Since it makes up 90% of primary liver malignancies, hepatocellular carcinoma (HCC) is the most common histological subtype of liver cancer and has a high death rate. It ranks as the third most frequent cause of cancer-related mortality globally [1], and is especially common in Asia, Africa and southern Europe [2]. HCC is known as the “king of cancers” because of its biological characteristics, such as high malignancy, strong invasiveness and easy metastasis. The main occurrence factors of HCC include genetic and epigenetic changes, chronic hepatitis B virus or hepatitis C virus exposure, aflatoxin exposure, smoking, obesity and diabetes [1, 3]. The incidence of HCC associated with metabolic dysfunction-associated fatty liver disease (MAFLD) is increasing in Western countries [4], and the high recurrence and metastasis rates of HCC lead to serious adverse prognoses [5]. Although patients with HCC have a wide range of treatment options, including liver transplantation, surgical resection, percutaneous ablation and radiotherapy, as well as standard treatment methods such as transarterial and systemic therapy [6], more than 70% of advanced cases are not suitable for transplantation or surgery [7]. Current studies on the dysregulation of key signaling pathways, epigenetic modifications, and the tumor microenvironment in HCC progression have provided novel insights for intervention strategies [8, 9, 10]. However, the current understanding of the molecular mechanisms underlying HCC progression and metastasis remains incomplete. In-depth investigation of its pathological mechanisms is crucial for identifying early biomarkers and improving treatment approaches.

Lon protease 1 (LONP1) is a highly conserved adenosine triphosphate (ATP)-dependent protease encoded by nuclear DNA and located in the mitochondrial matrix. This protein mediates the selective degradation of misfolded or oxidation-damaged polypeptides in the mitochondrial matrix, maintains the mitochondrial protein balance and regulates adaptive responses to cellular stress [11, 12]. Mitochondrial dysfunction is a signature phenomenon in cancer biology and may be one of the reasons for the transformation of mitochondrial energy metabolism in tumor tissues from aerobic respiration to glycolysis, a process that requires less oxygen. Dysfunction of LONP1 directly leads to the failure of mitochondrial protein degradation, and the accumulation of abnormal mitochondrial proteins leads to a series of types of cells and tissue damage. Previous studies have shown that the upregulation of LONP1 helps lung fibroblasts adapt to acute stress and is important for maintaining normal cell viability [13] and overcoming hypoxia and metabolic and proteotoxic stress associated with the oncogenic transformation of tumor cells [14]. Silencing LONP1 leads to apoptosis and mitochondrial damage in colon cancer cells, and LONP1 is involved in epithelial‒mesenchymal transition (EMT) in colon cancer cells [15]. It has shown that some long non-coding RNAs (lncRNAs) impair CD4+ T-cell activation by targeting LONP1, leading to a poor prognosis in patients with gastric cancer [16]. Helicobacter pylori partially induce LONP1 expression and overgrowth of gastric cancer cells through HIF1$\alpha$ regulation [17].

An iron-dependent type of regulatory cell death linked to an excessive buildup of lipid peroxides is known as ferroptosis [18, 19], and limiting cytotoxic lipid peroxidation protects cells against ferroptosis. Current studies on the mechanism of ferroptosis have confirmed that inhibiting the activity of System Xc^– and glutathione peroxidase 4 (GPX4) can induce ferroptosis, which is inhibited by erastin and RSL3, respectively [20, 21]. GPX4 is the main enzyme that catalyzes the neutralization of phospholipid hydroperoxides (PLOOH) in mammalian cells, peroxidation of lipid hydrogen and reduction to the corresponding alcohol. GPX4 gene deletion is known to cause lipid peroxidation-dependent cell death in mouse embryonic fibroblasts [22], and uncontrolled lipid peroxidation is a marker of ferroptosis. Acyl-coa synthase long chain family member 4 (ACSL4) is an important driver of ferroptosis [23, 24] and is regulated by multiple signaling pathways [25, 26]. The expression of ACSL4 in the triple-negative breast cancer cell line subpopulation is related to its sensitivity to ferroptosis inducers [23], and this correlation also exists in clear cell renal cancer cells [24]. p53 may promote ferroptosis by inhibiting the transcription of solute carrier family 7 member 11 (SLC7A11), the subunit of System Xc^–, and thus exerting its tumor suppressor function [27]. Arachidonate 12-lipoxygenase (ALOX12) has been reported to be essential for tumor protein 53 (p53)-dependent ferroptosis induced by peroxide [28]. It has reported that PE binding protein-1 (PEBP1) can interact with some lipoxygenases (LOXs) and participate in the regulation of ferroptosis [29]. The protein encoded by the aconitase 2 (ACO2) gene belongs to the cis-aconitase/isopropylmalate (IPM) isomerase family. This protein, which is encoded in the nucleus and plays a role in mitochondria, is a mitochondrial matrix protein that catalyzes the mutual conversion of citric acid to isocitric acid through cisaconite acid in the second step of the tricarboxylic acid cycle (TCA). It is involved mainly in the anabolism and catabolism of organisms, such as DNA repair and replication, as well as protein synthesis and degradation, and its expression products are widely distributed in the heart, kidney and other tissues and organs. Studies have shown that pathogenic gene variation or disordered expression of ACO2 is involved in neurodegenerative syndromes such as optic neuropathy [30] and Parkinson’s disease [31]. Abnormal expression of the Fe-S cluster protein ACO2 leads to mitochondrial dysfunction, and abdominal aortic aneurysm [32]. In addition, mitochondrial dysfunction leads to metabolic changes and affects the activation process of macrophages [33].

Ferroptosis is closely related to HCC development, but its mechanism of action remains unclear [34, 35]. Our study revealed that silencing LONP1 leads to the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS) in liver cancer cells, suggesting that abnormal LONP1 expression is associated with ferroptosis in these cells. However, the role of LONP1 in iron-related death in liver cancer is unknown. In this project, the expression, biological function and mechanism of LONP1 were studied in the field of HCC. These findings help to elucidate the new mechanism of HCC development and metastasis, provide new ideas for the treatment and diagnosis of HCC, and may also provide important reference value for similar studies of other tumors or diseases.

HCC is the fourth leading cause of cancer-related death and continues to increase worldwide, representing a major challenge for global health care. The biological process of HCC is complex and involves a variety of factors, and regulation of liver cancer cell differentiation and angiogenesis [36]. However, owing to the insidious onset of HCC, most HCC patients are already in the advanced stage when diagnosed and can only receive systemic therapy [37]. Systemic antitumor therapy is the best treatment for patients with advanced liver cancer. With the approval of several new drugs for the treatment of advanced HCC and the establishment of checkpoint inhibitor-based therapy as the standard of care [38], the therapeutic landscape for advanced HCC is more diverse than ever before.

In this study, statistical analysis of RNAseq samples of the LONP1 gene in the database revealed that the gene was differentially expressed in a variety of malignant tumors and their adjacent or normal tissues and was significantly highly expressed in HCC, suggesting that the protein encoded by the LONP1 gene plays an important role in tumor occurrence and development. We subsequently analyzed the expression level of the LONP1 protein in the liver cancer microtissue chip, and the results were mutually confirmed with the RNA-seq data, indicating that LONP1 was significantly highly expressed in liver cancer. Finally, these results were also verified via RT-qPCR and IHC experiments. Additionally, the development of therapeutic strategies targeting LONP1 could potentially alleviate the clinical pathology in HCC patients. After identifying the significant correlation between LONP1 and the occurrence and development of liver cancer, we further explored the function and mechanism of this gene at the cellular and animal levels.

Given the high expression of LONP1 in HCC, we first investigated whether the proliferation and migration of HCC cells were affected by LONP1 expression. To support long-term functional studies, we adopted a lentivirus interference strategy to construct stable Huh7 cell lines with low knockdown and overexpression of LONP1. The results showed that knocking down LONP1 inhibited the proliferation and migration of Huh7 cells, whereas overexpressing LONP1 increased the proliferation and migration of Huh7 cells. We subsequently verified these findings at the animal level, and the results showed that the inhibition of LONP1 expression significantly inhibited the growth of Huh7 tumors in vivo. We found that LONP1 is a tumor-promoting factor in HCC. These results are consistent with our previous findings and further elucidate the clinical value and significance of targeting LONP1.

This suggests that the aberrant expression of LONP1 in HCC may be associated with the ferroptosis process in tumor cells. Furthermore, LONP1 may interact with other mitochondrial proteins to regulate cell ferroptosis. Subsequently, we investigated the impact of the LONP1 gene on the ferroptosis process. siRNA strategy was used in Huh7 cells to lower the expression of LONP1, and changes in the ferroptosis-related indicators GSH/GSSG ratio and MDA accumulation were detected. The results revealed that when LONP1 was silenced as a single factor, the MDA contents were increased in Huh7 cells, but the GSH/GSSG ratio remained unchanged. However, the GSH/GSSG ratio was significantly increased when erastin treatment was combined with LONP1 silencing, suggesting that the ability of LONP1 to promote ferroptosis was weakened when the cells were severely damaged. These experimental results are consistent with existing studies in the field of chronic kidney diseases, and inhibition of the LONP1 gene can aggravate kidney injury and mitochondrial dysfunction [39].

In terms of the mechanism of action of LONP1, many studies have reported that LONP1 plays a role in protecting cell homeostasis by regulating mitochondrial function. LONP1 is a mitochondrial protease that is involved in the degradation of abnormal proteins in mitochondria due to oxidative damage or misfolding and is extremely important for maintaining the normal function of mitochondria [40, 41]. LONP expression induced by LONP1 in the liver can counteract the silencing effect of mitochondrial transcription factor A (TFAM), thereby maintaining the copy number of mitochondrial DNA (mtDNA) and mitochondrial function [42]. The inhibition of mitochondrial LONP1 activity by specific siRNAs can affect liver function [43, 44]. In addition, abnormalities in LONP1 are also associated with the development of other tumors [15], and are involved in cardiac stress [45]. As ACO2 is a mitochondrial protein, it plays a key role in TCA cycle, maintenance of iron homeostasis, oxidative stress defense, and integrity of mtDNA [46]. In this study, we found that knocking down LONP1 in Huh7 cells can promote the upregulated expression of ACO2. Correspondingly, the overexpression of LONP1 inhibited the expression of ACO2. ACO2 is a key mitochondrial protein involved in the TCA cycle, suggesting that LONP1 may influence mitochondrial function by modulating ACO2 expression, thereby regulating the proliferation and migration of liver cancer cells. Studies have shown that ACO2 affects the tumorigenicity of non-small cell lung cancer by regulating iron homeostasis [47]. ACO2 dysfunction can promote mitochondrial dysfunction, thus affecting the occurrence and development of different diseases [48]. In addition, we propose other hypotheses concerning the molecular mechanism of the cellular function and related signaling pathways of LONP1 [49, 50, 51, 52]. GPX4 converts reduced GSH to GSSG, thereby protecting cells against ferroptosis by limiting cytotoxic lipid peroxidation [53]. The GPX4 pathway is further regulated by amino acid antitransporters with xCT/SLC7A11 participation [54, 55, 56]. Importantly, the ferroptosis inducer erastin works by directly inhibiting the function of this heterodimer. In this study, the inhibition of LONP1 expression and erastin treatment resulted in an increased GSH/GSSG ratio and increased MDA accumulation, indicating that the pathway of GSH to GSSG transformation was blocked and that GPX4 may be inactivated. In other words, LONP1 may be involved in the regulation of ferroptosis via this pathway, thereby promoting cell proliferation, migration, and tumor formation. Based on relevant detection indicators, it is plausible that LONP1 exerts its effects through GPX4. It is important to note that no interactions between GPX4 and ACO2 have been reported in the literature. This mechanism remains underexplored and will be a key focus of our future studies. And we will enhance our investigation of mitochondrial function and elucidate the potential mechanisms underlying the LONP1/ACO2 axis in greater detail. LONP1 can also be used to predict the prognosis, immunotherapy response and sorafenib sensitivity of HCC patients [57]. p53, as a key tumor suppressor, plays a significant role in regulating apoptosis, autophagy and ferroptosis. Previous studies have shown that p53 can affect the ferroptosis process by regulating the expression of SLC7A11[27].

This study reveals a novel mechanism by which LONP1 inhibits ferroptosis in liver cancer by specifically degrading ACO2. This discovery expands our understanding of the carci9genic effect of LONP1 and shows similarities and differences with other types of cancer. Unlike the direct regulation of ferroptosis by LONP1 in this study, research in malignant tumors such as pancreatic cancer, breast cancer and ovarian cancer mostly emphasizes that LONP1 promotes tumor survival and proliferation by maintaining mitochondrial protein homeostasis, energy metabolism (such as stabilizing the key enzyme isocitrate dehydrogenase 2 (IDH2) of the TCA cycle) and resisting apoptosis (such as degrading pro-apoptotic factors). For instance, in glioblastoma, LONP1 has been reported to maintain mitochondrial genomic stability by eliminating oxidative damage proteins, but its association with the ferroptosis pathway has not been elaborated in depth. In colorectal cancer (though excluded but used as a comparison point) and lung cancer, the pro-cancer effect of LONP1 is often associated with antioxidant stress (such as regulating the nuclear factor erythroid 2-related factor 2 (NRF2) pathway) and metabolic reprogramming. Therefore, this study found that LONP1 directly alleviates ferroptosis by targeting and degrading ACO2. This not only provides a new perspective for the occurrence of liver cancer but also highlights the tissue or environmental specificity of LONP1’s carcinogenic function—in liver cancer, its unique ability to counteract ferroptosis may constitute a key carcinogenic driving force. This is significantly different from the mode in which LONP1 mainly supports basal metabolism and anti-apoptosis in other cancers, suggesting the potential of tissue-specific therapeutic targeting strategies.

In our study, LONP1 alleviated ferroptosis by degrading ACO2, thereby promoting the occurrence of HCC. However, the role of LONP1 is not limited to intracellular metabolic regulation; its role in the tumor immune microenvironment is also worthy of attention. Furthermore, the mechanism by which lncRNAs regulate LONP1 in gastric cancer [16] suggests that there may be similar pathways associated with HCC. These lncRNAs may further affect the activity of immune cells in the HCC microenvironment by regulating the expression of LONP1. Therefore, future research should delve deeply into the specific role of LONP1 in immune regulation in HCC, so as to gain a more comprehensive understanding of its multiple mechanisms in the occurrence and development of hepatocellular carcinoma.

In this study, highly expressed LONP1 in HCC participated in the regulation of ferroptosis, promoted the proliferation and migration of Huh7 cells, and thus affected tumor formation. Silencing its expression in HCC may play an important role in providing a new way to stop the progression of clinical HCC. Validation using bioinformatics data and clinical tissue microarrays from our research group suggests that LONP1 may exert its oncogenic effects through ACO2. We hypothesize that LONP1, at normal expression levels, primarily functions to maintain mitochondrial function in normal liver tissue. However, in cancerous tissues with overexpressed LONP1, the excess LONP1 may dissociate from the mitochondria and regulate the expression of ACO2, thereby inhibiting ferroptosis in liver cancer cells. A limitation of this study is that we have not explored the molecular regulatory mechanisms between LONP1, ACO2, and GPX4. Due to time constraints, we regret that we are unable to present research findings on this aspect at this time. Additionally, the biological regulatory roles of LONP1 and ACO2 in normal liver tissue have not been fully investigated.

By preventing ferroptosis and cuproptosis through the breakdown of ACO2, LONP1 stimulates HCC migration and proliferation. LONP1 targeting may be a useful therapeutic approach to stop the formation of HCC.

The datasets used or analyzed during the current study are available from the corresponding authors on reasonable request.

YZ, LW: Designed the experiments, Performed the experiments; Collected and analyzed data; Wrote the paper. ZP, WM, TW: technique support. BW, ZY: Designed the experiments and supervision. XL, EC: Designed the experiments, Supervision, and Funding acquisition. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

This retrospective study was approved by the Institutional Review Board of Peking University Shenzhen Hospital (Approval Number: [2022]No.(164)). Given the retrospective nature of the study and the use of de-identified patient data, the requirement for informed consent was waived by the IRB. The study was conducted in accordance with the ethical standards of the Declaration of Helsinki and its later Follow-up review (Approval Number: [2022]No.(164-Extension 1)). The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Peking University Shenzhen Hospital (protocol code 2023-959) for studies involving animals. All animal experiments and operations strictly adhere to the experimental protocols approved by the Laboratory Animal Ethics Committee of Peking University Shenzhen Hospital, as well as national and international laboratory animal welfare guidelines. All experimental animals were euthanized by trained researchers proficient in the cervical dislocation method.

We would like to express our gratitude to all those who helped me during the writing of this manuscript, thanks to all the peer reviewers for their opinions and suggestions.

This work was supported by the National Natural Science Foundation of China (82303446), the Shenzhen High-level Hospital Construction Fund and Peking University Shenzhen Hospital Scientific Research Fund (KYQD2023303), GuangDong Basic and Applied Basic Research Foundation (2023A1515220200), Medical Scientific Research Foundation of Guangdong Province of China (A2024351) and the Science and Technology Development Fund Project of Shenzhen (JCYJ20190809095011463).

The authors declare no conflict of interest.