Marker For Hepatocellular Carcinoma Research Articles

Vitronectin (VTN): A Novel Diagnostic and Prognostic Marker for Hepatocellular Carcinoma (HCC) on Top of Chronic Hepatitis C Virus Related Diseases

Vitronectin (VTN): A Novel Diagnostic and Prognostic Marker for Hepatocellular Carcinoma (HCC) on Top of Chronic Hepatitis C Virus Related Diseases

Glypican-3 (GPC-3) Structural Analysis and Cargo in Serum Small Extracellular Vesicles of Hepatocellular Carcinoma Patients.

Glypican-3 (GPC-3) is a heparin sulfate proteoglycan located extracellularly and anchored to the cell membrane of transformed hepatocytes. GPC-3 is not expressed in normal or cirrhotic liver tissue but is overexpressed in hepatocellular carcinoma (HCC). Because of this, GPC-3 is one of the most important emerging immunotargets for treatment and as an early detection marker of HCC. To determine if GPC-3 domains associated with serum small extracellular vesicles (sEVs) could be used as an HCC diagnostic marker, we predicted in silico GPC-3 structural properties and tested for the presence of its full-length form and/or cleaved domains in serum sEVs isolated from patients with HCC. Structural analysis revealed that the Furin cleavage site of GPC-3 is exposed and readily accessible, suggesting the facilitation of GPC-3 cleavage events. Upon isolation of sEVs from both hepatocytes, culture media and serum of patients with HCC were studied for GPC-3 content. This data suggests that Furin-dependent GPC-3 cleaved domains could be a powerful tool for detection of initial stages of HCC and serve as a predictor for disease prognosis.

Artificial intelligence-based reticulin proportionate area - anovel histological outcome predictor in hepatocellular carcinoma.

Reticulin stain is used routinely in the histological evaluation of hepatocellular carcinoma (HCC). The goal of this study was to assess whether the histological reticulin proportionate area (RPA) in HCCs predicts tumour-related outcomes. We developed and validated a supervised artificial intelligence (AI) model that utilises a cloud-based, deep-learning AI platform (Aiforia Technologies, Helsinki, Finland) to specifically recognise and quantify the reticulin framework in normal livers and HCCs using routine reticulin staining. We applied this reticulin AI model to a cohort of consecutive HCC cases from patients undergoing curative resection between 2005 and 2015. A total of 101 HCC resections were included (median age = 68 years, 64 males, median follow-up time = 49.9 months). AI model RPA reduction of > 50% (compared to normal liver tissue) was predictive of metastasis [hazard ratio (HR) = 3.76, P = 0.004, disease-free survival (DFS, HR = 2.48, P < 0.001) and overall survival (OS), HR = 2.80, P = 0.001]. In a Cox regression model, which included clinical and pathological variables, RPA decrease was an independent predictor of DFS and OS and the only independent predictor of metastasis. Similar results were found in the moderately differentiated HCC subgroup (WHO grade 2), in which reticulin quantitative analysis was an independent predictor of metastasis, DFS and OS. Our data indicate that decreased RPA is a strong predictor of various HCC-related outcomes, including within the moderately differentiated subgroup. Reticulin, therefore, may represent a novel and important prognostic HCC marker, to be further explored and validated.

Comprehensive analysis of KLF2 as a prognostic biomarker associated with fibrosis and immune infiltration in advanced hepatocellular carcinoma

PurposeMost Hepatocellular carcinoma (HCC) patients are in advanced or metastatic stage at the time of diagnosis. Prognosis for advanced HCC patients is dismal. This study was based on our previous microarray results, and aimed to explore the promising diagnostic and prognostic markers for advanced HCC by focusing on the important function of KLF2.MethodsThe Cancer Genome Atlas (TCGA), Cancer Genome Consortium database (ICGC), and the Gene Expression Comprehensive Database (GEO) provided the raw data of this study research. The cBioPortal platform, CeDR Atlas platform, and the Human Protein Atlas (HPA) website were applied to analyze the mutational landscape and single-cell sequencing data of KLF2. Basing on the results of single-cell sequencing analyses, we further explored the molecular mechanism of KLF2 regulation in the fibrosis and immune infiltration of HCC.ResultsDecreased KLF2 expression was discovered to be mainly regulated by hypermethylation, and indicated a poor prognosis of HCC. Single-cell level expression analyses revealed KLF2 was highly expressed in immune cells and fibroblasts. The function enrichment analysis of KLF2 targets indicated the crucial association between KLF2 and tumor matrix. 33-genes related with cancer associated fibroblasts (CAFs) were collected to identify the significant association of KLF2 with fibrosis. And SPP1 was validated as a promising prognostic and diagnostic marker for advanced HCC patients. CXCR6 CD8+ T cells were noted as a predominant proportion in the immune microenvironment, and T cell receptor CD3D was discovered to be a potential therapeutic biomarker for HCC immunotherapy.ConclusionThis study identified that KLF2 is an important factor promoting HCC progression by affecting the fibrosis and immune infiltration, highlighting its great potential as a novel prognostic biomarker for advanced HCC.

A novel approach for the analysis of single-cell RNA sequencing identifies TMEM14B as a novel poor prognostic marker in hepatocellular carcinoma

A fundamental goal in cancer-associated genome sequencing is to identify the key genes. Protein–protein interactions (PPIs) play a crucially important role in this goal. Here, human reference interactome (HuRI) map was generated and 64,006 PPIs involving 9094 proteins were identified. Here, we developed a physical link and co-expression combinatory network construction (PLACE) method for genes of interest, which provides a rapid way to analyze genome sequencing datasets. Next, Kaplan‒Meier survival analysis, CCK8 assays, scratch wound assays and Transwell assays were applied to confirm the results. In this study, we selected single-cell sequencing data from patients with hepatocellular carcinoma (HCC) in GSE149614. The PLACE method constructs a protein connection network for genes of interest, and a large fraction (80%) of the genes (screened by the PLACE method) were associated with survival. Then, PLACE discovered that transmembrane protein 14B (TMEM14B) was the most significant prognostic key gene, and target genes of TMEM14B were predicted. The TMEM14B-target gene regulatory network was constructed by PLACE. We also detected that TMEM14B-knockdown inhibited proliferation and migration. The results demonstrate that we proposed a new effective method for identifying key genes. The PLACE method can be used widely and make outstanding contributions to the tumor research field.

CD38 marks the exhausted CD8+ tissue-resident memory T cells in hepatocellular carcinoma.

Despite recent advances in immunotherapy for hepatocellular carcinoma (HCC), the overall modest response rate underscores the need for a better understanding of the tumor microenvironment (TME) of HCC. We have previously shown that CD38 is widely expressed on tumor-infiltrating leukocytes (TILs), predominantly on CD3+ T cells and monocytes. However, its specific role in the HCC TME remains unclear. In this current study, we used cytometry time-of-flight (CyTOF), bulk RNA sequencing on sorted T cells, and single-cell RNA (scRNA) sequencing to interrogate expression of CD38 and its correlation with T cell exhaustion in HCC samples. We also employed multiplex immunohistochemistry (mIHC) for validating our findings. From CyTOF analysis, we compared the immune composition of CD38-expressing leukocytes in TILs, non-tumor tissue-infiltrating leukocytes (NIL), and peripheral blood mononuclear cells (PBMC). We identified CD8+ T cells as the dominant CD38-expressing TILs and found that CD38 expression was significantly higher in CD8+ TRM in TILs than in NILs. Furthermore, through transcriptomic analysis on sorted CD8+ TRM from HCC tumors, we observed a higher expression of CD38 along with T cell exhaustion genes, including PDCD1 and CTLA4, compared to the circulating memory CD8 T cells from PBMC. This was validated by scRNA sequencing that revealed co-expression of CD38 with PDCD1, CTLA4, and ITGAE (CD103) in T cells from HCC tumors. The protein co-expression of CD38 and PD-1 on CD8+ T cells was further demonstrated by mIHC on HCC FFPE tissues, marking CD38 as a T cell co-exhaustion marker in HCC. Lastly, the higher proportions of CD38+PD-1+ CD8+ T cells and CD38+PD-1+ TRM were significantly associated with the higher histopathological grades of HCC, indicating its role in the aggressiveness of the disease. Taken together, the concurrent expression of CD38 with exhaustion markers on CD8+ TRM underpins its role as a key marker of T cell exhaustion and a potential therapeutic target for restoring cytotoxic T cell function in HCC.

FKBP11 upregulation promotes proliferation and migration in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related deaths world over. Early diagnosis and effective treatment monitoring significantly improves patients' outcomes. FKBP11 gene is highly expressed in HCC and could play a role in its development, early diagnosis and treatment. This study aimed to evaluate the expression of FKBP11 in HCC, its correlation with patients' clinical characteristics and potential role in HCC development. Expression was determined by bioinformatics analysis, quantitative real-time PCR, western blot, and immunohistochemistry. CCK-8, Transwell and wound healing assays were used to investigate involvement in HCC development. FKBP11 was significantly upregulated in HCC cells, tissues and blood (all p< 0.001). Its receiver operator characteristic (ROC) curve had an AUC of 0.864 (95% CI: 0.823-0.904), at a sensitivity of 0.86 and specificity of 0.78 indicating a good diagnostic potential in HCC. Its expression was markedly reduced after surgery (p< 0.0001), indicating a potential application in HCC treatment follow-up. Knockdown of FKBP11 in HCC cells attenuated proliferation and migration, suggesting a possible role in HCC pathogenesis. This study thus found that FKBP11 is upregulated in HCC, and the upregulation promotes HCC development. FKBP11 levels are significantly reduced post-surgery and could be a potential diagnostic and prognostic marker for HCC.

Development and validation of a non-invasive cfDNA targeted sequencing assay for early-stage hepatocellular carcinoma detection using cfDNA methylation and fragmentomics.

4128 Background: Hepatocellular carcinoma (HCC) is one of the most common cancers in China, and one of the leading causes of cancer-related deaths in the country. With a 5-year survival rate of only 15-20%, early detection is crucial to improve the treatment and survival of HCC patients. Currently, alpha-fetoprotein (AFP) is commonly used as a serum marker for HCC, but it is not a sufficiently specific and can cause false positive readings due to elevated levels caused by other liver conditions. An alternative method is to use circulating free DNA (cfDNA) released by tumor cells as cancer-screening targets, which has been shown to be a more sensitive and specific biomarkers for HCC detection. This study aims to develop a non-invasive screening assay based on cfDNA features to improve the detection of early-stage HCC. Methods: Candidate methylation markers for HCC detection were collected and evaluated using GEO, TCGA and in-house datasets, 1601 of which were incorporated into a targeted sequencing panel named HcSeer. Multiple types of cfDNA features were constructed from the sequencing data, which included methylation-related features such as methylation haplotype blocks (MHBs) and methylated haplotype fraction (MHF), and fragmentomics features such as end motif and CNV. For model building, Cancer and healthy plasma samples were randomly divided into a training and a testing set at a 2:1 ratio. A two-step deep neural network model was built to classify HCC using selected features of both types. Results: We previously enrolled a total of 401 plasma samples (200 healthy, 201 HCC) for model construction and the performance of the HcSeer model have been documented. An independent validation cohort of 421 plasma samples (280 healthy, 141 HCC) was currently collected from different centers. In this independent validation, the HcSeer model achieved an AUC of 0.98 with a sensitivity of 96.5% at a specificity of 96.4%. Importantly, HcSeer maintained a high sensitivity for HCC across all stages: 94.3%, 96%, 100% and 100% for stage I – IV cases, respectively. When compared to AFP, HcSeer achieved a significantly higher sensitivity of 94% than AFP’s 55% in 137 HCC cases having AFP level tested. When AFP level was combined with the HcSeer model, the sensitivity for HCC further increased to 96%. Conclusions: This study demonstrated that the DNA methylation and fragmentomics patterns of cfDNA can accurately distinguish HCC and healthy plasma samples, particularly in the early stages of HCC. The combination of the HcSeer and AFP further improved the accuracy of the prediction. Although this study was limited in sample size, it clearly showed the potential of the HcSeer assay for accurate HCC detection in blood.

Identification of N7-methylguanosine-related lncRNAs for the risk stratification of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The N7-methylguanosine (m7G) modification is related to the biological processes and regulation of various diseases. This study investigated the role and predictive value of m7G-related long non-coding RNAs (lncRNAs) in HCC. HCC patients were clustered by consensus clustering, and a prognostic signature was developed using Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression analysis. The immune landscape and clinicopathological features of the distinct clusters and subgroups were investigated. A total of 32 m7G-related lncRNAs were confirmed to be prognostic lncRNAs. Two molecular clusters showed significant differences in terms of their clinicopathological features, prognoses, and immune checkpoint gene (ICG) expression levels. Cluster II was associated with upregulated ICG expression and poor overall survival (OS). The Cancer Genome Atlas training cohort was then used to create an m7G-related lncRNA signature for predicting OS. The signature exhibited excellent predictive performance in the training, test, and all cohorts. The high-risk patients had worse clinical outcomes than the low-risk patients. Further study revealed that this signature was an independent prognostic indicator, and a predictive nomogram was developed based on the clinicopathological features and risk score. In addition, we discovered that this model was correlated with ICG expression and tumor immune cell infiltration. Our findings demonstrated that m7G-related lncRNAs are associated with the tumor immune landscape and prognosis and can serve as independent prognostic markers for HCC. These findings provide new insights into the functions of m7G-related lncRNAs in HCC.

Combined bulk RNA and single-cell RNA analyses reveal TXNL4A as a new biomarker for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a high mortality rate worldwide. The dysregulation of RNA splicing is a major event leading to the occurrence, progression, and drug resistance of cancer. Therefore, it is important to identify new biomarkers of HCC from the RNA splicing pathway. We performed the differential expression and prognostic analyses of RNA splicing-related genes (RRGs) using The Cancer Genome Atlas-liver hepatocellular carcinoma (LIHC). The International Cancer Genome Consortium (ICGC)-LIHC dataset was used to construct and validate prognostic models, and the PubMed database was used to explore genes in the models to identify new markers. The screened genes were subjected to genomic analyses, including differential, prognostic, enrichment, and immunocorrelation analyses. Single-cell RNA (scRNA) data were used to further validate the immunogenetic relationship. Of 215 RRGs, we identified 75 differentially expressed prognosis-related genes, and a prognostic model incorporating thioredoxin like 4A (TXNL4A) was identified using least absolute shrinkage and selection operator regression analysis. ICGC-LIHC was used as a validation dataset to confirm the validity of the model. PubMed failed to retrieve HCC-related studies on TXNL4A. TXNL4A was highly expressed in most tumors and was associated with HCC survival. Chi-squared analyses indicated that TXNL4A expression positively correlated positively with the clinical features of HCC. Multivariate analyses revealed that high TXNL4A expression was an independent risk factor for HCC. Immunocorrelation and scRNA data analyses indicated that TXNL4A was correlated with CD8 T cell infiltration in HCC. Therefore, we identified a prognostic and immune-related marker for HCC from the RNA splicing pathway.

An electrochemiluminescence resonance energy transfer biosensor based on Luminol-LDH and CuS@Pt for detection of alpha-fetoprotein

Alpha-fetoprotein (AFP) is the best diagnostic marker for hepatocellular carcinoma (HCC) and plays an important role in the general surveillance of the population. Therefore, the establishment of an ultra-sensitive AFP assay is essential for the early screening and clinical diagnosis of HCC. In this work, we designed a signal-off biosensor for ultra-sensitive detection of AFP based on an electrochemiluminescent resonance energy transfer (ECL-RET) strategy using luminol intercalated layered bimetallic hydroxide (Luminol-LDH) as an ECL donor and Pt nanoparticles-grown on copper sulfide nanospheres (CuS@Pt) as ECL acceptor. The (Au NPs/Luminol-LDH)n multilayer nanomembrane synthesized by our intercalation and layer-by-layer electrostatic assembly process not only effectively immobilizes luminol but also significantly enhances the ECL signal. The CuS@Pt composite has well visible light absorption ability and can burst the light emitted from luminol by ECL-RET. The biosensor showed good linearity in the range from 10−5 ng mL−1 to 100 ng mL−1 and a minimum detection limit of 2.6 fg mL−1. Therefore, the biosensor provides a novel and efficient strategy for the detection of AFP, which is important for the early screening and clinical diagnosis of HCC.

Characteristic of molecular subtypes based on PANoptosis-related genes and experimental verification of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a type of liver cancer that originates from liver cells. It is one of the most common types of liver cancer and a leading cause of cancer-related death worldwide. Early detection and treatment can improve the HCC prognosis. Therefore, it is necessary to further improve HCC markers and risk stratification. PANoptosome is a cytoplasmic polymer protein complex that regulates a proinflammatory programmed cell death pathway called "PANoptosis". The role of PANoptosis in HCC remains unclear. In this study, the molecular changes of PANoptosis related genes (PAN-RGs) in HCC were systematically evaluated. We characterized the heterogeneity of HCC by using consensus clustering to identify two distinct subtypes. The two subtypes showed different survival rate, biological function, chemotherapy drug sensitivity and immune microenvironment. After identification of PAN-RG differential expression genes (DEGs), a prognostic model was established by Cox regression analysis using minimum absolute contraction and selection operator (LASSO), and its prognostic value was verified by Cox regression analysis, Kaplan-Meier curve and receiver operating characteristic (ROC) curve. Our own specimens were also used to further validate the prognostic significance and possible clinical value of the selected targets. Subsequently, we conducted a preliminary discussion on the reasons for the influence of the model on the prognosis through TME analysis, drug resistance analysis, TMB analysis and other studies. This study provides a new idea for individualized and precise treatment of HCC.

Soluble CD163 is a predictor of fibrosis and hepatocellular carcinoma development in nonalcoholic steatohepatitis

BackgroundNonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. The serum level of soluble CD163 (sCD163), a macrophage activation marker, is associated with liver tissue changes; however, its prognostic value is unknown. Here, we determined the utility of sCD163 as a marker for hepatocellular carcinoma (HCC) and prognostic marker for NAFLD.MethodsThis retrospective study obtained data regarding serum sCD163 levels, liver histology, and background factors associated with NAFLD in 287 patients (men/women, 140/147; average age, 53 ± 14 years) with NAFLD who underwent liver biopsy. Repeated liver biopsies of 287 patients with NAFLD (5.0 ± 2.7 years) were compared regarding serum sCD163 levels and liver tissue changes (stage, grade, steatosis, and NAFLD activity score).ResultsSerum sCD163 levels increased with the progression of liver fibrosis and inflammation (both P < 0.05) and were particularly helpful in distinguishing cases of Grade 4 fibrosis (P < 0.001). Levels of sCD163 significantly decreased in patients with NAFLD exhibiting alleviated fibrosis and inflammation (P < 0.05). We could also predict the development of HCC and associated mortality based on serum sCD163 levels at the time of NAFLD diagnosis. Serum sCD163 levels were higher in patients with HCC than in patients without HCC (1074 ± 379 ng/ml vs. 669 ± 261 ng/ml; P < 0.0001), and the same trend was observed for mortality.ConclusionsThe serum sCD163 level reflects the progression of fibrosis and inflammation in liver tissues, showing much promise as a noninvasive biomarker for nonalcoholic steatohepatitis and NAFLD as well as a possible predictor of HCC development and patient prognosis.

VPS72, a member of VPS protein family, can be used as a new prognostic marker for hepatocellular carcinoma.

Vacuolar protein sorting (VPS) plays a crucial role in intracellular molecular transport between organelles. However, studies have indicated a correlation between VPSs and tumorigenesis and the development of several cancers. Nevertheless, the association between VPSs and hepatocellular carcinoma (HCC) remains unclear. By analyzing databases such as The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC), we investigated the differences in VPSs expression between normal tissue and HCC transcriptomes. Furthermore, we examined the relationship between VPSs expression and overall survival (OS) in patients with HCC. Univariate and multivariate Cox analyses were employed to assess the prognostic value of VPS72 as an independent factor, and the correlation between VPS72 and the tumor immune microenvironment was also analyzed. We observed significant overexpression of 28 VPSs in HCC tissues compared to normal tissues. The mRNA expression of VPSs displayed a negative correlation with OS, while exhibiting a positive correlation with tumor grade and stage. Additionally, both univariate and multivariate Cox analyses identified VPS72 as a potential independent risk factor for HCC prognosis. Overexpression of VPS72 demonstrated a positive correlation with various clinicopathological factors associated with poor prognosis, as well as the infiltration levels of immune cells. Therefore, our research shows that VPSs participate in HCC occurrence and development, especially VPS72, which may act as a potential target for HCC treatment and prognosis biomarker.

Screening prognostic markers for hepatocellular carcinoma based on pyroptosis-related lncRNA pairs

BackgroundPyroptosis is closely related to cancer prognosis. In this study, we tried to construct an individualized prognostic risk model for hepatocellular carcinoma (HCC) based on within-sample relative expression orderings (REOs) of pyroptosis-related lncRNAs (PRlncRNAs).MethodsRNA-seq data of 343 HCC samples derived from The Cancer Genome Atlas (TCGA) database were analyzed. PRlncRNAs were detected based on differentially expressed lncRNAs between sample groups clustered by 40 reported pyroptosis-related genes (PRGs). Univariate Cox regression was used to screen out prognosis-related PRlncRNA pairs. Then, based on REOs of prognosis-related PRlncRNA pairs, a risk model for HCC was constructed by combining LASSO and stepwise multivariate Cox regression analysis. Finally, a prognosis-related competing endogenous RNA (ceRNA) network was built based on information about lncRNA–miRNA–mRNA interactions derived from the miRNet and TargetScan databases.ResultsHierarchical clustering of HCC patients according to the 40 PRGs identified two groups with a significant survival difference (Kaplan–Meier log-rank, p = 0.026). Between the two groups, 104 differentially expressed lncRNAs were identified (|log2(FC)|> 1 and FDR < 5%). Among them, 83 PRlncRNA pairs showed significant associations between their REOs within HCC samples and overall survival (Univariate Cox regression, p < 0.005). An optimal 11-PRlncRNA-pair prognostic risk model was constructed for HCC. The areas under the curves (AUCs) of time-dependent receiver operating characteristic (ROC) curves of the risk model for 1-, 3-, and 5-year survival were 0.737, 0.705, and 0.797 in the validation set, respectively. Gene Set Enrichment Analysis showed that inflammation-related interleukin signaling pathways were upregulated in the predicted high-risk group (p < 0.05). Tumor immune infiltration analysis revealed a higher abundance of regulatory T cells (Tregs) and M2 macrophages and a lower abundance of CD8 + T cells in the high-risk group, indicating that excessive pyroptosis might occur in high-risk patients. Finally, eleven lncRNA–miRNA–mRNA regulatory axes associated with pyroptosis were established.ConclusionOur risk model allowed us to determine the robustness of the REO-based PRlncRNA prognostic biomarkers in the stratification of HCC patients at high and low risk. The model is also helpful for understanding the molecular mechanisms between pyroptosis and HCC prognosis. High-risk patients may have excessive pyroptosis and thus be less sensitive to immune therapy.

Dihydroartemisinin inhibited interleukin-18 expression by decreasing YAP1 in hepatocellular carcinoma cells

BackgroundYes-associated protein 1 (YAP1) is highly expressed in liver cancer and has been used as an independent prognostic marker for hepatocellular carcinoma (HCC), while inhibition of YAP1 slows down the progression of HCC. Interleukin-18 (IL-18) also tends to be highly expressed in liver cancer. Previous research has proved that dihydroartemisinin (DHA) plays an important role in HCC treatment by reducing YAP1 expression. However, the relationship between YAP1 and IL-18 has not been reported in HCC, especially during DHA therapy. ObjectiveThe purpose of this study was to clarify the relationship between YAP1 and IL-18 in HCC cells, and to explicit the role of IL-18 in the treatment of HCC by DHA. Methods and resultsWe found that YAP1 and IL-18 were highly expressed in patients with hepatocellular carcinoma by bioinformatics analysis. Moreover, YAP1 was positively correlated with IL18 in liver cancer. YAP1 and IL18 correlated with immune cell infiltration, notably T cell exhaustion. YAP1 knockdown decreased IL-18 expression, while YAP1 overexpression increased the IL-18 expression in HCC cells. DHA reduced IL-18 expression through YAP1 in HCC cells. Further, DHA reduced the growth of Hepa1–6 cells subcutaneous xenograft tumors by inhibiting the expression of YAP1 and IL-18. However, DHA improved IL-18 in serum and adjacent tissues from DEN/TCPOBOP-induced liver tumor model in C57BL/6 mice. ConclusionYAP1 was positively correlated with IL-18 in HCC. DHA reduced the expression of IL-18 by inhibiting YAP1 and plays a role in the treatment of HCC. Our study suggested that IL-18 is a potential target for the treatment of HCC, and DHA is a promising drug for HCC therapy. Data availabilityThe dataset that supports the findings of this study is available from the corresponding author upon reasonable request.

Silencing of RAB42 down-regulated PD-L1 expression to inhibit the immune escape of hepatocellular carcinoma cells through inhibiting the E2F signaling pathway

ObjectiveTo investigate the mechanistic role of RAB42 and corresponding regulatory path in hepatocellular carcinoma (HCC). MethodsThe expression of RAB42 in HCC tissue was checked by RT-qPCR and immunohistochemical staining assay. Cell proliferation was checked by colony formation and CCK-8 assay. Cell apoptosis and cycle distribution were analyzed with flow cytometry. The relevance of RAB42 and PD-L1 was analyzed from TCGA database. The binding of E2F1 to PD-L1 was detected by JASPAR database, luciferase and ChIP assay. The expression of PD-L1, cell apoptosis- and E2F pathway-related proteins were checked by western blotting. ResultsRAB42 was highly expressed in HCC tissue. RAB42 silencing could inhibit proliferation and induce G1 phase arrest and apoptosis of HCC cells. TCGA database disclosed that PD-L1 was highly associated with RAB42 expression. Silencing of RAB42 could retard PD-L1 expression in HCC cells. GSEA analysis showed RAB42 could activate E2F signaling pathway. Silencing of RAB42 could observably weaken the expression of E2F1, CDK1 and CDC20 in HCC cells. JASPAR database predicted the binding site between E2F1 and PD-L1, and E2F1 overexpression could promote PD-L1 expression. Overexpression of E2F1 could reverse the biological function of RAB42 silencing in HCC cells. ConclusionSilencing of RAB42 could down-regulate PD-L1 expression to inhibit immune escape through inhibiting E2F signaling pathway in HCC cells. RAB42 may become a novel clinical diagnostic and therapy marker for HCC.

NIFK, an independent prognostic biomarker of hepatocellular carcinoma, is correlated with immune infiltration

BackgroundThe molecular mechanisms that lead to hepatocellular carcinoma (HCC), a highly common malignant tumor, are currently unclear. In fact, while the nucleolar protein that interacts with the FHA domain of pKi-67 (NIFK) is known to promote lung cancer progression, its specific role in HCC remains unknown. ResultsIn HCC tissues, NIFK was significantly overexpressed in comparison with normal tissues. In The Cancer Genome Atlas (TCGA) database, NIFK expression showed a good prediction value according to an ROC curve and it was linked to poor progression-free interval, disease-specific survival and overall survival. Furthermore, the level of NIFK expression in HCC was significantly correlated with tumor stage, AFP (ng/mL), OS event, DSS event and PFI event. Based on GSEA evaluations, differentially expressed NIFK genes exhibited enrichment for fatty acid metabolism, oxidative phosphorylation as well as cancer, cell cycle, MAPK, TGF, WNT and NOTCH signaling pathways. The TIMER database analysis further revealed positive associations between NIFK expression and the immune cells, such as dendritic cells, neutrophils, macrophages, CD4+ T cells, CD8+ T cells and B cells. Also, the NIFK expression was positively correlated with immune checkpoints (PD1/PD-L1 and CTLA4). The experimental verification determined that NIFK knockdown could thwart HCC cellular properties of proliferation, metastasis and invasiveness. Univariate and multivariate Cox hazard regression validated NIFK expression as an independent prognostic marker in HCC. ConclusionsNIFK has theragnostic potential as a separate prognostic factor or novel biomarker involved in the immune infiltration of HCC.How to cite: Cheng F, Yuan L, Wu Z, et al. NIFK, an independent prognostic biomarker of hepatocellular carcinoma, is correlated with immune infiltration. Electron J Biotechnol 2023;63. https://doi.org/10.1016/j.ejbt.2023.01.003.

MALT1 paracaspase is overexpressed in hepatocellular carcinoma and promotes cancer cell survival and growth

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, therapeutic management of HCC remains a challenge, emphasizing the importance of exploring novel targets. MALT1 paracaspase is a druggable signaling molecule whose dysregulation has been linked to hematological and solid tumors. However, the role of MALT1 in HCC remains poorly understood, leaving its molecular functions and oncogenic implications unclear. Here we provide evidence that MALT1 expression is elevated in human HCC tumors and cell lines, and that correlates with tumor grade and differentiation state, respectively. Our results indicate that ectopic expression of MALT1 confers increased cell proliferation, 2D clonogenic growth, and 3D spheroid formation in well differentiated HCC cell lines with relatively low MALT1 levels. In contrast, stable silencing of endogenous MALT1 through RNA interference attenuates these aggressive cancer cell phenotypes, as well as migration, invasion, and tumor-forming ability, in poorly differentiated HCC cell lines with higher paracaspase expression. Consistently, we find that pharmacological inhibition of MALT1 proteolytic activity with MI-2 recapitulates MALT1 depletion phenotypes. Finally, we show that MALT1 expression is positively correlated with NF-kB activation in human HCC tissues and cell lines, suggesting that its tumor promoting functions may involve functional interaction with the NF-kB signaling pathway. This work unveils new insights into the molecular implications of MALT1 in hepatocarcinogenesis and places this paracaspase as a potential marker and druggable liability in HCC.

Abstract 2679: Combination of CDK 4/6 inhibitor palbociclib with rapamycin synergistically inhibits growth of hepatocellular carcinoma in preclinical models

Abstract Introduction: The cell cycle inhibitor palbociclib has been shown to have a cytostatic effect on hepatocellular carcinoma (HCC) cells via the induction of quiescence and senescence, though it has limited cytocidal effect. We hypothesized that arrested HCC cells upregulate mTOR signaling to stay metabolically active and investigated the effect of combining palbociclib with the mTOR inhibitor rapamycin on preclinical models of HCC. Methods: We identified commonly altered molecular markers in HCC, including cell cycle related genes, using single sample gene set enrichment analysis (ssGSEA) of 13 paired (tumor and adjacent non-tumor) RNA-seq and 15 paired proteomic data sets as well as 50 paired RNA-seq data sets from TCGA. Subsequently, we studied the in-vitro effects of palbociclib and rapamycin by evaluating induction of senescence, inhibition of cell growth with fixed ratio combination treatment as well as Western Blotting to establish mechanistic effects. We tested the in-vivo anti-tumor efficacy of palbociclib plus rapamycin in athymic nude mice with growing xenograft tumors. We then generated immunocompetent murine HCC models using hydrodynamic transfection of oncogenes ΔN90-β-catenin and c-Met, and measured plasma AFP levels as a marker of HCC to assess the efficacy of treatment with palbociclib and/or rapamycin. Results: ssGSEA revealed significant positive enrichment of several cell cycle related gene sets in all our HCC cases. Palbociclib treatment in vitro showed an increase in senescence associated β-galactosidase staining as well as increased levels of intrinsic CDK inhibitors p16, p15, p21 and p27, indicating senescence. Fixed ratio treatment with palbociclib and rapamycin established a synergistic interaction between the drugs, as indicated by a combination index of less than 1. We also observed an increase in levels of proteins of the mTOR pathway (p-mTOR, p-4eBP1, p-p70 and p-s6) after palbociclib treatment, followed by suppression of the levels on addition of rapamycin. In the xenograft HCC model, combination treatment with palbociclib and rapamycin resulted in slower tumor growth and significantly increased time to critical tumor volume (2000 mm3) (log rank, p &amp;lt;0.001). In the hydrodynamic transfection HCC model, we observed a significantly lower mean AFP level in the combination treatment group, along with lower liver weights which were closer to the liver weights of normal mice. Conclusion: HCC cells overexpress genes and proteins that drive cell cycle progression. Palbociclib induces senescence in HCC cells along with an associated upregulation in mTOR signaling, and combination with rapamycin has a synergistic effect on inhibition of tumor growth in vitro as well as in vivo, possibly by abrogating both actively growing and quiescent/senescent HCC cells. The combination of the two drugs may have potential as a novel therapy for HCC. Citation Format: Ankur Tiwari, Araceli Huerta, Hakim Bouamar, Debodipta Das, Francisco Cigarroa, LuZhe Sun. Combination of CDK 4/6 inhibitor palbociclib with rapamycin synergistically inhibits growth of hepatocellular carcinoma in preclinical models [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 2679.