Tumor Progression In Hepatocellular Carcinoma Research Articles

MicroRNA-642a-5p Targets DNA Damage-Inducible Transcript 4 to Suppress Hepatitis B Virus Hepatoma Carcinoma Cell

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors in clinical practice, with hepatitis B virus (HBV) being the most common risk factor for HCC. MicroRNAs (miRNAs) have emerged as a new marker for disease diagnosis and molecularly targeted therapies; however, the mechanism of miR-642a-5p in HBV-associated HCC remains unclear. Objectives: The aim of this study was to investigate the expression of miR-642a-5p, which targets DNA damage-inducible transcript 4 (DDIT4), in HBV-associated HCC, and its effect on the proliferation, migration, and invasion of HBV-positive HCC cells. Methods: miR-642a-5p in the serum of patients with HBV-associated liver cancer (LC), as well as miR-642a-5p and DDIT4 mRNA in LC tissues and cells, and HBV DNA in HBV-positive cells were detected. The targeting of DDIT4 by miR-642a-5p and the progression of cells were also examined. All cell experiments were repeated five times. Results: The results indicated that levels of miR-642a-5p were decreased, while levels of DDIT4 were increased in the serum, tissues, and cells of HBV-positive HCC patients. Overexpression of miR-642a-5p inhibited the progression of HBV-positive HCC cells, suppressed HBV DNA replication, cell proliferation, and invasion, and promoted apoptosis in HepG2.2.15 cells. Conclusions: In addition, miR-642a-5p directly targeted DDIT4, and knockdown of DDIT4 reversed the effects of miR-642a-5p upregulation, promoting the progression of HBV-positive HCC cells. In conclusion, miR-642a-5p is expressed at low levels in HBV-associated HCC and inhibits HBV DNA replication and tumor progression in HBV-positive HCC by targeting DDIT4. This study provides a foundation for molecular targeted therapy in HBV-positive HCC.

ACSL4 promotes malignant progression of Hepatocellular carcinoma by targeting PAK2 transcription

Long-chain fatty acyl-Coa ligase 4 (ACSL4) is an important enzyme that converts fatty acids to fatty acyl-Coa esters, there is increasing evidence for its role in carcinogenesis. However, the precise role of ACLS4 in hepatocellular carcinoma (HCC) is not clearly understood. In the present study, we provide evidence that ACSL4 expression was specifically elevated in HCC and is associated with poor clinical outcomes. ACSL4 significantly promotes the growth and metastasis of HCC both in vitro and in vivo. RNA sequencing and functional experiments showed that the effect of ACSL4 on HCC development was heavily dependent on PAK2. ACSL4 expression is well correlated with PAK2 in HCC, and ACSL4 even transcriptionally increased PAK2 gene expression mediated by Sp1. In addition, emodin, a naturally occurring anthraquinone derivative, inhibited HCC cell growth and tumor progression by targeting ACSL4. In summary, ACSL4 plays a novel oncogene in HCC development by regulating PAK2 transcription. Targeting ACSL4 could be useful in drug development and therapy for HCC.

Lipid metabolism-related long noncoding RNA RP11-817I4.1 promotes fatty acid synthesis and tumor progression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of tumors. The influence of lipid metabolism disruption on the development of HCC has been demonstrated in published studies. To establish an HCC prognostic model for lipid metabolism-related long non-coding RNAs (LMR-lncRNAs) and conduct in-depth research on the specific role of novel LMR-lncRNAs in HCC. Correlation and differential expression analyses of The Cancer Genome Atlas data were used to identify differentially expressed LMR-lncRNAs. Quantitative real-time polymerase chain reaction analysis was used to evaluate the expression of LMR-lncRNAs. Nile red staining was employed to observe intracellular lipid levels. The interaction between RP11-817I4.1, miR-3120-3p, and ATP citrate lyase (ACLY) was validated through the performance of dual-luciferase reporter gene and RIP assays. Three LMR-lncRNAs (negative regulator of antiviral response, RNA transmembrane and coiled-coil domain family 1 antisense RNA 1, and RP11-817I4.1) were identified as predictive markers for HCC patients and were utilized in the construction of risk models. Additionally, proliferation, migration, and invasion were reduced by RP11-817I4.1 knockdown. An increase in lipid levels in HCC cells was significantly induced by RP11-817I4.1 through the miR-3120-3p/ACLY axis. LMR-lncRNAs have the capacity to predict the clinical characteristics and prognoses of HCC patients, and the discovery of a novel LMR-lncRNAs, RP11-817I4.1, revealed its role in promoting lipid accumulation, thereby accelerating the onset and progression of HCC.

Critical Roles of the Sphingolipid Metabolic Pathway in Liver Regeneration, Hepatocellular Carcinoma Progression and Therapy.

The sphingolipid metabolic pathway, an important signaling pathway, plays a crucial role in various physiological processes including cell proliferation, survival, apoptosis, and immune regulation. The liver has the unique ability to regenerate using bioactive lipid mediators involving multiple sphingolipids, including ceramide and sphingosine 1-phosphate (S1P). Dysregulation of the balance between sphingomyelin, ceramide, and S1P has been implicated in the regulation of liver regeneration and diseases, including liver fibrosis and hepatocellular carcinoma (HCC). Understanding and modulating this balance may have therapeutic implications for tumor proliferation, progression, and metastasis in HCC. For cancer therapy, several inhibitors and activators of sphingolipid signaling, including ABC294640, SKI-II, and FTY720, have been discussed. Here, we elucidate the critical roles of the sphingolipid pathway in the regulation of liver regeneration, fibrosis, and HCC. Regulation of sphingolipids and their corresponding enzymes may considerably influence new insights into therapies for various liver disorders and diseases.

The combination of CD8 and TIM3 expression to predict survival outcomes in hepatocellular carcinoma.

543 Background: CD8+ T cells have anti-tumor immunity in a variety of cancers including hepatocellular carcinoma (HCC). Tumor-infiltrating CD8+ T cells express immune checkpoint proteins and focus on a therapeutic targeting. T cell immunoglobulin and mucin domain protein 3 (TIM3) has been reported to be associated with suppression of anti-tumor immunity, poor prognosis, and tumor progression in HCC, resulting in up-regulation on exhausted CD8+ T cells. As the exhausted T cells showed a decrease of effector cytokine production and cytolytic activity, leading to the tumor progression, TIM3 is one of the most important exhaustion markers. In the present study, we investigated the role of CD8 and TIM3 as a prognostic biomarker in HCC. Methods: We analyzed 474 HCC patients with clinical and transcriptomic data from The Cancer Genome Atlas (TCGA: n=359) and GSE 76427 (n=115) to examine associations between CD8, TIM3, and programmed death receptor-1/programmed cell death ligand 1 (PD-1/PD-L1) expression and survival outcomes. Each gene was divided into low- and high-expression groups using the tertiles of gene expression. Results: The patients with high CD8 and low TIM3 expression were associated with better overall survival (OS) (P=0.021 and P=0.025, respectively) in TCGA, while PD-1/PD-L1 were not associated with OS (P=0.306 and P=0.318, respectively). Moreover, the patients with high CD8 and low TIM3 expression were validated in GSE dataset (P=0.014 and P=0.009, respectively). The patients with high CD8 and low TIM3 expression were associated with improved OS compared with the overall HCC patient (P=0.008). Among the patients with high CD8 expression, low TIM3 expression (non-exhausted CD8+ T cells) was associated with better OS (P=0.009), but PD-1/PD-L1 were not associated with OS (P=0.491 and P=0.772, respectively). Finally, multivariate analysis showed that the patients withnon-exhausted CD8+ T cells were significantly superior for the survival outcomes (Hazard ratio=2.80, 95% CI=1.27–6.19, P=0.01). Conclusions: High CD8 and low TIM3 expression is associated with better OS in HCC than PD-1/PD-L1, suggesting that CD8 and TIM3 are useful biomarkers for predicting HCC prognosis. Restoring exhausted T cells via the suppression of TIM3 is a promising strategy for cancer treatment, which become a breakthrough in the cancer immunotherapy.

The role and mechanism of HIF-1α-mediated glypican-3 secretion in hypoxia-induced tumor progression in hepatocellular carcinoma

ObjectiveTo explore the expression and secretion mechanism of glypican-3 (GPC3) in hepatocellular carcinoma (HCC) cells under hypoxic conditions, and its role in tumor progression. MethodsHuh7 cells with and without the knockdown of hypoxia-inducible factor 1-alpha (HIF-1α) were cultured under 1% O2 for varying durations to induce hypoxia. The expression levels of GPC3, HSP70, CD63, STX11 and SYT7 in the cytoplasm and exosomes of Huh7 cells were evaluated by western blotting and immunofluorescence. GPC3 protein expression was further measured in cells treated with GW4869 under hypoxic conditions. Huh7 cells and human umbilical vein endothelial cells (HUVECs) were cultured with the exosomes extracted from the control and GPC3-knockdown cells, the cell proliferation, migration, epithelial-mesenchymal transition (EMT), invasion, and in vitro angiogenesis were analyzed. Tumor xenografts were established to assess the role of GPC3-deficient exosomes in tumor growth. ResultsHypoxic culture conditions downregulated GPC3, STX11 and SYT7 protein levels in the Huh7 cells and upregulated GPC3 mRNA, and also increased GPC3 protein expression in the exosomes. HIF-1α knockdown, as well as treatment with GW4869, upregulated GPC3 protein in the Huh7 cells grown under 1% O2, but downregulated exosomal GPC3. Furthermore, exosomes derived from the GPC3-knockdown cells inhibited the proliferation and migration of Huh7 cells, decreased the expression of N-cadherin, vimentin, β-catenin, c-Myc and cyclin D1, and increased that of E-cadherin. Likewise, the GPC3-deficient exosomes also suppressed the invasion and tube formation ability of the HUVECs compared to that of control cells. Consistent with the in vitro results, the GPC3-deficient exosomes also repressed tumor growth in vivo. ConclusionHypoxia promoted secretion of exosomal GPC3 through the activation of HIF-1α. GPC3-deficient exosomes inhibited the proliferation, migration and EMT of HCC cells via the Wnt/β-catenin signaling pathway, and suppressed the angiogenic potential of HUVECs. This provided a novel understanding of the role of exosomal GPC3 in HCC progression.

HSP90, as a functional target antigen of a mAb 11C9, promotes stemness and tumor progression in hepatocellular carcinoma

BackgroundIdentification of promising targeted antigens that exhibited cancer-specific expression is a crucial step in the development of novel antibody-targeted therapies. We here aimed to investigate the anti-tumor activity of a novel monoclonal antibody (mAb) 11C9 and identify the antibody tractable target in the hepatocellular cancer stem cells (HCSCs).MethodsThe identification of the targeted antigen was conducted using SDS-PAGE, western blot, mass spectrometry, and co-immunoprecipitation. Silence of HSP90 was induced by siRNA interference. Positive cells were sorted by fluorescence-activated cell sorting. Double-immunofluorescent (IF) staining and two-color flow cytometry detected the co-expression. Self-renewal, invasion, and drug resistance were assessed by sphere formation, matrigel-coated Transwell assay, and CCK-8 assay, respectively. Tumorigenicity was evaluated in mouse xenograft models. RNA-seq and bioinformatics analysis were performed to explore the mechanism of mAb 11C9 and potential targets.ResultsMAb 11C9 inhibited invasion and self-renewal abilities of HCC cell lines and reversed the cisplatin resistance. HSP90 (~ 95 kDa) was identified as a targeted antigen of mAb 11C9. Tissue microarrays and online databases revealed that HSP90 was overexpressed in HCC and associated with a poor prognosis. FACS and double-IF staining showed the co-expression of HSP90 and CSCs markers (CD90 and ESA). In vitro and in vivo demonstrated the tumorigenic potentials of HSP90. The inhibition of HSP90 by siRNA interference or 17-AAG inhibitor both decreased the number of invasion, sphere cells, and CD90+ or ESA+ cells, as well as reversed the resistance. Bioinformatics analysis and western blot verified that HSP90 activated Wnt/β-catenin signaling.ConclusionsThe study preliminarily revealed the anti-tumor activity of mAb 11C9. More importantly, we identified HSP90 as a targeted antigen of mAb 11C9, which functions as an oncogene in phenotype shaping, stemness maintenance, and therapeutic resistance by activating Wnt/β-catenin signaling.

TrkB inhibition of DJ-1 degradation promotes the growth and maintenance of cancer stem cell characteristics in hepatocellular carcinoma

Although TrkB may be associated with the pathogenesis of various cancer by upregulation, how upregulation of TrkB led to tumor progression in hepatocellular carcinoma (HCC) and the signaling mechanisms by which TrkB induces motility, invasion, metastasis, drug resistance, and acquisition of self-renewal traits has remained unclear. Here, we demonstrated that TrkB was significantly upregulated in highly metastatic HCC cells and HCC patients. Also, the increased TrkB levels were significantly correlated with tumor stages and poor survival of HCC patients. Furthermore, the upregulated TrkB expression enhances the metastatic ability of HCC cells through reduced anoikis sensitivity, induced migration, and colony formation. Most strikingly, TrkB markedly enhances the activation of STAT3 by preventing DJ-1 degradation through the formation of the TrkB/DJ-1 complex. This signaling mechanism is responsible for triggering cellular traits of highly aggressive HCC. The activation of the EMT program of HCC via increasing DJ-1 stability by TrkB induces the gain of cancer stem cell states and chemoresistance via the upregulation of stem cells cell markers and ABC transporters. Also, TrkB-mediated inhibition of DJ-1 degradation promotes tumor formation and metastasizes to other organs in vivo. Our observations illustrate that TrkB is a prognostic and therapeutic targeting in promoting aggressiveness and metastasis of HCC.

LncRNA NEAT1 suppresses cellular senescence in hepatocellular carcinoma via KIF11-dependent repression of CDKN2A.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Therapeutic options for advanced HCC are limited, which is due to a lack of full understanding of pathogenesis. Cellular senescence is a state of cell cycle arrest, which plays important roles in the pathogenesis of HCC. Mechanisms underlying hepatocellular senescence are not fully understood. LncRNA NEAT1 acts as an oncogene and contributes to the development of HCC. Whether NEAT1 modulates hepatocellular senescence in HCC is unknown. The role of NEAT1 and KIF11 in cellular senescence and tumor growth in HCC was assessed both in vitro and in vivo. RNA pulldown, mass spectrometry, Chromatin immunoprecipitation (ChIP), luciferase reporter assays, RNA FISH and immunofluorescence (IF) staining were used to explore the detailed molecular mechanism of NEAT1 and KIF11 in cellular senescence of HCC. We found that NEAT1 was upregulated in tumor tissues and hepatoma cells, which negatively correlated with a senescence biomarker CDKN2A encoding p16INK4a and p14ARF proteins. NEAT1 was reduced in senescent hepatoma cells induced by doxorubicin (DOXO) or serum starvation. Furthermore, NEAT1 deficiency caused senescence in cultured hepatoma cells, and protected against the progression of HCC in a mouse model. During senescence, NEAT1 translocated into cytosol and interacted with a motor protein KIF11, resulting in KIF11 protein degradation and subsequent increased expression of CDKN2A in cultured hepatoma cells. Furthermore, KIF11 knockdown caused senescence in cultured hepatoma cells. Genetic deletion of Kif11 in hepatocytes inhibited the development of HCC in a mouse model. Conclusively, NEAT1 overexpression reduces senescence and promotes tumor progression in HCC tissues and hepatoma cells, whereas NEAT1 deficiency causes senescence and inhibits tumor progression in HCC. This is associated with KIF11-dependent repression of CDKN2A. These findings lay the foundation to develop potential therapies for HCC by inhibiting NEAT1 and KIF11 or inducing senescence.

Development of a novel tumor microenvironment-related radiogenomics model for prognosis prediction in hepatocellular carcinoma.

The tumour microenvironment (TME) has occupied a potent position in the tumorigenesis and tumor progression of hepatocellular carcinoma (HCC). Radiogenomics is an emerging field that integrates imaging and genetic information, thus offering a novel class of non-invasive biomarkers with diagnostic, prognostic, and treatment response. However, optimal evaluation methodologies for radiogenomics in patients with HCC have not been well established. Therefore, this study aims to develop a radiogenomics models, associating contrast-enhanced computed tomography (CECT) based radiomics features and transcriptomics data with TME, to increase predictive precision for overall survival (OS) in patients with HCC. Transcriptome profiles of 365 patients with HCC from The Cancer Genome Atlas (TCGA)-HCC cohort were used to obtain TME-related genes by differential expression analysis. TME-related radiomics features of 53 patients with HCC from The Cancer Imaging Archive (TCIA)-HCC cohort matched with the TCGA-HCC cohort were screened via correlation analysis. Furthermore, a radiogenomics score-based prognostic model was constructed using the least absolute shrinkage and selection operator (LASSO) Cox regression analysis in the TCIA-HCC cohort. Finally, the ability to predict prognosis and the value of the model in identifying the abundance of immune cell infiltration were investigated. A radiogenomics prognostic model was developed, which incorporated 1 radiomics feature [original_gray-level co-occurrence matrix (glcm)_inverse difference normalized (Idn)] and 3 genes [spen paralogue and orthologue C‑terminal domain containing 1 (SPOCD1); killer cell lectin like receptor B1 (KLRB1); G protein-coupled receptor 182 (GPR182)]. The model performed satisfactorily in the training and test sets [1-year, 2-year, 3-year area under the curve (AUC) of 0.81, 0.85 and 0.87 in the training set, respectively; and 0.73, 0.83, and 0.84 in the test set, respectively]. Moreover, the model showed that higher radiogenomics scores were associated with worse OS and lower levels of immune infiltration. The novel CECT-based radiogenomics model may provide valuable insights for prognostic stratification and TME assessment of patients with HCC.

Scribble promotes fibrosis-dependent mechanisms of hepatocarcinogenesis by p53/PUMA-mediated glycolysis

Backgrounds and aimsLiver cancer is the sixth most common type of cancer and the fifth leading cause of cancer mortality worldwide. Scribble has been shown to function as a neoplastic tumor suppressor gene in most tumors. Our previous studies reported that down-regulation or mislocalization of Scribble was sufficient to initiate mammary tumorigenesis and NSCLC. Recently, it was reported that Scribble was highly expressed in hepatocellular carcinoma (HCC). We aim to study how it was up-regulated and the contradictory role of Scribble in HCC. Methods and resultsUsing a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis system, we showed that Scribble was over-expressed and which may protect the mice against hepatic fibrosis. Unexpectedly, we found out the potential for Scribble to act as a tumor driver at the advanced stage of N-nitrosodiethylamine (DEN) plus CCl4 induced HCC mice model in vivo. In addition, we observed even higher expression of Scribble in HCC tumors harboring elevated levels of wild-type p53. Most importantly, nuclear translocated Scribble could interact with p53, which lead to enhanced stability and transcriptional activity of p53. Mechanistically, our data suggested that Scribble might drive HCC progression by promoting metabolic regulation of p53 through p53-upregulated modulator of apoptosis (PUMA)-mediated Warburg effect. ConclusionsOur data identified the molecular basis of hepatic fibrosis-specific gene expression of polarity gene, such as Scribble. Interestingly, with the progression from fibrosis to cirrhosis to HCC, its nuclear translocation promoted a wild-type p53-mediated cancer metabolic switch and tumor progression in HCC. Taken together, we demonstrated that Scribble was up-regulated and served a protective role in liver fibrosis, while also apparently acting as a tumor driver in fibrosis-dependent hepatocarcinogenesis.

A comprehensive analysis of the potential role of necroptosis in hepatocellular carcinoma using single-cell RNA Seq and bulk RNA Seq.

Necroptosis plays an essential role in oncogenesis and tumor progression in hepatocellular carcinoma (HCC). This study aimed to investigate the role of necroptosis in the development and progression of HCC. Specifically, we constructed a prognostic prediction model using necroptosis-associated genes (NAGs) to predict patient outcomes. Using data from The Cancer Genome Atlas (TCGA) database, we analyzed gene expression and clinical data. We identified a 5-gene model associated with NAGs and explored genetic features and immune cell infiltration using the CIBERSORT algorithm. In addition, we conducted single-cell RNA sequencing to investigate the potential role of necroptosis in HCC. We constructed a 5-gene prognostic model based on NAGs that demonstrated excellent predictive accuracy in both training and validation sets. Using multifactorial cox regression analysis, we confirmed the risk score derived from the model as an independent predictor of prognosis, surpassing other clinical characteristics. Patients with high risk scores had significantly worse prognosis than those with low risk scores. To enhance the clinical utility of the necroptosis score, we constructed an accurate nomogram. Additionally, we compared metabolic pathway and immune microenvironment differences between HCC tumors with high and low risk scores. Our single-cell RNA sequencing analyses revealed that necroptosis in HCC was primarily associated with a specific subset of macrophages. Our study revealed the presence of two distinct necroptosis subtypes in HCC and developed a robust prognostic model with exceptional predictive accuracy. We observed significantly higher infiltration of M0 macrophages in the high-risk group. We propose that rescuing cytochrome c metabolism in HCC could serve as a potential therapeutic strategy. Furthermore, at a single-cell resolution, our analysis identified myeloid cells as the primary cells exhibiting necroptosis. Specifically, macrophages expressing CD5L, CETP, and MARCO, which may belong to a subset of tissue-resident macrophages, were found to be highly susceptible to necroptosis. These findings suggest the involvement of this specific macrophage subset in potential antitumor therapies. Our study provides novel insights into predicting patient prognosis and developing personalized therapeutic approaches for HCC.

MiR-3677-3p promotes development and sorafenib resistance of hepatitis B-related hepatocellular carcinoma by inhibiting FOXM1 ubiquitination.

Being encoded by hepatitis B, hepatitis B X (HBx) protein plays crucial roles in hepatitis B-related hepatocellular carcinoma (HCC) occurrence, development, and metastasis. miRNAs also function in the progression of hepatitis B-related HCC. Hence, the objective of this study was to explore the impacts of miR-3677-3p on tumor progression and sorafenib resistance in hepatitis B-related HCC and the related underlying mechanisms. Our research revealed that miR-3677-3p and FOXM1 was up-regulated and FBXO31 was down-regulated in HBV+ HCC cells and tumor tissues from nude mice. After miR-3677-3p overexpression, cell proliferative, invasive, and migrating potentials and stemness-related protein (CD133, EpCAM, and OCT4) levels were enhanced, and cell apoptosis was reduced in Huh7 + HBx/SR cells and HepG2.2.15/SR cells. Besides, miR-3677-3p promoted the drug resistance of Huh7 + HBx/SR cells and HepG2.2.15/SR cells to sorafenib and lifted IC50. In vivo experiments, miR-3677-3p down-regulation suppressed the tumor growth in the hepatitis B HCC nude mouse models. Mechanistically, miR-3677-3p targeted and negatively-regulated FBXO31 and FBXO31 could enrich FOXM1 protein. miR-3677-3p down-regulation or FBXO31 overexpression promoted the ubiquitylation of FOXM1. In a word, miR-3677-3p bound to FBXO31 and inhibited FBXO31 expression, thus curtailing the ubiquitylation degradation of FOXM1, contributing to HCC development and sorafenib resistance.

Construction of an M2 macrophage-related prognostic model in hepatocellular carcinoma.

M2 macrophages play a crucial role in promoting tumor angiogenesis and proliferation, as well as contributing to chemotherapy resistance and metastasis. However, their specific role in the tumor progression of hepatocellular carcinoma (HCC) and their impact on the clinical prognosis remain to be further elucidated. M2 macrophage-related genes were screened using CIBERSORT and weighted gene co-expression network analysis (WGCNA), while subtype identification was performed using unsupervised clustering. Prognostic models were constructed using univariate analysis/least absolute shrinkage selector operator (LASSO) Cox regression. In addition, Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and mutation analysis were used for further analysis. The relationship between the risk score and tumor mutation burden (TMB), microsatellite instability (MSI), the efficacy of transcatheter arterial chemoembolization (TACE), immunotype, and the molecular subtypes were also investigated. Moreover, the potential role of the risk score was explored using the ESTIMATE and TIDE (tumor immune dysfunction and exclusion) algorithms and stemness indices, such as the mRNA expression-based stemness index (mRNAsi) and the DNA methylation-based index (mDNAsi). In addition, the R package "pRRophetic" was used to examine the correlation between the risk score and the chemotherapeutic response. Finally, the role of TMCC1 in HepG2 cells was investigated using various techniques, including Western blotting, RT-PCR and Transwell and wound healing assays. This study identified 158 M2 macrophage-related genes enriched in small molecule catabolic processes and fatty acid metabolic processes in HCC. Two M2 macrophage-related subtypes were found and a four-gene prognostic model was developed, revealing a positive correlation between the risk score and advanced stage/grade. The high-risk group exhibited higher proliferation and invasion capacity, MSI, and degree of stemness. The risk score was identified as a promising prognostic marker for TACE response, and the high-risk subgroup showed higher sensitivity to chemotherapeutic drugs (e.g., sorafenib, doxorubicin, cisplatin, and mitomycin) and immune checkpoint inhibitor (ICI) treatments. The expression levels of four genes related to the macrophage-related risk score were investigated, with SLC2A2 and ECM2 showing low expression and SLC16A11 and TMCC1 exhibiting high expression in HCC. In vitro experiments showed that TMCC1 may enhance the migration ability of HepG2 cells by activating the Wnt signaling pathway. We identified 158 HCC-related M2 macrophage genes and constructed an M2 macrophage-related prognostic model. This study advances the understanding of the role of M2 macrophages in HCC and proposes new prognostic markers and therapeutic targets.

Hepatocellular Carcinoma Incidence Threshold for Surveillance in Virologically Cured Hepatitis C Individuals

Guidelines recommend biannual surveillance for hepatocellular carcinoma (HCC) in hepatitis C individuals with cirrhosis if the HCC incidence rate is above 1.5 per 100 person-years (PY). However, the incidence threshold for surveillance in individuals who achieve a virologic cure is unknown. We estimated the HCC incidence rate above which routine HCC surveillance is cost-effective in this growing population of virologically cured hepatitis C individuals with cirrhosis or advanced fibrosis. We developed a Markov-based microsimulation model of the natural history of HCC in individuals with hepatitis C who achieved virologic cure with oral direct-acting antivirals. We used published data on the natural history of hepatitis C, competing risk post virologic cure, HCC tumor progression, real-world HCC surveillance adherence, contemporary HCC treatment options and associated costs, and utilities of different health states. We estimated the HCC incidence above which biannual HCC surveillance using ultrasound and alpha-fetoprotein would be cost-effective. In virologically cured hepatitis C individuals with cirrhosis or advanced fibrosis, HCC surveillance is cost-effective if HCC incidence exceeds 0.7 per 100 PY using $100,000 per quality-adjusted life year willingness-to-pay. At this HCC incidence, routine HCC surveillance would result in 2650 and 5700 additional life years per 100,000 cirrhosis and advanced fibrosis persons, respectively, compared with no surveillance. At $150,000 willingness-to-pay, surveillance is cost-effective if HCC incidence exceeds 0.4 per 100 PY. Sensitivity analysis showed that the threshold mostly remained below 1.5 per 100 PY. The contemporary HCC incidence threshold is much lower than the previous 1.5% incidence value used to guide HCC surveillance decisions. Updating clinical guidelines could improve the early diagnosis of HCC.

Increased expression of autophagy-related gene 5 indicates poor prognosis in patients with Hepatocellular carcinoma.

As an critical component of the autophagic machinery, autophagy-related gene 5 (ATG5) is essential for autophagosome formation. Autophagy participates in various malignant tumor transformation and progression, but the role of ATG5 involved in hepatocellular carcinoma (HCC) remains to be illustrated. The expression of ATG5 was evaluated in 89 pairs of liver tumor and adjacent normal tissues. The correlation between ATG5 expression and clinicopathological characteristics or prognosis were evaluated. Moreover, clinical special analysis was conducted in the young patients group and the old patients group respectively, and nomograms estimating overall survival and disease-free survival were investigated. Our study demonstrated that the ATG5 level was increased in tumor specimens than adjacent normal tissues. Upregulated ATG5 level had positive association with serum α-fetoprotein (AFP). Moreover, cases with higher ATG5 level had poorer disease-free survival (DFS) and overall survival (OS) than those with lower ATG5 level. Multivariate analysis showed that overexpression of ATG5 predicts poor OS and DFS. Further analysis indicated that cases with higher ATG5 level had poorer OS and DFS in the young patients and solitary tumor number subgroup. The nomogram models suggested that it had a coherence between nomogram prediction and actual situation. Together these findings revealed that ATG5 promotes progression of HCC, making it a potentially biomarker in diagnosis and therapeutic target of hepatocellular carcinoma. This article is protected by copyright. All rights reserved.

Abstract 1489: Overexpression of ribosomal L22-like1 (RPL22L1) enhances cancer stem cell properties and promotes tumor progression in hepatocellular carcinoma (HCC)

Abstract The abnormality of ribosomal biogenesis is found to be involved in carcinogenesis and progression in various types of cancer. RPL22L1 gene is a highly homologous paralog of RPL22, both of which belong to the L22E family of ribosomal proteins and take part in encoding a cytoplasmic ribosomal protein that is a component of the 60S subunit of ribosomes. Our previous work has established a CRISPR/Cas9 genome-wide knockout screening profile and developed a two-component mixture model to identify cancer-specific essential genes in 436 cancer cell lines. Bioinformatics analysis reveals that RPL22L1 acts as a feature gene to predict cancer-specific essential genes in human cancer. Previous studies suggest that RPL22L1 is correlated with tumor progression and adverse prognosis in several cancer types, while its role in hepatocellular carcinoma (HCC) is unknown. In the present study, we found that RPL22L1 is highly expressed in embryonic stem cells (ES) and liver progenitor cells, which were acquired from a vitro hepatic differentiation model originating from ES cells. Hence, we hypothesized that RPL22L1 enhances cancer stem cell properties in HCC. Functional assays showed that RPL22L1 would promote sphere formation in vitro. qRT-PCR and Western Blotting results indicated that RPL22L1 could promote stemness-related gene expressions such as CD133, CD44, and SOX9 in HCC cell lines. Overexpression of RPL22L1 increased aldehyde dehydrogenase (ALDH) activity, which was decreased when RPL22L1 was silenced with short hairpin RNA (shRNA). Extreme limiting dilution assay showed that RPL22L1 overexpression stimulated self-renewal ability in immunodeficient mice. Furthermore, higher RPL22L1 expression can also promote cell proliferation and epithelial-mesenchymal-transition, as well as drug resistance to 5-Fluorouracil (5-FU) and cisplatin (CDDP). Silver nitrate staining demonstrated that RPL22L1 overexpression correlated with a larger size and number of nucleoli. The expression of RPL22L1 was also associated with adverse prognosis in HCC clinical samples. In conclusion, our study reveals aberrant ribosomal protein activity in HCC and the role of RPL22L1 in promoting HCC tumor progression. Citation Format: JinLin Huang, Zhenyang Guo, Jie Luo, Beilei Liu, Lanqi Gong, Xiaona Fang, Dora Lai-wan Kwong, Xinyuan Guan. Overexpression of ribosomal L22-like1 (RPL22L1) enhances cancer stem cell properties and promotes tumor progression in hepatocellular carcinoma (HCC) [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 1489.

Schisandrin B inhibits tumor progression of hepatocellular carcinoma by targeting the RhoA/ROCK1 pathway.

Schisandrin B (Sch. B) performs various pharmacological properties, including anticancer activities. However, the pharmacological mechanisms of Sch. B in hepatocellular carcinoma (HCC) are not fully elucidated. We investigated the impact and mechanism on progression in HCC, and to provide new experimental evidence for HCC treatment. To determine the inhibitory effect of Sch. B on HCC in vivo, 32 Balb/c nude mice were used to prepare the tumor-bearing mice model by subcutaneously inoculating HCC cells (Huh-7). As tumor volume grew to 100 mm3, mice were randomly divided into Saline (control group), 100 mg/kg Sch. B group (Sch. B-L), 200 mg/kg Sch. B group (Sch. B-M), and 400 mg/kg Sch. B group (Sch. B-H) (n=8). Saline or different concentration Sch. B was used to treat mice via gavage administration for 21 days. After mice were euthanized, tumor weight and volume were evaluated. Cell apoptosis was detected by TUNEL. Ki-67 and PCNA were detected by immunohistochemical staining. The RhoA and Rho-associated protein kinase 1 (ROCK1) were determined by western blot. In vitro experiment, Huh-7 cell were treated by Sch. B at 40, 30, 20, 10, 5, 1, and 0 µM to detect cell proliferation by Cell Counting Kit-8 (CCK-8). Huh-7 cells were divided as a control group, Sch. B group, and Sch. B + RhoA overexpression (Sch. B + RhoA) group. RhoA and ROCK1 were examined. The colony formation assay and flow cytometry were used to detect cell proliferation and apoptosis. The wound healing and Transwell assays were used for cell metastasis detection. Our results showed 100, 200 and 400 mg/kg Sch. B significantly reduced tumor weight and volume. And 200 and 400 mg/kg Sch. B increased apoptosis, and reduced Ki-67 and PCNA levels, inhibited the RhoA and ROCK1 in vivo (P<0.05). In vitro experiment, Sch. B inhibited Huh-7 cell proliferation at concentration more than 10 μM (P<0.05). Sch. B decreased cell duplication, promoted apoptosis and blocked migration and invasion of Huh-7 (P<0.05). Sch. B inhibited RhoA and ROCK1 level as compared with control group (P<0.05). RhoA overexpression reversed the effect of Sch. B (P<0.05). Sch. B inhibits Huh-7 cells progression via RhoA/ROCK1 pathway. The results provide new evidence for the clinical treatment of HCC.

Integrated Analysis of N1-Methyladenosine Methylation Regulators-Related lncRNAs in Hepatocellular Carcinoma

N1-methyladenosine (m1A) and long non-coding RNAs (lncRNAs) play significant roles in tumor progression in hepatocellular carcinoma (HCC). However, their association with HCC is still unclear. In this study, lncRNAs related to m1A were extracted from the mRNA expression matrix in The Cancer Genome Atlas (TCGA) database. Five m1A-related lncRNAs (AL031985.3, NRAV, WAC-AS1, AC026412.3, and AC099850.4) were identified based on lasso Cox regression and they generated a prognostic signature of HCC. The prognostic signature was identified as an independent prognosis factor in HCC patients. Moreover, the prognostic signature achieved better performance than TP53 mutation status or tumor mutational burden (TMB) scores in the stratification of patient survival. The immune landscape indicated that most immune checkpoint genes and immune cells were distributed differently between both risk groups. A higher IC50 of chemotherapeutics (sorafenib, nilotinib, sunitinib, and gefitinib) was observed in the high-risk group, and a lower IC50 of gemcitabine in the low-risk group, suggesting the potential of the prognostic signature in chemosensitivity. In addition, fifty-five potential small molecular drugs were found based on drug sensitivity and NRAV expression. Together, five m1A-related lncRNAs generated a prognostic signature that could be a promising prognostic prediction approach and therapeutic response assessment tool for HCC patients.

Combinatorial targeting of immune checkpoints and epigenetic regulators for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. The five-year survival rate of patients with unresectable HCC is about 12%. The liver tumor microenvironment (TME) is immune tolerant and heavily infiltrated with immunosuppressive cells. Immune checkpoint inhibitors (ICIs), in some cases, can reverse tumor cell immune evasion and enhance antitumor immunity. Rapidly evolving ICIs have expanded systemic treatment options in advanced HCC; however, single-agent ICIs achieve a limited 15-20% objective response rate in advanced HCC. Therefore, other combinatorial approaches that amplify the efficacy of ICIs or suppress other tumor-promoting pathways may enhance clinical outcomes. Epigenetic alterations (e.g., changes in chromatin states and non-genetic DNA modifications) have been shown to drive HCC tumor growth and progression as well as their response to ICIs. Recent studies have combined ICIs and epigenetic inhibitors in preclinical and clinical settings to contain several cancers, including HCC. In this review, we outline current ICI treatments for HCC, the mechanism behind their successes and failures, and how ICIs can be combined with distinct epigenetic inhibitors to increase the durability of ICIs and potentially treat "immune-cold" HCC.