PANoptosis in hepatocellular carcinoma: Underlying mechanisms

Abstract

PANoptosis is an inflammatory programmed cell death pathway possessing critical characteristics of apoptosis, pyroptosis, and necroptosis. It is regulated by PANoptosome complexes, involves interaction between these three key programmed cell death pathways, yet is distinct from any alone. PANoptosis holds vital significance in liver-related diseases, particularly hepatocellular carcinoma (HCC). This article summarizes research on the mechanism and treatments of PANoptosis in HCC. Current research has partially elucidated PANoptosis-related mechanisms in HCC and identified several molecules modulating it. Therapeutic strategies targeting PANoptosis hold significant promise. Investigations into these critical molecules have led to the development of traditional targeted drug therapies and emerging strategies like nanotechnology-based immunocombination therapies. However, there are still challenges in the mechanistic and pharmacological studies of PANoptosis in HCC, including the bidirectional regulation of key apoptotic factors, specific molecular mechanisms, and preclinical models. This article offers a new orientation for studying the pathogenesis and potential therapeutic strategies for HCC.

Key Words: PANoptosis; Hepatocellular carcinoma; Programmed cell death; Mechanisms; Treatment strategies

Core Tip: This article summarizes the specific mechanisms of PANoptosis in hepatocellular carcinoma (HCC) and explores potential therapeutic strategies, highlighting the promise of targeting PANoptosis for HCC treatment. While modulating PANoptosis in liver cancer cells can influence tumor progression, excessive activation of PANoptosis can trigger an inflammatory storm. We summarize current research on the mechanisms and therapies related to PANoptosis in HCC, aiming to provide possible treatment strategies for the disease.

INTRODUCTION

The state of health or disease of a multicellular organism is closely associated with the life and death of individual cells, and the organism needs to remove risk factors through programmed cell death (PCD), as PCD is an active system that helps the organism grow and survive[1]. Pyroptosis, apoptosis and necroptosis are the most prominent forms of PCD that protect the organism from internal and external risk factors[2,3]. In healthy conditions, apoptosis is the primary mechanism of cell death, which is a non-inflammatory, cysteine-dependent process mediated by specific caspases and acts in a cascade fashion[4]. Pyroptosis is a PCD characterized by cell lysis, release of inflammatory components, and caspase-1-dependent plasma membrane pore formation[5]. In contrast, necroptosis is PCD with a necrotic morphology that does not activate caspases but through the receptor interacting serine/threonine kinase 1 and receptor interacting serine/threonine kinase 3 complex signaling pathways[6]. In 2019, Malireddi et al[7] proposed that the concurrent occurrence of pyroptosis, apoptosis, and necroptosis in the same cell population is referred to as PANoptosis. PANoptosis is essentially a dynamically regulated and highly coordinated mechanism of programmed inflammatory cell death. The activation and formation of the PANoptosome is the most vital link in initiating PANoptosis, despite the presence of additional factors, for instance, injury, infection or self-defects. The PANoptosome can activate all three pro-inflammatory cell death processes and contains essential compounds required for pyroptosis, apoptosis, and necroptosis. When an infection or other impediment blocks one or more PCD pathways, PANoptosis provides the host with the means to trigger alternative cell death defense mechanisms[7].

Hepatocellular carcinoma (HCC) represents the most prevalent form of primary liver cancer, originating from hepatocytes and constituting approximately 80%-90% of primary liver malignancies[8]. It ranks as the fourth leading cause of cancer-related mortality worldwide. Patients with early-stage HCC may receive radical treatment, intermediate-stage may receive localized catheter treatment, and advanced-stage receive systemic therapies like multi-kinase inhibitors and immune checkpoint inhibitors. However, a significant proportion of these patients eventually develop acquired resistance to these treatments[9]. Over 50% of patients on sorafenib develop resistance within the first year of treatment, and acquired resistance affects a substantial subset (approximately 30%-40%) of initial responders to immune checkpoint inhibitors[10,11]. The pathophysiology involves factors including fatty liver, immune cells, and the tumor micro-environment[12]. The most significant risk factor arises from chronic inflammatory etiologies leading to chronic necrotizing inflammation[13]. HCC primarily manifests in chronic liver disease and cirrhosis, responsible for 70% to 90% of cases, mainly caused by alcoholic liver disease, hepatitis B, hepatitis C, and nonalcoholic steatohepatitis[14]. HCC results from persistent tissue injury mediated through pathogenic mechanisms encompassing extrinsic and intrinsic influences like alterations in the cellular microenvironment, viral/non-viral agents, and hereditary susceptibility[15]. Cancer-associated genetic modifications promote proliferation and reduce apoptosis. Hepatitis B virus, hepatitis C virus infection, and fatty liver contribute to chronic inflammation and HCC development[16].

PANoptosis plays a role in many tumors, as research has demonstrated that PANoptosis exhibits high activity in colorectal cancer, gastric cancer, breast cancer, lung cancer, esophageal cancer, chronic large B-cell lymphoma and adrenocortical carcinoma, but exhibits low to moderate activity in HCC and low activity in acute myeloid leukemia (AML)[17-25]. HCC is the most predominant type of primary HCC. Therefore, understanding the role of PANoptosis in HCC is crucial for exploring new therapeutic strategies. With the understanding of the molecular mechanisms underlying PANoptosis, its role in HCC is now being discovered. Drugs that target key PANoptosis molecules, which play significant roles in HCC, alongside some new therapeutic strategies, have been shown to be effective in inhibiting HCC development. For example, kanglexin (KLX) ameliorates HCC progression by mediating Z-DNA-binding protein 1 (ZBP1) transcription and ubiquitination to increase ZBP1-induced PANoptosis, and ultrasmall enzyodynamic PANoptosis nano-inducers enable ultrasound-amplified HCC therapy and lung metastasis inhibition[26,27].

MOLECULAR MECHANISM OF PANOPTOSIS

Central molecular pathways of PANoptosis

The central molecular pathways of PANoptosis (PANoptosome) form a cytoplasmic polymeric protein complex that plays a crucial role in regulating PANoptosis. Almost all PANoptosomes contain apoptosis-associated speck-like protein containing a CARD, Fas-associated death structural domain protein (FADD), caspase-8, ZBP1, melanoma-inhibiting factor 2 (AIM2), and core molecular machinery that mediates the execution of focal death, apoptosis, pyroptosis and necroptosis, but the specific components vary depending on the triggers[28]. The PANoptosome encompasses critical molecules involved in the cellular pathways of pyroptosis, apoptosis, and necroptosis, which have the capacity to initiate all three forms of cell death[29]. There are three classes of proteins that comprise the PANoptosome: (1) Adaptor proteins, such as ASC and FADD; (2) Sensors of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), such as ZBP1, AIM2, NLR family pyrin domain containing (NLRP) 12 and NLRP3; and (3) Catalytic effector proteins, such as receptor interacting serine/threonine kinase (RIPK) 1, caspase-1, RIPK3, and caspase-8. Four molecularly distinct PANoptosome subtypes have been characterized to date, comprising the ZBP1-PANoptosome, AIM2-PANoptosome, NLRP12-PANoptosome, and RIPK1-PANoptosome[30].

Among them, ZBP1 is the upstream sensor of PANoptosis, and it mediates cellular PANoptosis in inflammatory and infectious diseases. In the context of influenza A virus infections, ZBP1 plays a crucial role in the recognition of Z-RNA associated with the virus. Influenza A viruses can induce type I interferon (IFN) signaling, which in turn triggers ZBP1 upregulation. However, there are cell type differences in the mechanism of ZBP1 induction, and toll-like receptor (TLR) and RNA sensor RIG-I signaling are jointly involved in the induction process in immune cells, whereas RNA sensor RIG-I signaling is essential for ZBP1 up-regulation in non-immune cells. This recognition initiates the NLRP3 inflammasome activation, followed by caspase-1 activation to catalyze the hydrolytic maturation of precursor interleukin (IL)-1β and precursor IL-18, and to cleave gasdermin D (GSDMD) to release its pore-forming N-terminal structural domain (N-GSDMD). Meanwhile, caspase-3 and caspase-7 activation cleaves GSDME to produce pore-forming N-GSDME, while mixed lineage kinase domain-like protein (MLKL) is activated by phosphorylation. Together, N-GSDMD, N-GSDME, and phosphorylated MLKL mediate the formation of plasma membrane pores and promote the extracellular release of mature IL-1β, IL-18, other cytokines, and DAMPs. Furthermore, the interaction between ZBP1 and RIPK3 can be augmented by the presence of caspase-6. In infectious diseases, NLRP3, ASC, RIPK1, RIPK3, caspase-6, and caspase-8, which are important components of the PCD pathway, are involved in ZBP1-mediated PANoptosome assembly[30-32]. ZBP1 is a crucial component in the formation of the DNA-damaged PANoptosome, which activates endogenous retroviruses and acts as a ZBPI ligand to mediate PANoptosis. Double-stranded RNAs are induced upon DNA damage and bind to ZBP1 via the Zα domain to mediate DNA-damage-induced PANoptosis. PANoptosome assembly does not contain RIPK1. dsRNA is induced upon DNA damage and binds to ZBP1 via the Zα domain, mediating DNA damage-induced PANoptosis[33]. ZBP1’s role in coronavirus disease 2019 pathophysiology was supported by a necropsy-based analysis of leukocytes from coronavirus disease 2019-related deaths, which showed that ZBP1 protein expression was higher than that of individuals who recovered. The study indicated that viral infection-induced liver injury, as evidenced by elevated liver enzymes alanine aminotransferase/aspartate aminotransferase and histopathological changes, was significantly attenuated in ZBP1 knockout mice. Biochemical analysis of the presence of ZBP1-PANoptosome assembly was performed by bone marrow-derived macrophages[32].

The AIM2-PANoptosome is induced during infection with herpes simplex virus 1 and F. novicida to induce cellular PANoptosis. Melanoma-deficiency factor 2 (AIM2) acts upstream to regulate pyrin and ZBP1 expression, and it interacts with downstream pyrin, ZBP1, and ASC to form the AIM2-PANoptosome (containing AIM2, pyrin, ZBP1, ASC, FADD, caspase-1, caspase-8, RIPK1, and RIPK3). Concurrently, both herpes simplex virus 1 and F. novicida specifically induced the assembly of the AIM2-PANoptosome through their interaction with the Zα domain of ZBP1, which subsequently led to the inhibition of Rho GTPase activity. In addition, ZBP1 interacts with and localizes to the apoptosis-associated speck-like protein and acts as a key recognition molecule in inflammasome initiation, promoting specific cleavage by caspase-1 protease and ultimately triggering the process of programmed inflammatory cell death[34]. Molecular analysis of tumor tissue samples from HCC patients revealed that response to treatment was associated with elevated deoxyribonuclease 1 class 3 (DNASE1 L3) expression and AIM2 inflammasome pathway activation markers (e.g., GSDMD-NT) and provided strong molecular evidence through co-immunoprecipitation, Western blot, and co-localization analyses of key molecules (e.g., AIM2, ASC, caspase-8)[35].

NLRP12 is an immune sensor that senses molecular danger signals of hemoglobin or some hemolytic diseases associated with pathogens and activates cell death and inflammatory responses. In systems containing hemoglobin supplemented with innate immune stimuli (PAMPs) or inflammatory cytokines [tumor necrosis factor (TNF)], NLRP12 can form a PANoptosome with inflammatory vesicles via caspase-8/RIPK3 - i.e. NLRP12-PANoptosome (composed of NLRP12, NLRP3, RIPK3, ASC, caspase-1, and caspase-8). Cell death during hemoglobin plus PAMPS under high serum conditions is dependent only on the NLRP12-PANoptosome, which is upregulated by TLR-mediated interferon regulatory factor 1, thus driving NLRP12 expression and a series of cascading reactions. Activation of TLR2/4 signaling induces NLRP12 expression via interferon regulatory factor 1, which in turn drives the formation of inflammatory vesicles, a component of the larger NLRP12-PANoptosome apoptotic vesicles, ultimately leading to IL-1β and IL-18 maturation. In animal experiments, NLRP12 deficiency reduced lethality in a hemolysis model by protecting mice from acute kidney injury. NLRP12-PANoptosome in a mouse model of liver inflammation treated with hemoglobin and PAMPs were directly visualized in liver sections using immunofluorescence confocal microscopy to observe key molecules, such as NLRP12, NLRP3, ASC, caspase-8, and RIPK3, forming significant cytoplasmic aggregates[36].

RIPK1-PANoptosome (composed of RIPK1, RIPK3, NLRP3, ASC, caspase-1, and caspase-8) assembly occurs upon binding of Yersinia pestis or transforming growth factor-β-activated kinase 1 (TAK1) to lipopolysaccharide (LPS). Spontaneous activation of necrosis due to PIPK1 deficiency is further enhanced under Yersinia pestis infection. At the same time, RIPK1 is a key mediator of TAK1 inhibition in PANoptsis in this condition. TAK1 normally inhibits TNF production; however, upon TAK1 inhibition in the context of LPS stimulation, TNF-mediated autocrine signaling is promoted, leading to which further induction and assembly of the RIPK1-PANoptosome. RIPK1 interacts directly with several cysteoaspartic enzymes, including caspase-8. Caspase-8 plays a central effector role in TAK1 inhibition, Yersinia pestis-induced apoptosis, and GSDMD-mediated cell death, while RIPK1 is involved in the regulation of these death pathways. This functional link is reflected in the formation of the PANoptosome complex by RIPK1, which integrates caspase-8, suggesting a synergistic role in cell death signaling pathways[30,37].

NLR family CARD domain containing 5 serves as a significant innate immune sensor in the context of inflammation and hemolysis, possessing the capability to trigger inflammatory cell death upon interaction with certain specific ligands. It has been shown that NLRC5 induces PANoptosome formation and cellular PANoptosis during nicotinamide adenine dinucleotide+ depletion, and TLRs can regulate NLR family CARD domain containing 5 expression and reactive oxygen species production to trigger cellular PANoptosis[38]. In addition to the PANoptosomes that have been identified, the existence of other PANoptosomes still requires further research. Notably, the assembly of many PANoptosomes occurs during bacterial or viral infections. Although there are obvious differences in the initiation of PANoptosome assembly under specific conditions, the key molecules involved in the assembly still exhibit strong interconnections. Therefore, we speculate whether certain key molecules may integrate PANoptosis under different conditions, forming a PANoptosis regulatory network.

SIMILARITIES AND DIFFERENCES BETWEEN PANOPTOSIS AND APOPTOSIS, NECROPTOSIS, AND PYROPTOSIS

Apoptosis, necroptosis, and pyroptosis represent the three primary mechanisms of PCD. These pathways play a vital role in sustaining metabolic homeostasis and are linked to the progression of various diseases, illustrating intricate mechanisms of action. In initial studies, these three pathways have been regarded as different PCD pathways based on the differences in the key components of their responses (Figure 1). However, with further research, increasing studies hae shown that the three pathways of apoptosis, necroptosis, and focal death are inextricably linked to each other. The concept of PANoptosis was conceived in 2019, and means “pan-cellular death”.

Figure 1 Molecular mechanisms of pyroptosis, necroptosis, and apoptosis. ASC: Apoptosis-associated speck-like protein containing a CARD; BAX: Bcl-2 associated X protein; DD: Death domain; FADD: Fas-associated protein with death domain; GSDMD: Gasdermin D; N-GSDMD: N-terminal fragment of gasdermin D; MLKL: Mixed lineage kinase domain like pseudokinase; NEK7: Never in mitosis gene A-related kinase 7; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3: NLR family pyrin domain containing 3; PAMPs: Pathogen-associated molecular patterns; RIPK1: Receptor-interacting serine/threonine kinase 1; RIPK3: Receptor interacting serine/threonine kinase 3; ROS: Reactive oxygen species; TLR: Toll-like receptor; TNF-α: Tumor necrosis factor-alpha; TRADD: Tumor necrosis factor receptor-associated death domain protein.

Apoptosis

Apoptosis is characterized by apoptotic vesicle formation and cysteine-aspartic protease (caspase)-dependent control[38]. Execution occurs through two primary pathways: (1) the extrinsic pathway initiated by TNF receptor superfamily ligand binding, which induces receptor trimerization. This conformational change recruits FADD to form the death-inducing signaling complex, facilitating procaspase-8 activation to caspase-8. Active caspase-8 subsequently cleaves RIPK1 or directly activates effector caspases; and (2) the intrinsic (mitochondrial) pathway activated by stimuli including DNA damage, oxidative stress, metabolic disruption, and viral infection[39,40]. This pathway is governed by BCL2 apoptosis regulator (BCL-2) family pro-apoptotic members [e.g., BCL-2-associated X protein (BAX), BCL2 antagonist/killer, BH3 interacting domain death agonist][41,42], which permeabilize the mitochondrial outer membrane. Mitochondrial outer membrane releases cytochrome c into the cytosol, where it binds Apaf-1 and deoxyadenosine triphosphate to form the apoptosome. This complex activates procaspase-9. Both pathways converge to activate executioner caspases-3/7, which proteolytically cleave downstream targets. This cascade ensures precise apoptotic signaling under diverse stress conditions[43].

Non-coding RNAs (ncRNAs) play an indispensable role in the apoptotic regulatory network of HCC. Among the most extensively studied ncRNAs with critical regulatory functions are microRNAs (miRNAs) and long ncRNAs (lncRNAs). These molecules regulate apoptosis through distinct yet interrelated molecular mechanisms. Firstly, miRNAs (typically about 22 nucleotides in length) primarily exert post-transcriptional regulation by sequence-specifically binding the 3’ untranslated region of target mRNAs. When binding exhibits high complementarity, miRNAs can mediate direct cleavage and degradation of the target mRNA. More commonly, however, miRNAs recruit repressor complexes through incomplete complementarity, thereby inhibiting mRNA translation without significantly affecting mRNA stability. Within the extrinsic apoptotic pathway, miR-491-5p enhances HCC cell sensitivity to TNF-α-induced apoptosis by downregulating its target[44]. In the intrinsic pathway, miRNAs such as miR-34a and the miR-29 family activate apoptosis by directly targeting the 3’ untranslated regions of anti-apoptotic BCL-2 family members (e.g., BCL-2, MCL1 apoptosis regulator), leading to their downregulation while concurrently upregulating pro-apoptotic proteins (e.g., BAX)[45]. This sensitizes HCC cells to stressors like hypoxia, serum deprivation, and chemotherapeutic agents. Critically, key pro-apoptotic miRNAs (e.g., miR-34a, miR-29, let-7) are frequently downregulated in HCC, resulting in the overexpression of their target anti-apoptotic proteins, suppression of apoptosis, and ultimately promoting cancer cell survival, treatment resistance, and HCC progression[46,47]. Secondly, lncRNAs (usually > 200 nucleotides) exhibit more complex structures and diverse functions. A common mechanism by which lncRNAs regulate apoptosis involves acting as competitive endogenous RNAs or “sponges” that sequester miRNAs in the cytoplasm. For example, lncRNA nuclear paraspeckle assembly transcript 1 inhibits HCC cell proliferation, migration, and invasion while promoting apoptosis[48]. LncRNA cancer susceptibility 9 promotes HCC proliferation and migration while inhibiting apoptosis by downregulating miR-424-5p. Collectively, miRNAs and lncRNAs form a multi-layered, highly interconnected ncRNA regulatory network. This network deeply integrates with both classical caspase-dependent and caspase-independent apoptotic pathways, jointly determining cellular fate[49].

Epigenetic modifications exert precise temporal and spatial regulation over the expression of key apoptosis-related genes. These mechanisms can be categorized into three primary modes of action: Firstly, DNA methylation regulates apoptosis. For instance, cyclin dependent kinase inhibitor 2A, a key regulator of G1-phase cell cycle arrest, is frequently hypermethylated and inactivated in various cancers[50]. This inactivation leads to epigenetic silencing of p16, an important tumor suppressor commonly repressed in HCC[51]. Secondly, covalent histone modifications modulate apoptosis in HCC. These include modifications such as H3K4me3, H3K27ac, H3K9ac, and H3K4me2[52]. Elevated levels of H3K27me3 and H3K4me3 have been associated with poor prognosis and aggressive HCC behavior. Protein acetylation is critically involved in HCC pathogenesis. Histone deacetylases can silence multiple tumor suppressor genes (TSGs; e.g., CDH1, FOXO3, p53), thereby enhancing cancer cell survival[53,54]. Sirtuin 6 promotes HCC cell survival by downregulating BAX expression, while SIRT7 knockdown significantly increases the proportion of HCC cells in G1/S phase[55]. Furthermore, SIRT7 promotes HCC cell survival by targeting the transcription factor FOXO3[56]. Certain histone deacetylase inhibitors can activate the intrinsic apoptotic pathway by regulating the transcription of pro-apoptotic genes, including BIM, BH3 interacting domain death agonist, and BAD[57]. Thirdly, chromatin remodeling complexes [e.g., switch (SWI)/sucrose non-fermentable (SNF), imitation switch, chromodomain-helicase-DNA-binding, inositol requiring 80 families] alter chromatin compaction and nucleosome positioning. enhancer of zeste 2 polycomb repressive complex 2 subunit, another crucial chromatin remodeler, is significantly upregulated in diseased liver tissue and fundamentally alters chromatin structure. Silencing β-catenin antagonists can activate the Wnt/β-catenin signaling pathway, promoting HCC progression. The SWI/SNF complex, which possesses chromatin remodeling functions, is also frequently implicated in liver tumorigenesis. Overexpression of the SWI/SNF subunit SMARCD1 promotes HCC growth by activating the mammalian target of rapamycin (mTOR) signaling pathway[58].

Pyroptosis

One type of PCD that is closely related to the inflammatory response is pyroptosis. The activation of inflammatory vesicles and the formation of membrane holes by GSDMD are characteristics of this process. Members of the caspase family must be involved for the process to occur, and it is mainly carried out via classical, non-classical, and alternate pathways[59]. In the conventional pathway, either DAMPs or PAMPs trigger the activation of the inflammatory vesicle complex. This complex includes the adaptor protein ASC, the zymogen pro-caspase-1, and sensor proteins such NLRP3, AIM2, or pyrin[60]. ASC serves as a link between the upstream sensor and pro-caspase-1 via its PYD and CARD structural domains, thereby facilitating caspase-1 activation. When caspase-1 is activated, it cleaves GSDMD, releasing N-GSDMD and creating a hole in the plasma membrane. This process ultimately results in the secretion of cellular components, including IL-1β and IL-18[61]. On the other hand, the nonclassical pathway consists of direct activation of caspase-4/5 (human) or caspase-11 (mouse) by intracellular LPS, which likewise induces pyroptosis by cleaving the GSDMD[62,63]. Moreover, alternative mechanisms, including the cleavage of GSDMD mediated by caspase-3, caspase-8, or granzyme B, contribute to the regulation of pyroptosis, thereby enhancing the complexity of this particular cell death pathway[5].

Necroptosis

Necroptosis displays morphological features similar to those observed in necrosis. However, it is fundamentally integrated within the molecular and mechanical frameworks of the apoptotic signaling pathway[62]. This mechanism is primarily regulated by the receptor-interacting protein kinase RIPK3 and its effector protein MLKL. Within the diverse array of signaling pathways, the TNF/tumor necrosis factor receptor pathway is the most extensively researched in relation to the induction of necroptosis[64]. The death-inducing signaling complex is assembled in the classic apoptotic pathway when the death receptor interacts with its matching ligand. This complex subsequently activates caspase-8 and cleaves RIPK1, thereby initiating the process of apoptosis; however, in necroptosis, caspase-8 activity is inhibited, leading to the assembly of RIPK1 and RIPK3 into the necrotic body with the assistance of heat shock protein 90 and its cochaperone, the CDC37 complex[65,66]. This complex phosphorylates MLKL’s activation loop, which promotes oligomerization and translocation to the plasma membrane, where it generates transmembrane pores. This mechanism eventually compromises membrane integrity and induces necroptosis[67]. In addition, pattern recognition receptors, TLRs, and ZBP1 can directly activate RIPK3 and induce necroptosis, bypassing the RIPK1-dependent pathway[67]. These mechanisms reveal a molecular switch for cellular reprogramming towards necroptosis through signaling pathways when apoptosis is blocked, emphasizing the central regulatory role of RIPK1 in the dynamic balance between apoptosis and necroptosis.

PANoptosis is defined as the simultaneous occurrence of localized cell death, apoptosis, and necroptosis within a single cell population. It represents a meticulously coordinated and dynamically regulated process of inflammatory cell death[68]. The characteristics in question cannot be adequately accounted for by pyroptosis, apoptosis, or necroptosis in isolation; instead, they should be understood as a composite phenomenon that encompasses elements of all three processes. The characteristic of PANoptosis, which links and has mechanisms distinct from those of pyroptosis, apoptosis, or necroptosis individually, provides the body with an alternative coping strategy in the face of infection. However, pathological activation of the PANoptosis pathway can trigger excessive inflammatory responses, leading to a cascade of amplified cytokine release. If unregulated, this pathological process leads to progressive parenchymal cell necrosis and imbalance of organ microenvironmental homeostasis, which may ultimately lead to a fatal regression to multisystem failure[69].

HCC has been the subject of extensive attention by researchers. The initiation of apoptosis in cancer cells is inextricably linked to cancer treatment, and resistance to cell death in HCC is widely recognized as its central feature, which is a key factor contributing to the lack of success of conventional cancer treatment[70]. Cancer treatment, immunotherapy, and radiotherapy all rely heavily on PCD. In recent years, with the in-depth investigation of tumor molecular mechanisms, the complex regulatory role of PANoptosis-related genes (PRGs) in HCC development has steadily been disclosed. Several studies have shown that such genes not only affect the immunotherapeutic response by regulating the immunosuppressive state of the tumor microenvironment but also significantly reduce the sensitivity of chemotherapeutic drugs by activating the anti-apoptotic signaling pathway, leading to poor prognosis[46,47]. In the following, we describe the relationship between PANoptosis and HCC from the aspects of PANoptosis-related genes, PANoptosis-related RNA, and PANoptosis-related immune markers.

PANOPTOSIS-RELATED GENES

Research on ZBP1 in HCC remains limited. ZBP1 expression was found in HCC patients treated with IFN. Wang et al[71] developed a vasculogenic mimicry-associated scoring model (vasculogenic mimicry score) incorporating three risk factors: Secreted phosphoprotein 1, ADAM metallopeptidase with thrombospondin type 1 motif 5, and ZBP1, revealing their potential as prognostic biomarkers for HCC. Their analysis demonstrated that higher ZBP1 expression was associated with improved patient prognosis. Experimentally, ZBP1 expression was significantly higher in non-malignant hepatocytes compared to HCC tumor cells. ZBP1 may promote local inflammatory responses within the tumor microenvironment by inducing necroptosis. Collectively, ZBP1, along with secreted phosphoprotein 1 and ADAM metallopeptidase with thrombospondin type 1 motif 5, appears to drive HCC tumor progression[71].

RIPK3 is involved in necroapoptosis, primarily by phosphorylating MLKL, rather than relying on caspase in the apoptotic pathway, and this activation can shift cells from apoptosis to necroapoptosis, playing a key role in cell death and HCC progression. Specifically in HCC, RIPK3 plays a dual role. On the one hand, it exerts an oncogenic function: Its downregulation significantly correlates with poor patient prognosis, whereas experimental deletion [e.g., in diethylnitrosamine (DEN)-induced models] drives tumor formation, suggesting a basal tumor suppressor role. This protective effect is primarily achieved through necro-apoptotic pathway activation, which both kills cancer cells directly and activates the anti-tumor immune response through the release of DAMPs. On the other hand, RIPK3 also plays a pro-tumorigenic role: DAMPs released from RIPK3-mediated necroptosis can induce persistent chronic inflammation, which in turn promotes hepatic fibrosis, tumor microenvironment remodeling, ultimately accelerating HCC progression[72].

On the other hand, AIM2 protein significantly decreases in HCC cell lines and clinical samples and may further play a role in HCC growth and metastasis by affecting immune cell infiltration. In vitro, overexpression of hepatitis B virus X (HBx) protein inhibited AIM2 expression at both the protein and mRNA levels, and HBx promoted AIM2 degradation through ubiquitination. AIM2 deletion or reduction induced epithelial-mesenchymal transition activation, which was closely associated with tumor cell migration, vascular invasion, differentiation, tumor capsule defects, and low post-surgical survival[73-75]. The molecular components of the NLRP3 inflammasome are reduced or lost in HCC but increased during cirrhosis; only when cirrhosis progresses to HCC is the molecular component of the NLRP3 inflammasome reduced, and this reduction is closely related to advanced HCC stages as well as the poorly differentiated pathological condition. NLRP3 is also induced by S. Maltophilia in the tumor microbiota of patients with HCC and further accelerates HCC progression via the TLR4/nuclear factor kappa-B (NF-κB) pathway. S. Maltophilia in the tumor microbiota of HCC patients further accelerates cirrhosis through the TLR4/NF-κB pathway to drive HCC progression. In contrast, NLRP3 deficiency increases the probability of natural killer group 2D-major histocompatibility complex class I-related chain A interactions, as major histocompatibility complex class I-related chain A stripping by HCC cells can be inhibited in the absence of NLPR3, thus making the death of HCC by natural killer cells more effective[76,77].

In addition to upstream sensor molecules, other molecules also exert influence through various mechanisms. In a mouse model of Otulin (OTU) deubiquitinase with linear linkage specificity ablation in liver parenchymal cells, FADD gene ablation completely rescued mice from developing hepatic inflammation, apoptosis, hepatitis caused by compensatory hepatocyte proliferation, hepatic fibrosis, and further progression to HCC; knockdown of RIPK1 significantly protected mice from this spectrum of severe liver disease. FADD and RIPK1 can interact to affect tumor development[78]. Tripartite motif containing 21 (TRIM21) expression was elevated in HCC tissue cells. TRIM21-mediated RIPK1 ubiquitination could be promoted by PPDPF to activate NF-κB, resulting in improved apoptosis and compensatory cellular proliferation in mice, further inhibiting HCC development[79]. TRIM21 can facilitate HCC progression by undermining the tumor-suppressive functions of the p62-kelch-like ECH-associated protein 1 - NFE2 like BZIP transcription factor 2 (p62-Keap1-Nrf2) antioxidant signaling pathway. In contrast, mice with a deletion of the TRIM21 gene exhibited a reduction in hepatocarcinogenesis induced by DEN exposure compared to their wild-type counterparts[80]. In addition, TRIM21 can affect the structural stability of vacuolar protein sorting 72 homolog (VPS72) protein through VPS72 interaction, and the enhancement of VPS72 protein expression can further enhance HCC initiation and progression in vivo. TRIM21 affects HCC by interacting with VPS72[81]. Some reports illustrate that when activated, caspase-8 induces further activation of caspase-3, leading to apoptosis and achieving tumor growth inhibition[82]. However, it has also been reported to indicate that caspase-8 inhibits Fas-induced apoptosis and promotes tumor migration through a complex (containing RIPK1, FADD, cFLIP, caspase-8)[83]. Based on the detection of several PRGs, as well as clinical samples, it was found that TRIM21, HMOX1, S100A9, TRAF3, and TMC7 had elevated expression in HCC tumor tissues, whereas C-X-C motif chemokine receptor 2 and Ral guanine nucleotide dissociation stimulator like 4 had decreased expression in HCC tumor tissues[74]. TNF-α and IFN-γ initiate a cascade of events that lead to PANoptosis by collaboratively activating pivotal death execution proteins, including GSDME, caspase-8, caspase-3, caspase-7, and MLKL, which is a critical component of necrotic apoptosis. This form of inflammatory PCD demonstrates considerable anti-tumor efficacy across various tumor lineages[25,84]. From the perspective of microenvironmental regulation, PRGs influence tumor angiogenesis, inflammatory response, and immunosuppressive status by regulating eosinophil infiltration, and this complex regulatory network ultimately contributes to the malignant biological characteristics associated with tumor growth and metastasis[74].

The findings from these studies suggest that specific genes and fundamental molecules related to PANoptosis exhibit intricate functions in HCC development and progression. Some molecules are solely involved in promoting or suppressing HCC, while others, such as TRIM21 and caspase-8, exhibit dual regulatory effects. Additionally, as sensors of the PANoptosome, AIM2 differs from ZBP1, NLRP3, and RIPK1, which generally promote HCC, as its specific activation can inhibit HCC development. Therefore, we propose that PANoptosis exerts bidirectional regulation in HCC. Targeted inhibition or promotion of PANoptosis, depending on its context-specific induction mechanisms, may provide a therapeutic strategy for HCC (Figure 2).

Figure 2 Role of PANoptosis in hepatocellular carcinoma. Mechanisms of PANoptosis in hepatocellular carcinoma. Factors that inhibit the hepatocellular carcinoma (HCC) process are covered in green, like hepatitis B virus X (HBx) protein. Factors that can promote the HCC process are covered in red. Triple motif protein 21 (TRIM21) and caspase-8 exhibit both positive and negative regulatory mechanisms in HCC. Through positive regulation, TRIM21 can inhibit tumor suppression by inhibiting the p62 sequestosome 1-kelch-like ECH-associated protein 1-nuclear factor (erythroid-derived 2)-like 2 (p62-Keap1-Nrf2) antioxidant pathway. In contrast, its negative regulation involves two inhibitory pathways, receptor-interacting serine/threonine kinase 1 and vacuolar protein sorting 72 (VPS72), which suppress HCC progression. Caspase-8, while enhancing the tumor-suppressive activity of caspase-3, can also promote HCC development through interactions with Fas-associated protein with death domain (FADD) and receptor-interacting serine/threonine kinase 1 (RIPK1). AIM2: Absent in melanoma 2; NLRP3: NLR family pyrin domain containing 3; ZBP1: Z-DNA binding protein 1.

PANOPTOSIS-RELATED LNCRNAS

In addition to the key molecules related to PANoptosis, ncRNAs play an important role in the pan-apoptotic process of HCC. He et al[85] identified 13 prognostic characteristic lncRNAs in HCC through analysis of The Cancer Genome Atlas-Liver Hepatocellular Carcinoma and Gene Expression Omnibus datasets. Among these, FAM99A, LINC01703, AL445524.1, and LINC01093 showed significant associations with multiple immune cell types[85]. A separate bioinformatics study identified 105 PANoptosis-related lncRNAs (PRLs) associated with HCC[86]. While AC026412.3, AC026356.1, and LINC01224 were reported in association with HCC prognosis, their specific mechanistic roles in tumorigenesis remain unexplored[87-89]. Notably, AC026412.3 exhibited the highest risk coefficient among all PRLs. Furthermore, a high PRL score correlated with an increased mutation rate of TP53 and a decreased mutation rate of CTNNB1. Given that combined detection of TP53 and CTNNB1 mutations facilitates early HCC identification[87], these lncRNAs represent promising novel biomarkers and therapeutic targets for HCC[90].

PANOPTOSIS-RELATED IMMUNE MARKERS

Cheng et al[91] employed single cell sequencing to construct a tumor heterogeneity atlas for HCC, identifying four cell subtypes most strongly associated with PANoptosis. Notably, reduced abundance of the PANoptosis-linked apolipoprotein (APO)⁺ endothelial cells (ECs) significantly correlated with poor HCC prognosis. Cell communication analysis suggested that APO⁺ ECs may modulate T-cell proliferation and differentiation via regulation of human leukocyte antigen molecule expression, potentially contributing to improved patient outcomes. Immunofluorescence staining localized APO⁺ ECs predominantly to peritumoral tissues, with markedly lower expression within tumor parenchyma. In vitro functional assays demonstrated that APO H⁺ human umbilical vein ECs significantly suppressed HCC proliferation. Collectively, these findings implicate APO family members in HCC pathogenesis. Future research should evaluate their potential as diagnostic biomarkers or therapeutic targets. Clinically, quantifying APO family expression could stratify patients and predict responses to therapies like immunotherapy or targeted agents[91]. Utilizing The Cancer Genome Atlas-Liver Hepatocellular Carcinoma data, Wang et al[92] developed a PRG signature comprising six genes (CYP26B1, CKAP2, SLAMF6, S100A9, IL7R, and TRIM54) that significantly predicted HCC prognosis. These genes are implicated across multiple cancer types. Immune microenvironment analysis revealed elevated ESTIMATE, Immune, and Stromal Scores in the low-risk group. Significant differences in immune cell infiltration and immune-related gene expression were observed between risk groups. This PRG signature demonstrates biomarker potential for guiding individualized HCC therapy[92].

PANOPTOSIS IN HCC TREATMENT

PANoptosis has demonstrated breakthrough potential in the field of HCC therapy. The traditional single-mode cell death pathway is susceptible to tumor heterogeneity and microenvironmental regulation, resulting in limited therapeutic efficacy. PANoptosis provides an innovative strategy to overcome drug resistance in HCC treatment by synergistically activating the three programmed death pathways of apoptosis, pyroptosis, and necroptosis, resulting in a multidimensional anti-tumor effect. From the perspective of microenvironmental regulation, PANoptosis-associated genes affect tumor angiogenesis, inflammatory response, and immunosuppressive status by regulating the level of eosinophil infiltration, and this multidimensional regulatory network ultimately leads to the malignant biological behavior of tumor growth and metastasis[74].

In a mouse model of HCC, TNF-α enhanced tumor cell-induced adaptive immune resistance against IFN-γ by up-regulating the expression of B7 homolog 1, a homologue of programmed death-ligand 1, a mechanism that may promote immune escape through inhibition of T cell function[44]. The novel anthraquinone derivative KLX can up-regulate ZBP1 and bind to it directly in an in vitro model of HCC, which can induce cellular PANoptosis by altering the conformation of ZBP1, decreasing the affinity of ZBP1 for the E3 ubiquitin ligase cyclic node finger protein 180, and decreasing the stability of ubiquitylation of ZBP1, thus increasing ZBP1 stability. Meanwhile, KLX can also induce PANoptosis by increasing ZBP1 expression through transcription factor isoform D10. KLX has also been shown to inhibit tumor growth in an HCC cell xenograft model with less toxicity than oxaliplatin[26]. OTU is a protein important for hepatoprotection and plays an important role in preventing hepatocyte apoptosis. In OTU knockout mice, hepatocyte apoptosis was primarily driven by FADD, caspase-8 and RIPK1 kinases. The study showed that OTU could protect hepatocytes by inhibiting FADD/RIPK1-related-mediated hepatocyte apoptosis, preventing some liver inflammation and HCC[78]. DNASE1 L3 is a member of the deoxyribonuclease 1 family that cleaves nuclear chromosomal DNA and induces AIM2 pathway activation via dsDNA production, which in turn triggers PANoptosis. Analysis of available datasets showed that prognostic improvement with sorafenib and anti-programmed death receptor 1 monotherapy was positively correlated with DNASE1 L3. In the context of HCC tissues from patients undergoing treatment with sorafenib in conjunction with programmed death receptor 1 monoclonal antibodies, there was a notable increase in DNASE1 L3 expression levels. Accelerated DNA cleavage occurred in HCC cells with the presence of DNASE1 L3 overexpression after sorafenib treatment, and the level of cytoplasmic dsDNA accumulation was also higher than that in HCC tissues of patients not treated with sorafenib[35]. Bioinformatics analysis demonstrated that shown that silencing of HMOX1 decreases sorafenib resistance in sorafenib-resistant HCC cells, while HMOX1 overexpression produced the opposite effect. Moreover, HMOX1 silencing decreased the mRNA expression of ABC transporter proteins in sorafenib-resistant HCC cells, thereby increasing HCC cell sensitivity to sorafenib[93]. However, there is no evidence to confirm the specific role of Fusobacterium nucleatum outer membrane vesicle (Fn-OMV) in combination with human alphaherpesvirus 1-based tumor lysing virus (oHSV), adenosine deaminase 1 (ADAR1), IFNs and nuclear export inhibitors in HCC in tissues, as well as their specific involvement in HCC occurrence and progression. However, the potential implications of these factors may offer novel perspectives for the treatment of HCC associated with PANoptosis.

Apart from some targeted PANoptosis tumor treatment strategies that have been specifically confirmed to play a role in vivo or in vitro in HCC, these studies have only demonstrated their impact on tumor development by affecting PANoptosis-related molecules, and there is still a lack of specific research on their effects in HCC. PPDPF has been demonstrated to have a significant role in HCC. In a DEN-induced mouse model, PPDPF can inhibit HCC development by regulating TRIM21’s ubiquitination of RIPK1. Mechanistically, PPDPF catalyzes RIPK1 K63-linked ubiquitination by interacting with RIPK1 and recruiting the E3 Ligase TRIM21. In contrast, in PPDPF knockout mice, liver cell apoptosis and compensatory proliferation increased[79]. It has been found that Fn-OMV can increase the expression of the PANoptosis execution proteins GSDMD, GSDME and MLKL. oHSV enhances IFN-stimulated gene expression, in which case endogenous Z-RNA accumulates and activates ZBP1. Combining Fn-OMV with oHSV enhances the inflammatory response, improves the effect of ICB, and exhibits greater antitumor immunogenicity than oHSV alone[94]. Binding of nuclear export inhibitors and IFN binding can also activate ZBP1-mediated cellular PANoptosis. ZBP1 expression can be regulated by the editor ADAR1, which acts as an editor to maintain in vivo RNA homeostasis. ADAR1 binds to the Za2 structural domain of ZBP1, thereby preventing ZBP1-induced cellular PANoptosis by binding to RIPK3, whereas ADAR1 inhibition activates ZBP1-mediated cellular PANoptosis, resulting in the inhibition of tumorigenesis[95].

In addition to traditional drug therapies, some nanotherapies related to PANoptosis are increasingly being investigated. With improved targeting efficiency, longer circulation time, and superior chemical and physical properties compared to conventional materials, nanomaterials can greatly enhance the efficacy and safety of therapy in regulating tumor cell metabolism and remodeling the TME with immunosuppression. In addition, nanotechnology can enable synergistic enhancement of immunotherapy and PANoptosis. Currently, there is a novel ultra-small nano-enzyme Bi2Sn2O7, which produces an enhanced enzyme kinetic effect through ultrasound-responsive catalysis, for the treatment of HCC and inhibition of lung metastasis. Bi2Sn2O7 breaks through the tumor antioxidant defense threshold by targeting and destroying tumor cell mitochondria, interfering with energy metabolism homeostasis and continuously amplifying intracellular reactive oxygen species levels. This dual mechanism of action effectively activates the PANoptosis signaling network, achieving synergistic anti-tumor effects by cleaving key molecules such as caspase-3 (apoptosis pathway), NLRP3 inflammatory vesicle activation and GSDMD protein cleavage (pyroptosis pathway), and RIPK3/MLKL phosphorylation cascade (necroptosis pathway). Experimental studies have confirmed that external ultrasound radiation can lead to a significant increase in the efficiency of enzyme kinetic therapy[96]. Fullerenols are nanomaterials containing multiple hydroxyl groups, which, unlike few numbers of hydroxyl groups, can disrupt lysosomal phospholipid membranes and form structural voids that cause calcium ions and proteolytic enzymes to leak out of the lysosome into the cytoplasm, further affecting mitochondrial function and inducing endoplasmic reticulum stress. mTOR inhibitors can block mTOR-mediated signaling, and mTOR inhibitors can block mTOR-mediated signaling and enhance autophagic flux before autophagy is blocked. The combination of fullerenols containing multiple hydroxyl groups and mTOR inhibitors further disrupts lysosomal, mitochondrial, and endoplasmic reticulum interactions, exacerbates organelle homeostasis, activates cytosolic PANoptosis, and produces potent tumor suppression in in vitro and in vivo assays (zebrafish and mouse models)[97]. A therapeutic strategy to activate tumor immune reprogramming by ultrasound nanomedicines has been proposed, which centers on the use of exocysts secreted by nano/genetically engineered cells to induce highly immunogenic cellular PANoptosis in tumor cells, in which DAMPs are continuously released to activate the innate immune response cycle, which is iteratively reinforced to drive the effective activation of antigen-specific T cells and establish a systemic immune defense mechanism by means of the cyclic GMP-AMP synthase-STING signaling pathway, leading to increased efficacy of cancer immunotherapy. Furthermore, it leads to establishment of a systemic immune defense mechanism with the help of the cyclic GMP-AMP synthase-STING signaling pathway, resulting in improved efficacy of cancer immunotherapy. When combined with immune checkpoint blockade therapy, tumor immune microenvironment reconstruction can have a synergistic effect of intrinsic and adaptive immunity (Table 1)[98].

Table 1 PANoptosis and cancer therapeutic strategies.

Potential therapeutic strategy	Name	Effect	Mechanism	Ref.
Traditional laboratory therapy	TNF-α/IFN-γ	-	Triggers a PANoptosis cascade, exhibiting anti-tumor activity	[44]
		+	May promote immune escape by inhibiting T-cell function	-
	KLX	-	Induces PANoptosis by altering ZBP1 conformation.	[26]
	PPDPF	-	Regulates TRIM21 to ubiquitinate RIPK1, further inhibiting HCC progression	[78]
	OTULIN	-	Inhibits FADD/RIPK1-mediated hepatocyte apoptosis, protecting liver cells and preventing liver inflammation and HCC	[77]
	DNASE1 L3	-	Activates the AIM2 pathway, thereby inducing PANoptosis	[35]
	HMOX1	+	Silencing reduces HCC resistance to sorafenib	[92]
	Fn-OMV/oHSV	-	Combined use enhances inflammatory responses and improves immune checkpoint blockade (ICB) efficacy	[93]
	ADAR1	+	Blocking ADAR1 activates ZBP1-mediated PANoptosis, inhibiting tumorigenesis	[94]
Novel nanotherapies	Bi2Sn2O7	-	Through ultrasound-responsive catalytic effects, generates enhanced enzyme kinetic effects for HCC treatment and inhibition of later-stage lung metastasis; also targets mitochondrial disruption, disrupting energy metabolism and amplifying ROS levels	[95]
	Fullerenols	-	Affects mitochondrial function and induces endoplasmic reticulum stress	[96]

ADAR1: Adenosine deaminase, RNA-specific 1; AIM2: Absent in melanoma 2; Bi2Sn2O7: Bismuth tin oxide; DNASE1 L3: Deoxyribonuclease 1 class 3; FADD: Fas-associated protein with death domain; Fn-OMV: Fusobacterium nucleatum outer membrane vesicle; HCC: Hepatocellular carcinoma; HMOX1: Heme oxygenase 1; ICB: Immune checkpoint blockade; IFN-γ: Interferon-gamma; KLX: Kanglexin; oHSVs: Human alphaherpesvirus 1-based oncolytic viruses; OS: Oxidative stress; OTULIN: OTU deubiquitinase with linear linkage specificity; PPDPF: Pancreatic progenitor cell differentiation and proliferation factor; RIPK1: Receptor-interacting serine/threonine kinase 1; TNF-α: Tumor necrosis factor-alpha; TRIM21: Tripartite motif containing 21; ZBP1: Z-DNA binding protein 1.

CONCLUSION

PANoptosis is a novel PCD concept proposed in 2019 that integrates three classical PCD pathways, focal death, apoptosis, and necroptosis, containing key molecules of all three. PCD has been gaining traction in tumor research, and therapeutic techniques that target PANoptosis may be more effective than a single PCD route. PANoptosis plays a key role in a variety of illnesses, particularly cancer. Therefore, therapeutic techniques that target and regulate molecules in the PANoptosis pathway hold considerable potential. In terms of the molecular mechanism of PANoptosis, four types of PANoptosomes-ZBP1-PANoptosome, AIM2-PANoptosome, RIPK1-PANoptosome, and NLRP12-PANoptosome-have been identified and shown to be involved in some PANoptosis mechanisms. Many targeted drug therapies for these key pathway components have also emerged, as well as the application of nanotechnology in addition to traditional therapeutic approaches. Some of the traditional PANoptosis-targeted drugs can inhibit tumorigenesis and progression, but there are still problems, including systemic adverse effects, tumor cell resistance, low drug bioavailability, and difficulty in clinical translation. Although nanotechnology can take advantage of the excellent physicochemical characteristics of tumor tissues and can be used in conjunction with ultrasound and other adjuvant therapies to characteristically activate stronger PANoptosis, the toxicity and biosafety of nanomaterials are concerns.

Future therapeutic strategies for PANoptosis-related HCC can be further studied in the following aspects. First, in terms of the mechanism of HCC-specific PANoptosis, it is necessary to analyze the composition dynamics of the PANoptosome in HCC and identify the interaction patterns of core molecules such as ZBP1, RIPK1/RIPK3, and caspase family in the HCC microenvironment. Additional questions include: What molecules are contained in the HCC cell-specific PANoptosome complex structure? How does metabolic reprogramming and epigenetic modification regulate PANoptosome activation? What is the mechanism of the association between TME and immune escape. At present, PANoptosis and TME analysis is restricted to bioinformatics. How do cytokines released by immune cells through PANoptosis remodel immunosuppressive TME? Secondly, in the development of precise intervention strategies targeting PANoptosis, dual-target inhibitors (such as RIPK1-caspase-8 and ZBP1-NLRP3) should be explored to overcome compensatory drug resistance caused by single-pathway inhibition. It is also possible to target HCC-specific PRGs through nanomaterials or other delivery systems, and to explore the synergistic mechanism of PANoptosis and immunotherapy by establishing PRG feature-based prediction models for immunotherapy efficacy. Finally, in the clinic, new biomarkers can be developed by using multi-omics techniques to identify PANoptosis-related cell subsets in TME as prognostic markers. Furthermore, the activation of PANoptosis needs to be accurately controlled in terms of treatment, as over-activation could cause create an inflammatory storm in the body, while over-suppression could affect surrounding healthy tissues.