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Background Ferroptosis is the process of cell death triggered by lipid peroxides, and inhibition of glutathione (GSH) synthesis leads to ferroptosis. Liver cancer progression is closely linked to ferroptosis suppression. However, the mechanism by which inhibition of GSH synthesis suppresses potential ferroptosis of liver cancer cells and whether ferroptosis-related liver cancer biomarkers have a promising diagnostic value remain unknown. Methods Ribonucleotide reductase regulatory subunit M2 (RRM2) levels were measured using an enzyme linked immunosorbent assay (ELISA), quantitative RT-PCR (qPCR), immunoblotting (IB) and immunochemistry (IHC). Cell viability and cell death were measured by a CellTiter-Glo luminescent cell viability assay and staining with SYTOX Green followed by flow cytometry, respectively. Metabolites were measured using the indicated kits. The Interaction between glutathione synthetase (GSS) and RRM2 was measured using immunofluorescence (IF), co-immunoprecipitation (co-IP) and the proximal ligation assay (PLA). The diagnostic value was analyzed using the area under the receiver operating characteristic curve (AUC-ROC). Bioinformatics analysis was performed using the indicated database. Results RRM2 showed specifically elevated levels in liver cancer and inhibited ferroptosis by stimulating GSH synthesis via GSS. Mechanistically, phosphorylation of RRM2 at the Threonine 33 residue (T33) was maintained at normal levels to block the RRM2–GSS interaction and therefore protected RRM2 and GSS from further proteasome degradation. However, under ferroptotic stress, RRM2 was dephosphorylated at T33, thus the RRM2–GSS interaction was promoted. This resulted in the translocation of RRM2 and GSS to the proteasome for simultaneous degradation. Clinically, serum RRM2 was significantly associated with serum alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), albumin (ALB) and total bilirubin. The AUC-ROC for the combination of RRM2 with AFP was 0.947, with a sensitivity of 88.7% and a specificity of 97.0%, which indicates better diagnostic performance compared to either RRM2 or AFP alone. Conclusion RRM2 exerts an anti-ferroptotic role in liver cancer cells by sustaining GSH synthesis. Serum RRM2 will be useful as a biomarker to evaluate the degree to which ferroptosis is suppressed and improve diagnostic efficiency for liver cancer.

The regulation of homeostasis in the Ubiquitin (Ub) proteasome system (UPS) is likely to be important for the development of liver cancer. Tribbles homolog 2 (TRIB2) is known to affect Ub E3 ligases (E3s) in liver cancer. However, whether TRIB2 regulates the UPS in other ways and the relevant mechanisms are still unknown. Here, we reveal that TRIB2 decreased Ub levels largely by stimulating proteasome degradation of Ub. In the proteasome, proteasome 20S subunit beta 5 (PSMB5) was critical for the function of TRIB2, although it did not directly interact with TRIB2. However, poly (rC) binding protein 2 (PCBP2), which was identified by mass spectrometry, directly interacted with both TRIB2 and PSMB5. PCBP2 was a prerequisite for the TRIB2 induction of PSMB5 activity and decreased Ub levels. A significant correlation between TRIB2 and PCBP2 was revealed in liver cancer specimens. Interestingly, TRIB2 suppressed the K48-ubiquitination of PCBP2 to increase its level. Therefore, a model showing that TRIB2 cooperates and stimulates PCBP2 to reduce Ub levels was established. Additionally, the reduction in Ub levels induced by TRIB2 and PCBP2 was dependent on K48-ubiquitination. PCBP2 was one of the possible downstream factors of TRIB2 and their interaction relied on the DQLVPD element of TRIB2 and the KH3 domain of PCBP2. This interaction was necessary to maintain the viability of the liver cancer cells and promote tumor growth. Mechanistically, glutathione peroxidase 4 functioned as one of the terminal effectors of TRIB2 and PCBP2 to protect liver cancer cells from oxidative damage. Taken together, the data indicate that, in addition to affecting E3s, TRIB2 plays a critical role in regulating UPS by modulating PSMB5 activity in proteasome to reduce Ub flux, and that targeting TRIB2 might be helpful in liver cancer treatments by enhancing the oxidative damage induced by therapeutic agents.

Background Lung adenocarcinoma (LUAD)  is the most common subtype of lung cancer. Patient prognosis is poor, and the existing therapeutic strategies for LUAD are far from satisfactory. Recently, targeting N6-methyladenosine (m 6 A) modification of RNA has been suggested as a potential strategy to impede tumor progression. However, the roles of m 6 A modification in LUAD tumorigenesis is unknown. Methods Global m 6 A levels and expressions of m 6 A writers, erasers and readers were evaluated by RNA methylation assay, dot blot, immunoblotting, immunohistochemistry and ELISA in human LUAD, mouse models and cell lines. Cell viability, 3D-spheroid generation, in vivo LUAD formation, experiments in cell- and patient-derived xenograft mice and survival analysis were conducted to explore the impact of m 6 A on LUAD. The RNA-protein interactions, translation, putative m 6 A sites and glycolysis were explored in the investigation of the mechanism underlying how m 6 A stimulates tumorigenesis. Results The elevation of global m 6 A level in most human LUAD specimens resulted from the combined upregulation of m 6 A writer methyltransferase 3 (METTL3) and downregulation of eraser alkB homolog 5 (ALKBH5). Elevated global m 6 A level was associated with a poor overall survival in LUAD patients. Reducing m 6 A levels by knocking out METTL3 and overexpressing ALKBH5 suppressed 3D-spheroid generation in LUAD cells and intra-pulmonary tumor formation in mice. Mechanistically, m 6 A-dependent stimulation of glycolysis and tumorigenesis occurred via enolase 1 (ENO1). ENO1 mRNA was m 6 A methylated at 359 A, which facilitated it’s binding with the m 6 A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and resulted in enhanced translation of ENO1. ENO1 positively correlated with METTL3 and global m 6 A levels, and negatively correlated with ALKBH5 in human LUAD. In addition, m 6 A-dependent elevation of ENO1 was associated with LUAD progression. In preclinical models, tumors with a higher global m 6 A level showed a more sensitive response to the inhibition of pan-methylation, glycolysis and ENO activity in LUAD. Conclusions The m 6 A-dependent stimulation of glycolysis and tumorigenesis in LUAD is at least partially orchestrated by the upregulation of METTL3, downregulation of ALKBH5, and stimulation of YTHDF1-mediated ENO1 translation. Blocking this mechanism may represent a potential treatment strategy for m 6 A-dependent LUAD.

Tribbles homolog 2 (TRIB2) is known to boost liver tumorigenesis via regulating Ubiquitin (Ub) proteasome system (UPS). At least two ways are involved, i.e., acts as an adaptor protein to modulate ubiquitination functions of certain ubiquitin E3 ligases (E3s) and reduces global Ub levels via increasing the proteolysis activity of proteasome. Recently, we have identified the role of TRIB2 to relieve oxidative damage via reducing the availability of Ub that is essential for the ubiquitination and subsequent degradation of Glutathione peroxidase 4 (GPX4). Although GPX4 is a critical antioxidant factor to protect against ferroptosis, the exact evidence showing that TRIB2 desensitizes ferroptosis is lacking. Also, whether such function is via E3 remains unclear. Here, we demonstrated that deletion of TRIB2 sensitized ferroptosis via lifting labile iron in liver cancer cells. By contrast, overexpression of TRIB2 led to the opposite outcome. We further demonstrated that transferrin receptor (TFRC) was required for TRIB2 to desensitize the cells to ferroptosis. Without TFRC, the labile iron pool could not be reduced by overexpressing TRIB2. We also found that beta-transducin repeat containing E3 ubiqutin protein ligase (βTrCP) was a genuine E3 for the ubiquitination of TFRC, and TRIB2 was unable to decline labile iron level once upon βTrCP was knocked out. In addition, we confirmed that the opposite effects on ferroptosis and ferroptosis-associated lipid reactive oxygen species (ROS) generation resulted from knockout and overexpression of TRIB2 were all indispensible of TFRC and βTrCP. Finally, we demonstrated that TRIB2 exclusively manipulated RSL3- and erastin-induced-ferroptosis independent of GPX4 and glutathione (GSH). In conclusion, we elucidated a novel role of TRIB2 to desensitize ferroptosis via E3 βTrCP, by which facilitates TFRC ubiquitiation and finally decreases labile iron in liver cancer cells.

Background Ferroptosis, a form of regulated cell death, is an important topic in the field of cancer research. However, the signalling pathways and factors that sensitise tumour cells to ferroptosis remain elusive. Methods We determined the level of ferroptosis in cells by measuring cell death and lipid reactive oxygen species (ROS) production. The expression of RB1‐inducible coiled‐coil 1 (RB1CC1) and related proteins was analyzed by immunoblotting and immunohistochemistry. Immunofluorescence was used to determine the subcellular localization of RB1CC1. We investigated the mechanism of RB1CC1 nuclear translocation by constructing a series of RB1CC1 variants. To examine the ferroptosis‐ and RB1CC1‐dependent transcriptional program in tumour cells, chromatin immunoprecipitation sequencing was performed. To assess the effect of c‐Jun N‐terminal kinase (JNK) agonists on strenthening imidazole ketone erastin (IKE) therapy, we constructed cell‐derived xenograft mouse models. Mouse models of hepatocellular carcinoma to elucidate the importance of Rb1cc1 in IKE‐based therapy of liver tumourigenesis. Results RB1CC1 is upregulated by lipid ROS and that nuclear translocation of phosphorylation of RB1CC1 at Ser537 was essential for sensitising ferroptosis in tumour cells. Upon ferroptosis induction, nuclear RB1CC1 sharing forkhead box (FOX)‐binding motifs recruits elongator acetyltransferase complex subunit 3 (ELP3) to strengthen H4K12Ac histone modifications within enhancers linked to ferroptosis. This also stimulated transcription of ferroptosis‐associated genes, such as coiled‐coil–helix–coiled‐coil–helix domain containing 3 (CHCHD3), which enhanced mitochondrial function to elevate mitochondrial ROS early following induction of ferroptosis. FDA‐approved JNK activators reinforced RB1CC1 nuclear translocation and sensitised cells to ferroptosis, which strongly suggested that JNK is upstream of RB1CC1. Nuclear localisation of RB1CC1 correlated with lipid peroxidation in clinical lung cancer specimens. Rb1cc1 was essential for ferroptosis agonists to suppress liver tumourigenesis in mice. Conclusions Our findings indicate that RB1CC1‐associated signalling sensitises tumour cells to ferroptosis and that targeting RB1CC1 may be beneficial for tumour treatment. Nuclear translocation of RB1CC1 sensitises tumour cells to ferroptosis. Phosphorylation at S537 by JNK is prerequisite for RB1CC1 to translocate into nucleus. RB1CC1 recruits ELP3 to reinforce H4K12Ac within ferroptosis‐associated enhancers, and RB1CC1 activates transcriptional reprogramming to enhance mitochondrial function. RB1CC1 is an indispensible target for ferroptosis‐based anti‐tumour therapy

Yes-associated protein (YAP) activation is crucial for tumor formation and development, and its stability is regulated by ubiquitination. ISGylation is a type of ubiquitination like post-translational modification, whereas whether YAP is ISGylated and how ISGylation influences YAP ubiquitination-related function remains uncovered. In addition, YAP can activate glucose metabolism by activating the hexosamine biosynthesis pathway (HBP) and glycolysis, and generate a large number of intermediates to promote tumor proliferation. However, whether YAP stimulates the pentose phosphate pathway (PPP), another tumor-promoting glucose metabolism pathway, and the relationship between this stimulation and ISGylation needs further investigation. Here, we found that YAP was ISGylated and this ISGylation inhibited YAP ubiquitination, proteasome degradation, interaction with-beta-transducin repeat containing E3 ubiquitin-protein ligase (βTrCP) to promote YAP stability. However, ISGylation-induced pro-YAP effects were abolished by YAP K497R (K, lysine; R, arginine) mutation, suggesting K497 could be the major YAP ISGylation site. In addition, YAP ISGylation promoted cell viability, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) tumor formation. YAP ISGylation also increased downstream genes transcription, including one of the key enzymes of PPP, 6-phosphogluconolactonase (6PGL). Mechanistically, YAP promoted 6PGL transcription by simultaneously recruiting SMAD family member 2 (SMAD2) and TEA domain transcription factor 4 (TEAD4) binding to the 6PGL promoter to activate PPP. In clinical lung adenocarcinoma (LUAD) specimens, we found that YAP ISGylation degree was positively associated with 6PGL mRNA level, especially in high glucose LUAD tissues compared to low glucose LUAD tissues. Collectively, this study suggested that YAP ISGylation is critical for maintaining its stability and further activation of PPP. Targeting ISGylated YAP might be a new choice for hyperglycemia cancer treatment.

Detecting exosomal markers using laser desorption/ionization time‐of‐flight mass spectrometry (LDI‐TOF MS) is a novel approach for examining liquid biopsies of non‐small cell lung cancer (NSCLC) samples. However, LDI‐TOF MS is limited by low sensitivity and poor reproducibility when analyzing intact proteins directly. In this report, gold nanoparticles/cellulose nanocrystals (AuNPs/CNC) is introduced as the matrix for direct analysis of intact proteins in NSCLC serum exosomes. AuNPs/CNC with “dual dispersion” effects dispersed and stabilized AuNPs and improved ion inhibition effects caused by protein aggregation. These features increased the signal‐to‐noise ratio of [M+H]+ peaks by two orders of magnitude and lowered the detection limit of intact proteins to 0.01 mg mL–1. The coefficient of variation with or without AuNPs/CNC is measured as 10.2% and 32.5%, respectively. The excellent reproducibility yielded a linear relationship (y = 15.41x – 7.983, R2 = 0.989) over the protein concentration range of 0.01 to 20 mg mL–1. Finally, AuNPs/CNC‐assisted LDI‐TOF MS provides clinically relevant fingerprint information of exosomal proteins in NSCLC serum, and characteristic proteins S100 calcium‐binding protein A10, Urokinase plasminogen activator surface receptor, Plasma protease C1 inhibitor, Tyrosine‐protein kinase Fgr and Mannose‐binding lectin associated serine protease 2 represented excellent predictive biomarkers of NSCLC risk. A strategy to reduce AuNP cluster and protein aggregation in the sample using AuNPs/CNC, which improved the signal sensitivity and reproducibility of LDI‐TOF MS analysis for intact proteins is introduced. Through AuNPs/CNC assisted LDI‐TOF MS analysis, exosome protein fingerprint can effectively distinguished between healthy individuals and NSCLC patients. Moreover, five exosomal proteins represented excellent predictive biomarkers of NSCLC risk.

Dynamic changes occurring in the lung microbiota can impact the initiation, progression, and prognosis of lung cancer (LC). Consequently, the development of suitable intratumoral microbiota analysis methods is crucial. Although matrix‐assisted laser desorption/ionization mass spectrometry (MALDI MS) involves straightforward operations and provides precise results, the “direct smear method” limits the identification of bacterial subspecies. Furthermore, the issue of inadequate quantification with MALDI MS renders it unsuitable for direct analysis of intratumoral bacteria. To address these challenges, a novel ionic liquid in this study is employed, called norharmane conjugated to 2,5‐dihydroxybenzoic acid (Nor@DHB) for the direct detection of intratumoral bacteria using MALDI MS. Because gram‐negative bacteria are dominant within cancer cells, lipid A is selected as the chemical fingerprint for bacterial identification. The results demonstrated that using Nor@DHB can enhance the lipid A signal by an order of magnitude and achieved a good linear relationship within a concentration range of 0.01–80 ng mL−1. Here, this method is successfully applied to the direct analysis of lipid A in actual clinical samples. Subsequent machine learning and nomogram models further confirmed the correlation between characteristic lipid A ions and LC patient clinicopathological features, which are further validated through both in vitro and in vivo experiments. Integrated matrix‐assisted laser desorption/ionization mass spectrometry workflow quantifies intratumoral microbes in lung cancer through lipopolysaccharide/lipid A extraction from biopsies. The novel ionic matrix called norharmane conjugated to 2,5‐dihydroxybenzoic acid enhances lipid A ionization, enabling direct detection of bacterial genera‐specific signatures. Synthetic lipid A calibration and clinical correlation in 50 patients validate this culture‐free method linking microbial load to cancer pathophysiology.

Increasing evidence has suggested the critical implication of microRNAs (miRNAs) in the initiation and progression of non-small cell lung cancer (NSCLC). Previous studies have shown the tumor-suppressive function of miR-1305 in cancer; however, the role of miR-1305 in NSCLC has not been fully understood. The expression of miR-1305 in NSCLC was detected by RT-qPCR. The influence of miR-1305 on the growth of NSCLC cells was determined via Cell Counting Kit 8 (CCK-8), colony formation and FACS analysis. The targets of miR-1305 were predicted with the miRDB database. Luciferase reporter assay was performed to investigate the binding between miR-1305 and 3'-UTR of MDM2. Western blot was applied to check the expression of MDM2 with miR-1305. Here, we found that miR-1305 was down-regulated in NSCLC tissues and cell lines. Decreased miR-1305 was significantly correlated with the metastasis and poor prognostics of NSCLC patients. Overexpression of miR-1305 inhibited the proliferation and migration and promoted the apoptosis of NSCLC cells. Bioinformatics and luciferase assay uncovered that the mouse/murine double minute 2 (MDM2) was a target of miR-1305. miR-1305 bound the 3'-untranslated region (UTR) of MDM2 and decreased the expression of MDM2 in NSCLC cells. As MDM2 was a negative regulator of p53, decreased MDM2 by miR-1305 up-regulated the abundance of p53 in NSCLC cells. Restoration of MDM2 markedly attenuated the suppressive role of miR-1305 in the proliferation and migration of NSCLC cells. The findings provided novel mechanism of miR-1305/MDM2 signaling in regulating the progression of NSCLC, suggesting miR-1305 as a promising target for the treatment of NSCLC.