Background and Aims: Molecular classification is a promising tool for prognosis prediction and optimizing precision therapy for hepatocellular carcinoma (HCC). Here, we aimed to develop a molecular classification of HCC based on the fatty acid degradation (FAD) pathway, fully characterize it, and evaluate its ability in guiding personalized therapy. Approach and Results: We performed RNA sequencing (RNA-seq), PCR-array, lipidomics, metabolomics, and proteomics analysis of 41 HCC patients, in which 17 patients received anti-PD-1 therapy. Single-cell RNA sequencing (scRNA-seq) was performed to explore the tumor microenvironment. Nearly 60 publicly-available multi-omics datasets were analyzed. The associations between FAD subtypes and response to sorafenib, transarterial chemoembolization (TACE), immune checkpoint inhibitor (ICI) were assessed in patient cohorts, patient-derived xenograft (PDX), and spontaneous mice models. A novel molecular classification named F subtype (F1, F2, and F3) was identified based on the FAD pathway, distinguished by clinical, mutational, epigenetic, metabolic, and immunological characteristics. F1 subtypes exhibited high infiltration with immunosuppressive microenvironment. Subtype-specific therapeutic strategies were identified, in which F1 subtypes with the lowest FAD activities represent responders to compounds YM-155 and Alisertib, sorafenib, anti-PD1, anti-PD-L1, and atezolizumab plus bevacizumab (T + A) treatment, while F3 subtypes with the highest FAD activities are responders to TACE. F2 subtypes, the intermediate status between F1 and F3, are potential responders to T + A combinations. We provide preliminary evidence that the FAD subtypes can be diagnosed based on liquid biopsies. Conclusions: We identified three FAD subtypes with unique clinical and biological characteristics, which could optimize individual cancer patient therapy and help clinical decision-making. Export

Background and Aims: TGF-β induces multiple structural and functional changes in quiescent HSCs, including an increase in proliferation, mitochondrial mass, and matrix deposition. HSC transdifferentiation requires significant bioenergetic capacity, and it is not known how TGF-β–mediated transcriptional upregulation is coordinated with the bioenergetic capacity of HSCs. Approach and Results: Mitochondria are key bioenergetic organelles, and here, we report that TGF-β induces release of mitochondrial DNA (mtDNA) from healthy HSCs through voltage-dependent anion channels (VDACs), with the formation of an mtDNA-CAP on the external mitochondrial membrane. This stimulates organization of cytosolic cyclic GMP-AMP synthase (cGAS) onto the mtDNA-CAP and subsequent activation of the cGAS-STING-IRF3 pathway. TGF-β is unable to induce conversion of HSCs from a quiescent to a transdifferentiated phenotype in the absence of mtDNA, VDAC, or stimulator of interferon genes (STING). Transdifferentiation by TGF-β is blocked by a STING inhibitor, which also reduces liver fibrosis prophylactically and therapeutically. Conclusions: We have identified a pathway that requires the presence of functional mitochondria for TGF-β to mediate HSC transcriptional regulation and transdifferentiation and therefore provides a key link between bioenergetic capacity of HSCs and signals for transcriptional upregulation of genes of anabolic pathways. Export

Hepatocellular carcinoma (HCC) is a typical tumor that undergoes metabolic reprogramming, differing from normal liver tissue in glucose, lipid, nucleic acid, and amino acid metabolism. Although ammonia is a toxic metabolic by-product, it has also been recently recognized as a signaling molecule to activate lipid metabolism, and it can be a nitrogen source for biosynthesis to support tumorigenesis. In this study, we revealed that β-catenin activation increases ammonia production in HCC mainly by stimulating glutaminolysis. β-Catenin/LEF1 activated the transcription of the glutamate dehydrogenase GLUD1, which then promoted ammonia utilization to enhance the production of glutamate, aspartate, and proline as evidenced by 15NH4Cl metabolic flux. β-Catenin/TCF4 induced the transcription of SLC4A11, an ammonia transporter, to excrete excess ammonia. SLC4A11 was upregulated in HCC tumor tissues, and high SLC4A11 expression was associated with poor prognosis and advanced disease stages. Loss of SLC4A11 induced HCC cell senescence in vitro by blocking ammonia excretion and reduced β-catenin–driven tumor growth in vivo. Furthermore, elevated levels of plasma ammonia promoted the progression of β-catenin mutant HCC, which was impeded by SLC4A11 deficiency. Downregulation of SLC4A11 led to ammonia accumulation in tumor interstitial fluid and decreased plasma ammonia levels in HCC with activated β-catenin. Altogether, this study indicates that β-catenin activation reprograms ammonia metabolism and that blocking ammonia excretion by targeting SLC4A11 could be a promising approach to induce senescence in β-catenin mutant HCC. Significance: Ammonia metabolism reprogramming mediated by aberrant activation of β-catenin induces resistance to senescence in HCC and can be targeted by inhibiting SLC4A11 as a potential therapy for β-catenin mutant liver cancer.

Tumor-promoting inflammation is an emerging hallmark of cancer, which participates in both cancer progression and immune escape. Hepatocellular carcinoma (HCC) is a typical inflammation-related cancer with an extremely poor prognosis. Frankincense and myrrh are anti-inflammation agents commonly used in clinic. The purpose of this study is to investigate whether extract of frankincense and myrrh (FM) downregulates inflammatory microenvironment of HCC and thereby restores antitumor immune responses. The water-decocting FM was obtained and quantified. HCC cell lines HCCLM3 and Hepa1-6 were used to evaluate the efficacy of FM targeting NF-κB and STAT3 signaling with western blot and qRT-PCR analysis. CD8+NKG2D+ cells were derived from human peripheral blood and were used for evaluation of immune cells-mediated inflammation and oncolysis on HCCLM3 cells. The antitumor efficacy of FM was investigated both in immune compromised and immune competent mice bearing subcutaneous HCC. Mice received daily oral gavage of FM at 60 mg/kg. Immune activity within tumor microenvironment (TME) was assessed by ELISpot assay and flow cytometry, respectively. Depletion of CD8+ T cells or NK cells was achieved by intraperitoneal injection of respective neutralizing antibody. FM significantly inhibited the activation of NF-κB and STAT3 signaling in HCC cells induced by cytokines (TNF-α or IL-6) and in co-culture system with CD8+NKG2D+ cells. Furthermore, FM sensitized HCC cells to CD8+NKG2D+ cells-mediated oncolysis. In HCC-bearing mice, FM at a non-toxic dose failed to reduce tumor growth in immune compromised mice, whereas it significantly inhibited tumor growth and prolonged life span in immune competent mice. While the number of IFN-γ-producing cells within TME was increased in mice treated with FM, the infiltration of CD8+ T cells and NK cells was not increased. Finally, we identified that depletion of CD8+ T cells rather than NK cells abrogated the antitumor activity of FM. Our results show for the first time that CD8+ T cells mediate the antitumor activity of FM at a non-toxic dose. This may provide new insights to this ancient mysterious prescription in cancer therapy, which offers a novel and practical therapeutic strategy and the possibilities of combined immunotherapy for HCC as well as other inflammation-related cancers in clinic.

Background: The Warburg effect, also termed “aerobic glycolysis”, is one of the most remarkable and ubiquitous metabolic characteristics exhibited by cancer cells, representing a potential vulnerability that might be targeted for tumor therapy. Ketogenic diets (KDs), composed of high-fat, moderate-protein and low carbohydrates, are aimed at targeting the Warburg effect for cancer treatment, which have recently gained considerable attention. However, the efficiency of KDs was inconsistent, and the genotypic contribution is still largely unknown. Methods: The bulk RNA-seq data from The Cancer Genome Atlas (TCGA), single cell RNA sequencing (scRNA-seq), and microarray data from Gene Expression Omnibus (GEO) and Cancer Cell Line Encyclopedia (CCLE) were collected. A joint analysis of glycolysis and ketone bodies metabolism (KBM) pathway was performed across over 10,000 tumor samples and nearly 1,000 cancer cell lines. A series of bioinformatic approaches were combined to identify a metabolic subtype that may predict the response to ketogenic dietary therapy (KDT). Mouse xenografts were established to validate the predictive utility of our subtypes in response to KDT. Results: We first provided a system-level view of the expression pattern and prognosis of the signature genes from glycolysis and KBM pathway across 33 cancer types. Analysis by joint stratification of glycolysis and KBM revealed four metabolic subtypes, which correlated extensively but diversely with clinical outcomes across cancers. The glycolytic subtypes may be driven by TP53 mutations, whereas the KB-metabolic subtypes may be mediated by CTNNB1 (β-catenin) mutations. The glycolytic subtypes may have a better response to KDs compared to the other three subtypes. We preliminarily confirmed the idea by literature review and further performed a proof-of-concept experiment to validate the predictive value of the metabolic subtype in liver cancer xenografts. Conclusions: Our findings identified a metabolic subtype based on glycolysis and KBM that may serve as a promising biomarker to predict the clinical outcomes and therapeutic responses to KDT.

Immune checkpoint inhibitors produce durable antitumor effects in various cancers, but not all patients respond. High tumor mutational burden (TMB) is a known predictor of clinical benefit. In this study, we focused on the MHC-I-dependent neoantigen presentation pathway to enhance predictive capabilities beyond TMB. Using pan-cancer immunogenomic analyses of somatic mutation data from The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC), we analyzed 33 cancer types. Objective response rates (ORRs) to PD-1/PD-L1 inhibitors were evaluated in relation to immune characteristics, including TMB, neoantigen load, MHC-I gene expression, and CD8+ T cell fraction. Spearman’s rank correlation was used to assess these relationships. TMB showed the strongest correlation with ORR (r = 0.783, P = 2.17 × 10⁻⁵). However, integrating TMB, HLA-A expression, and CD8+ T cell fraction significantly improved predictive accuracy (r = 0.865, P = 1.80 × 10⁻⁶). Validation in external cohorts confirmed these findings, revealing notable differences in MHC-I pathway activity between responders and non-responders to immunotherapy. Our results demonstrate that the MHC-I antigen presentation pathway is strongly associated with response to PD-1/PD-L1 inhibitors. Importantly, combining antigen expression, processing, presentation, and recognition features provides superior predictive power compared to TMB alone. This integrated approach could improve treatment outcome predictions and advance personalized immunotherapy strategies.