Molecular characteristics of fatty acid metabolic reprogramming in hepatocellular carcinoma and their implications for immunotherapy

The challenges of hepatocellular carcinoma (HCC) pathogenesis, diagnosis, treatment, and prognosis evaluation are obvious. Hepatocyte-specific fatty acid (FA) metabolic reprogramming is an important marker of liver carcinogenesis and progression; elucidating its mechanism will help unravel the complexity of HCC pathogenesis. Noncoding RNAs (ncRNAs) play important roles in HCC development. Moreover, ncRNAs are important mediators of FA metabolism and are directly involved in the reprogramming of FA metabolism in HCC cells. Here we review significant new advances in understanding the mechanisms regulating HCC metabolism by focusing on ncRNA-mediated post-translational modifications of metabolic enzymes, metabolism-related transcription factors, and other proteins in associated signaling pathways. We also discuss the great therapeutic potential of targeting ncRNA-mediated FA metabolism reprogramming in HCC.

The role of MDSCs in hepatocellular carcinoma – in vivo veritas?

The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications

Background: Accumulating evidence suggests that aberrant cholesterol metabolism plays a pivotal role in modulating immune function and tumor progression. Among various enzymes involved in cholesterol metabolism, squalene epoxidase (SQLE) stands out as a rate-limiting enzyme in cholesterol synthesis, and its aberrant expression has been found to have clinical relevance in several cancer types. However, the immunomodulation role of SQLE remains largely unexplored. Here our goal was to investigate the role of SQLE on the hepatocellular carcinoma (HCC) immune microenvironment and explore a new strategy that strengthens immunotherapy in HCC. Methods: Multiplex immunohistochemical staining and spectral flow cytometry were applied to describe the immune landscape of human and mouse HCC based on SQLE expression level. Additionally, human peripheral blood-derived CD8+T cells were co-cultured with SQLE-edited HCC cell lines to assess differences in cellular proliferation and immune function. By using metabolomics and transcriptomics analyses, we investigated the regulatory mechanisms of SQLE on CD8+ T cell immune function. Furthermore, we evaluated the combined anti-tumor strategy of SQLE inhibitor terbinafine with anti-PD1 treatment in an HCC mouse model that is refractory to PD1 monotherapy. Results: Our results describe a distinct HCC immune ecosystem mediated by SQLE and identify tumoral CD8+ T cells as a critical immune component closely associated with SQLE expression. Both terbinafine-inhibited and genetically silenced SQLE in HCC cells promoted CD8+ T cell proliferation and activation via enhanced T cell receptor (TCR) signaling. Through conditional screening, metabolomic profiling, and liquid chromatography-mass spectrometry (LC-MS) quantification, we discovered that oxysterol 27-hydroxycholesterol, produced by SQLE-expressing HCC cells, functions as a signaling metabolite that modulates CD8+ T cells by supporting cholesterol requirements during TCR signal activation. Significantly, the in vivo model underscores the necessity of tumor-infiltrating CD8+ T cells for the therapeutic efficacy of terbinafine and supports a promising therapeutic strategy combining terbinafine with anti-PD1. Conclusion: Our study indicates that targeting cancer SQLE can rescue CD8+ T cell immune function by overcoming cholesterol restrictions in the HCC tumor environment, and illustrates a promising strategy by targeting a metabolic vulnerability to strengthen the antitumor efficacy of immunotherapy in HCC. Citation Format: Shuang Qiao, Hao Zou, Jing Xu, Limin Zheng. Potentiating the antitumor response of CD8T cells by targeting SQLE-mediated cholesterol metabolism reprogramming in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A022.

Fatty acid (FA) reprogramming has a significant role in liver cancer. However, the contribution of FA metabolism reprogramming to the heterogeneity of hepatocellular carcinoma (HCC) has not been established. Bioinformatics analysis using single-cell sequencing, a non-negative matrix factorization (NMF) algorithm, and survival analyses were used to investigate FA metabolism reprogramming in HCC patients. Molecular targets and the progress of drug discovery were also analyzed and discussed. Among 13 types of HCC cells, epithelial cells exhibited the highest score for FA metabolic aberrance, while certain lymphocytes, such as B cells, CD8Tcm cells, and Treg cells, exhibited the lowest score. Furthermore, epithelial cells displayed significant diversity in FA metabolism with a wide distribution range (−0.2 to 0.8). Additionally, a low level of FA metabolism was associated with poor prognosis in HCC patients (log-rank test, P=0.0089). Higher oxidase expression was correlated with a lower risk of oncogenesis and higher overall survival. However, enzymes involved in synthesis, oxidation, storage, and release exhibited considerable phenotypic diversity in HCC. FA metabolism reprograming was shown to be significantly correlated with the heterogeneity of HCC, which is characterized by a diversity of cancerous cells and enzymes.

Simple SummaryMyeloid-derived suppressor cells restrict the effectiveness of immune-checkpoint inhibitors for a subset of patients mainly through thwarting T cell infiltration into tumor sites. Treatments targeting MDSCs have shown potent inhibitory effects on multiple tumors, including hepatocellular carcinoma. In this review, we summarize the pathological mechanisms of MDSCs and their clinical significance as prognostic and predictive biomarkers for HCC patients, and we provide the latest progress of MDSCs-targeting treatment in HCC.One of the major challenges in hepatocellular carcinoma (HCC) treatment is drug resistance and low responsiveness to systemic therapies, partly due to insufficient T cell infiltration. Myeloid-derived suppressor cells (MDSCs) are immature marrow-derived cell populations with heterogeneity and immunosuppression characteristics and are essential components of the suppressive tumor immune microenvironment (TIME). Increasing evidence has demonstrated that MDSCs are indispensable contributing factors to HCC development in a T cell-dependent or non-dependent manner. Clinically, the frequency of MDSCs is firmly linked to HCC clinical outcomes and the effectiveness of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs). Furthermore, MDSCs can also be used as prognostic and predictive biomarkers for patients with HCC. Therefore, treatments reprograming MDSCs may offer potential therapeutic opportunities in HCC. Here, we recapitulated the dynamic relevance of MDSCs in the initiation and development of HCC and paid special attention to the effect of MDSCs on T cells infiltration in HCC. Finally, we pointed out the potential therapeutic effect of targeting MDSCs alone or in combination, hoping to provide new insights into HCC treatment.

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.

Acyl-CoA thioesterase 9 (ACOT9) is a critical regulator of cellular utilization of fatty acids by catalysing the hydrolysis of acyl-CoA thioesters to non-esterified fatty acid and coenzyme A (CoA). Recently, ACOT9 was reported to participate in the pathogenesis of non-alcoholic liver disease (NAFLD), which arises from aberrant lipid metabolism and serves as a risk factor for hepatocellular carcinoma (HCC). However, the functions of ACOT9 in carcinogenesis and aberrant lipid metabolism in HCC remain unexplored. Here, we found that ACOT9 expression is significantly elevated in HCC at least partially due to the down-regulation of miR-449c-3p. Upregulation of ACOT9 is closely associated with poor prognosis for patients with HCC. Knockdown of ACOT9 expression in HCC cells significantly decreased cell proliferation, colony formation, migration and invasion, mainly through suppression of G1-to-S cell cycle transition and epithelial-to-mesenchymal transition (EMT). By contrast, forced ACOT9 expression promoted HCC growth and metastasis. In addition, we found that ACOT9 reprogrammed lipid metabolism in HCC cells by increasing de novo lipogenesis. Furthermore, we demonstrated that increased lipogenesis was involved in ACOT9-promoted HCC growth and metastasis. Altogether, we demonstrate that ACOT9 plays a critical oncogenic role in the promotion of tumour growth and metastasis by reprogramming lipid metabolism in HCC, indicating ACOT9 as a potential therapeutic target in treatment of HCC.

Background: Tumor-associated macrophages (TAMs) play a crucial role in the tumor microenvironment (TME), and the metabolic activities of both tumor cells and TAMs have an impact on the TME. Moreover, the expression of MICA in tumor cells is closely associated with immune cells in hepatocellular carcinoma (HCC). However, it remains unclear whether MICA expression correlates with TAMs and influences the switch in macrophage phenotype by mediating metabolic alterations. Methods: Various biostatistical tools, qPCR, and IHC staining experiments were utilized to analyze data from The Cancer Genome Atlas (TCGA) and collected HCC tumor tissues. Single-cell RNA sequencing (scRNA-seq) analyses and a co-culture model of HCC cells with macrophages were performed to validate the findings from the biostatistical analyses. Results: Through the intersection of differentially expressed genes (DEGs), metabolism-related genes (MRGs), and co-expression genes (CEGs) with MICA in HCC, the EHHADH gene was identified. Gene set enrichment analyses were conducted to further confirm the role of EHHADH. EHHADH expression is decreased in HCC tumors and can serve as a prognostic biomarker for HCC. Expressions of MICA and EHHADH exhibited significant correlations with various phenotypic macrophages and exerted opposing effects on M1-like and M2-like macrophages infiltrating HCC. The underlying metabolic and molecular mechanisms revealed that MICA in tumor cells induced M2-like polarization through the PPAR/EHHADH pathway, which regulates the fatty acid oxidation (FAO) in macrophages. Conclusions: The metabolic gene EHHADH, which is associated with MICA, led to alterations in M2-like macrophages by promoting heightened fatty acid uptake and augmenting levels of FAO within macrophages.

Background Aberrant methylation and metabolic perturbations may deepen our understanding of hepatocarcinogenesis and help identify novel biomarkers for diagnosing hepatocellular carcinoma (HCC). We aimed to develop an HCC model based on a multi-omics. Research design and methods Four hundred patient samples (200 with HCC and 200 with hepatitis B virus-related liver disease (HBVLD)) were subjected to liquid chromatography-mass spectrometry and multiplex bisulfite sequencing. Integrative analysis of clinical data, CpG data, and metabolome for the 20 complete imputation datasets within a for-loopwas used to identify biomarker. Results Totally, 1,140 metabolites were annotated, of which 125 were differentially expressed. Lipid metabolism reprogramming in HCC, resulting in phosphatidylcholines (PC) significantly downregulated, partly due to the altered mitochondrial beta-oxidation of fatty acids with diverse chain lengths. Age, sex, serum-fetoprotein levels, cg05166871,cg14171514, cg18772205, PC (O-16:0/20:3(8Z, 11Z, 14Z)), and PC (16:1(9Z)/P-18:0) were used to develop the HCC model. The model presented a good diagnostic and an acceptable predictive performance. The cumulative incidence of HCC in low- and high-risk groups of HBVLD patients were 1.19% and 21.40%, respectively (p = 0.0039). Conclusions PCs serve as potential plasma biomarkers and help identify patients with HBVLD at risk of HCC who should be screened for early diagnosis and intervention.

Hepatocellular carcinoma (HCC), an aggressive malignancy with a dismal prognosis, poses a significant public health challenge. Recent research has highlighted the crucial role of lipid metabolism in HCC development, with enhanced lipid synthesis and uptake contributing to the rapid proliferation and tumorigenesis of cancer cells. Lipids, primarily synthesized and utilized in the liver, play a critical role in the pathological progression of various cancers, particularly HCC. Cancer cells undergo metabolic reprogramming, an essential adaptation to the tumor microenvironment (TME), with fatty acid metabolism emerging as a key player in this process. This review delves into intricate interplay between HCC and lipid metabolism, focusing on four key areas: de novo lipogenesis, fatty acid oxidation, dysregulated lipid metabolism of immune cells in the TME, and therapeutic strategies targeting fatty acid metabolism for HCC treatment.

Targeting metabolic reprogramming in hepatocellular carcinoma to overcome therapeutic resistance: A comprehensive review

The deregulation of fatty acid metabolism plays a crucial role in cancer. However, the prognostic value of genes involved in the metabolism in hepatocellular carcinoma (HCC) remains largely unknown. We first constructed a multi-fatty acid metabolic gene prognostic model of HCC based on The Cancer Genome Atlas (TCGA) and further validated it using the International Cancer Genome Consortium (ICGC) database. The model was integrated with the clinical parameters, and a nomogram was built and weighted. Moreover, immune cell infiltration of the tumor microenvironment was investigated. A prognostic model was constructed using 6 selected fatty acid metabolism-related genes, and HCC patients were divided into high- and low-risk groups. Receiver operating characteristic curve (ROC) analysis, principal component analysis (PCA), and t-distributed stochastic neighbor embedding (t-SNE) analysis showed the optimal performance of the model. The concordance index (C-index), ROC curve, calibration plot and decision curve analysis (DCA) all confirmed the satisfactory predictive capacity of the nomogram. The analysis of immune cell infiltration in HCC patients revealed a correlation with different risk levels. Our findings indicate that a prognostic model based on fatty acid metabolism-related genes has superior predictive capacities, which provides the possibility for further improving the individualized treatment of patients with HCC.

The CRAFITY score: A promising prognostic predictor for patients with hepatocellular carcinoma treated with tyrosine kinase inhibitor and immunotherapy combinations

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells