Modulating cell death pathways can further alter the tumor milieu and reduce immune resistance. Future research should continue to explore these dynamic interactions, aiming to develop novel drugs, optimize therapeutic regimens, and implement personalized treatment strategies that consider both the tumor microenvironment and mechanisms of immune escape (Table 2).

Table 2.

Applications of programmed cell death pathways in immunotherapy for TNBC

Cell death pathway involved	Drugs or molecules	Mechanism	References
Apoptosis	PD-1/PD-L1 inhibitors	Inhibiting the interaction between PD-1 and PD-L1 boosts T cell activity, enabling immune cells to more effectively identify and destroy tumor cells, thereby encouraging tumor cell apoptosis	[376]
	anti-CTLA-4 antibody	Inhibiting the CTLA-4 immune checkpoint pathway decreases Treg cell numbers, boosts T cell activation and proliferation, and encourages tumor cell apoptosis	[377]
	IFN-γ	IFN-γ induces endoplasmic reticulum stress thereby hindering the formation of autophagosomes, resulting in the misfolded proteins, damaged organelles and other materials wrapped in autophagosomes not being degraded in a timely manner, interfering with the normal metabolism and function of cells, and triggering apoptotic signaling	[378]
	PARP inhibitor	Causes DNA damage, halts the cell cycle, and triggers apoptosis in cancer cells while boosting the immune system’s capacity to target tumor cells	[379, 380]
	TNF-α	Binding of TNF-α to TNFR1 induces caspase-8 activation, which ultimately leads to apoptosis	[381]
	M1 macrophages	Induces iNOS to produce large amounts of NO and increases toxic effects on tumor cells to promote apoptosis	[382]
Pyroptosis	NLRP3	The NLRP3 inflammasomes trigger caspase-1 to initiate gasdermin D-dependent pyroptosis and facilitate the release of IL-1β and IL-18	[227, 383]
	CDK inhibitors	CDK inhibitor treatment increases the levels of caspase-3 and N-terminal fragments cleaved by GSDME	[384]
	TMAO	Inducing endoplasmic reticulum stress kinase PERK triggers pyroptosis in tumor cells, which boosts CD8 T cell-mediated anti-tumor immunity against TNBC in living organisms	[385]
	GM@LR	GM@LR delivers plasmid expressing GSDME and MnCO into TNBC cells to activate caspase-3, thereby converting apoptosis to pyroptosis in 4T1 cells. In addition, Mn2+ promotes the maturation of DCs through activation of the STING signaling pathway leading to the infiltration of large numbers of cytotoxic lymphocytes	[386]
Necroptosis	Shikonin	Increased expression of RIPK3, p-RIPK3, and MLKL worked together to induce necroptosis in tumor cells, effectively triggering ICD, boosting CD8 and CD4 T cell infiltration in the tumor, and suppressing Treg cells.	[281]
	RIPK3	An alternative pathway, independent of necroapoptosis, activates PGAM5 to control NKT cell activity and enhance NKT cell-driven anti-tumor immunity	[387]
	Caspase-8	Activation of specific NK cells and CD8 T cells to improve recognition of tumor antigens	[388]
	GSDMC	GSDMC increases PARPi sensitivity in multiple cancer types by expanding memory CD8T cells in lymphoid tissues and tumors	[389]
Autophagy	PD-1/PD-L1 inhibitors	Combined administration with endostatin has a synergistic effect, leading to a decrease in the levels of IL-17 and TGF-β1, an increase in the secretion of IFN-γ, a reduction in the accumulation of MDSCs, and a reversal of the inhibition of CD8+ T cells. The expressions of vascular endothelial growth factor (VEGF), CD34, and CD31 are significantly downregulated, while apoptosis in tumor cells and autophagy mediated by the PI3K/AKT/mTOR pathway are upregulated	[390]
	HCQ	Increase the visibility of colon cancer antigens triggered by ICD inducers, and boost ICD-based anti-tumor immunity in both laboratory and live settings	[391]
	DOX	Reducing tenascin-C protein levels alongside PD-1 inhibitors elevates CD4 and CD8 tumor-infiltrating lymphocytes (TILs) and significantly boosts granzyme B release	[392]
	DMKG	This results in a heightened release of antigens and inflammatory factors, activating DCs. It can also enhance the infiltration of CD8+ T cells into the tumor area, decrease the proportion of Tregs following radiotherapy, and remodel the tumor immune environment	[393]
Ferroptosis	GPX4 inhabitors	Combined use with immune checkpoint inhibitors can promote ferroptosis	[21]
	CD8 T cell	The discharge of perforin-granzyme and Fas-FasL can trigger cell death and boost the lipid peroxidation reaction specific to ferroptosis in tumor cells	[387]
	HIFU	Induce the expression of ferroptosis-related genes such as HOX1, GST, and SQSTM to enhance drug accumulation and penetration in tumors, and stimulate effective ferroptosis-mediated anti-tumor immunity	[394]

Regulators of cell death

Targeting specific regulators of programmed cell death offers a promising strategy for advancing TNBC therapy. Agents targeting the apoptotic machinery, such as Bcl-2 and Bcl-xL inhibitors, counteract anti-apoptotic signaling and promote tumor cell apoptosis [278]. Regulators of pyroptosis, including small-molecule inflammasome inhibitors like MCC950 and OLT1177, can suppress inflammation and enhance the effectiveness of TNBC treatment [279]. In addition to apoptosis and pyroptosis, necroptosis, autophagy, and ferroptosis pathways are gaining recognition as viable therapeutic targets [280, 281]. For example, drugs targeting necroptosis can trigger cell death by influencing pathways involving key molecules like RIPK1, RIPK3, and MLKL [282, 283]. Although small molecule inhibitors specific to these pathways remain limited, manipulating their core signaling components can still promote tumor cell death. For instance, TNF-α-driven activation of RIPK3 through the PI3K/AKT axis induces necroptosis, while ZBP1 cooperates with MLKL to potentiate this process in cancer therapy [284, 285]. Autophagy is modulated by regulators such as Beclin 1. The mTOR signaling pathway inhibits Beclin 1, but mTOR inhibitors, such as rapamycin, can restore autophagic activity and promote tumor cell degradation [286, 287]. Moreover, agents, such as chloroquine and hydroxychloroquine, which disrupt autophagosome-lysosome fusion, have been tested in TNBC clinical trials, as their inhibition of autophagy can induce tumor cell death [288, 289]. Research on ferroptosis-inducing drugs is rapidly evolving. Compounds such as Erastin and its derivatives initiate ferroptosis by inhibiting System Xc-, thereby reducing cystine uptake and GSH synthesis, which leads to lipid peroxidation and oxidative cell death [219, 220, 290]. Sulfasalazine induces ferroptosis by targeting the mitochondrial voltage-dependent anion channel (VDAC), disrupting mitochondrial function, releasing iron ions, and exacerbating oxidative stress [14, 291]. Additionally, RSL3, a direct inhibitor of GPX4 [292, 293], reduces the enzyme’s activity and promotes lipid peroxidation, thereby enhancing ferroptotic death in TNBC cells.

Despite significant advances, research on cell death regulators continues to face substantial challenges, particularly with the emergence of therapeutic resistance. Future investigations must prioritize understanding the molecular underpinnings of resistance and developing strategies to overcome it. One promising approach involves combination therapies that simultaneously target multiple cell death pathways, thereby enhancing therapeutic efficacy and circumventing resistance. For instance, cisplatin, which is a widely used chemotherapeutic agent in lung and ovarian cancer, induces cell death by interfering with DNA repair mechanisms but is often limited by the onset of resistance [294, 295]. Co-administering cisplatin with ferroptosis inducers such as Erastin or Sulfasalazine has shown promise in overcoming this resistance. These agents enhance tumor suppression by modulating iron metabolism and lipid peroxidation, ultimately triggering ferroptosis [296, 297]. Similarly, TRAIL induces necroptosis in colorectal and breast cancers but also faces therapeutic resistance [298, 299]. This limitation can be addressed by combining TRAIL with chloroquine, which inhibits autophagy in tumor cells, thereby sensitizing them to necroptosis and enhancing anti-tumor activity [300, 301]. In gastric and pancreatic cancers, oxaliplatin has been shown to activate necroptosis-related pathways [302, 303]. When combined with the GPX4 inhibitor RSL3, oxaliplatin’s efficacy is further improved by promoting ferroptosis through the inhibition of antioxidant defenses [304]. These multimodal strategies underscore the therapeutic potential of targeting multiple cell death pathways simultaneously to address resistance. However, in the context of TNBC, tumor heterogeneity significantly impacts drug responsiveness, and pharmacokinetics may vary due to metabolic differences across organs [305]. Furthermore, the dense cellular architecture and complex extracellular matrix of TNBC hinder drug penetration [306, 307]. Therefore, identifying novel molecular targets and developing precisely targeted therapies are crucial to overcoming these barriers and enhancing the therapeutic efficacy of cell death modulators (Table 3).

Table 3.

Application of different cell death regulators in TNBC

Cell death pathway involved	Drugs	Mechanism	Reference
Apoptosis	ABT-737	ABT-737 attaches to Bcl-2/Bcl-xL/Bcl-w, stopping them from interacting with Bax and Bak, which in turn lifts the suppression of apoptosis	[395]
	LCL161	LCL161 blocks IAPs, lifting their inhibition of apoptosis, and alters the tumor microenvironment by drawing macrophages to the tumor location	[396]
	APR-246	When p53 is activated, it increases the levels of pro-apoptotic proteins, such as Bax and PUMA, and decreases the levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL	[397, 398]
	Pembrolizumab	Blocking the PD-1/PD-L1 immune checkpoint activates CTLs and triggers cancer cell apoptosis via T cell-mediated cytotoxicity	[376, 399]
	Epothilone B	Induction of cancer cell apoptosis via the PI3K/AKT/mTOR signaling pathway enhances the apoptotic effects of ABT-737	[286]
Pyroptosis	Cisplatin	Activation of the MEG3/NLRP3/caspase-1/GSDMD pathway in TNBC induces pyroptosis to exert anti-tumor effects	[383]
	AZU1	Regulation of the pNF-κB/NLRP3/caspase-1/GSDMD axis in TNBC promotes pyroptosis	[400]
	MCC950	By binding to the NACHT domain of NLRP3, its oligomerization and activation are prevented, which inhibits its function and makes TNBC cells more sensitive to PTX	[279]
	CY-09	Inhibiting the Walker B motif of NLRP3 reduces its activity, which in turn suppresses IL-1β/EMT/Wnt/β-catenin signaling and makes TNBC cells more sensitive to GDC-0941	[401]
	OLT1177	Inhibition of NLRP3-induced IL-1β prevents the progression of TNBC	[402]
Autophgy	CQ	Preventing the fusion of autophagosomes with lysosomes results in the buildup of autophagosomes inside the cell, which encourages the death of tumor cells	[288, 289]
	Toosendanin	Raising lysosomal pH inhibits autolysosome maturation, thereby suppressing late-stage autophagy in TNBC cells	[403]
	circEGFR	The translocation of annexin A2 (ANXA2) to the plasma membrane in TNBC cells facilitates the dissociation of the transcription factor TFEB from the ANXA2-TFEB complex, subsequently promoting autophagy. Concurrently, circEGFR acts as a competing endogenous RNA (ceRNA) by directly binding to miR-224-5p, thereby inhibiting its expression and mitigating the suppression of autophagy mediated by the miR-224-5p/ATG13/ULK1 axis	[404]
	UBC12	Enhancing TRIM25 sumoylation boosts its interaction with TFEB, which promotes the transcription and activation of genes related to autophagy	[405]
Necroptosis	ZBP1	When glucose is deprived, mtDNA is released into the cytoplasm, where it attaches to ZBP1, triggering MLKL activation in a manner dependent on Bcl-2 family proteins and NOXA	[406]
	Curcuma longa	Enhanced expression of TRAIL under acidic conditions induces necroptotic apoptosis in a RIPK1- and RIPK3-dependent manner	[407]
Ferroptosis	Erastin	Blocking System Xc- on the cell membrane lowers cystine absorption, which results in reduced GSH production inside the cell and weakened antioxidant defenses, thus encouraging lipid peroxidation and ferroptosis	[218, 219]
	Sulfasalazine	Blocking VDAC in mitochondria impairs their function, leading to the release of iron ions and heightened oxidative stress	[408, 409]
	RSL3	Inhibition of GPX4 activity elevates intracellular lipid peroxidation levels, thereby promoting ferroptosis	[292, 293]
	Liproxstatin-1	In TNBC cells, the activation of cPLA2α leads to the activation of ACSL4, which facilitates the attachment of PUFAs to coenzyme A, thereby raising lipid peroxidation levels and inducing ferroptosis	[410]
	Eupaformosanin	Ubiquitination of mutant p53 induces apoptosis and ferroptosis in TNBC cells	[320]

Clinical translation of cell death pathways in the treatment of TNBC

In the context of TNBC, investigating mechanisms of cell death not only offers a pivotal direction for identifying therapeutic targets but also establishes the groundwork for the development of novel therapies. Nevertheless, much of the research in this area remains at the preclinical stage [41].

Within preclinical studies, animal models and organoid models are indispensable for assessing potential therapeutic agents and targets [308, 309]. The traditional immunodeficient xenograft mouse model, a well-established animal model for TNBC research, effectively simulates the growth, metastasis, and therapeutic response of TNBC in vivo. By inoculating immunodeficient mice with human-derived TNBC cell lines (such as MDA-MB-231 and BT-549), researchers can evaluate the effects of potential drugs or targets on tumor growth, metastasis, and survival outcomes in mice [310, 311]. For example, administering the ferroptosis inducer Erastin to mice inoculated with TNBC cells demonstrated that Erastin facilitates TNBC cell death by inhibiting the expression of GPX4, thereby providing preliminary evidence for the potential application of ferroptosis in TNBC therapy [312]. In recent years, organoid models, as innovative three-dimensional cell culture systems, have increasingly emerged as a pivotal platform for TNBC research due to their enhanced ability to accurately replicate the biological characteristics of patient tumors [313]. These organoids not only preserve the heterogeneity inherent in the patient’s tumor^[314]but can also be co-cultured with immune cells to develop models that closely mimic the in vivo tumor immune microenvironment. This capability provides unique insights into the effects of cell death mechanisms on tumor immunity [315]. In the context of screening ferroptosis inducers, research has identified significant variability in the sensitivity of TNBC organoids derived from different patients to these inducers, thereby supporting the advancement of personalized treatment strategies [21]. Compared with animal models, organoid models present distinct advantages in terms of cost-effectiveness, reduced experimental duration, and suitability for high-throughput screening [316]. Nonetheless, while these models can replicate certain biological aspects of TNBC, they still fall short of capturing the intricate physiological and pathological environment of humans, posing a challenge for the direct translation of preclinical findings into clinical practice. Furthermore, the pronounced heterogeneity of TNBC complicates the identification of universal treatment strategies. Consequently, accurately classifying and treating patients on the basis of their specific cell death characteristics remains a formidable challenge in clinical translation.

In summary, preclinical investigations into cell death mechanisms in TNBC serve as a crucial conduit for translating foundational research into clinical practice. Animal models provide initial evidence of the therapeutic potential of cell death mechanisms through in vivo simulations, while organoid models, with their capacity for personalized and efficient screening, offer substantial support for the development of precision therapeutic strategies. Nonetheless, discrepancies between models and human biology, alongside the inherent heterogeneity of TNBC, continue to impede clinical translation. Future endeavors should prioritize the optimization of research models and the enhancement of our understanding of cell death mechanisms to facilitate the progression of related therapies from preclinical research to clinical application. This advancement holds the promise of offering new survival prospects for TNBC patients and achieving the clinical translation of basic research(Table 4).

Table 4.

Different research models for five types of PCD in TNBC

Types of model research	Cell death pathway involved	Targeted drugs/strategies	Mechanism	References
Animal model	Apoptosis	USP19	USP19 interacts with deubiquitinating enzymes to stabilize the chaperone regulator BAG6. BAG6 promotes the ubiquitination and degradation of BCL2, a process that elevates ER calcium levels, thereby inducing ER stress and increasing cellular apoptosis	[411]
		Overexpression of PTPN14	Overexpression of PTPN14 acts as a key regulatory factor in dephosphorylation in estrogen receptor-negative breast cancer (ER- BC) resistance, inducing anoikis and inhibiting proliferation in TNBC cells by suppressing the PI3K/AKT and ERK signaling pathways. This process has no significant impact on normal breast cells	[412]
		lasiokaurin	LA effectively inhibits the activation of the PI3K/Akt/mTOR and STAT3 signaling pathways, inducing cell cycle arrest, apoptosis, and DNA damage in TNBC cells, while also suppressing cellular migration	[413]
		A1155463 + R547	Targeting BCL-XL enhances the sensitivity of TNBC cells to CDK1/2/4 inhibition by synergistically depleting survival signals and the RTK/AKT pathway, while restoring the tumor-suppressive function of FOXO3a. This results in the suppression of cellular viability, accompanied by the accumulation of DNA damage and the induction of apoptosis	[414]
		PBCH	PBCH is a hyaluronic acid-modified nanocomposite targeting CD44, serving as an oxidative stress amplifier. In the acidic tumor microenvironment, it first releases Ca2⁺, leading to mitochondrial dysfunction and apoptosis. The PdAg nanoenzyme within the composite can also be activated by near-infrared (NIR) light, mediating a burst of ROS and promoting apoptosis through immunogenic cell death	[415]
	Pyroptosis	CP@Gel combined with DAC	Following the injection of CP@Gel, CP continuously releases to catalyze the self-generation of ROS, thereby activating caspase-3. Pre-treatment with DAC enhances the protein levels of GSDME by inhibiting its methylation, inducing robust pyroptosis and anti-tumor immunity, and eliminating residual cancer cells that survive owing to apoptosis resistance	[364]
	Autophagy	CQ	CQ inhibits autophagy, and when combined with the PI3K inhibitor ipatasertib and the AKT inhibitor taselisib, it alleviates the therapeutic limitations induced by autophagy activation in response to PI3K/AKT inhibition, thereby enhancing the anti-tumor effects of conventional chemotherapy	[416]
		STA1	In TNBC, the loss of autophagy leads to the high expression of STA1. SAT1 activates a negative regulatory mechanism of autophagy by stabilizing mTOR mRNA, promoting cell proliferation and metastasis in TNBC. Therefore, targeting STA1 may represent a key strategy for targeted therapy in TNBC	[417]
		ADT-OH	ADT-OH improves autophagic flux by inhibiting autophagosome formation, while simultaneously downregulating dynactin subunit 1 and upregulating mitochondrial fusion proteins to induce mitochondrial elongation. Additionally, it reduces mitochondrial autophagy flux and inhibits mitochondrial function, ultimately suppressing migration and invasion in TNBC cells	[418]
		PC3-15	By inhibiting UbcH5b and its mediated p62 ubiquitination, autophagy induced by lapatinib is suppressed, thereby increasing the sensitivity of TNBC to lapatinib	[419]
	Necroptosis	NP-ALT	Inhibition of tyrosine phosphorylation of p27Kip1 suppresses CDK4/6 and CDK2, inducing ROS-dependent necroptosis. This mechanism subsequently blocks the proliferation of HR + breast cancer cells and CDK4i-resistant cell types, including TNBC	[420]
	Ferroptosis	JQ1 + BTZ	Low-dose combination therapy targeting Brd4 and the proteasome (JQ1 + BTZ) triggers lysosomal metabolic reprogramming, promoting the accumulation of LIP. Simultaneously, glutamine catabolism provides substrates for the TCA cycle, exacerbating lipid peroxidation	[421]
		Curcumenol	Cur reduces mitochondrial membrane potential and promotes the accumulation of lipid ROS. It also reverses the inhibitory effect of ferrostatin-1 on ferroptosis. By inhibiting the SLC7A11/NF-κB/TGF-β pathway, Cur effectively blocks the progression of TNBC	[422]
		HFPN	Targeted modulation of the core kinase Aurora A in TNBC disrupts the tumor redox balance, promoting ferroptosis in tumor cells, thereby enhancing the efficacy of radiotherapy in TNBC	[423]
		ZC3H13	ZC3H13-mediated m6A modification promotes ferroptosis through the KCNQ1OT1/TRABD axis, thereby reducing DOX resistance in TNBC	[424]
		OST-01	Downregulation of LRP8 reduces selenium uptake, lowering the expression of the selenium-dependent protein GPX4. This diminishes the cellular antioxidant capacity, increases lipid peroxidation, and elevates malondialdehyde levels, ultimately inducing ferroptosis in breast cancer cells and inhibiting tumor growth	[425]
Organoid model	Apoptosis	Restoration of CircRNA-CREIT expression	CircRNA-CREIT promotes the ubiquitination and degradation of PKR by binding to it and recruiting HACE1, thereby inhibiting the PKR/eIF2α signaling pathway and stress granule assembly. The released RACK1 activates the MTK1/JNK pathway, upregulating pro-apoptotic proteins such as Bim, while simultaneously enhancing doxorubicin-induced DNA damage and inhibiting autophagy. This synergistically activates the caspase cascade, leading to apoptosis	[314]
		Nicotinamide	NAM reduces mitochondrial membrane potential and ATP production, while enhancing the activity of reverse electron transport, fatty acid β-oxidation, and glycerophospholipid/sphingolipid metabolic pathways in TNBC. This collectively increases ROS levels, subsequently triggering apoptosis	[426]
		YZ-836P	Targeted degradation of PRMT5 via PROTACs relieves its inhibition of the p53 pathway, inducing G1-phase cell cycle arrest and significantly promoting apoptosis in TNBC cells	[361]
		LXG6403	Targeting lysyl oxidases first disrupts the extracellular matrix (ECM), leading to the inactivation of FAK/Src signaling. This subsequently promotes chemotherapy-induced ROS production and DNA damage, ultimately resulting in G1/S-phase cell cycle arrest and apoptosis	[427]
		CDDD11-8	Selective inhibition of CDK9 downregulates the mRNA and protein levels of oncogenes, such as Myc and MCL1, leading to cell cycle arrest and induction of apoptosis in TNBC cells	[428]
	Autophagy	lncRNA DDIT4-AS1	PTX chemotherapy increases the expression of DDIT4-AS1. Silencing its expression inhibits the activation of autophagy, thereby sensitizing TNBC cells to PTX treatment	[429]
		JNK-IN-8	Inhibition of mTOR blocks the phosphorylation of TFEB, inducing the nuclear translocation of both TFEB and TFE3. This upregulates their target genes, promoting lysosomal biogenesis and autophagy, ultimately triggering cell death in TNBC cells	[430]
		CDDO-Me	By binding to EGFR and promoting its interaction with KEAP1, K63 ubiquitination of EGFR is induced, leading to its degradation via the autophagy-lysosome pathway. This process blocks the inhibitory effect of the PI3K/AKT/mTOR pathway on autophagy, activates autophagic flux, and synergistically suppresses the progression of TNBC	[431]
		cGAS-STING	In the context of serum deprivation, the inhibition of the cGAS-STING axis leads to cell death through an autophagy-dependent mechanism	[432]
	Ferroptosis	α-Hederin	Activation of IRF1 and promotion of its nuclear translocation directly bind to the GPX4 promoter, inhibiting its transcription and reducing GPX4 protein levels. This weakens the glutathione system’s capacity to detoxify lipid peroxides, triggering iron-dependent lipid peroxidation and inducing ferroptosis in TNBC	[433]
		IN10018 combined with crizotinib	Upregulation of p53 inhibits G2/M phase cell cycle arrest, enhances apoptosis by increasing the Bax/Bcl-2 ratio, and induces ferroptosis through the regulation of the SLC7A11/GSH/GPX4 pathway	[434]
		PSMD14	Silencing PSMD14 reduces its deubiquitinase-mediated stabilization of SF3B4, disrupting the SF3B4/HNRNPC complex that mediates m6A modification and exon inclusion of FADS1 mRNA, leading to downregulation of FADS1. Combined with AA supplementation, this results in decreased FADS1-mediated unsaturated fatty acid production, weakening the glutathione system’s capacity to counter lipid peroxidation, and inducing ferroptosis in TNBC	[435]
Clinical research model	Apoptosis	TAK-228 combined with Alisertib	TAK-228 enhances the anti-tumor effects of alisertib in vivo. Alisertib induces cell cycle arrest at the G2/M phase, while TAK-228 inhibits the mTORC1/2 pathway, disrupting cellular metabolism and severing survival signals in senescent cells, thereby promoting apoptosis	[436]
		Omeprazole	By inhibiting the expression of FASN and reducing fatty acid synthesis, the mTORC1 pathway is suppressed, leading to the activation of the mitochondrial apoptotic pathway and promoting cell cycle arrest. This ultimately inhibits the growth and survival of TNBC cells	[437]
		Tigatuzumab	Nab-paclitaxel induces cell death through microtubule stabilization and the mitochondrial apoptotic pathway, while tigatuzumab triggers the extrinsic apoptotic pathway by activating DR5. The combination of these two agents achieves synergistic enhancement through the cross-activation of the caspase cascade, cell cycle synchronization, and modulation of the immune microenvironment	[438]
	Autophagy	TRF2	Overexpression of TRF2 following PTX exposure results in a reduction of LC3 levels, which is associated with the accumulation of p62SQSTM1, thereby inhibiting the autophagic response induced by paclitaxel treatment	[439]
	Pyroptosis	IRE1α RNase	Inhibition of IRE1α RNase activity synergizes with taxane-induced NLRP3 inflammasome-mediated pyroptosis, promoting the conversion of PD-L1-negative “cold tumors” in TNBC to a PD-L1-high expressing immunogenic phenotype. This significantly enhances the sensitivity of TNBC to PD-1 inhibitor therapy	[16]

Integrated therapeutic strategies for TNBC

TNBC is a highly heterogeneous and aggressive subtype of breast cancer, posing substantial challenges for treatment. Recent research efforts have focused on integrating cell death modulators with conventional therapies such as chemotherapy, radiotherapy, and immunotherapy to achieve personalized treatment approaches that enhance tumor cell death and therapeutic efficacy [281, 317].

Chemotherapeutic agents, such as doxorubicin, induce apoptosis in TNBC cells through the activation of the ERK1/2 signaling pathway, which enhances treatment sensitivity and reduces resistance [318]. Olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, has demonstrated efficacy in BRCA-mutated TNBC by impairing DNA repair and inducing apoptosis [319]. Other approaches involve the induction of pro-apoptotic genes or the inhibition of anti-apoptotic proteins. For example, Eupaformosanin restores p53 function, promoting both apoptosis and ferroptosis [174, 320]. In addition, targeting Bcl-2 using siRNAs or inhibitors, such as Venetoclax, increases TNBC cell sensitivity to standard treatments like paclitaxel and cisplatin, highlighting a promising avenue for cancer therapy [321].

Inducing pyroptosis also holds potential for TNBC treatment. Oxaliplatin, a platinum-based chemotherapeutic, elevates ROS production by inhibiting mitochondrial respiration, forcing cancer cells to depend on glycolysis. This metabolic stress activates the MAPK pathway and enhances ROS-related gene expression, triggering pyroptosis [322–324]. Doxorubicin similarly elevates ROS levels and causes DNA damage, releasing mitochondrial DNA and DAMPs, which in turn activate the NLRP3 inflammasome and promote necroptosis [325, 326]. These processes can potentiate the effects of other treatments by increasing tumor cell death and reducing resistance, particularly when apoptosis modulators are used in combination with chemotherapy.

Targeting necroptosis through modulation of proteins, such as RIPK1, RIPK3, and MLKL, offers another promising strategy. Although direct targeting of RIPK1 and RIPK3 is limited due to their roles in normal cellular processes, the mitochondrial protein Smac can inhibit inhibitor of apoptosis proteins (IAPs), thereby enabling RIPK1 and RIPK3 activation and promoting necroptosis [327–330]. Smac mimetics such as LCL161 induce apoptosis via caspase activation while also indirectly activating necroptotic pathways [331]. Ongoing research aims to exploit necroptosis to improve TNBC sensitivity to chemotherapy [332].

Autophagy modulation has also emerged as a key strategy to enhance chemotherapy sensitivity and reduce resistance. Inhibiting ULK1 using MRT68921 disrupts autophagy initiation, leading to the accumulation of cytotoxic substances and increased tumor cell death [333, 334]. Additionally, targeting the PI3KC1-Akt-mTORC1 signaling pathway can inhibit autophagy [335]. Metformin activates AMPK, which downregulates mTORC1 activity and promotes autophagy by suppressing the PI3K/Akt pathway [336, 337]. NVP-BEZ235, a dual PI3K/mTOR inhibitor, also facilitates autophagy and holds promise for TNBC therapy [338]. Although autophagy can enhance the effects of anti-cancer drugs, there are currently no direct small molecule modulators for Beclin 1, a key autophagy regulator in TNBC [339]. However, agents such as 3-methyladenine, used in conjunction with chemotherapy, inhibit PI3KC3 and indirectly affect the Beclin-1-PI3KC3 complex, providing a potential avenue for regulation [340]. Researchers are exploring drugs targeting the autophagy pathway to enhance traditional therapies and improve TNBC patient outcomes.

Targeting ferroptosis by managing iron balance and altering tumor cell metabolism can enhance chemosensitivity [341]. Targeting TFR1 within the iron transport pathway has been shown to trigger ferroptosis [342]. Roxadustat enhances iron uptake and ferroptosis by stabilizing HIF and upregulating TFR1 expression [343, 344]. Moreover, GPX4 inhibitors such as RSL3 and FIN56 promote ferroptosis by inhibiting glutathione peroxidase activity and increasing ROS production [345, 346]. Buthionine sulfoximine (BSO), a synthetic amino acid, suppresses γ-glutamylcysteine synthetase (γ-GCS), thereby reducing glutathione synthesis, promoting lipid peroxidation, and inducing ferroptosis in TNBC cells [347, 348].

The need for personalized treatment approaches is particularly urgent in TNBC, given its high degree of inter- and intra-tumor heterogeneity [349]. Tailoring therapy on the basis of individual genotypic, phenotypic, and pathological characteristics can significantly enhance treatment precision and efficacy. Advances in genomics, transcriptomics, and proteomics have enabled a more comprehensive understanding of TNBC biology, facilitating the development of targeted and personalized treatment strategies. These include novel molecular targets such as ICIs, which provide greater specificity and reduced toxicity compared with traditional chemotherapeutics [350, 351]. A deeper understanding of cell death mechanisms and their associated drug actions will pave the way for more effective and safer treatments.

In summary, the future of TNBC therapy lies in the integration of cell death modulators with personalized treatment regimens. By combining multiple therapeutic modalities and tailoring strategies to individual patients, it is possible to improve treatment outcomes and survival rates. Future research should prioritize the identification of novel targets, development of innovative drugs, optimization of clinical trial design, and refinement of therapeutic protocols.

Challenges and future directions

TNBC treatment continues to face significant challenges, particularly in the areas of drug resistance and heterogeneous patient responses. Resistance to therapy not only compromises treatment efficacy but also contributes to disease progression, while individual variability results in widely differing clinical outcomes [352]. Overcoming these challenges requires innovative research strategies centered on the discovery of novel molecular targets, the refinement of therapeutic approaches, and the development of personalized treatment regimens. Advanced technologies now offer the ability to tailor therapies more precisely, with the potential to improve therapeutic efficacy while minimizing adverse effects.

Owing to the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2), TNBC does not respond to endocrine therapy or HER2-targeted agents, making chemotherapy and certain targeted therapies the primary treatment options [353]. However, prolonged administration of agents that regulate cell death pathways may lead to the development of resistance. A thorough understanding of resistance mechanisms is therefore essential. Combining cell death modulators with other modalities—such as chemotherapy or immunotherapy—may allow for synergistic effects that attack tumor cells through multiple pathways, thereby helping to overcome resistance [41]. To more effectively eliminate resistant tumor cell populations, it is crucial to identify novel molecular targets and design therapies that specifically disrupt these resistance mechanisms. Emerging technologies in genomics and proteomics provide powerful tools to characterize the molecular profiles of resistant tumors, facilitating the development of individualized treatment strategies.

Furthermore, TNBC is not a single disease entity but consists of at least six distinct molecular subtypes, each exhibiting unique biological features and differential responses to therapy [354]. The heterogeneity of these subtypes necessitates the use of personalized treatment approaches informed by comprehensive genetic profiling. Incorporating genotypic and phenotypic data, along with patient-specific clinical characteristics, is essential to improve therapeutic outcomes [355]. Future research should focus on elucidating TNBC heterogeneity through the application of cutting-edge technologies such as single-cell RNA sequencing, metabolomics, and transcriptomics. These approaches can aid in the identification of predictive biomarkers and therapeutic targets, ultimately enhancing the precision and effectiveness of treatment. By leveraging these insights, it may be possible to significantly improve the safety, efficacy, and personalization of TNBC therapies, thereby optimizing patient outcomes.

Current research efforts are increasingly focused on elucidating the pathogenesis and signaling pathways involved in TNBC, with the goal of developing novel therapeutics or repurposing existing drugs. Although preclinical models have yielded promising results—for example, the histone deacetylase inhibitor WW437 and the RNA helicase EIF4A inhibitor Zotatifin—these findings have not yet translated effectively into clinical application, largely due to species-specific differences between human and animal models, as well as a lack of robust clinical data [356, 357]. Future studies should prioritize the identification and validation of novel molecular targets, given the critical role of cell death regulation in the initiation, progression, and therapeutic response of TNBC. Investigating these targets could pave the way for innovative and more effective treatment modalities. The integration of emerging technologies such as artificial intelligence (AI) and high-throughput screening has the potential to revolutionize the drug discovery process. AI enables the analysis of vast biological datasets to identify potential targets, while high-throughput screening facilitates the rapid and systematic assessment of these targets’ biological relevance and therapeutic potential. Addressing the therapeutic challenges posed by TNBC requires a comprehensive understanding of resistance mechanisms, patient-specific variability, and the development of novel, multifaceted therapeutic strategies.

Conclusions

This review underscores the central importance of cell death pathways in TNBC and advocates for the development of integrated therapeutic strategies to address the multifaceted challenges associated with this aggressive cancer subtype. We call for a reassessment of the role of cell death in disease progression and therapeutic response, with the aim of generating new insights into both basic research and clinical practice.

Traditionally considered as discrete processes, apoptosis, pyroptosis, necroptosis, ferroptosis, and autophagy are now recognized to be interrelated through intricate signaling networks. These interactions complicate therapeutic targeting, as the induction of one type of cell death—such as apoptosis—may inadvertently trigger others, including pyroptosis or ferroptosis. This interconnectedness may reflect compensatory feedback mechanisms or the activation of shared signaling pathways. Consequently, focusing exclusively on a single form of cell death may limit therapeutic efficacy. We propose an integrative approach that treats cell death as a coordinated and dynamic process, highlighting the importance of cross-talk between different forms.

By targeting one pathway, it may be possible to modulate others, thereby amplifying the therapeutic impact. We emphasize the importance of integrated therapeutic strategies, as cell death modulators that target a single pathway often encounter resistance. Combining such modulators with other treatment modalities—particularly immunotherapy—can enhance their overall efficacy in TNBC. Immune checkpoint inhibitors, for instance, activate the host immune response, promote tumor cell apoptosis, influence iron metabolism to induce ferroptosis, and may also trigger pyroptosis through the upregulation of inflammatory mediators, thereby indirectly affecting autophagy. In cellular immunotherapy, CAR-T and TCR-T cells recognize tumor-associated antigens and release cytotoxic molecules that initiate apoptosis. NK cells can induce both apoptosis and pyroptosis, activate dendritic cells, and further promote necroptosis and ferroptosis. Given the high degree of heterogeneity in TNBC, personalized therapeutic strategies should be developed on the basis of individual patient profiles. This precision medicine approach holds the potential to improve treatment efficacy while minimizing adverse effects. Moreover, a deeper understanding of cell death mechanisms can aid in disease progression monitoring and early intervention strategies.

The clinical translation of cell death mechanisms in TNBC continues to pose a substantial challenge, as initial investigations predominantly utilize animal and organoid models. Animal models, such as immunodeficient xenograft mice, are instrumental in replicating in vivo tumor growth and treatment responses, while organoid models maintain the heterogeneity of patient tumors and facilitate high-throughput screening. These models offer preliminary evidence of the therapeutic potential inherent in cell death pathways. Nonetheless, significant challenges persist, such as the discrepancies between model systems and human physiology, alongside the pronounced heterogeneity of TNBC, which impede the clinical translation of preclinical findings. To address these issues, future research should focus on optimizing these models and enhancing the comprehension of underlying molecular mechanisms.By addressing these challenges, we aim to facilitate the progression of cell death-related therapies from preclinical research to clinical application, thereby offering novel therapeutic prospects for patients with TNBC.

In summary, we highlight the crucial role of cell death pathways in the treatment of TNBC and advocate for an integrated, systems-level approach that challenges traditional single-pathway paradigms. By targeting interconnected signaling networks, we propose a comprehensive therapeutic strategy that enhances our understanding of disease mechanisms and facilitates the development of innovative treatments. Such an approach aims to expand our knowledge of cell death regulation, uncover novel drug targets, and support the personalization of cancer therapies. Ultimately, modulating multiple forms of programmed cell death may improve tumor cell cytotoxicity and overcome therapeutic resistance, potentially driving breakthroughs in the treatment of complex malignancies such as TNBC.

Abbreviation

TNBC

Triple-negative breast cancer

BC

Breast cancer

ER

Estrogen receptor

PR

Progesterone receptor

HER2

Human epidermal growth factor receptor 2

ACD

Accidental cell death

PCD

Programmed cell death

LCD

Lysosome-dependent cell death

PAMPs

Pathogen-associated molecular patterns

DAMPs

Damage-associated molecular patterns

TNF

Tumor necrosis factor

FasL

Fas ligand

TRAIL

TNF-related apoptosis inducing ligand

FasRs

Fas receptors

TNFR1

Tumor necrosis factor receptor 1

DR4

Death receptor 4

DR5

Death receptor 5

DISC

Death-inducing signal complex

ROS

Reactive oxygen species

Cytc

Cytochrome C

ATR

ATM and rad3-related

CCL2

C–C motif chemokine ligand 2

EMT

Epithelial mesenchymal transition

CSC

Cancer stem cell

CTL

Cytotoxic T-lymphocyte

NLRP3

NOD-like receptor thermal protein domain associated protein 3

AIM2

Absent in melanoma 2

ASC

Apoptosis-associated speck-like protein containing a CARD

PYD

Pyrin domain

HIN200

Hematopoietic interferon-inducible nuclear 200 proteins

CARD

Caspase recruitment domain

LPS

Lipopolysaccharides

DC

Dendritic cell

RIPK

Receptor interacting protein kinase

MLKL

Mixed lineage kinase domain-like

HMGB1

High mobility group box-1 protein

ATGs

Autophagy-related genes

mTORC1

Mechanistic target of rapamycin complex 1

PI3K

Phosphatidylinositol 3 kinase

LC3

Microtubule associated protein light chain 3

PE

Phosphatidylethanolamine

STX17

Syntaxin-17

SNAP29

Synaptosome associated protein 29

VAMP8

Vesical-associated membrane protein 8

TFR1

Transferrin receptor

FPN1

Ferroportin 1

LIP

Labile iron pool

GPX4

Glutathione peroxidase 4

GSH

Glutathione

IRP

Iron regulatory protein

Nrf2

Nuclear factor erythroid 2-related factor 2

FADD

Fas-associated protein with death domain

ZDHHC1

Zinc finger DHHC-type containing 1

AML

Acute myeloid leukemia

HSPA5

Heat shock protein family A member 5

LSD1

Lysine-specific demethylase 1

TRAF6

TNF receptor-associated factor 6

MDR

Multiple drug resistance

AC

Anomanolide C

TIM-3

T cell immunoglobulin and mucin domain-containing protein 3

TAMs

Tumor-associated macrophages

CAR-T

Chimeric antigen receptor T cell immunotherapy

TCR-T

Receptor-engineered T cell

MDSCs

Myeloid-derived suppressor cells

VDAC

Voltage-dependent anion channel

MAPK

Mitogen-activated protein kinase

ERK

Extracellular regulated protein kinases

JNK

C-Jun n-terminal kinase

IAPs

Inhibitor of apoptosis proteins

γ-GCS

γ-Glutamylcysteine synthetase

RSL3

RAS-selective lethal 3

Author contributions

Y.L. carried out manuscript writing and chart drawing; J.H. and J.C. carried out manuscript review and editing; T.C. and W.L. carried out figure drawing; Z.Y. and F.Z. carried out supervision and funding acquisition.

Funding

This research was supported by the Sichuan Science and Technology Program (2022YFS0623) and the Natural Science Foundation of Southwest Medical University (2022QN079).

Data availability

Data sharing does not apply to this article as no new datasets were created or analyzed in this study. All information is derived from publicly published articles.

Declarations

Ethics approval and consent to participate

Since this article does not pertain to research involving humans or animals, the review and/or approval of an ethics committee is not required.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships.