Adjacent Normal Liver Tissues Research Articles

Head-to-head study of [18F]FAPI-04 PET/CT and [18F]FDG PET/CT for non-invasive assessment of liver cancer and its immunohistochemical markers.

To compare the performance of [18F]FDG and [18F]FAPI-04 in PET/CT evaluation for liver cancer lesions, with a further exploration of the associations between PET semiquantitative data and immunohistochemical markers to liver cancer. Patients with suspected malignant liver lesions (MLL) underwent [18F]FDG and [18F]FAPI-04 PET/CT scanning. Liver lesions were visually classified as positive or negative based on their uptake level exceeding that of adjacent normal liver tissue. SUVmax and tumor-to-background ratio (TBR) were recorded for semi-quantitative analysis. Sensitivity, specificity and accuracy of each tracer were determined using pathological findings as the gold standard. Furthermore, immunohistochemical analysis provided the molecular characteristics of all MLLs. Comprehensive analysis explored correlations between these molecular markers and PET semiquantitative parameters (SUVmax andTBR) to identify potential associations. The study enrolled 44 patients, with 39 confirmed cases of MLL, comprising 28 hepatocellular carcinomas (HCC) and 11 intrahepatic cholangiocarcinomas (ICC). For MLL detection, [18F]FAPI-04 and [18F]FDG exhibited sensitivities of 84.6% (33/39) and 76.9% (30/39), specificitiesy of 60% (3/5) and 100%(5/5), and accuracy of 81.8% (36/44) and 79.5%(35/44). Across all liver lesions, [18F]FAPI-04 significantly surpassed [18F]FDG in SUVmax(10.54 ± 6.72 VS. 7.68 ± 6.79) and TBR(4.35 ± 3.78 Vs. 3.17 ± 3.05). Notably, [18F]FAPI-04 displayed markebly elevated SUVmax in benign liver lesions (BLLs) (P = 0.032), HCCs (P = 0.005), and ICCs (P = 0.011). Lesions with hepatocyte negativity (P = 0.023), CD34 negativity(P = 0.044), and high Ki67 expression (> 30%) (P = 0.001) had higher SUVmax on [18F]FAPI-04. Additionally, ARG-1-negative lesions demonstrated higher TBR on [18F]FAPI-04 than ARG-1-positive lesions(P = 0.018). No significant SUVmax/TBR differences were observed with [18F]FDG based on these markers. A linear relationship was identified between Ki67 scores and SUVmax of [18F]FAPI-04 (R = 0.603, P < 0.001). [18F]FAPI-04 exhibits superior performance over [18F]FDG in PET/CT evaluation of liver cancer, characterized by increased sensitivity and SUVmax/TBR. Significant correlations with molecular markers, including Ki67, suggest [18F]FAPI-04's potential for characterizing liver cancer subtypes and assessing tumor proliferation. However, further research is required to validate these findings and their clinical significance. NCT05485792, Registered 01 August 2022.

Stage dependent microbial dynamics in hepatocellular carcinoma and adjacent normal liver tissues

The interactive pathway of the gut-liver axis underscores the significance of microbiome modulation in the pathogenesis and progression of various liver diseases, including hepatocellular carcinoma (HCC). This study aims to investigate the disparities in the composition and functionality of the hepatic microbiota between tumor tissues and adjacent normal liver tissues, and their implications in the etiology of HCC. We conducted a comparative analysis of the hepatic microbiome between adjacent normal liver tissues and tumor tissues from HCC patients. Samples were categorized according to the modified Union for International Cancer Control (mUICC) staging system into Non-tumor, mUICC stage I, mUICC stage II, and mUICC stage III groups. Microbial richness and community composition were analyzed, and phylogenetic profiles were examined to identify significantly altered microbial taxa among the groups. Predicted metabolic pathways were analyzed using PICRUSt2. Our analysis did not reveal significant differences in microbial richness and community composition with the development of HCC. However, phylogenetic profiling identified significantly altered microbial taxa among the groups. Sphingobium, known for degrading polychlorinated biphenyls (PCBs), exhibited a significantly negative correlation with clinical indices in HCC patients. Conversely, Sphingomonas, a gut bacterium associated with various liver diseases, showed a positive correlation. Predicted metabolic pathways suggested a correlation between atrazine degradation and valine, leucine, and isoleucine biosynthesis with mUICC stage and tumor size. Our results underscore the critical link between hepatic microbial composition and function and the HCC tumor stage, suggesting a potentially pivotal role in the development of HCC. These findings highlight the importance of targeting the hepatic microbiome for therapeutic strategies in HCC.

Integrated ubiquitomics characterization of hepatocellular carcinomas.

Patients with aggressive HCC have limited therapeutic options. Therefore, a better understanding of HCC pathogenesis is needed to improve treatment. Genomic studies of HCC have improved our understanding of cancer biology. However, the ubiquitomic characteristics of HCC remain poorly understood. We aimed to reveal the ubiquitomic characteristics of HCC and provide clinical feature biomarkers of the aggressive HCC that may be used for diagnosis or therapy in the clinic. The comprehensive proteomic, phosphoproteomic, and ubiquitomic analyses were performed on tumors and adjacent normal liver tissues from 85 patients with HCC. HCCs displayed overexpression of drugable targets CBR1-S151 and CPNE1-S55. COL4A1, LAMC1, and LAMA4 were highly expressed in the disease free survival-poor patients. Phosphoproteomic and ubiquitomic features of HCC revealed cross talk in metabolism and metastasis. Ubiquitomics predicted diverse prognosis and clarified HCC subtype-specific proteomic signatures. Expression of biomarkers TUBA1A, BHMT2, BHMT, and ACY1 exhibited differential ubiquitination levels and displayed high prognostic risk scores, suggesting that targeting these proteins or their modified forms may be beneficial for future clinical treatment. We validated that TUBA1A K370 deubiquitination drove severe HCC and labeled an aggressive subtype of HCCs. TUBA1A K370 deubiquitination was at least partly attributed to protein kinase B-mediated USP14 activation in HCC. Notably, targeting AKT-USP14-TUBA1A complex promoted TUBA1A degradation and blocked liver tumorigenesis in vivo. This study expands our knowledge of ubiquitomic signatures, biomarkers, and potential therapeutic targets in HCC.

Optimizing of a suitable protocol for isolating tissue-derived extracellular vesicles and profiling small RNA patterns in hepatocellular carcinoma.

Extracellular vesicles (EVs) facilitate cell-cell interactions in the tumour microenvironment. However, standard and efficient methods to isolate tumour tissue-derived EVs are lacking, and their biological functions remain elusive. To determine the optimal method for isolating tissue-derived EVs, we compared the characterization and concentration of EVs obtained by three previously reported methods using transmission electron microscopy, nanoparticle tracking analysis, and nanoflow analysis (Nanoflow). Additionally, the differential content of small RNAs, especially tsRNAs, between hepatocellular carcinoma (HCC) and adjacent normal liver tissues (ANLTs)-derived EVs was identified using Arraystar small RNA microarray. The targets of miRNAs and tsRNAs were predicted, and downstream functional analysis was conducted using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, non-negative matrix factorization and survival prediction analysis. A differential centrifugation-based protocol without cell cultivation (NC protocol) yielded higher EV particles and higher levels of CD9+ and CD63+ EVs compared with other isolation protocols. Interestingly, the NC protocol was also effective for isolating frozen tissue-derived EVs that were indistinguishable from fresh tissue. HCC tissues showed significantly higher EV numbers compared with ANLTs. Furthermore, we identified different types of small RNAs in HCC tissue-derived EVs, forming a unique multidimensional intercellular communication landscape that can differentiate between HCC and ANLTs. ROC analysis further showed that the combination of the top 10 upregulated small RNAs achieved better diagnostic performance (AUC = .950 [.895-1.000]). Importantly, most tsRNAs in HCC tissue-derived EVs were downregulated and mitochondria-derived, mainly involving in lipid-related metabolic reprogramming. The NC protocol was optimal for isolating EVs from HCC, especially from frozen tissues. Our study emphasized the different roles of small-RNA in regulating the HCC ecosystem, providing insights into HCC progression and potential therapeutic targets.

Targeted silencing of SOCS1 by DNMT1 promotes stemness of human liver cancer stem-like cells

BackgroundHuman liver cancer stem-like cells (HLCSLCs) are widely acknowledged as significant factors in the recurrence and eradication of hepatocellular carcinoma (HCC). The sustenance of HLCSLCs’ stemness is hypothesized to be intricately linked to the epigenetic process of DNA methylation modification of genes associated with anticancer properties. The present study aimed to elucidate the stemness-maintaining mechanism of HLCSLCs and provide a novel idea for the clearance of HLCSLCs.MethodsThe clinical relevance of DNMT1 and SOCS1 in hepatocellular carcinoma (HCC) patients was evaluated through the GEO and TCGA databases. Cellular immunofluorescence assay, methylation-specific PCR, chromatin immunoprecipitation were conducted to explore the expression of DNMT1 and SOCS1 and the regulatory relationship between them in HLCSLCs. Spheroid formation, soft agar colony formation, expression of stemness-associated molecules, and tumorigenicity of xenograft in nude mice were used to evaluate the stemness of HLCSLCs.ResultsThe current analysis revealed a significant upregulation of DNMT1 and downregulation of SOCS1 in HCC tumor tissues compared to adjacent normal liver tissues. Furthermore, patients exhibiting an elevated DNMT1 expression or a reduced SOCS1 expression had low survival. This study illustrated the pronounced expression and activity of DNMT1 in HLCSLCs, which effectively targeted the promoter region of SOCS1 and induced hypermethylation, consequently suppressing the expression of SOCS1. Notably, the stemness of HLCSLCs was reduced upon treatment with DNMT1 inhibitors in a concentration-dependent manner. Additionally, the overexpression of SOCS1 in HLCSLCs significantly mitigated their stemness. The knockdown of SOCS1 expression reversed the effect of DNMT1 inhibitor on the stemness of HLCSLCs. DNMT1 directly binds to the SOCS1 promoter. In vivo, DNMT1 inhibitors suppressed SOCS1 expression and inhibited the growth of xenograft.ConclusionDNMT1 targets the promoter region of SOCS1, induces hypermethylation of its CpG islands, and silences its expression, thereby promoting the stemness of HLCSLCs.

Discovering urinary protein biomarkers for hepatocellular carcinoma in the population of patients with chronic hepatitis B: A single-center case-control study.

e16230 Background: Chronic hepatitis B (CHB) can result in hepatocellular carcinoma (HCC) imposing a substantial health and economic burden worldwide. Early detection of hepatitis B virus-related HCC (HBV-HCC) in CHB with potential biomarkers has emerged as an urgent and challenging task. Recent progression of urinary proteomics provides a promising method for HBV-HCC biomarker discovery. Methods: This single-center case-control study, conducted at Peking Union Medical College Hospital, enrolled 33 patients with HBV-HCC, 26 patients with CHB, 30 healthy controls, and 33 controls with other types of cancer. Urine proteomes were analyzed using liquid chromatography with tandem mass spectrometry by data-dependent acquisition or parallel reaction monitoring, respectively. Differential analysis was carried out with limma package in R software, and the origin of selected proteins was confirmed with immunohistochemistry of surgical specimens. The diagnostic performance of the biomarker panel was evaluated with area under receiver operator curves (AUROC). Results: 21 urinary proteins were significantly upregulated in HBV-HCC compared with CHB samples, playing key roles in vesicular transportation, cytoskeleton-extracellular matrix interaction, and tumor development according to enrichment analysis. Immunohistochemistry validated the differential expression of 4 urinary proteins (4UP: HNRNPM, ADGRG1, APOC1, and RALA) across cancerous and adjacent normal liver tissues. These proteins were specifically found in HBV-HCC but not in CHB, healthy control, or other cancer controls. The AUROCs of 4UP individually (0.642 for APOC1, 0.780 for HNRNPM, 0.755 for ADGRG1, 0.729 for RALA) were no worse than that of serum α-fetoprotein (AUROC = 0.751), and the 4UP panel unified via logistic regression showed an AUROC of 0.828. Conclusions: Urinary proteomics revealed HNRNPM, ADGRG1, APOC1, and RALA as biomarkers to distinguish HBV-HCC from CHB. The 4UP could be traced back to HBV-HCC tumor cells, shedding light on pathogenesis of HBV-HCC. [Table: see text]

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.

An Evaluation Analysis for Computed Tomography Image Quality of Primary Liver Cancer Lesions Based on Deep Learning Image Reconstruction.

Abdominal multi-slice helical computed tomography (CT) and contrast-enhanced scanning have been widely recognized clinically. The impact of the deep learning image reconstruction (DLIR) on the quality of dynamic contrast-enhanced CT imaging of primary liver cancer lesions was evaluated through comparison with the filtered back projection (FBP) and the new generation of adaptive statistical iterative reconstruction-V (ASIR-V). We evaluated the image noise of the lesion, fine structures inside the lesion, and diagnostic confidence in 48 liver cancer subjects. The CT values of the solid part of the lesion and the adjacent normal liver tissue and the systolic and diastolic blood pressure (SD) values of the right paravertebral muscle were measured. The muscle SD value was considered as the background noise of the image, and the signal noise ratio (SNR) and contrast signal-to-noise ratio (CNR) of the lesion and normal liver parenchyma were calculated. High consistency in the evaluation of image noise (Kappa = 0.717). The Kappa values for margin/pseudocapsule, fine structure within the lesion, and diagnostic confidence were 0.463, 0.527, and 0.625, respectively. Besides, the differences in SD, SNR and CNR data of reconstructed lesion images among the six groups were statistically significant. The contrast-enhanced CT image noise of DLIR-H in the portal venous phase is much lower than that of ASIR-V and FBP in primary liver cancer patients. In terms of the lesion structure display, the new reconstruction algorithm DLIR is superior.

Genomic and transcriptomic profiling of hepatocellular carcinoma reveals a rare molecular subtype.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, occurring predominantly in patients with underlying chronic liver disease and cirrhosis. Here, we describe a case of a 62-year-old man that was admitted to our hospital and diagnosed with HCC where the cancer has already metastasized to the retroperitoneum and peritoneum. In order to better characterize the HCC, both the cancerous liver tissue and the adjacent normal liver tissue of the patient were collected and subjected to a genomic, transcriptomic and proteomic analysis. Our patient carries a highly mutated HCC, which is characterized by both somatic mutation in the following genes ALK, CDK6, TP53, PGR. In addition, we observe several molecular alterations that are associated with potential therapy resistance, for example the expression of the organic-anion-transporting polypeptide (OATP) family members B1 and B3, that mediate the transport of the anticancer drugs, has been found decreased. Overall, our molecular profiling potentially classify the patient with poor prognosis and possibly displaying resistance to pharmacological therapy.

Biomarker Identification in Liver Cancers Using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) Imaging: An Approach for Spatially Resolved Metabolomics.

Liver cancers are characterized by interindividual and intratumoral heterogeneity, which makes early diagnosis and the development of therapies challenging. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging is a potent and sensitive MS ionization technique for direct, unaltered 2D and 3D imaging of metabolites in complex biological samples. Indeed, DESI gently desorbs and ionizes analyte molecules from the sample surface using an electrospray source of highly charged aqueous spray droplets in ambient conditions. DESI-MS imaging of biological samples allows untargeted analysis and characterization of metabolites in liver cancers to identify new biomarkers of malignancy. In this chapter, we described a detailed protocol using liver cancer samples collected and stored for histopathology examination, either as frozen or as formalin-fixed, paraffin-embedded specimens. Such hepatocellular carcinoma samples can be subjected to DESI-MS analyses, illustrating the capacity of spatially resolved metabolomics to distinguish malignant lesions from adjacent normal liver tissue.

Intelectin-1 is a novel prognostic biomarker for hepatocellular carcinoma.

The molecular mechanisms of hepatocellular carcinoma (HCC) are still not well understood. Gene microarray analysis showed that the expression of Intelectin-1 (ITLN-1) in tumor-adjacent normal liver tissue was 454.8 times higher than in the corresponding cancer tissue. ITLN-1 is a secreted soluble glycoprotein which has been reported to be associated with the occurrence and development of various tumor types. However, the prognostic significance of ITLN-1 in HCC remain unclear. Real-time fluorescence quantitative polymerase chain reaction was used to investigate 149 liver cancer cases for ITLN-1 mRNA expression. Immunohistochemistry and western blot analysis were used to ascertain protein expression of ITLN-1 in cancer and para-carcinomatous tissue, and further to evaluate the correlation between ITLN-1 mRNA expression and surgical prognosis after liver resection. The ITLN-1 mRNA and protein levels were significantly higher in adjacent normal liver tissues than HCC tissues. Real-time fluorescence quantitative polymerase chain reaction showed that the ITLN-1 expression was decreased in 78.5% (117/149) of HCC tissues compared with their corresponding adjacent liver tissues. Moreover, its low expression was significantly correlated with increased tumor size, tumor differentiation degree, degree of liver cirrhosis, capsule integrity, vascular invasion and tumor recurrence. Patients with high ITLN-1 expression had significantly better overall and recurrence-free survival after curative liver resection. Multivariate cox regression analysis showed that ITLN-1 was an independent predictor of surgical outcomes in HCC patients. The present study suggested that low ITLN-1 expression was associated with poor clinical outcome for HCC patients, indicating a novel biomarker for prognosis evaluation and a potential therapeutic target for HCC patients.

Gender dimorphism in hepatocarcinogenesis-DNA methylation modification regulated X-chromosome inactivation escape molecule XIST.

Sex disparities constitute a significant issue in hepatocellular carcinoma (HCC). However, the mechanism of gender dimorphism in HCC is still not completely understood. 5-Hydroxymethylcytosine (5hmC)-Seal technology was utilised to detect the global 5hmC levels from four female and four male HCC samples. Methylation of XIST was detected by Sequenom MassARRAY methylation profiling between HCC tissues (T) and adjacent normal liver tissues (L). The role of Tet methylcytosine dioxygenase 2 (TET2) was investigated using diethylnitrosamine (DEN)-administered Tet2-/- female mice, which regulated XIST in hepatocarcinogenesis. All statistical analyses were carried out by GraphPad Prism 9.0 and SPSS version 19.0 software. The results demonstrated that the numbers of 5hmC reads in the first exon of XIST from female HCC tissues (T) were remarkably lower than that in female adjacent normal liver tissues (L). Correspondingly, DNA methylation level of XIST first exon region was significantly increased in female T than in L. By contrast, no significant change was observed in male HCC patients. Compared to L, the expression of XIST in T was also significantly downregulated. Female patients with higher XIST in HCC had a higher overall survival (OS) and more extended recurrence-free survival (RFS). Moreover, TET2 can interact with YY1 binding to the promoter region of XIST and maintain the hypomethylation state of XIST. In addition, DEN-administered Tet2-/- mice developed more tumours than controls in female mice. Our study provided that YY1 and TET2 could interact to form protein complexes binding to the promoter region of XIST, regulating the methylation level of XIST and then affecting the expression of XIST. This research will provide a new clue for studying sex disparities in hepatocarcinogenesis. XIST was significantly downregulated in HCC tissues and had gender disparity. Methylation levels in the XIST first exon were higher in female HCC tissues, but no significant change in male HCC patients. The TET2-YY1 complex regulate XIST expression in female hepatocytes. Other ways regulate XIST expression in male hepatocytes.

Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

PurposeThis study was based on hepatocellular carcinoma (HCC) patients of early-stage to explore the diagnostic capability and possible production causes of anti-GNAS autoantibody. MethodsWe evaluated the frequency of anti-GNAS autoantibody in sera from patients with early-stage HCC by enzyme-linked immunosorbent assay (ELISA) and the expression of GNAS protein in early-stage HCC tissues by immunohistochemistry. Western blotting (WB) and real-time polymerase chain reaction (RT-PCR) were utilized to examine the expressions of GNAS protein and mRNA in cell lines. GEO and International Cancer Genome Consortium (ICGC) databases were inquired to explore mRNA expression and mutation of GNAS in HCC tissues. ResultsThe positive rates of anti-GNAS autoantibody in HCC patients at clinical stage I (78.1 %) and clinical stage II (57.1 %) were all significantly higher than that in healthy control (20 %). There was also a significant difference in GNAS protein expression between HCC and its adjacent normal liver tissues. The results from WB and RT-PCR showed a significant difference at the mRNA level but no statistical difference at the protein level between HCC and normal liver cell lines. The difference in mRNA level between HCC and adjacent normal liver tissues was verified to be significant. Furthermore, the ICGC database demonstrated a 10.6 % mutation frequency for GNAS in HCC patients. ConclusionThe coordination of elevated anti-GNAS autoantibody, high expression of GNAS in the mRNA and protein levels in HCC, and high frequency of GNAS mutation indicates that anti-GNAS autoantibody may be used as an early indicator of HCC.

Protein profiles reveal MSH6/MSH2 as a potential biomarker for hepatocellular carcinoma with microvascular invasion.

Microvascular invasion (MVI) is an independent risk factor for postoperative recurrence and metastasis in hepatocellular carcinoma (HCC). However, the specific protein expression profiles that differentiate HCC with MVI from those without MVI remain unclear. The profiles of proteins in early-stage HCC tissues and normal liver tissues were characterized by quantitative proteomics techniques. Immunohistochemical (IHC) staining was undertaken on tissue microarrays from 80 HCC patients to assess the expression of MSH2 and MSH6. Cell counting, colony formation, migration, and invasion assays were carried out in vitro. We identified 5164 proteins in both HCC tissues and adjacent normal liver tissues. Compared to HCC without MVI, 148 upregulated proteins and 97 downregulated proteins were found in HCC with MVI. Particularly noteworthy was the remarkable upregulation of MSH6/MSH2 among these dysregulated proteins in HCC with MVI. Further validation through bioinformatics prediction and IHC confirmed the elevated expression of MSH6/MSH2, which correlated with aggressive disease characteristics and poor prognosis. Receiver operating characteristic curve analyses revealed a substantial area under the curve of 0.761 (specificity 71.79%, sensitivity 73.17%) for the combined use of MSH6/MSH2. Knockdown of MSH6/MSH2 significantly inhibited HCC cell proliferation and invasion in vitro. Our study establishes MSH6 or MSH2 as an oncogene that is prominently overexpressed during HCC progression, which provides new targets for HCC with MVI.

Cavin1 activates the Wnt/β-catenin pathway to influence the proliferation and migration of hepatocellular carcinoma

Introduction and ObjectivesCavin1 is a cell membrane caveolin, with controversial function in different tumors. Meanwhile, the role of Cavin1 in hepatocellular carcinoma (HCC) progression remains unclear. In this study, we attempted to elucidate the significance of Cavin1 in HCC occurrence and progression. Materials and MethodsCavin1 content was examined in HCC tissues and paired adjacent normal liver tissues by qRT-PCR and IHC among 81 HCC patients. The Cavin1-mediated regulation of HCC proliferation and metastasis was assessed through in vitro and in vivo experiments. Finally, using GSEA, we found out Cavin1 could be a potential regulator of the Wnt pathway. The alterations of the Wnt pathway-related proteins were identified by Western Blot analysis. ResultsCavin1 was lower expressed in HCC, which implied poor survival outcomes in HCC patients. Phenotypic experiments revealed that Cavin1 strongly suppressed HCC proliferation and migration in vitro and in vivo. Besides, altered epithelial-mesenchymal transition (EMT)-related protein expressions were detected. Based on our GSEA analysis, Cavin1 activated the Wnt pathway, and Western Blot analysis revealed diminished β-catenin, c-Myc, and MMP9 contents upon Cavin1 overexpression. ConclusionsCavin1 suppresses HCC progression by modulating HCC proliferation and migration via inhibiting the Wnt/β-catenin axis activation.

ARF6 promotes hepatocellular carcinoma proliferation through activating STAT3 signaling

BackgroundHepatocellular Carcinoma (HCC) possesses the high mortality in cancers worldwide. Nevertheless, the concrete mechanism underlying HCC proliferation remains obscure. In this study, we show that high expression of ARF6 is associated with a poor clinical prognosis, which could boost the proliferation of HCC.MethodsImmunohistochemistry and western blotting were used to detect the expression level of ARF6 in HCC tissues. We analyzed the clinical significance of ARF6 in primary HCC patients. We estimated the effect of ARF6 on tumor proliferation with in vitro CCK8, colony formation assay, and in vivo nude mouse xenograft models. Immunofluorescence was conducted to investigate the ARF6 localization. western blotting was used to detect the cell cycle-related proteins with. Additionally, we examined the correlation between ARF6 and STAT3 signaling in HCC with western blotting, immunohistochemistry and xenograft assay.ResultsARF6 was upregulated in HCC tissues compared to adjacent normal liver tissues. The increased expression of ARF6 correlated with poor tumor differentiation, incomplete tumor encapsulation, advanced tumor TNM stage and poor prognosis. ARF6 obviously promoted HCC cell proliferation, colony formation, and cell cycle progression. In vivo nude mouse xenograft models showed that ARF6 enhanced tumor growth. Furthermore, ARF6 activated the STAT3 signaling and ARF6 expression was positively correlated with phosphorylated STAT3 level in HCC tissues. Furthermore, after intervening of STAT3, the effect of ARF6 on tumor-promoting was weakened, which demonstrated ARF6 functioned through STAT3 signaling in HCC.ConclusionsOur results indicate that ARF6 promotes HCC proliferation through activating STAT3 signaling, suggesting that ARF6 may serve as potential prognostic and therapeutic targets for HCC patients.

B4GALNT1 promotes hepatocellular carcinoma stemness and progression via integrin α2β1-mediated FAK and AKT activation

β-1,4-N-Acetyl-galactosaminyltransferase 1 (B4GALNT1) has been reported to contribute to the development of human malignancies. However, its role in hepatocellular carcinoma (HCC) remains uncharacterised. In this study, we aimed to elucidate the role of B4GALNT1 in HCC stemness and progression. Immunohistochemical staining was used to evaluate B4GALNT1 expression in HCC tissues and adjacent normal liver tissues. Flow cytometry analysis and sphere formation analysis were performed to investigate the role of B4GALNT1 in HCC stemness. Colony formation, Incucyte, wound-healing, Transwell migration, and invasion assays, and an animal model were used to study the role of B4GALNT1 in HCC progression. RNA-sequencing and co-immunoprecipitation were used to investigate the downstream targets of B4GALNT1. B4GALNT1 was upregulated in HCC and associated with poor clinical outcome of patients with the disease. Moreover, B4GALNT1 promoted HCC stemness, migration, invasion, and growth. Mechanistically, B4GALNT1 not only promoted the expression of the integrin α2β1 ligand THBS4, but also directly interacted with the β subunit of integrin α2β1 ITGB1 to inhibit its ubiquitin-independent proteasomal degradation, resulting in activation of FAK and AKT. Ophiopogonin D inhibited HCC stemness and progression by reducing ITGB1 and THBS4 expression and inhibiting FAK and AKT activation. Our study suggests the B4GALNT1/integrin α2β1/FAK/PI3K/AKT axis as a therapeutic target for the inhibition of HCC stemness and tumour progression. The role and regulatory mechanism of B4GALNT1 in HCC have not been studied previously. Here, we reveal that B4GALNT1 has a crucial role in HCC stemness and progression by activating the integrin α2β1/FAK/PI3K/AKT axis, providing a potential target for HCC therapy. In addition, we find Ophiopogonin D as a potential therapeutic drug for patients with HCC.

Long non-coding RNAs CCAT1 and CCAT2 in colorectal liver metastases are tumor-suppressive via MYC interaction and might predict patient outcomes.

Liver metastases severely reduce the long term survival of colorectal cancer patients. Long non-coding RNAs (lncRNAs) CCAT1 and CCAT2 have previously been found to be associated with impaired patient outcomes in primary colorectal cancer. We aimed to elucidate the role of CCAT1 and CCAT2 in colorectal liver metastases. Total RNA was isolated from 97 human tissue samples of colorectal liver metastases and adjacent normal liver tissue. Gene expression analysis was performed by RT-qPCR and Multiplex ELISA and correlated with patient characteristics and survival. Gene expression, cancer cell migration, invasion, and proliferation were studied after siRNA-mediated knockdown of CCAT1, CCAT2, and MYC in metastatic colorectal cancer cell lines Colo205 and HROC277Met2. Elevated expression levels of lncRNAs CCAT1 and CCAT2, and their common target MYC in colorectal liver metastases were associated with prolonged progression-free survival after liver resection. High expression of CCAT1 was likewise associated with prolonged overall survival. Knockdown of CCAT1, CCAT2, and MYC resulted in increased migratory and invasive potential in metastatic colorectal cancer cell lines. Gene expression analysis revealed alterations in constituents of Wnt signaling following knockdown. Our findings demonstrate tumor-suppressive functions of lncRNAs CCAT1 and CCAT2 in colorectal liver metastases. They suppress Wnt signaling directly and indirectly through target gene MYC and might prevent further metastatic spread from colorectal liver metastases.

AATF inhibition exerts antiangiogenic effects against human hepatocellular carcinoma.

Angiogenesis is a key factor in the growth and metastasis of hepatic tumors and thus a potential therapeutic target in hepatocellular carcinoma (HCC). In this study, we aim to identify the key role of apoptosis antagonizing transcription factor (AATF) in tumor angiogenesis and its underlying mechanisms in HCC. HCC tissues were analyzed for AATF expression by qRT-PCR and immunohistochemistry. Stable clones of control and AATF knockdown (KD) were established in human HCC cells. The effect of AATF inhibition on the angiogenic processes was determined by proliferation, invasion, migration, chick chorioallantoic membrane (CAM) assay, zymography, and immunoblotting techniques. We identified high levels of AATF in human HCC tissues compared to adjacent normal liver tissues, and the expression was found to be correlated with the stages and tumor grades of HCC. Inhibiting AATF in QGY-7703 cells resulted in higher levels of pigment epithelium-derived factor (PEDF) than controls due to decreased matric metalloproteinase activity. Conditioned media from AATF KD cells inhibited the proliferation, migration, and invasion of human umbilical vein endothelial cells as well as the vascularization of the chick chorioallantoic membrane. Furthermore, the VEGF-mediated downstream signaling pathway responsible for endothelial cell survival and vascular permeability, cell proliferation, and migration favoring angiogenesis was suppressed by AATF inhibition. Notably, PEDF inhibition effectively reversed the anti-angiogenic effect of AATF KD. Our study reports the first evidence that the therapeutic strategy based on the inhibition of AATF to disrupt tumor angiogenesis may serve as a promising approach for HCC treatment.

TMEM147 Correlates with Immune Infiltration and Serve as a Potential Prognostic Biomarker in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and is associated with high mortality. Transmembrane protein 147 (TMEM147) is a seven-transmembrane protein that may mediate immune regulation. However, the relevance of TMEM147 to immune regulation in HCC and the prognosis of HCC patients are unclear. We analyzed TMEM147 expression in HCC by using the Wilcoxon rank-sum test. Real time quantitative PCR (RT-qPCR) and Western blot analysis of tumor tissues and cell lines were used to verify TMEM147 expression in HCC. The influence of TMEM147 on HCC prognosis was assessed using Kaplan-Meier analysis, Cox regression analysis, and a prognostic nomogram. The functions of the TMEM147-related differentially expressed genes (DEGs) were identified by Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and gene set enrichment analysis (GSEA). In addition, we examined the associations between TMEM147 expression and immune infiltration using single-sample gene set enrichment analysis (ssGSEA) and immunofluorescence staining of HCC tissues. Our results showed that the expression of TMEM147 was significantly higher in human HCC tissues than in adjacent normal liver tissues, with similar findings in human HCC cell lines. High TMEM147 expression was correlated with T stage, pathological stage, histological grade, race, alpha-fetoprotein level, and vascular invasion in HCC. Moreover, we revealed that high TMEM147 expression was associated with shorter survival times and that TMEM147 could be a risk factor for overall survival, along with T stage, M stage, pathological stage, and tumor status. Mechanistic studies revealed that high TMEM147 expression was linked to the B lymphocyte, antigen response, IL6 signaling pathway, cell cycle, Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling pathway, and myelocytomatosis oncogene (MYC) targets. Correspondingly, TMEM147 expression was positively associated with the infiltration of immune cells, including Th2 cells, follicular helper T cells, macrophages, and NK CD56 bright cells in HCC. TMEM147 might be a biomarker for poor prognosis and is related to immune cell infiltration in HCC.