Human Hepatocellular Carcinoma Samples Research Articles

ZNF692 drives malignant development of hepatocellular carcinoma cells by promoting ALDOA-dependent glycolysis

Hepatocellular carcinoma (HCC) is one of the malignancies with the worst prognosis worldwide, in the occurrence and development of which glycolysis plays a central role. This study uncovered a mechanism by which ZNF692 regulates ALDOA-dependent glycolysis in HCC cells. RT-qPCR and western blotting were used to detect the expression of ZNF692, KAT5, and ALDOA in HCC cell lines and a normal liver cell line. The influences of transfection-induced alterations in the expression of ZNF692, KAT5, and ALDOA on the functions of HepG2 cells were detected by performing MTT, flow cytometry, Transwell, cell scratch, and colony formation assays, and the levels of glucose and lactate were determined using assay kits. ChIP and luciferase reporter assays were conducted to validate the binding of ZNF692 to the KAT5 promoter, and co-IP assays to detect the interaction between KAT5 and ALDOA and the acetylation of ALDOA. ZNF692, KAT5, and ALDOA were highly expressed in human HCC samples and cell lines, and their expression levels were positively correlated in HCC. ZNF692, ALDOA, or KAT5 knockdown inhibited glycolysis, proliferation, invasion, and migration and promoted apoptosis in HepG2 cells. ZNF692 bound to the KAT5 promoter and promoted its activity. ALDOA acetylation levels were elevated in HCC cell lines. KAT5 bound to ALDOA and catalyzed ALDOA acetylation. ALDOA or KAT5 overexpression in the same time of ZNF692 knockdown, compared to ZNF692 knockdown only, stimulated glycolysis, proliferation, invasion, and migration and reduced apoptosis in HepG2 cells. ZNF692 promotes the acetylation modification and protein expression of ALDOA by catalyzing KAT5 transcription, thereby accelerating glycolysis to drive HCC cell development.

TRIM29 modulates proteins involved in PTEN/AKT/mTOR and JAK2/STAT3 signaling pathway and suppresses the progression of hepatocellular carcinoma.

Tripartite motif-containing 29 (TRIM29), also known as the ataxia telangiectasia group D-complementing (ATDC) gene, has been reported to play an oncogenic or tumor suppressive role in developing different tumors. So far, its expression and biological functions in hepatocellular carcinoma (HCC) remain unclear. We investigated TRIM29 expression pattern in human HCC samples using quantitative RT-PCR and immunohistochemistry. Relationships between TRIM29 expression level, clinical prognostic indicators, overall survival (OS), and disease-free survival (DFS) were evaluated by Kaplan-Meier analysis and Cox proportional hazards model. A series of in vitro experiments and a xenograft tumor model were conducted to detect the functions of TRIM29 in HCC cells. RNA sequencing, western blotting, and immunochemical staining were performed to assess the molecular regulation of TRIM29 in HCC. We found that the mRNA and protein levels of TRIM29 were significantly reduced in HCC samples, compared with adjacent noncancerous tissues, and were negatively correlated with poor differentiation of HCC tissues. Survival analysis confirmed that lower TRIM29 expression significantly correlated with shorter OS and DFS of HCC patients. TRIM29 overexpression remarkably inhibited cell proliferation, migration, and EMT in HCC cells, whereas knockdown of TRIM29 reversed these effects. Moreover, deactivation of the PTEN/AKT/mTOR and JAK2/STAT3 pathways might be involved in the tumor suppressive role of TRIM29 in HCC. Our findings indicate that TRIM29 in HCC exerts its tumor suppressive effects through inhibition of the PTEN/AKT/mTOR and JAK2/STAT3 signaling pathways and may be used as a potential biomarker for survival in patients with HCC.

Elevated expression of HIGD1A drives hepatocellular carcinoma progression by regulating polyamine metabolism through c-Myc–ODC1 nexus

BackgroundHypoxia contributes to cancer progression through various molecular mechanisms and hepatocellular carcinoma (HCC) is one of the most hypoxic malignancies. Hypoxia-inducible gene domain protein-1a (HIGD1A) is typically induced via epigenetic regulation and promotes tumor cell survival during hypoxia. However, the role of HIGD1A in HCC remains unknown.MethodsHIGD1A expression was determined in 24 pairs of human HCC samples and para-tumorous tissues. Loss-of-function experiments were conducted both in vivo and in vitro to explore the role of HIGD1A in HCC proliferation and metastasis.ResultsIncreased HIGD1A expression was found in HCC tissues and cell lines, which was induced by hypoxia or low-glucose condition. Moreover, HIGD1A knockdown in HCC cells arrested the cell cycle at the G2/M phase and promoted hypoxia-induced cell apoptosis, resulting in great inhibition of cell proliferation, migration, and invasion, as well as tumor xenograft formation. Interestingly, these anti-tumor effects were not observed in normal hepatocyte cell line L02. Further, HIGD1A knockdown suppressed the expression of ornithine decarboxylase 1 (ODC1), a rate-limiting enzyme of polyamine metabolism under c-Myc regulation. HIGD1A was found to bind with the c-Myc promoter region, and its knockdown decreased the levels of polyamine metabolites. Consistently, the inhibitory effect on HCC phenotype by HIGD1A silencing could be reversed by overexpression of c-Myc or supplementation of polyamines.ConclusionsOur results demonstrated that HIGD1A activated c-Myc–ODC1 nexus to regulate polyamine synthesis and to promote HCC survival and malignant phenotype, implying that HIGD1A might represent a novel therapeutic target for HCC.

TGFβR1 inhibition drives hepatocellular carcinoma proliferation through induction of toll-like-receptor signalling.

Transforming growth factor (TGF)-β and toll-like receptors (TLRs) have been shown to independently modulate the proliferation of hepatocellular carcinoma (HCC). Since a direct cross-talk between these two signalling pathways in HCC has not been clearly described before, we aimed here to explore the possibility of such interaction. A human HCC tissue array (n = 20 vs. four control samples), human HCC samples (n = 10) and steatohepatitis-driven murine HCC samples (control, NASH and HCC; n = 6/group) were immunostained for TGFβR1, pSMAD2, TRAF6, IRAK1 and PCNA. The results were confirmed by immunoblotting. Effects of constant activation of the SMAD pathway by constitutive expression of ALK5 or knockdown of mediators of TLR signalling, IRAK1 and MyD88, on HCC proliferation, were investigated in the HCC cell line (HUH-7) after treatment with TGFβ1 cytokine or TGFβR1 kinase inhibitor (LY2157299) using PCNA and MTS assay. TGFβR1 expression is decreased in human and murine HCC and associated with downregulated pSMAD2, but increased IRAK1, TRAF6 and PCNA staining. TGFβR1 kinase inhibition abolished the cytostatic effects of TGFβ1 and led to the induction of IRAK1, pIRAK1 and elevated mRNA levels of TLR-9. Overexpression of ALK5 and knockdown of MyD88 or IRAK1 augmented the cytostatic effects of TGFβ1 on HUH-7. In another epithelial HCC cell line, that is, HepG2, TGFβR1 kinase inhibitor similarly elevated cellular proliferation. There is a balance between the canonical SMAD-driven tumour-suppressing arm and the non-canonical tumour-promoting arm of TGFβ signalling. Disruption of this balance, by inhibition of the canonical pathway, induces HCC proliferation through TLR signalling.

CRKL dictates anti-PD-1 resistance by mediating tumor-associated neutrophil infiltration in hepatocellular carcinoma

Background & AimsImmune checkpoint inhibitors (ICIs) resistance limits immunotherapy success in hepatocellular carcinoma (HCC). However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in HCC against anti-PD-1 therapy. MethodsGene expression in HCC specimens from 10 patients accepted anti-PD-1 therapy was identified by RNA-sequencing. A total of 342 and 62 HCC samples from TMA1 and TMA2, respectively, were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/Trp53fl/fl) were given hydrodynamic tail vein injections (HDTVi) of adeno-associated virus serotype 8 (AAV8) vector to overexpress CRKL. Mass cytometry by time of flight (CyTOF) was used to profile the proportion and status of different immune cell lineages in the mouse tumor tissues. ResultsCRKL was identified as a candidate anti-PD-1 resistant gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 therapy efficacy by mobilizing tumor-associated neutrophils (TANs) to block infiltration and function of CD8+ T cells. PD-L1+ TANs were found to be an essential subset of TANs that were regulated by CRKL expression and display an immunosuppressive phenotype. Mechanistically, CRKL inhibits adenomatous polyposis coli (APC)-mediated proteasomal degradation of β-catenin by competitively decreasing Axin1 binding, and thus fosters VEGFα and CXCL1 expression. Using human HCC samples, we verified the positive correlations of CRKL/β-catenin/VEGFα and CXCL1. Targeting CRKL using CRISPR-Cas9 gene editing (CRKL knockout) or its downstream regulators effectively restored the efficacy of anti-PD-1 therapy in an orthotopic mouse model and a patient-derived organotypic tumor spheroid (PDOTS) model. ConclusionsActivation of the CRKL/β-catenin/VEGFα and CXCL1 axis is a critical obstacle to successful anti-PD-1 therapy. Therefore, CRKL inhibitors combined with anti-PD-1 may be developed for the treatment of HCC. Impact and implicationsHere, we found CRKL was overexpressed in anti-PD-1 resistant HCC and CRKL upregulation promotes anti-PD-1 resistance in HCC. We identified that CRKL/β-catenin/VEGFα and CXCL1 axis upregulation contributes to anti-PD-1 tolerance through promoting tumor-associated neutrophils (TANs) infiltration. These findings support the strategy of bevacizumab-based ICIs combination therapy, and CRKL inhibitors combined with anti-PD-1 therapy may be developed for the treatment of HCC.

DNA polymerase beta connects tumorigenicity with the circadian clock in liver cancer through the epigenetic demethylation of Per1

The circadian-controlled DNA repair exhibits a strong diurnal rhythm. Disruption in circadian clock and DNA repair is closely linked with hepatocellular carcinoma (HCC) progression, but the mechanism remains unknown. Here, we show that polymerase beta (POLB), a critical enzyme in the DNA base excision repair pathway, is rhythmically expressed at the translational level in mouse livers. Hepatic POLB dysfunction dampens clock homeostasis, whereas retards HCC progression, by mediating the methylation of the 4th CpG island on the 5′UTR of clock gene Per1. Clinically, POLB is overexpressed in human HCC samples and positively associated with poor prognosis. Furthermore, the hepatic rhythmicity of POLB protein expression is orchestrated by Calreticulin (CALR). Our findings provide important insights into the molecular mechanism underlying the synergy between clock and food signals on the POLB-driven BER system and reveal new clock-dependent carcinogenetic effects of POLB. Therefore, chronobiological modulation of POLB may help to promote precise interventions for HCC.

Active AKT2 stimulation of SREBP1/SCD1-mediated lipid metabolism boosts hepatosteatosis and cancer

Due to soared obesity population worldwide, hepatosteatosis is becoming a major risk factor for hepatocellular carcinoma (HCC). Undertaken molecular events during the progression of steatosis to liver cancer are thus under intensive investigation. In this study, we demonstrated that high-fat diet potentiated mouse liver AKT2. Hepatic AKT2 hyperactivation through gain-of-function mutation of Akt2 (Akt2E17K) caused spontaneous hepatosteatosis, injury, inflammation, fibrosis, and eventually HCC in mice. AKT2 activation also exacerbated lipopolysaccharide and D-galactosamine hydrochloride-induced injury/inflammation and N-Nitrosodiethylamine (DEN)-induced HCC. A positive correlation between AKT2 activity and SCD1 expression was observed in human HCC samples. Activated AKT2 enhanced the production of monounsaturated fatty acid which was dependent on SREBP1 upregulation of SCD1. Blockage of active SREBP1 and ablation of SCD1 reduced steatosis, inflammation, and tumor burden in DEN-treated Akt2E17K mice. Therefore, AKT2 activation is crucial for the development of steatosis-associated HCC which can be treated with blockage of AKT2-SREBP1-SCD1 signaling cascade.

A prognostic model based on DNA methylation-related gene expression for predicting overall survival in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) continues to increase in morbidity and mortality among all types of cancer. DNA methylation, an important epigenetic modification, is associated with cancer occurrence and progression. The objective of this study was to establish a model based on DNA methylation risk scores for identifying new potential therapeutic targets in HCC and preventing cancer progression. Transcriptomic, clinical, and DNA methylation data on 374 tumor tissues and 50 adjacent normal tissues were downloaded from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma database. The gene expression profiles of the GSE54236 liver cancer dataset, which contains data on 161 liver tissue samples, were obtained from the Gene Expression Omnibus database. We analyzed the relationship between DNA methylation and gene expression levels after identifying the differentially methylated and expressed genes. Then, we developed and validated a risk score model based on the DNA methylation-driven genes. A tissue array consisting of 30 human hepatocellular carcinoma samples and adjacent normal tissues was used to assess the protein and mRNA expression levels of the marker genes by immunohistochemistry and qRT-PCR, respectively. Three methylation-related differential genes were identified in our study: GLS, MEX3B, and GNA14. The results revealed that their DNA methylation levels were negatively correlated with local gene expression regulation. The gene methylation levels correlated strongly with the prognosis of patients with liver cancer. This was confirmed by qRT-PCR and immunohistochemical verification of the expression of these genes or proteins in tumors and adjacent tissues. These results revealed the relationship between the level of relevant gene methylation and the prognosis of patients with liver cancer as well as the underlying cellular and biological mechanisms. This allows our gene signature to provide more accurate and appropriate predictions for clinical applications. Through bioinformatics analysis and experimental validation, we obtained three DNA methylation marker: GLS, MEX3B, and GNA14. This helps to predict the prognosis and may be a potential therapeutic target for HCC patients.

High USP32 expression contributes to cancer progression and is correlated with immune infiltrates in hepatocellular carcinoma

BackgroundUbiquitin-specific protease 32 (USP32) is a highly conserved gene that promotes cancer progression. However, its role in hepatocellular carcinoma (HCC) is not well understood. The aim of this project is to explore the clinical significance and functions of USP32 in HCC.MethodsThe expression of USP32 in HCC was evaluated using data from TCGA, GEO, TISCH, tissue microarray, and human HCC samples from our hospital. Survival analysis, PPI analysis and GSEA analysis were performed to evaluate USP32-related clinical significance, key molecules and enrichment pathways. Using the ssGSEA algorithm and TIMER, we investigated the relationships between USP32 and immune infiltrates in the TME. Univariate and multivariate Cox regression analyses were then used to identify key USP32-related immunomodulators and constructed a USP32-related immune prognostic model. Finally, CCK8, transwell and colony formation assays of HCC cells were performed and an HCC nude mouse model was established to verify the oncogenic role of USP32.ResultsUSP32 is overexpressed in HCC and its expression is an independent predictive factor for outcomes of HCC patients. USP32 is associated with pathways related to cell behaviors and cancer signaling, and its expression is significantly correlated with the infiltration of immune cells in the TME. We also successfully constructed a USP32-related immune prognostic model using 5 genes. Wet experiments confirmed that knockdown of USP32 could repress the proliferation, colony formation and migration of HCC cells in vitro and inhibit tumor growth in vivo.ConclusionUSP32 is highly expressed in HCC and closely correlates with the TME of HCC. It is a potential target for improving the efficacy of chemotherapy and developing new strategies for targeted therapy and immunotherapy in HCC.

Loss of Krüppel-like factor-10 facilitates the development of chemical-induced liver cancer in mice

BackgroundKrüppel-like factor 10 (KLF10) is involved in a positive feedback loop that regulates transforming growth factor β (TGFβ) signaling, and TGFβ plays an important role in the pathogenesis of liver disease. Here, we investigated whether KLF10 deletion affects the development of liver fibrosis and hepatocellular carcinoma (HCC).MethodsWe induced KLF10 deletion in C57BL/6 mice. Liver fibrosis was induced by feeding a diet high in fat and sucrose (high-fat diet [HFD]), whereas HCC was produced by intraperitoneal administration of N-diethylnitrosamine (DEN). An in vitro experiment was performed to evaluate the role of KLF10 in the cancer microenvironment using Hep3B and LX2 cells. An immunohistochemical study of KLF10 expression was performed using human HCC samples from 60 patients who had undergone liver resection.ResultsKLF10 deletion resulted in an increased DEN-induced HCC burden with significant upregulation of SMAD2, although loss of KLF10 did not alter HFD-induced liver fibrosis. DEN-treated mice with KLF10 deletion exhibited increased levels of mesenchymal markers (N-cadherin and SNAI2) and tumor metastasis markers (matrix metalloproteinases 2 and 9). KLF10 depletion in Hep3B and LX2 cells using siRNA was associated with increased invasiveness. Compared with co-culture of KLF10-preserved Hep3B cells and KLF10-intact LX2 cells, co-culture of KLF10-preserved Hep3B cells and KLF10-depleted LX2 cells resulted in significantly enhanced invasion. Low KLF10 expression in resected human HCC specimens was associated with poor survival.ConclusionThe results of this study suggest that loss of KLF10 facilitates liver cancer development with alteration in TGFβ signaling.

Diet-induced rewiring of the Wnt gene regulatory network connects aberrant splicing to fatty liver and liver cancer in DIAMOND mice

Several preclinical models have been recently developed for metabolic associated fatty liver disease (MAFLD) and associated hepatocellular carcinoma (HCC) but comprehensive analysis of the regulatory and transcriptional landscapes underlying disease in these models are still missing. We investigated the regulatory and transcriptional landscape in fatty livers and liver tumours from DIAMOND mice that faithfully mimic human HCC development in the context of MAFLD. RNA-sequencing and ChIP-sequencing revealed rewiring of the Wnt/β-catenin regulatory network in DIAMOND tumours, as manifested by chromatin remodelling and associated switching in the expression of the canonical TCF/LEF downstream effectors. We identified splicing as a major mechanism leading to constitutive oncogenic activation of β-catenin in a large subset of DIAMOND tumours, a mechanism that is independent on somatic mutations in the locus and that has not been previously shown. Similar splicing events were found in a fraction of human HCC and hepatoblastoma samples.

BMP4 upregulates glycogen synthesis through the SMAD/SLC2A1 (GLUT1) signaling axis in hepatocellular carcinoma (HCC) cells

BackgroundExcessive hepatic glycogen accumulation benefits tumorigenesis and cancer cell survival. We previously reported that BMP4 has the strongest ability to promote glycogenesis among the 14 BMPs in hepatocytes and augmented hepatocellular carcinoma (HCC) cell survival under hypoxia and hypoglycemia conditions by promoting the glycolysis pathway. However, the mechanism underlying BMP4’s effect on glycogenesis in HCC remains elusive.MethodsThe expression of BMP4 and SLC2A1 were acquired by analyzing the TCGA-LIHC dataset, as well as by immunohistochemical analysis of the 40 pairs of human HCC samples and para-tumor tissues. Gene expressions were detected by qPCR, immunoflurorescence staining, and Western blotting. Overexpression and silencing of BMP4 were accomplished through adenoviruses Ad-B4 and Ad-siB4 infection. Hepatic glycogen was detected by PAS staining. SLC2A1 (GLUT1) function was blocked by the inhibitor BAY-876. ChIP assay was used to determine the binding of SMADs to the promoter region of SLC2A1 in HCC cells. Lastly, the in vivo effect of BMP4-regulated SLC2A1 on HCC tumor growth was assessed in a xenograft model of HCC.ResultsThe elevated expression of BMP4 in HCC tumor tissues was highly correlated with hepatic glycogen accumulation in clinical samples. SLC2A1 was highly expressed in HCC tumor tissue and correlated with clinical stage and prognosis. Exogenous BMP4 augmented glycogen accumulation and upregulated the expression of glycogen synthesis-related genes in Huh7 and HepG2 cells, both of which were effectively blunted by SLC2A1inhibitor BAY-876. In mechanism, BMP4 activated SMAD5 to regulate the promoter of SLC2A1to enhance its expression. The in vivo xenograft experiments revealed that BMP4 promoted glycogen accumulation and tumor growth, which were effectively diminished by BAY-876.ConclusionThese results demonstrate that BMP4 upregulates glycogen synthesis through the SMAD/SLC2A1 (GLUT1) signaling axis in HCC cells, which may be exploited as novel therapeutic targets for HCC treatment.

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer.

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

MiR-182-5p Is Upregulated in Hepatic Tissues from a Diet-Induced NAFLD/NASH/HCC C57BL/6J Mouse Model and Modulates Cyld and Foxo1 Expression.

Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.

RNF125 attenuates hepatocellular carcinoma progression by downregulating SRSF1-ERK pathway.

Hepatocellular carcinoma (HCC) is one of the most deadly malignant cancers worldwide. Research into the crucial genes responsible for maintaining the aggressive behaviour of cancer cells is important for the clinical treatment of HCC. The purpose of this study was to determine whether the E3 ubiquitin ligase Ring Finger Protein 125 (RNF125) plays a role in the proliferation and metastasis of HCC. RNF125 expression in human HCC samples and cell lines was investigated using TCGA dataset mining, qRT‒PCR, western blot, and immunohistochemistry assays. In addition, 80 patients with HCC were studied for the clinical value of RNF125. Furthermore, the molecular mechanism by which RNF125 contributes to hepatocellular carcinoma progression was determined with mass spectrometry (MS), coimmunoprecipitation (Co-IP), dual-luciferase reporter assays, and ubiquitin ladder assays. We found that RNF125 was markedly downregulated in HCC tumour tissues, which was associated with a poor prognosis for patients with HCC. Moreover, the overexpression of RNF125 inhibited HCC proliferation and metastasis both in vitro and in vivo, whereas the knockdown of RNF125 exerted antithetical effects. Mechanistically, mass spectrometry analysis revealed a protein interaction between RNF125 and SRSF1, and RNF125 accelerated the proteasome-mediated degradation of SRSF1, which impeded HCC progression by inhibiting the ERK signalling pathway. Furthermore, RNF125 was detected to be the downstream target of miR-103a-3p. In this study, we identified that RNF125 is a tumour suppressor in HCC and inhibits HCC progression by inhibiting the SRSF1/ERK pathway. These findings provide a promising treatment target for HCC.

LOXL2 serves as a prognostic biomarker for hepatocellular carcinoma by mediating immune infiltration and vasculogenic mimicry.

The development of human hepatocellular carcinoma (HCC) is a multistep process that is accompanied by progressive changes in the liver microenvironment, including immune evasion and angiogenesis. Lysyl oxidase-like 2 (LOXL2) has been suggested to contribute to tumour progression and metastasis; however, the underlying mechanism remains unclear. The purpose of the present study was to explore the relationship between LOXL2 and immune infiltration and vasculogenic mimicry (VM) and to identify the role of LOXL2 in HCC diagnosis prognosis evaluation. The Cancer Genome Atlas (TCGA), UALCAN, GEPIA and Kaplan-Meier plotter databases were used to analyse LOXL2 expression and perform survival analysis. The Tumour Immune Estimation Resource (TIMER) was used to analyse immune cell infiltration, immune cell biomarkers and immune checkpoints. Immunohistochemistry (IHC) of 201 HCC samples was used to confirm the expression of LOXL2 and its relationship with VM. Coimmunoprecipitation (co-IP) and gain- and loss-of-function studies were performed to confirm the molecular mechanism of LOXL2 in VM. The expression of LOXL2 in HCC was higher than that in normal tissues at both the mRNA and protein levels. High expression of LOXL2 was associated with a poorer prognosis of HCC. The genetic alteration rate of LOXL2 was 5%. LOXL2 was positively related to immune cell infiltration and immune checkpoints (PD-1 and CTLA-4) in HCC. Co-IP showed that LOXL2 can interact directly with IQGAP1. Both gain- and loss-of-function studies showed that LOXL2 significantly induced cell migration, invasion and VM formation when IQGAP1 was upregulated. LOXL2 is involved in immune cell infiltration and promotes VM by upregulating IQGAP1. LOXL2 can be used as a novel biomarker for HCC diagnosis and prognosis prediction.

Ubiquitin ligase MARCH8 promotes the malignant progression of hepatocellular carcinoma through PTEN ubiquitination and degradation.

Membrane-associated ring-CH-type finger 8 (MARCH8) belongs to the MARCH family of membrane-bound E3 ubiquitin ligases. The N-terminus of MARCH family members contains the C4HC3 RING-finger domain, which can bind to E2 ubiquitin-conjugating enzymes, ubiquitinate substrate proteins, and thereby promote protein degradation through the proteasome pathway. The aim of this study was to determine the role of MARCH8 in hepatocellular carcinoma (HCC). We first analyzed the clinical relevance of MARCH8 based on The Cancer Genome Atlasdatabase. Immunohistochemical staining was used to detect MARCH8 expression in human HCC samples. Migration and invasion assays were conducted in vitro. Cell apoptosis and cell cycle distribution were analyzed by flow cytometry. The expression of phosphatase and tensin homologdeleted on chromosome 10(PTEN)-related markers was evaluated in HCC cells through Western blot analysis. MARCH8 was highly expressed in human HCC tissues, and its high expression was inversely correlated with patients' survival. Disrupting MARCH8 expression significantly inhibited the proliferation, migration, and cell cycle progression of HCC cells, while also promoting their apoptosis. In contrast, the overexpression of MARCH8 significantly enhanced cell proliferation. Mechanistically, our results showed that MARCH8 interacted with PTEN and suppressed the protein stability of PTEN by enhancing its ubiquitination level via the proteasome. MARCH8 also activated AKT in HCC cells and tumors. In vivo, overexpression of MARCH8 could promote the growth of hepatic tumors through the AKT pathway. MARCH8 may promote the malignant progression of HCC by promoting the ubiquitination of PTEN, thereby relieving the inhibitory effect of PTEN on the malignant phenotype of HCC cells.

Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with dreadful clinical outcome. Tyrosine kinase inhibitors and immune checkpoint inhibitors are the only United States Food and Drug Administration-approved therapeutic options for patients with advanced HCC with limited therapeutic success. Ferroptosis is a form of immunogenic and regulated cell death caused by chain reaction of iron-dependent lipid peroxidation. Coenzyme Q10 (CoQ10)/ferroptosis suppressor protein 1 (FSP1) axis was recently identified as a novel protective mechanism against ferroptosis. We would like to explore whether FSP1 could be a potential therapeutic target for HCC. FSP1 expression in human HCC and paired non-tumorous tissue samples were determined by reverse transcription-quantitative polymerase chain reaction, followed by clinicopathologic correlation and survival studies. Regulatory mechanism for FSP1 was determined using chromatin immunoprecipitation. The hydrodynamic tail vein injection model was used for HCC induction to evaluate the efficacy of FSP1 inhibitor (iFSP1) invivo. Single-cell RNA sequencing revealed the immunomodulatory effects of iFSP1 treatment. We showed that HCC cells greatly rely on the CoQ10/FSP1 system to overcome ferroptosis. We found that FSP1 was significantly overexpressed in human HCC and is regulated by kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 pathway. FSP1 inhibitor iFSP1 effectively reduced HCC burden and profoundly increased immune infiltrates including dendritic cells, macrophages, and T cells. We also demonstrated that iFSP1 worked synergistically with immunotherapies to suppress HCC progression. We identified FSP1 as a novel, vulnerable therapeutic target in HCC. The inhibition of FSP1 potently induced ferroptosis, which promoted innate and adaptive anti-tumor immune responses and effectively suppressed HCC tumor growth. FSP1 inhibition therefore represents a new therapeutic strategy for HCC.

Interplay Between Fatty Acids, Stearoyl-Co-A Desaturase, Mechanistic Target of Rapamycin, and Yes-Associated Protein/Transcriptional Coactivator With PDZ-Binding Motif in Promoting Hepatocellular Carcinoma

Nonalcoholic fatty liver disease has reached pandemic proportions with one of its most consequential complications being hepatocellular carcinoma (HCC). Nonalcoholic fatty liver disease-related HCC is becoming the leading indication for liver transplantation in the United States. Given the scarcity of available organs, early detection and prevention remain key in prevention and management of the disease. Over the years, the yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) pathway emerged as a key signal transduction pathway in the pathogenesis of HCC. In this review, we explore the interplay between the YAP/TAZ pathway as a point of convergence in HCC pathogenesis. We review the evidence of how lipid reprogramming and key lipid pathways, saturated and monounsaturated fatty acids (through the rate-limiting enzyme stearoyl Co-A desaturase), the mevalonic acid pathway (the role of statins), and mechanistic target of rapamycin all play critical roles in intricate and complex networks that tightly regulate the YAP/TAZ pro-oncogenic pathway. Nonalcoholic fatty liver disease has reached pandemic proportions with one of its most consequential complications being hepatocellular carcinoma (HCC). Nonalcoholic fatty liver disease-related HCC is becoming the leading indication for liver transplantation in the United States. Given the scarcity of available organs, early detection and prevention remain key in prevention and management of the disease. Over the years, the yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) pathway emerged as a key signal transduction pathway in the pathogenesis of HCC. In this review, we explore the interplay between the YAP/TAZ pathway as a point of convergence in HCC pathogenesis. We review the evidence of how lipid reprogramming and key lipid pathways, saturated and monounsaturated fatty acids (through the rate-limiting enzyme stearoyl Co-A desaturase), the mevalonic acid pathway (the role of statins), and mechanistic target of rapamycin all play critical roles in intricate and complex networks that tightly regulate the YAP/TAZ pro-oncogenic pathway. Primary liver cancer is the second leading cause of cancer death worldwide.1Sung H. Ferlay J. Siegel R.L. et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin. 2021; 71: 209-249Crossref PubMed Scopus (24505) Google Scholar Hepatocellular carcinoma (HCC), which comprises 90% of the cases, commonly presents in patients with hepatic fibrosis associated with chronic hepatitis B virus or hepatitis C virus, excessive alcohol consumption, or the metabolic syndrome (MetS). As described below, the genetic landscape of HCC is complex and includes mutations in the Wnt/β-catenin (CTNNB1) pathway, chromatin remodeling, telomere maintenance, and inactivation of p53. In the USA, liver cancer incidence rates have more than tripled since 1980, while the cancer-associated death rates have more than doubled during this time. In parallel, nonalcoholic fatty liver disease (NAFLD), a common condition in patients with obesity and type 2 diabetes, has reached pandemic proportions and continues to rise.2Estes C. Razavi H. Loomba R. et al.Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease.Hepatology. 2018; 67: 123-133Crossref PubMed Scopus (960) Google Scholar In the era of direct-acting antiviral therapy for hepatitis C virus and improved hepatitis B virus treatment, NAFLD-HCC is predicted to become the leading indication for liver transplantation.3Younossi Z.M. Stepanova M. Ong J. et al.Nonalcoholic steatohepatitis is the most rapidly increasing indication for liver transplantation in the United States.Clin Gastroenterol Hepatol. 2021; 19: 580-589.e5Abstract Full Text Full Text PDF PubMed Google Scholar As liver transplantation remains a scarce resource with recent evidence suggesting that HCCs are transplanted less often following changes in United States Organ Procurement Network policy,4Kwong A.J. Ghaziani T.T. Mehta N. Decreased urgency among liver transplantation candidates with hepatocellular carcinoma in the United States.Liver Transpl. 2022; 28: 725-727Crossref PubMed Scopus (2) Google Scholar understanding its underlying mechanisms is of upmost importance to develop and implement chemoprevention and early detection programs, as well as tailor treatment approaches. NAFLD and HCC are complex diseases that result from genetic and environmental interactions.5Eslam M. Valenti L. Romeo S. Genetics and epigenetics of NAFLD and NASH: clinical impact.J Hepatol. 2018; 68: 268-279Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar Many clinical potentially modifiable risk factors have been associated with NAFLD-HCC development, including features of the MetS, as defined by the clustering of type 2 diabetes, hypertension, dyslipidemia, and obesity, where each condition plays an additive or synergistic role in the pathogenesis of NAFLD-HCC.6Simon T.G. King L.Y. Chong D.Q. et al.Diabetes, metabolic comorbidities, and risk of hepatocellular carcinoma: results from two prospective cohort studies.Hepatology. 2018; 67: 1797-1806Crossref PubMed Scopus (68) Google Scholar,7Kanwal F. Kramer J.R. Mapakshi S. et al.Risk of hepatocellular cancer in patients with non-alcoholic fatty liver disease.Gastroenterology. 2018; 155: 1828-1837.e2Abstract Full Text Full Text PDF PubMed Google Scholar Although statins are commonly prescribed for the treatment of dyslipidemia and the MetS, their use has been shown to be protective in all etiologies of HCC,8Singh S. Singh P.P. Singh A.G. et al.Statins are associated with a reduced risk of hepatocellular cancer: a systematic review and meta-analysis.Gastroenterology. 2013; 144: 323-332Abstract Full Text Full Text PDF PubMed Google Scholar,9Simon T.G. Duberg A.S. Aleman S. et al.Lipophilic statins and risk for hepatocellular carcinoma and death in patients with chronic viral hepatitis: results from a nationwide Swedish population.Ann Intern Med. 2019; 171: 318-327Crossref PubMed Scopus (61) Google Scholar including in nonalcoholic steatohepatitis (NASH), the more severe form in the NAFLD spectrum.10Kaplan D.E. Serper M.A. Mehta R. et al.Effects of hypercholesterolemia and statin exposure on survival in a large national cohort of patients with cirrhosis.Gastroenterology. 2019; 156: 1693-1706.e12Abstract Full Text Full Text PDF PubMed Google Scholar,11Pinyopornpanish K. Al-Yaman W. Butler R.S. et al.Chemopreventive effect of statin on hepatocellular carcinoma in patients with nonalcoholic steatohepatitis cirrhosis.Am J Gastroenterol. 2021; 116: 2258-2269Crossref PubMed Scopus (7) Google Scholar The mechanisms involved in NAFLD-related HCC pathogenesis are therefore of major fundamental and translational importance but remain incompletely understood. In a large veteran population, the estimated incidence of HCC in the patients with NAFLD is 0.21/1000 person-years (PY), with the highest incidence occurring in the subpopulation of patients with cirrhosis (10.6/1000 PY).7Kanwal F. Kramer J.R. Mapakshi S. et al.Risk of hepatocellular cancer in patients with non-alcoholic fatty liver disease.Gastroenterology. 2018; 155: 1828-1837.e2Abstract Full Text Full Text PDF PubMed Google Scholar Simon et al6Simon T.G. King L.Y. Chong D.Q. et al.Diabetes, metabolic comorbidities, and risk of hepatocellular carcinoma: results from two prospective cohort studies.Hepatology. 2018; 67: 1797-1806Crossref PubMed Scopus (68) Google Scholar demonstrated similar findings in a European population-based cohort of biopsy-proven NAFLD and NASH, where HCC occurred at an incidence of 6.2/1000 PY for patients with underlying cirrhosis (95% confidence interval 4.2–8.8/1000 PY). The incidence of HCC was lower for patients with biopsy-proven simple steatosis (n = 5939) at 0.8/1000 PY and NASH without fibrosis (n = 1050) at 1.2/1000 PY.6Simon T.G. King L.Y. Chong D.Q. et al.Diabetes, metabolic comorbidities, and risk of hepatocellular carcinoma: results from two prospective cohort studies.Hepatology. 2018; 67: 1797-1806Crossref PubMed Scopus (68) Google Scholar The differences in incidence and disease progression likely reflect the heterogeneity in the NAFLD population, potentially nonmodifiable genetic differences, and their gene-environment interactions. There is evidence from previous genome-wide association studies demonstrating a predominance of NAFLD in Hispanic patients, followed by Caucasians and African Americans,12Romeo S. Kozlitina J. Xing C. et al.Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (2215) Google Scholar which have been further substantiated in the HCC population.13Trepo E. Caruso S. Yang J. et al.Common genetic variation in alcohol-related hepatocellular carcinoma: a case-control genome-wide association study.Lancet Oncol. 2022; 23: 161-171Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Unlike viral and ethanol-associated causes of HCC, NAFLD patients represent a distinct high-risk population where 20%–30% of all HCCs occur in the absence of cirrhosis.14Kanwal F. Kramer J.R. Li L. et al.Effect of metabolic traits on the risk of cirrhosis and hepatocellular cancer in nonalcoholic fatty liver disease.Hepatology. 2020; 71: 808-819Crossref PubMed Scopus (105) Google Scholar,15Benhammou J.N. Aby E.S. Shirvanian G. et al.Improved survival after treatments of patients with nonalcoholic fatty liver disease associated hepatocellular carcinoma.Sci Rep. 2020; 10: 9902Crossref PubMed Scopus (8) Google Scholar Although current guidance does not recommend HCC screening in patients without cirrhosis, the estimated disease burden globally16Huang D.Q. El-Serag H.B. Loomba R. Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention.Nat Rev Gastroenterol Hepatol. 2021; 18: 223-238Crossref PubMed Scopus (437) Google Scholar and health-care-associated costs17Allen A.M. Van Houten H.K. Sangaralingham L.R. et al.Healthcare cost and utilization in nonalcoholic fatty liver disease: real-world data from a large U.S. Claims Database.Hepatology. 2018; 68: 2230-2238Crossref PubMed Scopus (70) Google Scholar prompt the need for further research in this population, as outlined by recent professional society recommendations.18Kanwal F. Shubrook J.H. Younossi Z. et al.Preparing for the NASH epidemic: a call to action.Metabolism. 2021; 122: 154822Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Heritability of NAFLD and NASH has been suggested based on early observations of the clustering of disease in families and twins.19Schwimmer J.B. Celedon M.A. Lavine J.E. et al.Heritability of nonalcoholic fatty liver disease.Gastroenterology. 2009; 136: 1585-1592Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar,20Loomba R. Schork N. Chen C.H. et al.Heritability of hepatic fibrosis and steatosis based on a prospective twin study.Gastroenterology. 2015; 149: 1784-1793Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar In NAFLD-related HCC, teasing out inherited genetic contributions from chronic fibrosis/cirrhosis vs NAFLD-specific causes using genome-wide association studies remains a challenge and is still being investigated. Independent of its genetic predisposing factors, HCC often results from an accumulation of somatic mutations that lead to tumor initiation and progression. Studies have demonstrated that each HCC has about 40–60 somatic mutations, several of which converge on key molecular pathways.21Llovet J.M. Zucman-Rossi J. Pikarsky E. et al.Hepatocellular carcinoma.Nat Rev Dis Primers. 2016; 2: 16018Crossref PubMed Google Scholar Many mutations affect telomere maintenance (telomere reverse transcriptase activation); Wnt activation (mutation in CTNNB1 which encodes β-catenin, APC, and AXIN1); the tumor suppressor p53 gene (TP53); chromatin remodeling (including in the adenine-thymine-rich interactions-rich interaction domain 1A or ARID1A); activation of the PI3K/mechanistic target of rapamycin (mTORC1); and activation of receptor tyrosine kinases.21Llovet J.M. Zucman-Rossi J. Pikarsky E. et al.Hepatocellular carcinoma.Nat Rev Dis Primers. 2016; 2: 16018Crossref PubMed Google Scholar, 22Llovet J.M. Kelley R.K. Villanueva A. et al.Hepatocellular carcinoma.Nat Rev Dis Primers. 2021; 7: 6Crossref PubMed Scopus (1262) Google Scholar, 23Chiang D.Y. Villanueva A. Hoshida Y. et al.Focal gains of VEGFA and molecular classification of hepatocellular carcinoma.Cancer Res. 2008; 68: 6779-6788Crossref PubMed Scopus (480) Google Scholar, 24Guegan J.P. Lapouge M. Voisin L. et al.Signaling by the tyrosine kinase Yes promotes liver cancer development.Sci Signal. 2022; 15eabj4743Crossref PubMed Scopus (1) Google Scholar, 25Takeda H. Takai A. Eso Y. et al.Genetic landscape of multistep hepatocarcinogenesis.Cancers (Basel). 2022; 14: 568Crossref PubMed Scopus (3) Google Scholar The majority of mutations identified have been shown not to be actionable therapeutically.22Llovet J.M. Kelley R.K. Villanueva A. et al.Hepatocellular carcinoma.Nat Rev Dis Primers. 2021; 7: 6Crossref PubMed Scopus (1262) Google Scholar,26Hyman D.M. Taylor B.S. Baselga J. Implementing genome-driven oncology.Cell. 2017; 168: 584-599Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar Of the multiple pathways identified, certain ones deserve special attention because of their common occurrence. For instance, β-catenin activation through point mutations of CTNNB1 and mutation of TP53 occurs in up to 50% of HCC cases.25Takeda H. Takai A. Eso Y. et al.Genetic landscape of multistep hepatocarcinogenesis.Cancers (Basel). 2022; 14: 568Crossref PubMed Scopus (3) Google Scholar,27de La Coste A. Romagnolo B. Billuart P. et al.Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.Proc Natl Acad Sci U S A. 1998; 95: 8847-8851Crossref PubMed Scopus (979) Google Scholar,28Khalaf A.M. Fuentes D. Morshid A.I. et al.Role of Wnt/beta-catenin signaling in hepatocellular carcinoma, pathogenesis, and clinical significance.J Hepatocell Carcinoma. 2018; 5: 61-73Crossref PubMed Google Scholar HCCs can be subclassified into proliferation and nonproliferation classes with some overlaps in the mutational landscape, including in β-catenin activation.22Llovet J.M. Kelley R.K. Villanueva A. et al.Hepatocellular carcinoma.Nat Rev Dis Primers. 2021; 7: 6Crossref PubMed Scopus (1262) Google Scholar,29Zucman-Rossi J. Villanueva A. Nault J.C. et al.Genetic landscape and biomarkers of hepatocellular carcinoma.Gastroenterology. 2015; 149: 1226-1239.e4Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar An emerging area of interest is the interplay among lipid metabolism, mTORC1, and the Hippo pathway in HCC. These represent major foci of this article, which are discussed in subsequent sections. It is widely accepted that growing cells require unsaturated fatty acids and cholesterol as essential building blocks for biogenesis of cellular membranes. Early on, investigators observed that cancer cells also synthesize lipids.30Medes G. Thomas A. Weinhouse S. Metabolism of neoplastic tissue. IV. A study of lipid synthesis in neoplastic tissue slices in vitro.Cancer Res. 1953; 13: 27-29PubMed Google Scholar Lipids, and specifically fatty acids, have been shown to have an integral role in changing the tumor environment (TME) to meet the demands of rapid cell proliferation observed in cancer cells, where oxygen and nutrients are scarce.31Currie E. Schulze A. Zechner R. et al.Cellular fatty acid metabolism and cancer.Cell Metab. 2013; 18: 153-161Abstract Full Text Full Text PDF PubMed Scopus (1206) Google Scholar This hallmark of cancer cells has been termed “metabolic reprogramming,”32Beloribi-Djefaflia S. Vasseur S. Guillaumond F. Lipid metabolic reprogramming in cancer cells.Oncogenesis. 2016; 5e189Crossref PubMed Google Scholar and has been linked to clinical aggressiveness of many tumors.33de Gonzalo-Calvo D. Lopez-Vilaro L. Nasarre L. et al.Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study.BMC Cancer. 2015; 15: 460Crossref PubMed Scopus (127) Google Scholar This is supported by a large body of evidence that suppression of lipogenic pathways decreases cell growth in both in vitro and in vivo models.34Guillaumond F. Bidaut G. Ouaissi M. et al.Cholesterol uptake disruption, in association with chemotherapy, is a promising combined metabolic therapy for pancreatic adenocarcinoma.Proc Natl Acad Sci U S A. 2015; 112: 2473-2478Crossref PubMed Scopus (241) Google Scholar,35Svensson R.U. Parker S.J. Eichner L.J. et al.Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models.Nat Med. 2016; 22: 1108-1119Crossref PubMed Scopus (280) Google Scholar Understanding tumor lipid metabolism and the crosstalk between the lipidome and the pathways that drive HCC has therefore advanced the targeting of lipid pathways in cancer treatments and includes the use of fatty acid synthase (FASN) TVB-2640 for non-small-cell carcinoma (NCT03808558), high-grade sarcoma (NCT03032484), and triple-negative breast cancer (NCT03179904), which are all currently investigated in clinical trials. The study of the lipidome has only recently been extended to understanding the role of lipids in the pathogenesis of HCC. Most human studies have been conducted in viral etiologies of HCC and have focused on urine and serum lipid analyses with the aim to understand tumor biology and develop clinical biomarkers.36Xue R. Lin Z. Deng C. et al.A serum metabolomic investigation on hepatocellular carcinoma patients by chemical derivatization followed by gas chromatography/mass spectrometry.Rapid Commun Mass Spectrom. 2008; 22: 3061-3068Crossref PubMed Scopus (0) Google Scholar, 37Chen F. Xue J. Zhou L. et al.Identification of serum biomarkers of hepatocarcinoma through liquid chromatography/mass spectrometry-based metabonomic method.Anal Bioanal Chem. 2011; 401: 1899-1904Crossref PubMed Scopus (75) Google Scholar, 38Xiao J.F. Varghese R.S. Zhou B. et al.LC-MS based serum metabolomics for identification of hepatocellular carcinoma biomarkers in Egyptian cohort.J Proteome Res. 2012; 11: 5914-5923Crossref PubMed Scopus (100) Google Scholar, 39Shariff M.I. Ladep N.G. Cox I.J. et al.Characterization of urinary biomarkers of hepatocellular carcinoma using magnetic resonance spectroscopy in a Nigerian population.J Proteome Res. 2010; 9: 1096-1103Crossref PubMed Scopus (66) Google Scholar, 40Nahon P. Amathieu R. Triba M.N. et al.Identification of serum proton NMR metabolomic fingerprints associated with hepatocellular carcinoma in patients with alcoholic cirrhosis.Clin Cancer Res. 2012; 18: 6714-6722Crossref PubMed Scopus (61) Google Scholar, 41Chen T. Xie G. Wang X. et al.Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma.Mol Cell Proteomics. 2011; 10M110.004945Google Scholar, 42Passos-Castilho A.M. Carvalho V.M. Cardozo K.H. et al.Serum lipidomic profiling as a useful tool for screening potential biomarkers of hepatitis B-related hepatocellular carcinoma by ultraperformance liquid chromatography-mass spectrometry.BMC Cancer. 2015; 15: 985Crossref PubMed Scopus (16) Google Scholar Given that serum and tissue lipids measurements are not always concordant,43Parker B.L. Calkin A.C. Seldin M.M. et al.An integrative systems genetic analysis of mammalian lipid metabolism.Nature. 2019; 567: 187-193Crossref PubMed Scopus (62) Google Scholar other studies have focused on understanding the lipid profile of the HCCs and adjacent nontumor tissue controls.44Lu Y. Li N. Gao L. et al.Acetylcarnitine is a candidate diagnostic and prognostic biomarker of hepatocellular carcinoma.Cancer Res. 2016; 76: 2912-2920Crossref PubMed Scopus (58) Google Scholar,45Li Z. Guan M. Lin Y. et al.Aberrant lipid metabolism in hepatocellular carcinoma revealed by liver lipidomics.Int J Mol Sci. 2017; 18: 2550Crossref PubMed Scopus (57) Google Scholar Few lipidomic studies have been conducted in NAFLD-HCC patients. Lewinska et al46Lewinska M. Santos-Laso A. Arretxe E. et al.The altered serum lipidome and its diagnostic potential for non-alcoholic fatty liver (NAFL)-associated hepatocellular carcinoma.EBioMedicine. 2021; 73103661Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar recently studied the serum lipidome profile of patients with NAFLD-HCC (n = 27) and compared it to that of patients with ethanol and viral etiologies of HCC (n = 32), morbidly obese patients (n = 102), and healthy controls (n = 35). Compared to patients with ethanol and viral etiologies of HCC and obsese patients with NAFLD, NAFLD-HCC patients had higher triglycerides (47:0 and 45:1) and phosphatidylcholine (16:0/17:0, 18:2/0:0, 0:0/18:2) levels but lower monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids, and linoleic acid, after adjusting for age, sex, and body mass index.46Lewinska M. Santos-Laso A. Arretxe E. et al.The altered serum lipidome and its diagnostic potential for non-alcoholic fatty liver (NAFL)-associated hepatocellular carcinoma.EBioMedicine. 2021; 73103661Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar The authors propose that the lower MUFA and polyunsaturated fatty acids pool may be related to an increase in the uptake of these lipid species by the HCCs from the serum to sustain their growth and proliferation. Consistent with these findings, Muir et al,47Muir K. Hazim A. He Y. et al.Proteomic and lipidomic signatures of lipid metabolism in NASH-associated hepatocellular carcinoma.Cancer Res. 2013; 73: 4722-4731Crossref PubMed Scopus (110) Google Scholar identified that stearoyl-Co-A desaturase (SCD), the rate-limiting enzyme in the conversion of saturated fatty acids (SFAs) to MUFAs, which is preferentially found on the endoplasmic reticulum, was upregulated in human NAFLD-related HCC samples and Pten-null NASH-HCC murine models. While unsaturated fatty acids are required for cellular proliferation and membrane biogenesis, fatty acids also play important signaling functions that remain much less understood. As indicated above, SCD plays a critical role in regulating the ratio of unsaturated fatty acids/SFAs. MUFAs represent the precursors of the main components of cellular membranes and are crucially important in the pathogenesis of the MetS, NAFLD, NASH, and HCC pathogenesis.48Chiappini F. Coilly A. Kadar H. et al.Metabolism dysregulation induces a specific lipid signature of nonalcoholic steatohepatitis in patients.Sci Rep. 2017; 746658Crossref Scopus (110) Google Scholar, 49Walle P. Takkunen M. Mannisto V. et al.Fatty acid metabolism is altered in non-alcoholic steatohepatitis independent of obesity.Metabolism. 2016; 65: 655-666Abstract Full Text Full Text PDF PubMed Google Scholar, 50Lai K.K.Y. Kweon S.M. Chi F. et al.Stearoyl-CoA desaturase promotes liver fibrosis and tumor development in mice via a Wnt positive-signaling loop by stabilization of low-density lipoprotein-receptor-related proteins 5 and 6.Gastroenterology. 2017; 152: 1477-1491Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar This is supported by clinical studies demonstrating worse overall survival of patients with HCCs who demonstrate a high SCD expression, compared to HCCs with low SCD expression. Consistent with SCD playing a critical role in HCC tumor growth, Bansal et al51Bansal S. Berk M. Alkhouri N. et al.Stearoyl-CoA desaturase plays an important role in proliferation and chemoresistance in human hepatocellular carcinoma.J Surg Res. 2014; 186: 29-38Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar demonstrated the inverse relationship between SCD gene expression patterns and HCC tumor differentiation. Similarly, the role of SCD in HCC is further supported by an elevated serum saturated to unsaturated phosphatidylcholine (16:0/16:1) level in patients with cirrhosis and HCC.50Lai K.K.Y. Kweon S.M. Chi F. et al.Stearoyl-CoA desaturase promotes liver fibrosis and tumor development in mice via a Wnt positive-signaling loop by stabilization of low-density lipoprotein-receptor-related proteins 5 and 6.Gastroenterology. 2017; 152: 1477-1491Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar,52Cotte A.K. Cottet V. Aires V. et al.Phospholipid profiles and hepatocellular carcinoma risk and prognosis in cirrhotic patients.Oncotarget. 2019; 10: 2161-2172Crossref PubMed Scopus (4) Google Scholar Mechanistic studies of SCD and MUFAs in chronic liver disease point to selective effects of SCD on different resident liver cell types including M1 macrophage, hepatic stellate cells, and hepatocytes.53Kikuchi K. Tsukamoto H. Stearoyl-CoA desaturase and tumorigenesis.Chem Biol Interact. 2020; 316: 108917Crossref PubMed Scopus (0) Google Scholar These data suggest that the TME, supported by these other cell types, may play an integral role in HCC growth and aggressiveness. However, detailed human cell-type studies are lacking and have only recently been able to be identified with the advent of single-cell and single-nucleus RNA sequencing.54Slyper M. Porter C.B.M. Ashenberg O. et al.A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.Nat Med. 2020; 26: 792-802Crossref PubMed Scopus (1) Google Scholar, 55Alvarez M. Benhammou J.N. Darci-Maher N. et al.Human liver single nucleus and single cell RNA sequencing identify a hepatocellular carcinoma-associated cell-type affecting survival.Genome Med. 2022; 14: 50Crossref PubMed Scopus (2) Google Scholar As shown in Figure 1, SCD expression is regulated at multiple levels,56Benhammou J.N. Ko A. Alvarez M. et al.Novel lipid long intervening noncoding RNA, oligodendrocyte maturation-associated long intergenic noncoding RNA, regulates the liver steatosis gene stearoyl-coenzyme A desaturase as an enhancer RNA.Hepatol Commun. 2019; 3: 1356-1372Crossref PubMed Google Scholar including by the sterol regulatory element-binding protein 1 (SREBP1), the master transcription regulator of lipid biosynthesis, which has higher expression in HCC through mammalian target of rapamycin (mTOR) signaling. mTOR Functions as a catalytic subunit in 2 distinct multiprotein complexes, the mTORC1, characterized by the subunit Raptor (regulatory-associated protein of mTORC1), and mTORC2, characterized by the subunit Rictor (Raptor-independent companion of mTORC2). The heterodimer of the tumor suppressor TSC1 and TSC2 represses mTORC1 activity by acting as the GTPase-activating protein for Rheb, a potent activator of mTORC1 in its guanosine triphosphate-bound state (Figure 1). Using an interactive open-access database (www.proteinatlas.org/pathology), we found that higher expression of Rheb is significantly associated with poor survival in HCC cases (Figure 2A). In the presence of amino acids, activated Akt and/or an extracellular signal-regulated kinasesK/p90RSK phosphorylate and uncouple TSC1/TSC2 from Rheb, leading to Rheb-GTP accumulation and mTORC1 activation at lysosomal membranes, which then promotes cell growth.57Porstmann T. Santos C.R. Griffiths B. et al.SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth.Cell Metab. 2008; 8: 224-236Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar Ragulator complex proteins LAMTOR 1, 3, and 5, which are involved in amino acid sensing and mTORC1 activation, are also significantly associated with unfavorable prognosis (survival) in HCC (Figure 2B–D). The association of Rheb and LAMTORS 1, 3, and 5 with unfavorable prognosis emphasizes the importance of mTORC1 function in HCC development. In turn, mTORC1 increases lipogenesis through regulation of SREBP1 at several levels, including trafficking, processing, and transcription58Han J. Wang Y. mTORC1 signaling in hepatic lipid metabolism.Protein Cell. 2018; 9: 145-151Crossref PubMed Scopus (73) Google Scholar that culminate in its nuclear localization and transcriptional activation. In turn, nuclear SREBP1 induces the expression of lipogenesis genes, including SCD.Figure 2Kaplan-Meier plots for gene expression of the signaling network in HCC. Images were reproduced from the Human Protein Atlas (version 17) available from www.proteinatlas.org.59Uhlén M. Fagerberg L. Hallström B.M. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Google Scholar Transcriptomics data were available from 365 patients in total with 119 female and 246 male patients. A majority of patients (n = 235) were still alive at the time of data collection. The stage distribution was (i) 170 patients, stage (ii) 84 patients, stage (iii) 83 patients, stage (iv) 4 patients, and 24 patients with missing data information. The links to the specific genes shown are as follows:Show full caption(A) Rheb: https://www.proteinatlas.org/ENSG00000106615-RHEB/pathology/liver+cancer/(B) LAMTOR1: https://www.proteinatlas.org/ENSG00000149357-LAMTOR1/pathology/liver+cancer(C) LAMTOR3: https://www.proteinatlas.org/ENSG00000109270-LAMTOR3/pathology/liver+cancer(D) LAMBOR5: https://www.proteinatlas.org/ENSG00000134248-LAMTOR5/pathology/liver+cancer(E) SAV1: https://www.proteinatlas.org/ENSG00000151748-SAV1/pathology/liver+cancer(F) BIRC5: https://www.proteinatlas.org/ENSG00000089685-BIRC5/pathology/liver+cancer(G) TEAD2: https://www.proteinatlas.org/ENSG00000074219-TEAD2/pathology/liver+cancer(H) Mob4: https://www.proteinatlas.org/ENSG00000115540-MOB4/pathology/liver+cancer.View Large

LINC02870 facilitates SNAIL translation to promote hepatocellular carcinoma progression.

Exploring the roles of long noncoding RNAs (lncRNAs) in tumorigenesis and metastasis could contribute to the recognition of novel diagnostic and therapeutic targets. LINC02870 is a novel lncRNA, whose role in tumors has not been reported. Herein, we focused on the function and mechanism of LINC02870 in human hepatocellular carcinoma (HCC). We first carried out a pan-cancer study of LINC02870 expression and its relationship to prognosis, and LINC02870 was determined to be a possible oncogene in HCC. Upregulated expressions of LINC02870 were also found in our HCC samples compared to the para-tumor samples. Moreover, overexpression of LINC02870 promoted the growth, migration, and invasion of HCC cells. Subsequently, binding proteins of LINC02870 were identified by a number of in silico analyses, including correlation analysis, signaling network analysis, and survival analysis. Intriguingly, the most promising binding protein of LINC02870 was predicted and confirmed to be eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), an important component of the eukaryotic translation initiation factor 4F complex that initiates cap-dependent translation. Further investigation showed that LINC02870 increased the translation of SNAIL to induce malignant phenotypes in HCC cells. Additionally, HCC patients with higher expression levels of LINC02870 and EIF4G1 had shorter survival times than those with lower expression levels. Thus, our findings suggested that LINC02870 induced SNAIL translation and correlated with poor prognosis and tumor progression in HCC.