Model Of HCC Research Articles

A combined model of six serum microRNAs as diagnostic markers for hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is associated with high morbidity and mortality, and its poor prognosis is mainly due to the lack of an effective means of early diagnosis. This study aimed to identify a group of serum microRNAs (miRNAs) as potential biomarkers for the diagnosis of HCC. MethodsWe collected 190 HCC cases, 109 benign lesions of the liver, 40 cases of non-HCC tumors, and 130 healthy controls. The 469 participants were divided into training and validation sets. A literature search revealed 12 miRNAs closely associated with HCC. In the training set, significantly differentially expressed miRNAs (DEmiRNAs) were screened using real-time quantitative PCR, and a diagnostic model of HCC was constructed using logistic regression analysis. An independent validation was performed using a validation set. The identified DE miRNAs were subjected to target gene prediction and functional analyses. ResultsCompared to the controls, the levels of miR-21, miR-221, miR-801, and miR-1246 significantly decreased in HCC (P < 0.05), while the levels of miR-26a and miR-122 significantly increased (P < 0.05). A diagnostic model based on the six DE miRNAs was successfully constructed, with AUC values of 0.953 for the training set and 0.952 for the verification set. Finally, 100 target genes of the DE miRNAs were predicted and were significantly enriched in the B cell receptor, neurotrophin, ferroptosis, and EGFR tyrosine kinase inhibitor resistance signaling pathways. ConclusionsThe constructed diagnostic model based on six DE miRNA combinations has important clinical value for the early diagnosis of HCC

Targeting hepatocellular carcinoma growth and metabolism: A synergistic approach with para-toluenesulfonamide and radiofrequency ablation

Background and aimsHepatocellular carcinoma (HCC) is one of the most prevalent and lethal forms of cancer, posing challenges to standard treatment methods. Radiofrequency ablation (RFA) has emerged as a valuable technique for the local control of advanced HCC, especially in cases unsuitable for surgical resection. Para-toluenesulfonamide (PTS) has demonstrated antitumor properties in various cancers, yet its efficacy against HCC remains underexplored. The present study aims to investigate the therapeutic potential of PTS in HCC and examine potential synergistic effects with RFA. MethodsPTS was injected directly into subcutaneous tumor tissues and its effects on cell survival, gene expression, and metabolic activity in HCC cells were evaluated. The effect of PTS and RFA in combination on HCC growth was examined using a nude mouse model of subcutaneous HCC. Data were visualized using heat maps. ResultsPTS inhibited the survival of HCC cells in a dose-dependent manner, modulating the expression of key genes related to cell survival, apoptosis, epithelial–mesenchymal transition, and proliferation. The combination of PTS and RFA markedly enhanced the inhibition of HCC tumor growth and induced alterations in cellular metabolism. ConclusionCombining PTS and RFA may be a promising approach to improve the efficacy of HCC treatment.

Effects of mesenchymal stem cells versus curcumin on sonic hedgehog signaling in experimental model of Hepatocellular Carcinoma.

Sonic Hedgehog (SHH) is a fundamental signaling pathway that controls tissue reconstruction, stem cell biology, and differentiation and has a role in gut tissue homeostasis and development. Dysregulation of SHH leads to the development of HCC. The present study was conducted to compare the effects of mesenchymal stem cells (MSCs) and curcumin on SHH molecular targets in an experimental model of HCC in rats. One hundred rats were divided equally into the following groups: control group, HCC group, HCC group received MSCs, HCC group received curcumin, and HCC group received MSCs and curcumin. Histopathological examinations were performed, and gene expression of SHH signaling target genes (SHH, PTCH1, SMOH, and GLI1) was assessed by real-time PCR in rat liver tissue. Results showed that SHH target genes were significantly upregulated in HCC-untreated rat groups and in MSC-treated groups, with no significant difference between them. Administration of curcumin with or without combined administration of MSCs led to a significant down-regulation of SHH target genes, with no significant differences between both groups. As regards the histopathological examination of liver tissues, both curcumin and MSCs, either through separate use or their combined use, led to a significant restoration of normal liver pathology. In conclusion, SHH signaling is upregulated in the HCC experimental model. MSCs do not inhibit the upregulated SHH target genes in HCC. Curcumin use with or without MSCs administration led to a significant down-regulation of SHH signaling in HCC and a significant restoration of normal liver pathology.

Light&fast generative adversarial network for high-fidelity CT image synthesis of liver tumor

Background and objective:Hepatocellular carcinoma is a common disease with high mortality. Through deep learning methods to analyze HCC CT, the screening classification and prognosis model of HCC can be established, which further promotes the development of computer-aided diagnosis and treatment in the treatment of HCC. However, there are significant challenges in the actual establishment of HCC auxiliary diagnosis model due to data imbalance, especially for rare subtypes of HCC and underrepresented demographic groups. This study proposes a GAN model aimed at overcoming these obstacles and improving the accuracy of HCC auxiliary diagnosis. Methods:In order to generate liver and tumor images close to the real distribution. Firstly, we construct a new gradient transfer sampling module to improve the lack of texture details and excessive gradient transfer parameters of the deep model; Secondly, we construct an attention module with spatial and cross-channel feature extraction ability to improve the discriminator’s ability to distinguish images; Finally, we design a new loss function for liver tumor imaging features to constrain the model to approach the real tumor features in iterations. Results:In qualitative analysis, the images synthetic by our method closely resemble the real images in liver parenchyma, blood vessels, tumors, and other parts. In quantitative analysis, the optimal results of FID, PSNR, and SSIM are 75.73, 22.77, and 0.74, respectively. Furthermore, our experiments establish classification models for imbalanced data and enhanced data, resulting in an increase in accuracy rate by 21%–34%, an increase in AUC by 0.29 - 0.33, and an increase in specificity to 0.89. Conclusion:Our solution provides a variety of training data sources with low cost and high efficiency for the establishment of classification or prognostic models for imbalanced data.

Abstract LB005: Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics

Abstract Hepatocellular carcinoma (HCC) is an aggressive tumor, and treatment options for patients with unresectable HCC are limited to targeted therapies and immunotherapy. The activation and dysregulation of MYC have been implicated in multiple cancers, including HCC, where approximately 30% of human HCC samples show MYC gene amplification. However, it is challenging to target MYC directly. Therefore, we applied a novel approach to circumvent targeting MYC directly by leveraging Pepper’s proprietary transomic analysis platform, COMPASS, to identify novel targets that are predicted to mimic the effect of turning MYC off. Specifically, COMPASS unlocks functional drivers of disease to identify novel drug targets. To study the role of MYC in tumor biology, we utilize an HCC cell line with tunable MYC expression. We generate genomic, transcriptomic, proteomic, and phosphoproteomic data from samples with MYC-on and MYC-off and compare via COMPASS to identify novel targets that mimic turning MYC off. The MYC-conditional HCC cell line (EC4) allows the regulation of MYC expression via the tetracycline regulatory (Tet-Off) system. Four omic datasets were collected from each sample: genomics (next-generation sequencing, NGS), transcriptomics (NGS), proteomics (mass spectrometry), and phosphoproteomics (mass spectrometry). We employed the COMPASS target prioritization algorithm to identify and rank novel targets that mimic turning MYC off. Targets were then filtered to select those with an available pharmacological tool compound (PTC). The PTCs were used to evaluate the targets in the Huh-7 xenograft model of HCC. All targets were kinases or related to kinase activity and pathways. All PTCs inhibited tumor growth from 18.1-78.3%. One multi-tyrosine kinase inhibitor resulted in maximum tumor growth inhibition, resulting in stasis of tumor growth and increased survival compared with control vehicle-treated mice. We have previously shown how the COMPASS transomics analysis approach can identify novel drug targets for drug resistant EGFRm NSCLC. The data presented here on MYC-driven HCC further validates this approach for novel target identification. Ongoing work will investigate novel targets using gene silencing, as PTCs were unavailable for many of these novel high-ranked targets identified in this study. Further validation studies are ongoing against other difficult-to-treat MYC-driven cancers to identify novel clinical targets and drug candidates to circumvent the MYC pathway. Citation Format: Simon P. Fricker, Christopher J. Nicholson, Samuel J. Roth, Arudhir Singh, Caitlin Brown, Jon Hu, Petronela Buiga, Vishnu P. Kanakaveti, Anja Deutzmann, Dean Felsher, Samantha D. Strasser. Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB005.

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

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

Hepatoprotective effects of aspirin on diethylnitrosamine-induced hepatocellular carcinoma in rats by reducing inflammation levels and PD-L1 expression

Aspirin, as a widely used anti-inflammatory drug, has been shown to exert anti-cancer effects in a variety of cancers. PD-L1 is widely expressed in tumor cells and inhibits anti-tumor immunity. This study aims to clarify whether aspirin exerts its anti-hepatocellular carcinoma (HCC) effect by inhibiting PD-L1 expression. The rat model of HCC was established by drinking 0.01% diethylnitrosamine (DEN), and aspirin was given by gavage. The gross and blood biochemical indexes of rats were analyzed. CD4 and CD8 expression in liver tissues were investigated by immunohistochemistry. CCK8 assay was used to detect the inhibitory effect of aspirin on the proliferation of HCC cells. The regulatory effect of aspirin on PD-L1 expression was analyzed by western blot. As a result, the tumor number and liver weight ratio in the DEN + ASA group were lower than those in the DEN group (P = 0.006, P = 0.046). Compared with the DEN group, the expression of CD4 in the DEN + ASA group was significantly increased, while CD8 was decreased (all P < 0.01). Biochemical indexes showed that there were differences in all indexes between the DEN and control group (P < 0.05). The levels of DBIL, ALP, and TT in the DEN + ASA group were lower than those in the DEN group (P = 0.038, P = 0.042, P = 0.031). In the DEN group, there was an obvious fibrous capsule around the tumor, and the portal vein was dilated. The pathological changes were mild in the DEN + ASA group. Compared with the DEN group, the expression of PD-L1 in liver tissue of the DEN + ASA group was decreased (P = 0.0495). Cytological experiments further showed that aspirin could inhibit the proliferation and PD-L1 expression in Hep G2 and Hep 3B cells. In conclusion, aspirin can inhibit the proliferation of HCC cells and reduce tumor burden by reducing inflammation and targeting PD-L1.

TEDC2 plays an oncogenic role and serves as a therapeutic target of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies and tends to have a poor prognosis due to its insidious onset, difficulty in early diagnosis, and limited treatment options. Tubulin epsilon and delta complex 2 (TEDC2), also known as C16orf59, is implicated in maintaining centriole stability, but the involvement of TEDC2 in HCC remains unknown. This study aimed to investigate the expression profile and potential mechanisms of TEDC2 in HCC. MethodsMultiple RNA sequencing datasets were screened for differentially expressed genes in HCC, and the prognosis-related gene, TEDC2, was further screened as a target gene in this study. The expression of TEDC2 in public datasets and clinical specimens was analyzed, and the involvement of TEDC2 in HCC was investigated by bioinformatic analysis and in vitro experiments. ResultsTEDC2 levels were elevated in HCC compared to healthy livers. Overexpression of TEDC2 was positively correlated with pathologic stage and histologic grade. In addition, TEDC2 was found to be an independent prognostic predictor. An excellent prognostic model of HCC was successfully constructed with TEDC2 in combination with the TNM stage. Bioinformatic analysis revealed that overexpression of TEDC2 might be associated with impaired tumor immunity in HCC, as evidenced by increased infiltration of T helper 2 (Th2) cells and reduced infiltration of cytotoxic cells. Further studies showed that TP53 mutations regulated TEDC2 expression, and TEDC2 was significantly associated with drug sensitivity. Moreover, overexpression of TEDC2 promoted cell metastasis and proliferation in vitro. ConclusionThese findings initially suggested a crucial effect of TEDC2 overexpression on HCC tumor progression, suggesting its potential as a novel prognostic and therapeutic target in HCC.

SAT018 Investigate If Sex Differences In Microbiota Is Sufficient To Promote Bile Acid-Mediated Hepatocellular Carcinoma In Mice

Abstract Disclosure: A.E. Dean: None. S. Anakk: None. Liver cancer is one of the leading causes of cancer-related deaths with men having a higher prevalence than women. It is becoming clearer that changes in bile acid levels are seen in patients diagnosed with liver cancer. Although initially made in the liver, these primary bile acids can also be modified by bacteria in the intestines to form secondary bile acids. The secondary bile acids are more hydrophobic and cytotoxic and when in excess, can promote tumorigenesis. We, therefore, examined if microbial differences between males and females explain sex-dependent HCC development when bile acid metabolism is altered. In this study, we used a mouse model of bile acid excess and HCC, where the bile acid-related nuclear receptors, farnesoid X receptor (Fxr) and small heterodimer partner (Shp), are knocked out. This double knockout (DKO) model mimics the cancer incidence observed in humans, where male mice are more susceptible to spontaneous tumorigenesis than females at 12 months of age. The circulating bile acid profiles of the male DKO mice are more hydrophobic and have a greater proportion of secondary bile acids than the females. Since we observed changes in the bile acid composition, we next investigated if fecal and cecal microbial profiles of male and female DKO mice differed, along with their wild-type counterparts. 16s rRNA full-length sequencing was performed on fecal and cecal samples from 12-month-old mice, and analyses are currently ongoing. We expect that the microbes identified in the male DKO mice will reflect their bile acid compositions, as well as spontaneous tumorigenesis. Additionally, we are analyzing the 16s rRNA gene from mice treated with cholestyramine (CHR), a bile acid binding resin, that exhibit decreased tumorigenesis at 12 months of age. In this manner, we can correlate microbiota changes to the bile acid composition, as well as the extent of tumor burden. Thus, this study will be able to integrate how sex differences in liver cancer incidence can be secondary to microbial modification of bile acids. Presentation: Saturday, June 17, 2023

Establishment and validation of exhausted CD8+ T cell feature as a prognostic model of HCC.

The exhausted CD8+T (Tex) cells are a unique cell population of activated T cells that emerges in response to persistent viral infection or tumor antigens. Tex cells showed the characteristics of aging cells, including weakened self-renewal ability, effector function inhibition, sustained high expression of inhibitory receptors including PD-1, TIGIT, TIM-3, and LAG-3, and always accompanied by metabolic and epigenetic reprogramming. Tex cells are getting more and more attention in researching immune-related diseases and tumor immunotherapy. However, studies on Tex-related models for tumor prognosis are still lacking. We hope to establish a risk model based on Tex-related genes for HCC prognosis. Tex-related GEO datasets from different pathologic factors (chronic HBV, chronic HCV, and telomere shortening) were analyzed respectively to acquire differentially expressed genes (DEGs) by the 'limma' package of R. Genes with at least one intersection were incorporated into Tex-related gene set. GO, KEGG, and GSEA enrichment analyses were produced. Hub genes and the PPI network were established and visualized by the STRING website and Cytoscape software. Transcription factors and targeting small molecules were predicted by the TRUST and CLUE websites. The Tex-related HCC prognostic model was built by Cox regression and verified based on different datasets. Tumor immune dysfunction and exclusion (TIDE) and SubMap algorithms tested immunotherapy sensitivity. Finally, qRT-PCR and Flow Cytometry was used to confirm the bioinformatic results. Hub genes such as AKT1, CDC6, TNF and their upstream transcription factor ILF3, Regulatory factor X-associated protein, STAT3, JUN, and RELA/NFKB1 were identified as potential motivators for Tex. Tex-related genes SLC16A11, CACYBP, HSF2, and ATG10 built the HCC prognostic model and helped with Immunotherapy sensitivity prediction. Our study demonstrated that Tex-related genes might provide accurate prediction for HCC patients in clinical decision-making, prognostic assessment, and immunotherapy. In addition, targeting the hub genes or transcription factors may help to reverse T cell function and enhance the effect of tumor immunotherapy.

Blockade of T-cell receptor with Ig and ITIM domains elicits potent antitumor immunity in naturally occurring HBV-related HCC in mice.

Chronic HBV infection is the leading cause of HCC and a serious health problem in China, East Asia, and North African countries. Effective treatment of HBV-related HCC is currently unavailable. This study evaluated the therapeutic potential of T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade in HBV-related HCC. A mouse model of spontaneous HBV-related HCC was generated by replacing wild-type hepatocytes with HBsAg + hepatocytes (namely HBs-HepR mice). The tumors in HBs-HepR mice were inflammation-associated HCC, similar to HBV-related HCC in patients, which was distinguished from other HCC mouse models, such as diethylnitrosamine-induced HCC, TGF-β-activated kinase 1 knockout-induced HCC, HCC in a stelic animal model, or NASH-induced HCC. HCC in HBs-HepR mice was characterized by an increased number of CD8 + T cells, whereas the production of IL-2, TNF-α, and interferon-gamma (IFN-γ) by intrahepatic CD8 + T cells was decreased. Increased expression of TIGIT on CD8 + T cells was responsible for functional exhaustion. The therapeutic effect of TIGIT blockade was investigated at the early and middle stages of HCC progression in HBs-HepR mice. TIGIT blockade reinvigorated intrahepatic CD8 + T cells with increased TNF-α and IFN-γ production and an increased number of CD8 + T cells in tumors, thereby slowing the development of HCC in HBs-HepR mice. Blocking PD-L1 did not show direct therapeutic effects or synergize with TIGIT blockade. Blockade of TIGIT alone enhanced the antitumor activity of CD8 + T cells during the progression of HBV-related HCC in a spontaneous HCC mouse model.

Aucubin enhances the antitumor activity of cisplatin through the inhibition of PD-L1 expression in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer-associated mortality in the world. However, the anticancer effects of aucubin against HCC have yet to be reported. Cisplatin often decreased CD8+ tumor-infiltrating lymphocytes in the tumor microenvironment through increasing programmed death-ligand 1 (PD-L1) expression, which seriously affected the prognostic effect of cisplatin in the treatment of patients with HCC. Therefore, it is necessary to identify a novel therapeutic avenue to increase the sensitivity of cisplatin against HCC. PurposeThis study aims to evaluate the anti-tumor effect of aucubin on HCC, and also to reveal the synergistic effects and mechanism of aucubin and cisplatin against HCC. Study design and methodsAn H22 xenograft mouse model was established for the in vivo experiments. Cancer cell proliferation was detected by MTT assay. RT-qPCR was performed to analyze CD274 mRNA expression in vitro. Western blotting was employed to determine the expression levels of the PD-L1, p-Akt, Akt, p-β-catenin, and β-catenin in vitro. Immunofluorescence was carried out to examine β-catenin nuclear accumulation in HCC cells. Immunohistochemistry was used to detect tumoral PD-L1 and CD8α expression in xenograft mouse model. ResultsAucubin inhibits tumor growth in a xenograft HCC mouse model, but did not affect HCC cell viability in vitro. Aucubin treatment significantly inhibited PD-L1 expression through inactivating Akt/β-catenin signaling pathway in HCC cells. Overexpression of PD-L1 dramatically reversed aucubin-mediated tumoral CD8+ T cell infiltration and alleviated the antitumor activity of aucubin in xenograft mouse model. Moreover, Cisplatin could induce the expression of PD-L1 through the activation of the Akt/β-catenin signaling pathway in HCC cells, which can be blocked by aucubin in vitro. In xenograft mouse model, cisplatin treatment induced PD-L1 expression and alleviated the infiltration of CD8+ T lymphocytes in the tumor microenvironment. Aucubin not only abrogated cisplatin-induced PD-L1 expression but also enhanced the antitumor efficacy of cisplatin in a mouse xenograft model of HCC. ConclusionAucubin exerts antitumor activity against HCC and also enhances the antitumor activity of cisplatin by suppressing the Akt/β-catenin/PD-L1 axis.

Construction and validation of a prognostic model for hepatocellular carcinoma: Inflammatory ferroptosis and mitochondrial metabolism indicate a poor prognosis.

An increasing number of innovations have been discovered for treating hepatocellular carcinoma (HCC or commonly called HCC) therapy, Ferroptosis and mitochondrial metabolism are essential mechanisms of cell death. These pathways may act as functional molecular biomarkers that could have important clinical significance for determining individual differences and the prognosis of HCC. The aim of this study was to construct a stable and reliable comprehensive model of genetic features and clinical factors associated with HCC prognosis. In this study, we used RNA-sequencing (fragments per kilobase of exon model per million reads mapped value) data from the Cancer Genome Atlas (TCGA) database to establish a prognostic model. We enrolled 104 patients for further validation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analyses (KEGG) analysis were used for the functional study of differentially expressed genes. Pan-cancer analysis was performed to evaluate the function of the Differentially Expressed Genes (DEGs). Thirteen genes were identified by univariate and least absolute contraction and selection operation (LASSO) Cox regression analysis. The prognostic model was visualized using a nomogram. We found that eight genes, namely EZH2, GRPEL2, PIGU, PPM1G, SF3B4, TUBG1, TXNRD1 and NDRG1, were hub genes for HCC and differentially expressed in most types of cancer. EZH2, GRPEL2 and NDRG1 may indicate a poor prognosis of HCC as verified by tissue samples. Furthermore, a gene set variation analysis algorithm was created to analyze the relationship between these eight genes and oxidative phosphorylation, mitophagy, and FeS-containing proteins, and it showed that ferroptosis might affect inflammatory-related pathways in HCC. EZH2, GRPEL2, NDRG1, and the clinical factor of tumor size, were included in a nomogram for visualizing a prognostic model of HCC. This nomogram based on a functional study and verification by clinical samples, shows a reliable performance of patients with HCC.

Development and validation of a mitochondrial energy metabolism-related risk model in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most prevalent cancers and ranks third inmortality. Mitochondria are the energy manufacturers of cells. Disruption of mitochondrial energy metabolism pathways is strongly correlated with the onset and progression of HCC. Aberrant genes in mitochondrial energy metabolism pathways may represent a unique diagnostic and therapeutic targets that act as indicators for HCC. MethodsGene expression data from 374 HCC patients and 50 controls were acquired from TCGA database. A total of 188 mitochondrial energy metabolism-related genes (MMRGs) were obtained from KEGG PATHWAY database. A total of 368 patients with survival data were randomly split into training and validation groups in a 7: 3 ratio. Prognosis-related MMRGs were selected by univariate Cox and LASSO analyses. Kaplan–Meier and ROC curves were employed to analyze the model precision, whereas the validation set was used for model verification. Furthermore, clinical examinations, immune infiltration analysis, GSVA, and immunotherapy analysis were conducted in the high- and low-risk groups. Finally, the risk model was combined with the clinical variables of HCC patients to perform univariate and multivariate Cox regression analyses to obtain independent risk indicators and draw a nomogram. Therefore, we evaluated the accuracy of the predictions using calibration curves. ResultsA total of 6032 differentially expressed genes (DEGs) were detected in the HCC and control samples. After overlapping DEGs with 188 MMRGs, 42 mitochondrial energy metabolism-related DEGs (DEMMRGs) were identified. A 17 specific genes-based risk score model of HCC was created, which revealed effectiveness in each TCGA training and validation dataset. Moreover, patients categorized by risk scores exhibited distinct immune infiltration status, immunotherapy responsiveness, and functional properties. Finally, univariate and multivariate Cox regression analyses revealed that risk score and stage T were independent predictive variables. Based on the T stage and risk score, a nomogram for estimating the survival of HCC patients was created. The calibration curves demonstrated that the prediction model had a high level of accuracy. ConclusionsOur study constructed a mitochondrial energy metabolism-related risk model, that may be utilized to anticipate HCC prognosis and represent the immunological microenvironment of HCC.

Treatment Strategies and Mechanisms Associated with the Prevention of NASH-Associated HCC by a Toll-like Receptor 4 Inhibitor.

Means to prevent development of HCC or progression of small adenomas to HCC in patients with NASH are urgently needed to reduce the growing mortality due to HCC. We characterized the chemopreventive effect of oral administration of the TLR4 inhibitor TAK-242 in a model of NASH-associated HCC.

Screening of Prognostic Markers for Hepatocellular Carcinoma Patients Based on Multichip Combined Analysis.

Methods GSE (14520, 36376, 57957, 76427) datasets were accessed from GEO database. 55 differential mRNAs (DEGs) were obtained by differential analysis based on the datasets. GO and KEGG analysis results indicated that the DEGs were enriched in xenobiotic metabolic process and other pathways. Expression profiles and clinical data of TCGA-LIHC mRNAs were from TCGA database. We established a prognostic model of HCC through univariate and multivariate Cox risk regression analyses. ROC curve analysis was used to examine the prognostic model performance. GSEA analysis was performed between the high- and low-risk score sample groups. Results A 4-gene HCC prognostic model was constructed, in which the gene expressions correlated to HCC patients' survival. The AUC value presented 0.734 in the ROC analysis for the prognostic model. Conclusion The four-gene model could be introduced as an independent prognostic factors to assess HCC patients' survival status.

Abstract 2662: Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the world. Current treatments for HCC have limited efficacy. First-line HCC therapy atezolizumab with bevacizumab increased patient 6-month PFS from 37% to 55% relative to sorafenib, a multi-kinase inhibitor currently serving as a standard-of-care treatment. Although immunotherapies provide survival benefits, reported patient response and disease control rates were 27% and 74%, respectively, highlighting the need for novel therapeutic agents that can re-establish responsiveness to immunotherapies and enhance their therapeutic effects. Revolution Medicines has developed a class of selective mTORC1 inhibitors, termed ‘bi-steric’, which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-6272 is a representative bi-steric inhibitor that exhibits potent and selective (&amp;gt;30-fold) inhibition of mTORC1 over mTORC2. Several lines of evidence suggest that inhibition of the mTORC1 pathway may result in synthetic lethality of MYC-driven HCC. Thus, we hypothesized that MYC-driven HCC tumors are sensitive to mTORC1 inhibition and p-4EBP1 suppression. A single dose of RMC-6272 induced HCC regression in 80% of the Lap-tTA/Tet-O-MYC mouse model of MYC-driven HCC, whereas sorafenib did not cause tumor regression. Mechanistically, RMC-6272 inhibited the phosphorylation of mTORC1 downstream substrates S6k and 4EBP1 and depleted MYC protein levels. Given that the MYC oncogene enables immune evasion in HCC, we hypothesized that by reducing MYC, RMC-6272 can re-establish anti-tumor immune surveillance and responsiveness to α-PD-1 immunotherapy. Indeed, RMC-6272 induced tumor recruitment of dendritic cells, T cells, B cells, and natural killer (NK) cells, producing similar anti-tumor immunity as observed upon MYC genetic inactivation. Moreover, combining RMC-6272 with α-PD-1 resulted in deeper tumor regression and a more durable response as compared to either RMC-6272 or α-PD-1 alone in the MYC-driven HCC mouse model. By immunohistochemical staining and analysis, RMC-6272 in combination with α-PD-1, but not either agent alone, promoted tumor recruitment of CD4+ T cells and NK cells, immune cell degranulation, and the release of perforins and granzymes in MYC-driven HCC tumors. Our results show that the bi-steric mTORC1 inhibitor RMC-6272 synergizes with α-PD-1 to induce immune activation and the release of perforins and granzymes, resulting in sustained tumor regression in a MYC-driven HCC mouse model. The bi-steric mTORC1 inhibitor RMC-5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952). These preclinical data highlight a prospective anti-cancer therapeutic opportunity for mTORC1 selective inhibitors in combination with immune checkpoint inhibitors in MYC-driven cancers. Citation Format: Wadie D. Mahauad-Fernandez, Yu C. Yang, Ian Lai, Jangho Park, Lilian Yao, James W. Evans, Jacqueline A. Smith, Mallika Singh, Dean W. Felsher. Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2662.

Abstract 5286: RNase7-guided anti-ROS1 targeted therapy for hepatocellular carcinoma

Abstract Oncogenic activation of c-ros oncogene 1 (ROS1) kinase due to gene fusion by chromosomal rearrangements was found in multiple cancers. The anti-ROS1 fusion gene is a successful therapeutic target with a 72% objective response rate in ROS1-rearranged NSCLC patients. However, it has yet to be determined whether wild-type ROS1 plays a role in cancer development or could serve as a therapeutic target as its ligand was unknown. Our study unexpectedly identified that RNase7, a RNase superfamily protein, is specifically pulled down by ROS1 and acts as a ROS1 ligand. In HCC patient-derived xenograft and an orthotopic mouse model of HCC, we found that crizotinib, an FDA-approved ROS1 inhibitor, exhibits potent anti-tumor activity in the mice harboring RNase7-expressing tumors. In addition, clinical data analysis indicated that ROS1 and RNase7 co-expression occurs in about 30% of HCC patients and is highly correlated with poorer survival. The plasma level of RNase7 is positively correlated with phosphorylated ROS1 in HCC patients. Our current report provides a rationale to choose RNase7 as a potential plasma biomarker to stratify HCC patients for anti-ROS1 targeted therapy. Citation Format: Chunxiao Liu, Zhengyu Zha, Chenhao Zhou, Dihua Yu, Ning Ren, Mien-Chien Hung. RNase7-guided anti-ROS1 targeted therapy for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5286.

Abstract 1372: Macrophages impair antigen specific CD8+ T cell response against HCC in NAFLD mice

Abstract Background: Liver cancer is a leading cause of cancer-related deaths. Non-alcoholic fatty liver disease (NAFLD) has become a burgeoning etiology of liver cancer. Emerging data shows that NAFLD alters adaptive T cell immunity and has a profound influence on both liver cancer development and efficacy of immunotherapy. Tumor antigen-specific T cells recognize tumor and are crucial for controlling malignancy. However, their populations are often low, and detection requires special tools. This study utilized a novel mouse model of HCC to investigate how the tumor antigen-specific T cell response is affected in NAFLD. Methods: We generated a doxycycline-inducible MHC-I OVA257-264 and MHC-II OVA323-339 antigen-expressing HCC cell line which allowed us to investigate tumor antigen-specific T cell responses. The cell line was seeded orthotopically into the livers of diet-induced (methionine choline-deficient [MCD]) and genetic (Ob/Ob [Ob]) NAFLD mouse models. OVA257-264 specific CD8+ T cells were detected by flow cytometry using H-2Kb/OVA257-264 tetramer. Pharmacological depletion experiments were used to identify additional effector cells. Results: The creation of an inducible MHC-I and -II OVA antigen-expressing HCC cell line was validated in both in-vitro and in-vivo settings. Tumor expression of highly immunogenic OVA antigens caused a strong tumor suppression which was mostly mediated by CD8+ T cells in mice fed with normal diet. The inverse correlation between percentage of OVA257-264 specific CD8+ T cells with tumor size further supported their critical role in tumor control. In contrast, in NAFLD mice fed MCD diet as well as Ob/Ob mice, tumor OVA antigens induction failed to reduce liver tumor growth. The presence of OVA257-264 specific CD8+ T cells in NAFLD mice suggested that NAFLD did not inhibit the generation of tumor antigen-specific T cells. This was supported by the finding that OVA257-264 specific CD8+ T cells from NAFLD mice had similar cytotoxic potential, activation, degranulation, PD-1 expression, and effector memory phenotype compared to controls, suggesting an indirect mechanism for the impaired anti-tumor immunity. Immunoprofiling revealed a drastic increase of macrophages in tumor-bearing NAFLD livers. Depletion of macrophages with clodronate reversed the NAFLD caused resistance to tumor-antigen dependent tumor suppression. It was observed that there were minimal changes to the amount of TAS CD8+ T cells in doxycycline-treated mice irrespective of clodronate depletion. There was, however, higher markers of activation such as CD69 and TNFα in clodronate-depleted mice compared to controls. Conclusions: NAFLD impairs antigen-specific CD8+ T cell immunity against liver tumor. The effect was not due to reduced generation or intrinsic functional changes of tumor antigen-specific CD8+ T cells but caused by accumulated macrophages in the liver environment. Citation Format: John C. McVey, Benjamin L. Green, Benjamin Ruf, Tim F. Greten, Chi Ma. Macrophages impair antigen specific CD8+ T cell response against HCC in NAFLD mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1372.

Ring Finger Protein 125 Is an Anti-Proliferative Tumor Suppressor in Hepatocellular Carcinoma.

Simple SummaryHCC is a leading cause of cancer-related deaths worldwide. However, its tremendous inter- and intra-tumor heterogeneity has made it difficult to identify driver genes for HCC. Transposon mutagenesis is a versatile in vivo tool to identify cancer genes in tissues of interest. Herein, we analyzed transposon mutagenesis screening data in the liver and discovered a novel tumor suppressor gene, RNF125. This gene functions as a negative regulator of cell proliferation through transcriptional suppression of multiple genes important for cell proliferation and liver regeneration. This gene is frequently inactivated in human HCC and has a significant impact on patient prognosis. Our findings identify a new regulatory network of cell proliferation mediated by RNF125 and its contribution to HCC development and progression.Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide and the only cancer with an increasing incidence in the United States. Recent advances in sequencing technology have enabled detailed profiling of liver cancer genomes and revealed extensive inter- and intra-tumor heterogeneity, making it difficult to identify driver genes for HCC. To identify HCC driver genes, we performed transposon mutagenesis screens in a mouse HBV model of HCC and discovered many candidate cancer genes (SB/HBV-CCGs). Here, we show that one of these genes, RNF125 is a potent anti-proliferative tumor suppressor gene in HCC. RNF125 is one of nine CCGs whose expression was >3-fold downregulated in human HCC. Depletion of RNF125 in immortalized mouse liver cells led to tumor formation in transplanted mice and accelerated growth of human liver cancer cell lines, while its overexpression inhibited their growth, demonstrating the tumor-suppressive function of RNF125 in mouse and human liver. Whole-transcriptome analysis revealed that RNF125 transcriptionally suppresses multiple genes involved in cell proliferation and/or liver regeneration, including Egfr, Met, and Il6r. Blocking Egfr or Met pathway expression inhibited the increased cell proliferation observed in RNF125 knockdown cells. In HCC patients, low expression levels of RNF125 were correlated with poor prognosis demonstrating an important role for RNF125 in HCC. Collectively, our results identify RNF125 as a novel anti-proliferative tumor suppressor in HCC.