Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer-related mortality worldwide1,2. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulate progressive driver mutations3, with hepatocytes the most likely cell of origin2. However, the landscape of driver mutations in HCC is broadly independent of the underlying aetiologies4. Despite an increasing range of systemic treatment options for advanced HCC, outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations5,6. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC7. Here we generated a suite of genetically driven immunocompetent in vivo and matched in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance and metastasis. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with the human histopathology. In a proof-of-principle analysis, we verified response to standard-of-care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not previously been linked to HCC treatment. Cladribine acts in a highly effective subtype-specific manner in combination with standard-of-care therapy.

AGR2 is a pro-oncogenic protein overexpressed in multiple cancer types, and it promotes tumor progression. Therefore, it is regarded as a promising therapeutic target for cancer. We reported the development and antitumor mechanism of AGR2-specific monoclonal antibody 18A4. To elicit AGR2-guided synergistic antitumor response by redirecting cytotoxic T-cells, we developed first T-cell-engaging bispecific antibody (BsAb) targeting AGR2 and PD1 simultaneously. This novel BsAb efficiently targets AGR2-rich solid tumors. In this study, we elucidated the antitumor mechanisms of AGR2xPD1 BsAb in vitro and in vivo. Higher attachment of T-cells and T-cell-mediated cytotoxicity were seen in cancer cells in BsAb-treated co-culture group. BsAb enhanced T-cell activation when co-cultured with target cells, and the BsAb recruited T-cells to the AGR2-overexpressing cancer cells and induced T-cells to highly express cytolytic proteins. AGR2xPD1 BsAb enhanced co-localization of AGR2 and PD1 in AGR2-overexpressing tumor sites and mediated higher attachment and infiltration of CD3 + CD8 + cytotoxic T-cells into tumor microenvironment in mice. Additionally, AGR2xPD1 BsAb inhibited AGR2-induced angiogenesis and tumor growth. Furthermore, we demonstrate that AGR2 induced PDL1 upregulation through EGFR signaling pathway and inhibited by AGR2xPD1 BsAb. Our study reveals AGR2xPD1 BsAb could be a potential therapeutic agent for targeting AGR2-overexpressing solid tumors.

The pathogenesis of cancer is complicated, and different types of cancer often exhibit different gene mutations resulting in different omics profiles. The purpose of this study was to systematically identify cancer-specific biological pathways and potential cancer-targeting drugs. We collectively analyzed the transcriptomics and proteomics data from 16 common types of human cancer to study the mechanism of carcinogenesis and seek potential treatment. Statistical approaches were applied to identify significant molecular targets and pathways related to each cancer type. Potential anti-cancer drugs were subsequently retrieved that can target these pathways. The number of significant pathways linked to each cancer type ranged from four (stomach cancer) to 112 (acute myeloid leukemia), and the number of therapeutic drugs that can target these cancer related pathways, ranged from one (ovarian cancer) to 97 (acute myeloid leukemia and non-small-cell lung carcinoma). As a validation of our method, some of these drugs are FDA approved therapies for their corresponding cancer type. Our findings provide a rich source of testable hypotheses that can be applied to deconvolute the complex underlying mechanisms of human cancer and used to prioritize and repurpose drugs as anti-cancer therapies.

The steroidal glycoalkaloids (SGAs) produced in Solanaceae crops, including tomato, are antinutritional because of their cellular toxicity and resultant bitter taste to humans. To make fruits palatable, SGA profiles shift from bitter and toxic α-tomatine to nonbitter and nontoxic esculeoside A during the ripening process. However, the mechanisms regulating this conversion remain unclear. In this study, we showed that removal of toxic and bitter SGAs is under the control of DNA demethylation, ethylene, and key transcription factors by forming a feedback loop that governs the expression of key GLYCOALKALOID METABOLISM (GAME) genes during ripening. Moreover, the ethylene-inducible transcription factors NON-RIPENING, RIPENING INHIBITOR, and FRUITFULL1 coordinately regulate the expression of GAME31, GAME40, GAME5, and the glycoalkaloid transporter gene GORKY, whereas jasmonic acid-induced MYC2 modulates the transcription of GAME36. Furthermore, DNA demethylation mediated by the DEMETER-LIKE 2 drives SGA detoxification during tomato domestication.

Salinity stress severely affects rice growth and reduces its productivity. With No Lysine Kinases (WNKs) are serine/threonine kinases emerging as potential candidate genes due to their involvement in various abiotic stress tolerance responses. However, studies providing mechanistic insights into the roles of WNKs in plants remain scarce. In the present study, OsWNK9-overexpressing rice lines showed strong tolerance to salinity stress. Overexpression of OsWNK9 also triggered the accumulation of abscisic acid (ABA) and restored indole-3-acetic acid (IAA) concentrations in roots, triggering stomatal closure in shoots and maintaining cell expansion of the root epidermal cells when challenged with salt treatment. The overexpression lines showed increased activity of antioxidant enzymes, which further mitigated ROS-mediated cellular damage under salinity stress. We also identified that OsWNK9 interacts with Receptor for Activated Kinase C1A (RACK1A), ABA-8'-hydroxylase, and (Vacuolar Type ATPase) V-Type ATPase. Taken together, our findings suggest that OsWNK9 expression is warranted under salinity stress and exerts its effects by interacting with its downstream targets and by increased accumulation of ABA and IAA, thereby regulating seed germination, stomatal activity, improved root growth, and ionic homeostasis, which all contribute to significantly higher yield produced per plant under long term salinity stress.

Dendritic cells (DCs) play a crucial role in orchestrating immune responses by bridging innate and adaptive immunity. In vitro generation of DCs from mouse and human tissues such as bone marrow and peripheral blood monocytes, has been widely used to study their immunological functions. In chicken, DCs have mainly been derived from bone marrow cell cultures, with limited characterization from blood monocytes.

EGFR protein trafficking is critical for regulating multiple biological processes, including cell growth and survival. However, how EGFR protein homeostasis is maintained remains unclear. In this study, we show that a reduction in plasma membrane-associated EGFR triggers EGFR transcription by promoting pSTAT3 nuclear localization. Nucleus-localized pSTAT3 binds to the EGFR gene promoter to transactivate EGFR. Moreover, erlotinib, an EGFR tyrosine kinase inhibitor (TKI), can also increase pSTAT3 nuclear accumulation, resulting in increased EGFR transcription and erlotinib resistance. Importantly, pharmacological inhibition of pSTAT3 can significantly overcome the resistance of cancer cells to erlotinib. Together, these findings demonstrate that pSTAT3 is pivotal for maintaining EGFR protein homeostasis and suggest that activation of the pSTAT3-EGFR axis contributes to EGFR-TKI resistance.

Peptidylarginine deiminase 4 (PAD4) is an enzyme that modifies proteins by converting positively charged arginine residues to neutral citrulline residues. This process, termed citrullination, has been known to trigger NETosis, a neutrophil cell death pathway involving the release of neutrophil extracellular traps (NETs). Abnormal PAD4 activity and protein citrullination have been linked to various diseases, including those affecting the central nervous system. Herein we investigated the profile of PAD4 expression in an animal model of stroke induced by middle cerebral artery occlusion (MCAO). PAD4 levels were significantly elevated in the ischemic core and penumbra of the affected hemisphere at 3-6 and 6-48 h post-MCAO, respectively. Notably, NETosis induction, indicated by the upregulation of CitH3 (citrullinated histone H3, a NETosis marker), was observed between 48 and 96 h post-MCAO, peaking at 96 h. While PAD4 was present in most brain cell types of sham controls, strong PAD4 induction was primarily observed in neurons during the peak PAD4 induction period (12-24 h post-MCAO). Importantly, intranasal administration of the PAD4 inhibitor BB-Cl-amidine (BBCA) significantly reduced infarct volume and improved neurological and functional outcomes at 24 h post-MCAO, demonstrating a strong protective effect of PAD4 inhibition in ischemic stroke. Staining with an antibody that recognizing citrullinated proteins (F95) revealed an accumulation of these proteins, especially degenerating neurons, however, BBCA treatment significantly suppressed this accumulation in dying neurons. These findings indicate that PAD4-mediated protein citrullination in neurons plays a critical role in promoting ischemic brain damage. Furthermore, delayed administration of BBCA (at 48/72 h post-MCAO) suppresses the NETosis induction observed at 96 h post-MCAO, potentially ameliorating repair processes such as blood vessel regeneration. Collectively, these findings suggest a complex role of PAD4 in cerebral ischemia, with neuroprotective effects (NETosis-independent function) during the acute to subacute period and NETosis-suppressive effects at later time points.

TP53 mutations are linked to aggressive progression and chemoresistance in gastric cancer (GC). Frameshift mutation is the second most common mutation type of TP53. However, the consequences of this mutation type in GC were not well understood, and targeted therapies for cancer patients harboring frameshift mutations were also not established. Histone methylation significantly influences tumorigenesis in TP53-mutated cancers, and related inhibitors are emerging as specific therapeutic strategies.

Chitosan, a non-toxic and biodegradable compound, enhances plant growth and secondary metabolite production, presenting innovative approaches to mitigating plant stress. Salinity, a common abiotic stress, significantly impairs plant growth and development. This study investigates the effects of chitosan on the physiological, biochemical, and gene expression responses of salt-stressed Brassica napus L. exposed to NaCl concentrations of 0, 50, 100, and 150 mM. Chitosan was applied as a foliar spray at concentrations of 0, 5 and 10 mg/L. The research focuses on gene expression changes in P5CS, PIP, and PAL genes in the roots and shoots of Brassica napus, revealing notable alterations, particularly in PIP expression under saline conditions. The study also observed enhanced PAL enzyme activity, increased chlorophyll and proline levels, and changes in iron, potassium, and nitrogen content. These findings demonstrate chitosan's potential to improve plant resilience to salt stress. By modulating gene expression and enhancing physiological responses, chitosan presents a promising solution for enhancing plant tolerance to salinity, with valuable implications for agricultural practices.

DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO.

Plants frequently modulate their hormonal signaling pathways in response to stress, thereby regulating the synthesis of secondary metabolites and adapting to fluctuations in their surroundings. The APETALA2/ethylene-responsive factor (AP2/ERF) domain transcription factors are important in regulating abiotic stress tolerance. The accumulation of asperosaponin VI in the root was significantly enhanced under low temperature stress, which exhibited a correlation with the AP2/ERF family. However, the involvement of AP2/ERF in regulating asperosaponin VI biosynthesis under cold stress remains ambiguous. Under cold stress conditions below 10 °C, we observed the accumulation of asperosaponin VI and an increase in jasmonic acid (JA) levels. This response was attributed to the activation of the JA synthesis pathway induced by low temperatures. Additionally, a comprehensive analysis of the full-length transcriptome of Dipsacus asper identified a total of 80 DaAP2/ERF transcription factors, which exhibited significant homology with Arabidopsis thaliana and Citrus ERFs based on phylogenetic analysis. Furthermore, qRT-PCR analysis demonstrated that both cold stress and methyl jasmonate (MeJA) induction upregulated DaERF108 expression. The expression of DaERF108 is notably upregulated in the leaves and during the early stages of growth and development of D. asper, while subcellular localization analysis confirmed its presence in the nucleus. The overexpression of DaERF108 significantly enhanced the accumulation of oleanolic acid, a precursor of asperosaponin VI, and activated the triterpenoid biosynthesis pathway in Arabidopsis roots. Additionally, the overexpression of DaERF108 induced the activation of the terpenoid synthesis pathway under cold stress conditions. Notably, there was a positive correlation between DaERF108 expression and genes involved in asperosaponin VI biosynthesis, particularly with 3-hydroxy-3-methylglutaryl coenzyme A synthase (DaHMGS). The interaction between DaERF108 and the GCC-box element in the DaHMGS promoter was demonstrated by LUC and Y1H assays, leading to enhanced activity. These findings suggest that DaERF108 specifically binds to the G-box element, thereby regulating DaHMGS gene expression, activating the JA signaling pathway, and promoting asperosaponin VI biosynthesis in response to cold stress.

Adrenocortical Carcinoma is a rare and aggressive endocrine malignancy, that arises from cells of one of the three cortical layers of the adrenal gland. Radical surgery is the only curative treatment, even if recurrence rates are high. Therapeutic options are limited, with mitotane as the cornerstone of medical therapy. Despite 50 years of clinical use, the mechanism of action of mitotane has not yet been fully established, possibly due to the drug's susceptibility to interaction with confounding factors that reduce its biological activity. In the present study, we evaluated by RNAseq the effect of mitotane on gene expression in the H295R cell line, in an environment free of known confounding factors. Our approach allowed us to identify transcriptional deregulation of the ATF4/ATF3 axis, often involved in ER stress. These results were also validated by ddPCR in independent experiments. Mitotane-mediated ATF4 overexpression was also confirmed at the protein level. We observed how an incremental concentration of mitotane could deregulate main biological pathways. Further, we confirmed, both at RNAseq and ddPCR level, the mitotane-mediated downmodulation of genes such as STAR, CYP11A1, CYP21A2, and HSD3B2, highlighting its effect on steroid hormones biosynthesis. Through our approach, we identified biological pathways altered by mitotane in early response stages and with low drug concentrations. Some of these pathways could potentially be investigated in the future as functional biomarkers to monitor adrenocortical carcinoma treatment or as new pharmacological targets for this rare disease.

Identification and characterization of genes encoding herbicide-detoxifying enzymes is lacking in allohexaploid wheat (Triticum aestivum L.). Gene expression is frequently induced by herbicide safeners and implies the encoded enzymes serve a role in herbicide metabolism and detoxification. Cloquintocet-mexyl (CM) is a safener commonly utilized with halauxifen-methyl (HM), a synthetic auxin herbicide whose phytotoxic form is halauxifen acid (HA). Our first objective was to identify candidate HA-detoxifying genes via RNA-Seq by comparing untreated and CM-treated leaf tissue. On average, 81% of RNA-Seq library reads mapped uniquely to the reference genome and 76.4% of reads were mapped to a gene. Among the 103 significant differentially expressed genes (DEGs), functional annotations indicate the majority of DEGs encode proteins associated with herbicide or xenobiotic metabolism. This finding was further corroborated by gene ontology (GO) enrichment analysis, where several genes were assigned GO terms indicating oxidoreductase activity (34 genes) and transferase activity (45 genes). One of the significant DEGs is a member of the CYP81A subfamily of cytochrome P450s (CYPs; denoted as CYP81A-5A), which are of interest due to their ability to catalyze synthetic auxin detoxification. To investigate CYP expression induced by HM and/or CM, our second objective was to measure gene-specific expression of CYP81A-5A and its homoeologs (CYP81A-5B and CYP81A-5D) in untreated leaf tissue and leaf tissue treated with CM and HM over time using RT-qPCR. Relative to the reference gene (β-tubulin), basal CYP expression is high, expression among these CYPs varies over time, and expression for all CYPs is CM-inducible but not HM-inducible. Further analysis of CYP81A-5A, such as gene knock-out, overexpression experiments, or in vitro activity assays with purified enzyme are necessary to test the hypotheses that the encoded CYP detoxifies HA and that CM upregulates this reaction.

Chimeric Antigen Receptor T (CAR-T) cells offer a promising strategy for cancer treatment. These CAR-T cells are either autologous or allogeneic T cells that are genetically modified to express a chimeric antigen receptor targeting a specific tumor antigen. Ongoing research aims to optimize the CAR-T cell efficacy, including strategies to modulate their metabolism. One such approach involves inducing transgene expression by activating the GCN2 kinase signaling pathway through dietary deprivation of an essential amino acid. In this study, we investigated the general impact of a 6-hour leucine deprivation on primary activated human T cells using RNA-seq technology. Our analysis identified 3,431 differentially expressed genes between T cells cultured in regular medium and those cultured in leucine-deprived medium. Gene Set Enrichment Analysis revealed that "TNFα signaling via NFκB", "interferon-γ response", and "unfolded protein response" gene sets were positively enriched, while "mTORC1 signaling", "Myc targets", and "oxidative phosphorylation" gene sets were negatively enriched. To further evaluate the involvement of GCN2 kinase in regulating the differential gene expression during the 6-hour leucine deprivation, T cells were cultured with or without a GCN2 inhibitor. We found that 59% of the differentially expressed genes in our dataset were dependent on the kinase GCN2 (n = 2028), with 1,140 up-regulated and 888 down-regulated genes. These findings suggest a promising strategy to enhance CAR-T cell efficacy by combining short amino acid starvation with transient overexpression of a target gene.

Natural killer (NK) cell mediated cytotoxicity is a crucial form of anti-cancer immune response. Natural killer group 2 member D (NKG2D) is a prominent activating receptor of NK cell. UL16-binding protein 2 (ULBP2), always expressed or elevated on cancer cells, functions as a key NKG2D ligand. ULBP2-NKG2D ligation initiates NK cell activation and subsequent targeted elimination of cancer cells. Enhanced expression of ULBP2 on cancer cells leads to more efficient elimination of these cells by NK cells. Resveratrol (RES) is known for its multiple health benefits, while current understanding of its role in regulating cancer immunogenicity remains limited. This study aims to investigate how RES affects the expression of ULBP2 and the sensitivity of breast cancer (BC) cells to NK cell cytotoxicity, along with the underlying mechanisms.

Pharmacological targeting of Hedgehog (HH)/GLI has proven effective for certain blood, brain and skin cancers including basal cell carcinoma (BCC). However, limited response rates and the development of drug resistance call for improved anti-HH therapies that take synergistic crosstalk mechanisms and immune evasion strategies into account. In previous work, we demonstrated that cooperation of HH/GLI and Interleukin 6 (IL6)/STAT3 signaling drives BCC growth. Whether synergistic HH-IL6 signaling promotes BCC via the activation of immune evasion mechanisms remained unclear.

As a flavonoid compound, astragalin (AST) is found in a variety of medicinal plants. In clinical studies, AST has anti-inflammatory and analgesia effects on rheumatoid arthritis, bronchopneumonia diseases and so on, but its specific role and mechanism is still not clear. This study aimed to investigate the effect and molecular mechanism of AST on inflammatory pain and pain-related emotions in complete Freund's adjuvant (CFA) mice. In this study, we observed that AST significantly alleviated CFA-induced inflammatory pain and associated emotional disturbances in mice. The mechanism may be related to down-regulating mGluR5-mediated PKCλ-ERK1/2-FOXO6 signaling pathway in CFA mice. Treatment with the mGluR5-specific inhibitor MTEP resulted in the downregulation of proteins associated with the PKCλ-ERK1/2-FOXO6 pathway, similar to the effects observed with AST administration. These results suggested that AST might play a crucial role in the management of inflammatory pain and related emotions, shedding light on its underlying mechanism for treating such conditions.

Mitochondrial metabolism-regulated epigenetic modification is a driving force of aging and a promising target for therapeutic intervention. Mitochondrial malate dehydrogenase (MDH2), an enzyme in the TCA cycle, was identified as an anti-aging target through activity-based protein profiling in present study. The expression level of MDH2 was positively correlated with the cellular senescence in Mdh2 knockdown or overexpression fibroblasts. Glibenclamide (Gli), a classic anti-glycemic drug, was found to inhibit the activity of MDH2 and relieve fibroblast senescence in an MDH2-dependent manner. The anti-aging effects of Gli were also further validated in vivo, as it extended the lifespan and reduced the frailty index of naturally aged mice. Liver specific Mdh2 knockdown eliminated Gli's beneficial effects in naturally aged mice, reducing p16INK4a expression and hepatic fibrosis. Mechanistically, MDH2 inhibition or knockdown disrupted central carbon metabolism, then enhanced the methionine cycle flux, and subsequently promoted histone methylation. Notably, the tri-methylation of H3K27, identified as a crucial methylation site in reversing cellular senescence, was significantly elevated in hepatic tissues of naturally aged mice with Mdh2 knockdown. Taken together, these findings reveal that MDH2 inhibition or knockdown delays the aging process through metabolic-epigenetic regulation. Our research not only identified MDH2 as a potential therapeutic target and Gli as a lead compound for anti-aging drug development, but also shed light on the intricate interplay of metabolism and epigenetic modifications in aging.

Pancreatic cancer (PCa) has insidious onset, high malignancy and poor prognosis. Gemcitabine (GEM) is one of the first-line chemotherapy drugs for PCa. However, GEM resistance has always been a bottleneck problem leading to recurrence and death of PCa patients. RNA-binding proteins (RBPs) are important proteins that regulate transportation, splicing, stability and translation of RNA. Abnormal expression of RBPs often lead to a series of abnormal accumulation or degradation of downstream RNA resulting in various diseases. In our study, we utilized RIP seq, RIP-qPCR, in vitro and in vivo experiments and found that pumilio2 (PUM2) was high expression in PCa, and promoted GEM resistance of PCa by regulating mRNA stability of integrin Alpha 3 (ITGA3) and other genes in focal adhesion pathway, and there was positive feedback regulation between PUM2 and transcription factor early growth response gene 1 (EGR1), that is PUM2 binding to 3'UTR region of EGR1 mRNA, and EGR1 binding to promoter region of PUM2 gene. The discovery of EGR1/PUM2/ITGA3 axis provided a solid experimental basis for the selection of chemotherapy regiments for PCa patients and exploration of combined regimens to reverse GEM resistance in the future.