Human HCC Tissues Research Articles

Upregulation of miR-3130-5p Enhances Hepatocellular Carcinoma Growth by Suppressing Ferredoxin 1 : miR-3130-5p Enhances HCC Growth via Inhibiting FDX1.

Hepatocellular carcinoma [HCC] is a leading cause of cancer-related mortality worldwide, necessitating the exploration of novel therapeutic targets. Although accumulating studies have identified Ferredoxin 1 [FDX1], a key regulator of cuproptosis, as a candidate tumor suppressor and potential therapeutic target, its role and mechanism remain elusive in HCC. The FDX1 expression was investigated in human HCC tissues and cell lines. Potential microRNAs targeting FDX1 were predicted by bioinformatic analysis and validated using qPCR screening, a dual luciferase reporter assay, MiR-3130-5p and miR-1910-3p mimics and inhibitors, overexpression plasmids, and xenograft nude mouse model. The correlation between miR-3130-5p/FDX1 axis and HCC patient prognosis was analyzed by using Kaplan-Meier survival analysis. We demonstrated that the expression of FDX1 was downregulated in human HCC tissues and cell lines compared to non-cancerous counterparts, and the downregulation of FDX1 was associated with poor overall survival in HCC patients. Subsequent bioinformatic analysis and experimental validations showed that FDX1 expression was reduced by microRNA [miR]-3130-5p mimic while induced by miR-3130-5p inhibitor. Further, miR-3130-5p was upregulated in HCC tissues and cells, correlating with a poor prognosis of HCC patients. Besides, lentivirus-mediated overexpression of miR-3130-5p significantly enhanced HCC growth in xenograft nude mouse models. Mechanistically, it was demonstrated that miR-3130-5p inhibited FDX1 expression via binding to its 3' untranslated region [3' UTR], while overexpression of FDX1 counteracted the promoting effect of miR-3130-5p on HCC cell proliferation. These findings suggest the miR-3130-5p/FDX1 axis as a prognostic biomarker as well as a potential therapeutic target in HCC.

Loss of FAM172A gene prompts cell proliferation in liver regeneration.

The present study was designed to explore the function of FAM172A in liver regeneration and HCC. Mice were sacrificed after 70% partial hepatectomy (PH). RNA sequencing was performed on primary hepatocytes of WT and FAM172A-/- mice. We used HepG2 cells to construct cell lines with stably knockdown and overexpression of FAM172A. The expression of FAM172A in liver tissues was investigated by immunohistochemical staining, and we also used public database to perform survival analysis and prognostic model in HCC. Compared with WT mice after PH, normalized liver weight/body weight (LW/BW) ratio and the proliferating cell nuclear antigen (PCNA) protein level of FAM172A-/- mice elevated. The DEGs were mainly enriched in inflammatory response, tumor necrosis factor production, and wound healing. FAM172A knockdown enhanced the NFκB-TNFα and pERK-YAP1-Cyclin D1 axis. FAM172A peptide inhibited proliferation of primary hepatocytes. Moreover, the low expression of FAM172A in human HCC tissues implies a lower likelihood of survival and a valid diagnostic marker for HCC. Loss of FAM172A gene promotes cell proliferation by pERK-YAP1-Cyclin D1 and pNFκB-TNFα pathways during liver regeneration after PH. FAM172A may be a favorable diagnosis marker of HCC.

USP40 promotes hepatocellular carcinoma cell proliferation, migration and stemness by deubiquitinating and stabilizing Claudin1

BackgroundHepatocellular carcinoma (HCC) is a prevalent malignant tumor that poses a major threat to people’s lives and health. Previous studies have found that multiple deubiquitinating enzymes are involved in the pathogenesis of HCC. The purpose of this work was to elucidate the function and mechanism of the deubiquitinating enzyme USP40 in HCC progression.MethodsThe expression of USP40 in human HCC tissues and HCC cell lines was investigated using RT-qPCR, western blotting and immunohistochemistry (IHC). Both in vitro and in vivo experiments were conducted to determine the crucial role of USP40 in HCC progression. The interaction between USP40 and Claudin1 was identified by immunofluorescence, co-immunoprecipitation and ubiquitination assays.ResultsWe discovered that USP40 is elevated in HCC tissues and predicts poor prognosis in HCC patients. USP40 knockdown inhibits HCC cell proliferation, migration and stemness, whereas USP40 overexpression shows the opposite impact. Furthermore, we confirmed that Claudin1 is a downstream gene of USP40. Mechanistically, USP40 interacts with Claudin1 and inhibits its polyubiquitination to stabilize Claudin1 protein.ConclusionsOur study reveals that USP40 enhances HCC malignant development by deubiquitinating and stabilizing Claudin1, suggesting that targeting USP40 may be a novel approach for HCC therapy.

Downregulation of Roundabout guidance receptor 2 suppresses hepatocellular carcinoma progression by interacting with Y-box binding protein 1

Roundabout guidance receptor 2 (Robo2) is closely related to malignant tumors such as pancreatic cancer and liver fibrosis, but there is no relevant research on the role of Robo2 in HCC. The study will further explore the function and mechanism of Robo2 and its downstream target genes in HCC. Firstly, Robo2 protein levels in human HCC tissues and paired adjacent normal liver tissues were detected. Then we established HepG2 and Huh7 hepatoma cell lines with knock-down Robo2 by transfection with lentiviral vectors, and examined the occurrence of EMT, proliferation and apoptosis abilities in HCC cells by western blot, flow cytometry, wound healing assay and TUNEL staining. Then we verified the interaction between Robo2 and its target gene by Co-IP and immunofluorescence co-staining, and further explored the mechanism of Robo2 and YB-1 by rescue study. The protein expression level of Robo2 in HCC was considerably higher than that in the normal liver tissues. After successfully constructing hepatoma cells with knock-down Robo2, it was confirmed that down-regulated Robo2 suppressed EMT and proliferation of hepatoma cells, and accelerated the cell apoptosis. High-throughput sequencing and validation experiments verified that YB-1 was the downstream target gene of Robo2, and over-expression of YB-1 could reverse the apoptosis induced by Robo2 down-regulation and its inhibitory effect on EMT and proliferation. Robo2 deficiency inhibits EMT and proliferation of hepatoma cells and augments the cell apoptosis by regulating YB-1, thus inhibits the occurrence of HCC and provides a new strategy for the treatment of HCC.

Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis

BackgroundHepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation.MethodsBuilding upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells.ResultsSphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner.ConclusionsThis study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Dynamic regulation of EXO1 promotes the progression from liver fibrosis to HCC through TGF-β1/Smad signaling feedback loop.

HSCs are the main stromal cells in the process of liver fibrosis and accelerate HCC progression. Previous studies determined that highly expressed exonuclease 1 (EXO1) increases the malignant behavior of HCC cells and is closely related to liver cirrhosis. This study aimed to explore the roles and mechanisms of EXO1 in the development of liver cirrhosis and HCC. We fully demonstrated that EXO1 expression was positively correlated with liver fibrosis and cirrhotic HCC by combining bioinformatics, hepatic fibrosis mouse models, and human HCC tissues. The role of EXO1 in a murine HCC model induced by activated forms of AKT and Ras oncogenes (AKT/Ras) was investigated by employing an adeno-associated virus-mediated EXO1 knockdown technique. The knockdown of EXO1 promoted a regression of HCC in AKT/Ras mice and reduced the degree of liver fibrosis. Downregulated EXO1 inhibited LX-2 cell activation and inhibited the proliferation and migration of HCC cells. Moreover, conditioned medium of LX-2 cells with EXO1 overexpression increased the proliferation and migration of HCC cells, which was attenuated after EXO1 knockout in LX-2 cells. EXO1 knockdown attenuated the role of LX-2 in promoting HepG2 xenograft growth in vivo. Mechanistically, EXO1 promotes the activation of the downstream TGF-β-smad2/3 signaling in LX-2 and HCC cells. Interestingly, increased TGF-β-smad2/3 signaling had a feedback effect on EXO1, which sustains EXO1 expression and continuously stimulates the activation of HSCs. EXO1 forms a positive feedback circuit with TGF-β-Smad2/3 signaling and promotes the activation of HSCs, which accelerates HCC progression. Those findings indicate EXO1 may be a promising target for the diagnosis and treatment of cirrhotic HCC.

Loss of KDM6B epigenetically confers resistance to lipotoxicity in nonalcoholic fatty liver disease-related HCC.

NAFLD caused by abnormalities in hepatic lipid metabolism is associated with an increased risk of developing HCC. The molecular mechanisms underlying the progression of NAFLD-related HCC are not fully understood. We investigated the molecular mechanism and role of KDM6B downregulation in NAFLD-related HCC after the KDM6B gene was identified using microarray analysis as commonly downregulated in mouse NAFLD-related HCC and human nonhepatitis B and nonhepatitis C viral-HCC. The 5-hydroxymethylcytosine levels of KDM6B in HCC cells were determined using glycosylated hydroxymethyl-sensitive PCR. Microarray and chromatin immunoprecipitation analyses using KDM6B-knockout (KO) cells were used to identify KDM6B target genes. Lipotoxicity was assessed using a palmitate-treated cell proliferation assay. Immunohistochemistry was used to evaluate KDM6B expression in human HCC tissues. KDM6B expression levels in HCC cells correlated with the 5-hydroxymethylcytosine levels in the KDM6B gene body region. Gene set enrichment analysis revealed that the lipid metabolism pathway was suppressed in KDM6B-KO cells. KDM6B-KO cells acquired resistance to lipotoxicity (p < 0.01) and downregulated the expression of G0S2, an adipose triglyceride lipase/patatin like phospholipase domain containing 2 (ATGL/PNPLA2) inhibitor, through increased histone H3 lysine-27 trimethylation levels. G0S2 knockdown in KDM6B-expressed HCC cells conferred lipotoxicity resistance, whereas ATGL/PNPLA2 inhibition in the KDM6B-KO cells reduced these effects. Immunohistochemistry revealed that KDM6B expression was decreased in human NAFLD-related HCC tissues (p < 0.001), which was significantly associated with decreased G0S2 expression (p = 0.032). KDM6B-disrupted HCC acquires resistance to lipotoxicity via ATGL/PNPLA2 activation caused by epigenetic downregulation of G0S2 expression. Reduced KDM6B and G0S2 expression levels are common in NAFLD-related HCC. Targeting the KDM6B-G0S2-ATGL/PNPLA2 pathway may be a useful therapeutic strategy for NAFLD-related HCC.

Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2

BackgroundHepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages.MethodsConstitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3LCKO& IL-6−/−) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells.ResultsWe observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC.ConclusionIn summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

α-catenin interaction with YAP/FoxM1/TEAD-induced CEP55 supports liver cancer cell migration

BackgroundAdherens junctions (AJs) facilitate cell–cell contact and contribute to cellular communication as well as signaling under physiological and pathological conditions. Aberrant expression of AJ proteins is frequently observed in human cancers; however, how these factors contribute to tumorigenesis is poorly understood. In addition, for some factors such as α‐catenin contradicting data has been described. In this study we aim to decipher how the AJ constituent α‐catenin contributes to liver cancer formation.MethodsTCGA data was used to detect transcript changes in 23 human tumor types. For the detection of proteins, liver cancer tissue microarrays were analyzed by immunohistochemistry. Liver cancer cell lines (HLF, Hep3B, HepG2) were used for viability, proliferation, and migration analyses after RNAinterference-mediated gene silencing. To investigate the tumor initiating potential, vectors coding for α‐catenin and myristoylated AKT were injected in mice by hydrodynamic gene delivery. A BioID assay combined with mass spectrometry was performed to identify α‐catenin binding partners. Results were confirmed by proximity ligation and co-immunoprecipitation assays. Binding of transcriptional regulators at gene promoters was investigated using chromatin-immunoprecipitation.Resultsα‐catenin mRNA was significantly reduced in many human malignancies (e.g., colon adenocarcinoma). In contrast, elevated α‐catenin expression in other cancer entities was associated with poor clinical outcome (e.g., for hepatocellular carcinoma; HCC). In HCC cells, α‐catenin was detectable at the membrane as well as cytoplasm where it supported tumor cell proliferation and migration. In vivo, α‐catenin facilitated moderate oncogenic properties in conjunction with AKT overexpression. Cytokinesis regulator centrosomal protein 55 (CEP55) was identified as a novel α‐catenin-binding protein in the cytoplasm of HCC cells. The physical interaction between α‐catenin and CEP55 was associated with CEP55 stabilization. CEP55 was highly expressed in human HCC tissues and its overexpression correlated with poor overall survival and cancer recurrence. Next to the α‐catenin-dependent protein stabilization, CEP55 was transcriptionally induced by a complex consisting of TEA domain transcription factors (TEADs), forkhead box M1 (FoxM1), and yes-associated protein (YAP). Surprisingly, CEP55 did not affect HCC cell proliferation but significantly supported migration in conjunction with α‐catenin.ConclusionMigration-supporting CEP55 is induced by two independent mechanisms in HCC cells: stabilization through interaction with the AJ protein α‐catenin and transcriptional activation via the FoxM1/TEAD/YAP complex.

Abstract 1350: Kupffer cells secrete CXCL5 to promote liver cancer

Abstract Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leading cause of cancer related deaths. In addition to the genomic and transcriptomic heterogeneity of liver tumor cells which is recognized as a major driver in liver cancer progression, the liver immune system is also fundamental to liver carcinogenesis and presents a promising target for therapy. The liver immune response is orchestrated by cytokines and chemokines. Recent studies suggest that chemokines not only recruit immune cells but also regulate various liver functions. In partial hepatectomy, CXCL2 has been shown to promote hepatocyte proliferation. CXCL1, 2, 5 and 8 can induce endothelial cells chemotaxis to promote angiogenesis through binding to CXCR2. These diverse functions suggest that chemokines could play multifaceted roles in liver cancer development. However, chemokines that are commonly associated with liver cancer is still unknown.We analyzed HCC patient data from the GEO database, and we categorized the datasets based on HCC etiologies including HBV, HCV, alcoholic and NASH. We identified CXCL5 as the only chemokine consistently upregulated in HCC with different etiologies compared to healthy or cirrhotic livers. Immunohistochemistry (IHC) analysis reveals that CXCL5 was produced by immune cells but not tumor cells in human HCC tissues. To further study HCC associated CXCL5 expression, the liver-specific Pten deletion mouse model (PM mice) that recapitulates NAFLD-NASH-HCC progression was used. A gradual increase of hepatic CXCL5 expression is observed during HCC development, reaching nearly 100-fold upregulation of CXCL5 mRNA expression in 12-month-old PM mice livers carrying tumors. Examination of liver immune cell populations showed that macrophages were significantly enriched in Pten deleted livers bearing tumors than wild type livers without tumors. Flow cytometry and IHC analysis further identified Kupffer cells (KCs), the liver resident macrophages as the source of CXCL5 in tumor bearing livers using these mice. Since increased LPS is a prominent feature in most chronic liver diseases, we isolated and treated mouse KCs with LPS and found that LPS treatment robustly increased CXCL5 expression by nearly 20-fold. Interestingly, neither murine macrophage cell lines nor primary peritoneal macrophages displayed induced CXCL5 expression in response to LPS. These data suggest that induction of CXCL5 in KCs is likely a unique function of the KCs but not of other macrophages. To explore the function of CXCL5 in HCC development, we treated mouse hepatocytes and HCC cells with CXCL5 and showed that CXCL5 induces the proliferation of these cells. This effect is further blocked by the inhibition of CXCR2, the receptor of CXCL5, demonstrating the specificity for CXCL5 mediated effects. Together we show here for the first time that CXCL5 expression is a unique property of Kupffer cells and the induction of CXCL5 promotes HCC progression. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Curtis T. Okamoto, Bangyan L. Stiles. Kupffer cells secrete CXCL5 to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1350.

Abstract 250: Identifying drivers of liver cancer using CRISPR activation screening in vivo

Abstract Hepatocellular Carcinoma (HCC) is the most prevalent type of liver cancer and the third leading cause of cancer deaths worldwide. Drugs approved for first- and second-line therapy extend survival by only several months. Hence, there is still a pressing need for new and effective treatments. Sequencing technologies applied to human HCC tissues have identified hundreds of genes that undergo amplification and that may be correlated with mortality. Nevertheless, correlation does not equate to functionality. We aim to annotate gene function by employing an innovative approach with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and catalytically dead version of CRISPR-associated endonuclease (dCas9) in a CRISPR/dCas9 activation (CRISPRa) screen to identify driver genes of HCC tumorigenesis in mice. We deliver to the liver’s hepatocytes a gRNA library targeting genes of interest on plasmids that contain: (1) a transposon system for stable integration of DNA sequences into the cell genome, (2) Myc to drive hyperproliferation, as MYC is frequently over-expressed in human HCC, (3) synergistic activation mediator complex sequences for transcriptional activation of target genes, and (4) the gRNA sequence, the only variable sequence in the library. Using datasets from the Cancer Genome Atlas, we identified 51 genes that are both enriched and amplified in HCC patients. We included gRNAs targeting Tert, Vegfa, and Ccnd1, as control genes with known roles in driving HCC. In total, the screening library contained 290 gRNAs targeting 51 genes and 3 controls. After delivery of the plasmid library to the liver, the Myc gene provided a baseline level of oncogenic hyperproliferation, and the readout was gRNA prevalence pre- and post-proliferation via next-generation sequencing. We found that the combination of Myc and CRISPRa gRNA library led to a large number of expansive tumors in the liver as compared to Myc or the CRISPRa gRNA library only, which contained few to no tumors. gRNAs targeting control genes Ccnd1 and Vegfa were enriched as expected, validating our approach to annotate gene function during tumorigenesis. Additionally, we identified enrichment in gRNAs targeting genes Zbtb7b, Vps72, and Gba in multiple tumors, implicating a role in liver tumorigenesis. ZBTB7B and GBA expression levels are increased in patients with HCC recurrence as compared to patients in remission (data from BIOSTORM trial), while VPS72 is an unfavorable prognostic marker for HCC (data from The Human Protein Atlas). In this study, we have established a novel model of liver cancer that combines the Myc oncogene with a CRISPRa gRNA library to annotate driver genes in human HCC. Additionally, we identified several promising targets for the treatment of HCC. Ultimately, this study has the potential to impact on the care of patients with HCC in the US and worldwide. Citation Format: Alexandra Mariel Vázquez Salgado, Shirui He, Kirk J. Wangensteen. Identifying drivers of liver cancer using CRISPR activation screening in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 250.

The somatic mutational landscape and role of the ARID1A gene in hepatocellular carcinoma

The somatic mutational landscape and role of the ARID1A gene in hepatocellular carcinoma

KLF14 regulates the growth of hepatocellular carcinoma cells via its modulation of iron homeostasis through the repression of iron-responsive element-binding protein 2

BackgroundHepatocellular carcinoma (HCC) is a multifactor-driven malignant tumor with rapid progression, which causes the difficulty to substantially improve the prognosis of HCC. Limited understanding of the mechanisms in HCC impedes the development of efficacious therapies. Despite Krüpple-Like factors (KLFs) were reported to be participated in HCC pathogenesis, the function of KLF14 in HCC remains largely unexplored.MethodsWe generated KLF14 overexpressed and silenced liver cancer cells, and nude mouse xenograft models for the in vitro and in vivo study. Luciferase reporter assay, ChIP-qPCR, Co-IP, immunofluorescence were performed for mechanism research. The expression of KLF14 in HCC samples was analyzed by quantitative RT-PCR, Western blotting, and immunohistochemistry (IHC) analysis.ResultsKLF14 was significantly downregulated in human HCC tissues, which was highly correlated with poor prognosis. Inhibition of KLF14 promoted liver cancer cells proliferation and overexpression of KLF14 suppressed cells growth. KLF14 exerts its anti-tumor function by inhibiting Iron-responsive element-binding protein 2 (IRP2), which then causes transferrin receptor-1(TfR1) downregulation and ferritin upregulation on the basis of IRP-IREs system. This then leading to cellular iron deficiency and HCC cells growth suppression in vitro and in vivo. Interestingly, KLF14 suppressed the transcription of IRP2 via recruiting SIRT1 to reduce the histone acetylation of the IRP2 promoter, resulting in iron depletion and cell growth suppression. More important, we found fluphenazine is an activator of KLF14, inhibiting HCC cells growth through inducing iron deficiency.ConclusionKLF14 acts as a tumor suppressor which inhibits the proliferation of HCC cells by modulating cellular iron metabolism via the repression of IRP2. We identified Fluphenazine, as an activator of KLF14, could be a potential compound for HCC therapy. Our findings therefore provide an innovative insight into the pathogenesis of HCC and a promising therapeutic target.

LncRNA CEBPA-DT promotes liver cancer metastasis through DDR2/β-catenin activation via interacting with hnRNPC

BackgroundHepatocellular carcinoma (HCC) is the world’s third leading cause of cancer-related death; due to the fast growth and high prevalence of tumor recurrence, the prognosis of HCC patients remains dismal. Long non-coding RNA CEBPA-DT, a divergent transcript of the CCAAT Enhancer Binding Protein Alpha (CEBPA) gene, has been shown to participate in multiple tumor progression. However, no research has established its cancer-promoting mechanism in HCC yet.MethodsCEBPA-DT was identified in human HCC tissues through RNA sequencing. The expression level of CEBPA-DT was assessed by quantitative real-time PCR. The biological effects of CEBPA-DT were evaluated in vitro and in vivo through gain or loss of function experiments. RNA fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down assays were applied to investigate the downstream target of CEBPA-DT. Immunofluorescence, subcellular protein fractionation, western blot, and co-immunoprecipitation were performed to analyze the subcellular location of β-catenin and its interaction with Discoidin domain-containing receptor 2 (DDR2).ResultsCEBPA-DT was upregulated in human HCC tissues with postoperative distant metastasis and intimately related to the worse prognosis of HCC patients. Silencing of CEBPA-DT inhibited the growth, migration and invasion of hepatoma cells in vitro and in vivo, while enhancement of CEBPA-DT played a contrasting role. Mechanistic investigations demonstrated that CEBPA-DT could bind to heterogeneous nuclear ribonucleoprotein C (hnRNPC), which facilitated cytoplasmic translocation of hnRNPC, enhanced the interaction between hnRNPC and DDR2 mRNA, subsequently promoted the expression of DDR2. Meanwhile, CEBPA-DT induced epithelial-mesenchymal transition (EMT) process through upregulation of Snail1 via facilitating nuclear translocation of β-catenin. Using DDR2 inhibitor, we revealed that the CEBPA-DT induced the interaction between DDR2 and β-catenin, thus promoting the nuclear translocation of β-catenin to activate transcription of Snail1, contributing to EMT and HCC metastasis.ConclusionsOur results suggested that CEBPA-DT promoted HCC metastasis through DDR2/β-catenin mediated activation of Snail1 via interaction with hnRNPC, indicating that the CEBPA-DT-hnRNPC-DDR2/β-catenin axis may be used as a potential therapeutic target for HCC treatment.

Knockdown of PKMYT1 is associated with autophagy inhibition and apoptosis induction and suppresses tumor progression in hepatocellular carcinoma

Knockdown of PKMYT1 is associated with autophagy inhibition and apoptosis induction and suppresses tumor progression in hepatocellular carcinoma

LCMT1 indicates poor prognosis and is essential for cell proliferation in hepatocellular carcinoma

LCMT1 indicates poor prognosis and is essential for cell proliferation in hepatocellular carcinoma

Elevation of spermine remodels immunosuppressive microenvironment through driving the modification of PD-L1 in hepatocellular carcinoma

BackgroundSpermine is frequently elevated in tumor tissues and body fluids of cancer patients and is critical for cancer cell proliferation, migration and invasion. However, the immune functions of spermine in hepatocellular carcinoma progression remains unknown. In the present study, we aimed to elucidate immunosuppressive role of spermine in hepatocellular carcinoma and to explore the underlying mechanism.MethodsWhole-blood spermine concentration was measured using HPLC. Human primary HCC tissues were collected to examine the expression of CaSR, p-Akt, β-catenin, STT3A, PD-L1, and CD8. Mouse model of tumorigenesis and lung metastasis were established to evaluate the effects of spermine on hepatocellular carcinoma. Western blotting, immunofluorescence, real time PCR, digital Ca2+ imaging, and chromatin immunoprecipitation assay were used to investigate the underlying mechanisms by which spermine regulates PD-L1 expression and glycosylation in hepatocellular carcinoma cells.ResultsBlood spermine concentration in the HCC patient group was significantly higher than that in the normal population group. Spermine could facilitate tumor progression through inducing PD-L1 expression and decreasing the CD8+ T cell infiltration in HCC. Mechanistically, spermine activates calcium-sensing receptor (CaSR) to trigger Ca2+ entry and thereby promote Akt-dependent β-catenin stabilization and nuclear translocation. Nuclear β-catenin induced by spermine then activates transcriptional expression of PD-L1 and N-glycosyltransferase STT3A, while STT3A in turn increases the stability of PD-L1 through inducing PD-L1 protein N-glycosylation in HCC cells.ConclusionsThis study reveals the crucial function of spermine in establishing immune privilege by increasing the expression and N-glycosylation of PD-L1, providing a potential strategy for the treatment of hepatocellular carcinoma.7ng36xqHia8rCQn62itWxRVideo

NAD + salvage governs mitochondrial metabolism, invigorating natural killer cell antitumor immunity.

Natural killer (NK) cells are key players in tumor immunosurveillance, and metabolic adaptation manipulates their fate and functional state. The nicotinamide adenine dinucleotide (NAD + ) has emerged as a vital factor to link cellular metabolism and signaling transduction. Here, we identified NAD + metabolism as a central hub to determine the homeostasis and function of NK cells. NAD + level was elevated in activated NK cells. NAD + supplementation not only enhanced cytokine production and cytotoxicity but also improved the proliferation and viability of NK cells. Intriguingly, the salvage pathway was involved in maintaining NAD + homeostasis in activated NK cells. Genetic ablation or pharmacological blockade of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD + salvage pathway, markedly destroyed the viability and function of NK cells. Mechanistically, NAD + salvage dictated the mitochondrial homeostasis and oxidative phosphorylation activity to support the optimal function of NK cells. However, in human HCC tissues, NAMPT expression and NAD + level were significantly down-regulated in tumor-infiltrating NK cells, which negatively correlated with patient survival. And lactate accumulation in the tumor microenvironment was at least partially responsible for the transcriptional repression of NAMPT in NK cells. Further, deficiency of Nampt in NK cells accelerated the growth of HCC and melanoma. Supplementation of the NAD + precursor nicotinamide mononucleotide (NMN) significantly improved NK antitumor response in both mouse and human cell-derived xenografts. These findings reveal NAD + salvage as an essential factor for NK-cell homeostasis and function, suggesting a potential strategy for invigorating NK cell-based immunotherapy.

SLC38A6, regulated by EP300-mediated modifications of H3K27ac, promotes cell proliferation, glutamine metabolism and mitochondrial respiration in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common form of liver cancer. The incidence of HCC is increasing and effective prevention methods are needed. The solute carrier family 38 member 6 (SLC38A6) plays an important role in the metabolism of glutamine, which is a central nutrient for many cancers. However, the regulation and function of SLC38A6 in HCC are unclear. SLC38A6 levels in human HCC tissue arrays and cells were determined. SLC38A6 was silenced or overexpressed to determine its role in regulating cell viability, colony formation, cell cycle progression, glutamine metabolism and mitochondrial respiration. A luminescence assay was used to study the interaction between SLC38A6 and EP300. The interactions between SLC38A6, H3K27ac and EP300 were determined using chromatin immunoprecipitation assays. Quantitative RT-PCR and immunoblots were performed to measure mRNAs and proteins, respectively. SLC38A6 expression was higher in HCC compared with expression in normal tissue. Silencing SLC38A6 inhibited cell viability, colony formation, cell cycle progression, glutamine metabolism and mitochondrial respiration, while SLC38A6 overexpression had the opposite effects. Silencing SLC38A6 also inhibited tumor growth in vivo. Silencing EP300 significantly suppressed the interaction between H3K27ac and the SLC38A6 promoter, leading to decreased SLC38A6. SLC38A6 is regulated by EP300-mediated modifications of H3K27ac and promotes viability, colony formation, cell cycle progression, glutamine metabolism and mitochondrial respiration in HCC cells.

MicroRNA-146a promotes proliferation, migration, and invasion of HepG2 via regulating FLAP

Abnormal expression of 5-Lipoxygenase Activating Protein (FLAP) has been detected in many tumor cells. MicroRNAs (miRNAs) negatively regulate gene expression post-transcriptionally by binding to the 3'–untranslated region (3'–UTR) of the target mRNA sequences and have been shown to be involved in various types of cancers. Herein, we aimed to demonstrate the expression of miR-146a and FLAP in human HCC tissues and liver cancer cell lines. We demonstrated that miR-146a expression is overexpressed, while FLAP protein and mRNA are suppressed in hepatocellular carcinoma tissues and HepG2 cells compared to para-carcinoma tissues and HL–7702 cells. Dual luciferase reporter gene assay showed that miR-146a-5p can directly target FLAP mRNA. Knockdown of miR-146a also resulted in increased FLAP expression of cancer cells. Additionally, miR-146a silencing or restoration of FLAP led to a reduction of HepG2 cell proliferation, cell cycle progression, migration, and invasion. This study showed that miR-146a has a stimulatory role in HepG2 cells and promotes HepG2 cell migration and invasion by targeting FLAP mRNA. Thus, miR-146a may be a tumor promoter and a potential therapeutic target for the treatment of HCC patients.