Hepatocellular Cancer Progression Research Articles

RBM12 regulates the progression of hepatocellular cancer via miR-497–5p/CPNE1 Axis

BackgroundHepatocellular Carcinoma (HCC), also called hepatocellular cancer, has emerged as a highly prevalent malignancy globally. By binding to specific RNA via one or more spherical RNA Domains (RBDs) or RNA Motifs (RBMs), RNA Binding Proteins (RBPs) can affect RNA modification, splicing, localization, translation, and stability. MethodsThis paper builds on previous research by further investigating the impact of RBM12 on LC progression. In order to determine the effect of RBM12 expression on the prognosis of patients with hepatocellular cancer, we first investigated its expression in liver cancer cells (LCC) and tissues. The effect of RBM12 on the malignant biological behavior of LCC was subsequently detected using cytological experiments. To explore the upstream mechanism affecting RBM12, we predicted the miRNA targeting RBM12. According to the database, miR-497–5p was the best candidate gene. The double Luciferase reporter gene experiment was executed to validate the bounding of miR-497–5p with RBM12. ResultsAccording to the cytological experiments, a high RBM12 expression promoted the propagation, migration, and invasion of LCC and impeded liver cancer cell apoptosis. By secreting TGF-β1, RBM12 could induce the EMT process. The miR-497–5p expression is suppressed in hepatocellular cancer. As shown by the CCK8, plate cloning, Transwell, EDU, and other experiments, miR-497–5p suppressed RBM12 expression and tumor growth. The double Luciferase reporter gene system was utilized to verify the combination of miR-497–5p and RBM12. The CPNE1 is a downstream gene regulated by RBM12. A high CPNE1 expression was exhibited in LCC and tissues. The CPNE1 is essential in the process where RBM12 promotes the incidence and progression of liver cancer. ConclusionsBy elucidating the exact molecular mechanism through which RBM12 promotes the initiation and progression of LC, thus, the current investigation provides some reference for the clinical management of LC.

Circular RNA circBNC2 facilitates glycolysis and stemness of hepatocellular carcinoma through the miR-217/high mobility group AT-hook 2 (HMGA2) axis

Hepatocellular cancer (HCC) accounts for approximately 90% of primary liver carcinoma and is a significant health threat worldwide. Circular RNA basonuclin 2 (circBNC2) is implicated with the progression of several cancers. However, its roles in carcinogenesis and glycolysis are still unclear in HCC. In this study, the levels of circBNC2 and high mobility group AT-hook 2 (HMGA2) were highly expressed, while these of miR-217 were poorly expressed in HCC tissues and cells. Upregulation of circBNC2 was related to poor prognosis and tumor node metastasis (TNM) stage. Knockdown of circBNC2 inhibited the HCC progression. Moreover, knockdown of circBNC2 suppressed the levels of Ras, ERK1/2, PCNA, HK2, and OCT4. Notably, circBNC2 functioned as a molecular sponge of microRNA 217 (miR-217) to upregulate the HMGA2 expression. The inhibitory effects of the circBNC2 silence on the growth and stemness of HCC cells, and levels of PCNA, HK2 and OCT4 were aggravated by the miR-217 overexpression, but neutralized by the HMGA2 overexpression. Besides, silencing of circBNC2 blocked the tumor growth through upregulating the expression of miR-217 and downregulating the levels of HMGA2, PCNA2, HK2 and OCT4 in vivo. Thus, the current data confirmed that circBNC2 sponged miR-217 to upregulate the HMGA2 level, thereby contributing to the HCC glycolysis and progression. These findings might present novel insight into the pathogenesis and treatment of HCC.

Expression of Bone Morphogenetic Protein 14 in Liver Disease and Cancer

The activation of hepatic stellate cells (HSCs) is the key event of hepatic fibrosis. Furthermore, activated HSCs also play an important role in the progression of hepatocellular cancer (HCC). Bone morphogenetic protein 14 (BMP14) is a member of the TGF-β/BMP superfamily. So far, most studies have analyzed BMP14 in the context of bone and cartilage formation and homeostasis. The aim of this study was to assess the expression and function of BMP14 in liver fibrosis and HCC. The BMP14 expression increased during the in vitro activation of primary human HSCs and also in mouse models of liver fibrosis. In human HCC, as well as non-tumorous liver tissues, there was a significant correlation between the expression of BMP14 and alpha-smooth-muscle actin (α-SMA), an established marker for HSC activation. RNAi-mediated BMP14 suppression in activated HSCs resulted in the reduced expression of the transcription factors inhibitor of differentiation 1 (ID1) and ID2, known targets of BMP signaling. Interestingly, α-SMA and collagen expression was also reduced in BMP14-depleted cells, while treatment with recombinant BMP14 induced ID1, ID2, α-SMA and collagen expression. In human HCC cell lines, treatment with recombinant BMP14 induced proliferation, migratory activity and colony formation. In summary, our data indicate activated HSCs as a major cellular source of enhanced BMP14 expression in fibrotic liver disease and HCC, and show that BMP14 exhibits pro-fibrogenic as well as pro-tumorigenic effects. Future analyses will reveal the potential of this soluble growth factor as a therapeutic target or prognostic marker for the progression of fibrosis and HCC in patients with chronic liver disease.

Phosphorylation of UHRF2 affects malignant phenotypes of HCC and HBV replication by blocking DHX9 ubiquitylation

Hepatitis B virus (HBV) infection is one of main contributors to poor prognosis and rapid progression of hepatocellular cancer (HCC). We previously identified the important role of the phosphorylation of ubiquitin-like with PHD and ring finger domains (UHRF2) in HBV-associated HCC. In this study we identify upregulated UHRF2 protein levels in HBV-associated HCC cells and tissues. UHRF2 overexpression promotes the viability, proliferation, migration and invasiveness of HBV-positive HCC cell lines, and enhances HBV DNA replication. To obtain a comprehensive understanding of the interaction networks of UHRF2 and their underlying mechanism, this study suggests that UHRF2 facilitates the ubiquitin-proteasome-mediated proteolysis of DExD/H (Asp-Glu-Ala-His) -box helicase enzyme 9 (DHX9). However, phosphorylation of UHRF2 by HBx at S643 inhibits E3 ubiquitin ligase activity of UHRF2 and improves DHX9 protein stability. Furthermore, results suggest that HBx promotes phosphorylation of UHRF2 by the ETS1-CDK2 axis through the downregulation of miR-222-3p in HBV-associated HCC specimens and cells. Our findings suggest that HBx-induced phosphorylation of UHRF2 S643 acts as a “switch” in HBV-associated HCC oncogenesis, activating the positive feedback between phosphorylated UHRF2 and HBV, provide evidence that UHRF2 is a new regulator and a potential prognostic indicator of poor prognosis for HBV-associated HCC.

The role of calcineurin inhibitors in the progression of hepatocellular carcinoma after liver transplantation

Introduction. Orthotopic liver transplantation is the most radical method of treatment of hepatocellular carcinoma. The high recurrence rate limits the use of transplantation in patients with hepatocellular cancer. Immunosuppressive therapy may affect the frequency of oncoprogression after liver transplantationAim. To evaluate the role of immunosuppressive therapy in the postoperative progression of hepatocellular cancer in patients after liver transplantationMaterial and methods. The recurrence rate of hepatocellular cancer and tumor free survival in 104 patients after liver transplantation were analyzed. To evaluate the effect of the immunosuppression main component concentration on the postoperative progression of hepatocellular carcinoma, we studied the mean baseline concentration (C0) for the entire follow-up period for patients with a tumor-free period and the mean baseline concentration for patients with hepatocellular carcinoma progression, in whom only the duration of the tumor-free period was studied. According to the degree of tumor lesion, patients were distributed in accordance with the Milan criteria (based on the results of a pathologic and morphological examination of the recipient's explanted liver.Results. The values of the baseline blood level of tacrolimus>6.0 ng/ml and cyclosporine A>100 ng/ml is associated with a high rate of progression of hepatocellular cancer. Reducing the load of calcineurin inhibitors can reduce the incidence of cancer progression by at least 2 times. The values of 1-, 3- and 5-year relapse-free survival in patients with advanced cancer and low figures of the baseline blood level of calcineurin inhibitor are 82%, 70% and 70%, respectively.Conclusion. Minimization of immunosuppression is of crucial importance in the prevention of posttransplant progression of hepatocellular cancer, especially among patients with its common form.

Regulatory T-cells-related signature for identifying a prognostic subtype of hepatocellular carcinoma with an exhausted tumor microenvironment.

Regulatory T-Cells (Tregs) are important in the progression of hepatocellular cancer (HCC). The goal of this work was to look into Tregs-related genes and develop a Tregs-related prognostic model. We used the weighted gene co-expression network analysis (WGCNA) to look for Tregs-related genes in the TCGA, ICGC, and GSE14520 cohorts and then used the non-negative matrix factorization (NMF) algorithm to find Tregs-related subpopulations. The LASSO-Cox regression approach was used to determine Tregs-related genes, which were then condensed into a risk score. A total of 153 overlapping genes among the three cohorts were considered Tregs-related genes. Based on these genes, two Tregs-associated clusters that varied in both prognostic and biological characteristics were identified. When compared with Cluster 1, Cluster 2 was a TME-exhausted HCC subpopulation with substantial immune cell infiltration but a poor prognosis. Five Tregs-related genes including HMOX1, MMP9, CTSC, SDC3, and TNFRSF11B were finally used to construct a prognostic model, which could accurately predict the prognosis of HCC patients in the three datasets. Patients in the high-risk scores group with bad survival outcomes were replete with immune/inflammatory responses, but exhausted T cells and elevated PD-1 and PD-L1 expression. The results of qRT-PCR and immunohistochemical staining (IHC) analysis in clinical tissue samples confirmed the above findings. Moreover, the signature also accurately predicted anti-PD-L1 antibody responses in the IMvigor210 dataset. Finally, HMOX1, MMP9, and TNFRSF11B were expressed differently in Hep3B and Huh7 cells after being treated with a PD1/PD-L1 inhibitor. In conclusion, our study uncovered a Tregs-related prognostic model that could identify TME- exhausted subpopulations and revealed that PD1/PD-L1 inhibitors could alter the expression levels of HMOX1, MMP9, and TNFRSF11B in Hep3B and Huh7 cells, which might help us better understand Tregs infiltration and develop personalized immunotherapy treatments for HCC patients.

Hsa_circ_0000098 is a novel therapeutic target that promotes hepatocellular carcinoma development and resistance to doxorubicin

BackgroundCircular RNA (circRNA) is crucial to the progression of hepatocellular cancer (HCC). In addition, Mitochondrial calcium uniporter regulatory factor 1 (MCUR1) is commonly overexpressed in HCC to increase cellular ATP levels. Due to the highly aggressive characteristics of HCC, it is essential to identify new diagnostic biomarkers and therapeutic targets that may facilitate the diagnosis of HCC and the development of effective anti-HCC treatments.MethodsA series of in vitro and in vivo experiments were undertaken to investigate the biological importance and underlying mechanisms of circ_0000098 in HCC.ResultsThe expression of circ_0000098 was higher in HCC tissues compared to paired adjacent tissues. According to the receiver-operating characteristic curves, circ_0000098 functioned as a potential diagnostic tumor marker in HCC. Our experiments indicated that circ_0000098 served as a key oncogenic circRNA to increase HCC cell proliferation and invasion in vitro and HCC progression in vivo. Furthermore, mechanistic investigation demonstrated that by sequestering miR-383 from the 3′-UTR of MCUR1, circ_0000098 positively regulated MCUR1 expression in HCC cells and finally promoted HCC progression. On the other hand, inhibiting circ_0000098 in HCC cells could diminish doxorubicin (DOX) resistance by decreasing P-glycoprotein (P-gp, MDR1) expression and intracellular ATP levels. Either downregulation of MCUR1 or overexpression of miR-383 improved DOX sensitivity in HCC cells. Subsequently, a short hairpin RNA targeting circ_0000098 (referred to as sh-1) and doxorubicin (DOX) were encapsulated into platelets (PLTs), referred to as DOX/sh-1@PLT. Activated DOX/sh-1@PLT through HCC cells resulted in the creation of platelet-derived particles that were capable of delivering the DOX/sh-1 combination into HCC cells and promoting intracellular DOX accumulation. Furthermore, our in vivo experiments showed that DOX/sh-1@PLT can effectively reduce P-gp expression, promote DOX accumulation, and reverse DOX resistance.ConclusionsOur results demonstrated that circ_0000098 is an oncogenic circRNA that promotes HCC development through the miR-383/MCUR1 axis and targeting circ_0000098 with DOX/sh-1@PLT may be a promising and practical therapeutic strategy for preventing DOX resistance in HCC.

MYC Promotes LDHA Expression through MicroRNA-122-5p to Potentiate Glycolysis in Hepatocellular Carcinoma.

MYC is a notorious oncogene in a vast network of malignancies, whereas liver-specific microRNA- (miR-) 122-5p is downregulated in hepatocellular cancer (HCC). Here, we studied the possible correlation between these two and their involvement in glycolysis in HCC. MYC was overexpressed in HCC tissues and cells compared to normal liver tissues and normal hepatocytes NHC, which predicted a poor survival of HCC sufferers. Functional assays demonstrated that silencing of MYC inhibited the glycolysis in HCC cells, as evidenced by significantly weaker glucose consumption, lactate production, adenosine triphosphate (ATP) levels, and downregulated HK1 and HK2 protein expression. Moreover, MYC bound to the miR-122-5p promoter and repressed the miR-122-5p expression. Rescue experiments showed that miR-122-5p inhibitor rescued the diminished glycolysis after MYC silencing. In addition, lactate dehydrogenase (LDHA) was identified as a downstream target of miR-122-5p. The overexpression of LDHA mitigated the effects of si-MYC and miR-122-5p mimic on glycolysis of HCC cells, respectively. In conclusion, the MYC/miR-122-5p/LDHA axis modulates glycolysis in HCC cells and possibly affects HCC progression.

Long non-coding RNA SNHG1 activates glycolysis to promote hepatocellular cancer progression through the miR-326/PKM2 axis.

Hepatocellular cancer (HCC) is a lethal malignancy with extremely poor prognosis. In the present study, we aimed to investigate the role and underlying mechanism of SNHG1 in HCC progression. Combined with bioinformatics and experimental validation, we explored the clinical significance of SNHG1 in HCC. A Cell Counting Kit-8 assay, cell colony formation assay, and subcutaneous tumorigenesis experiments of nude mice were conducted to evaluate the pro-proliferative capacity of SNHG1. Glucose consumption and lactate production were measured to explore the regulatory role of SNHG1 in glycolysis. Nuclear-cytoplasmic separation, quantitative real-time polymerase chain reaction and Western blot assays, chromatin immunoprecipitation, and luciferase reporter and RNA immunoprecipitation assays were performed to investigate the molecular mechanisms of SNHG1 in HCC. SNHG1 expression was dramatically increased in HCC and positively correlated with poor prognosis. E2F1 bound to the SNHG1 promoter region to activate SNHG1 transcription. Furthermore, SNHG1 served as a molecular sponge for miR-326 to sequester the interaction of miR-326 and pyruvate kinase M2 (PKM2), facilitating the expression of PKM2. Activating PKM2 expression was revealed to be one of mechanisms of SNHG1 with respect to promoting glycolysis and the proliferation of HCC cells. E2F1-activated SNHG1 modulates the miR-326/PKM2 axis to facilitate glycolysis and the proliferation of HCC cells. Targeting SNHG1 could be a promising therapeutic option for HCC.

Histone Demethylase JMJD2D: A Novel Player in Colorectal and Hepatocellular Cancers.

Simple SummaryHistone demethylase JMJD2D is a multifunctional epigenetic factor coordinating androgen receptor activation, DNA damage repair, DNA replication, cell cycle regulation, and inflammation modulation. JMJD2D is also a well-established epigenetic facilitator in the progression of multiple malignant tumors, especially in colorectal cancer (CRC) and hepatocellular cancer (HCC). This review aims to summarize the mechanisms of JMJD2D in promoting CRC and HCC progression, which provides novel ideas for targeting JMJD2D in oncotherapy. JMJD2D promotes gene transcription by reducing H3K9 methylation and serves as a coactivator to enhance the activities of multiple carcinogenic pathways, including Wnt/β-catenin, Hedgehog, HIF1, JAK-STAT3, and Notch signaling; or acts as an antagonist of the tumor suppressor p53.Posttranslational modifications (PTMs) of histones are well-established contributors in a variety of biological functions, especially tumorigenesis. Histone demethylase JMJD2D (also known as KDM4D), a member of the JMJD2 subfamily, promotes gene transcription by antagonizing H3K9 methylation. JMJD2D is an epigenetic factor coordinating androgen receptor activation, DNA damage repair, DNA replication, and cell cycle regulation. Recently, the oncogenic role of JMJD2D in colorectal cancer (CRC) and hepatocellular cancer (HCC) has been recognized. JMJD2D serves as a coactivator of β-catenin, Gli1/2, HIF1α, STAT3, IRF1, TCF4, and NICD or an antagonist of p53 to promote the progression of CRC and HCC. In this review, we summarize the molecular mechanisms of JMJD2D in promoting the progression of CRC and HCC as well as the constructive role of its targeting inhibitors in suppressing tumorigenesis and synergistically enhancing the efficacy of anti-PD-1/PD-L1 immunotherapy.

Enhancer of zeste homolog 2 promotes hepatocellular cancer progression and chemoresistance by enhancing protein kinase B activation through microRNA-381-mediated SET domain bifurcated 1

ABSTRACT Metastasis and chemoresistance are the leading causes of death in patients with hepatocellular carcinoma (HCC). microRNAs (miRNAs or miRs) may be useful as diagnostic, therapeutic and prognostic markers for HCC. In this study, we set out to investigate the possible role of miR-381 in HCC development and chemoresistance along with the related mechanism. Microarray-based gene expression profiling was carried out to analyze the expression of SET domain bifurcated 1 (SETDB1) and histone methyltransferase enhancer of zeste homolog 2 (EZH2) followed by validation in clinical HCC tissues and cells. The potential binding between miR-381 and SETDB1 was found and verified. Then, the role of SETDB1 in HCC in relation to miR-381 and protein kinase B (AKT) pathway was explored through gain- and loss-of-function approaches. After expression determination of EZH2, SETDB1, miR-381, and AKT pathway-related factors, their reactions were analyzed and their functional roles in HCC progression and chemoresistance were investigated in vitro and in vivo. SETDB1 was aberrantly upregulated in clinical HCC tissues and cells. This upregulation activated AKT pathway by promoting its tri-methylation on K64. SETDB1 promoted the proliferation, migration and chemoresistance through the AKT pathway in HCC cells. In a xenograft mouse model, SETDB1 promoted HCC cell tumorigenesis in vivo by activating the AKT pathway. Furthermore, EZH2 suppressed miR-381 by catalyzing the activity of H3K27me3 on its promoter region. In conclusion, EZH2 suppressed miR-381 expression by promoting H3K27me3 activity on its promoter region to facilitate SETDB1 expression, thereby activating the AKT pathway to promote hepatocarcinogenesis and chemoresistance.

Mechanisms and clinical significance of TGF-β in hepatocellular cancer progression.

Despite progress in treating or preventing viral hepatitis, a leading cause of liver cancer, hepatocellular cancer (HCC) continues to be a major cause of cancer-related deaths globally. HCC is a highly heterogeneous cancer with many genetic alterations common within a patient's tumor and between different patients. This complicates therapeutic strategies. In this review, we highlight the critical role that the Smad-mediated transforming growth factor β (TGF-β) pathway plays both in liver homeostasis and in the development and progression of HCC. We summarize the mouse models that have enabled the exploration of the dual nature of this pathway as both a tumor suppressor and a tumor promoter. Finally, we highlight how the insights gained from evaluating pathway activity using transcriptional profiling can be used to stratify HCC patients toward rational therapeutic regimens based on the differences in patients with early or late TGF-β pathway activity or activated, normal, or inactivated profiles of this key pathway.

Abnormal Expression of Centromere Protein U Is Associated with Hepatocellular Cancer Progression.

Background Hepatocellular carcinoma (HCC) is one of the most common malignancies globally, but its molecular mechanism is unclear. Abnormal expression of centromere protein U (CENPU) is closely related to diverse human cancers. The purpose of this article was to evaluate the function and potential mechanisms of CENPU in HCC development. Methods We performed bioinformatics analysis of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Gene Expression Profiling Interactive Analysis (GEPIA), and Kaplan-Meier plotter databases to investigate the clinical significance and prognostic value of CENPU in HCC. Western blotting and immunohistochemical staining were used to measure protein expression, while reverse transcription-quantitative polymerase chain reaction (qRT-PCR) was used to determine mRNA expression. Cell Counting Kit8 (CCK-8) and colony formation assays were conducted to examine cell proliferation. Transwell and wound healing assays were used to assess cell migration and invasion. Gene set enrichment analysis (GSEA) was used to explore the potential signaling pathways of CENPU involved in HCC. Results High expression of CENPU in HCC was predicted by public database analysis and indicated a poor prognosis. CENPU expression was significantly higher in HCC tissues and cells than in normal tissues and cell. In vitro, CENPU promoted the proliferation, migration, and invasion of HCC cells. GSEA results indicated that CENPU was linked to the Notch signaling pathway, and our research supported this prediction. Conclusion CENPU promotes the malignant biological process of HCC and may be a promising target for HCC treatment.

UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11.

Simple SummaryHerein, the oncogenic role of UBE2M as an E2 NEDD8-conjugating enzyme was explored in hepatocellular carcinoma (HCC) cells, since neddylation plays a critical role in tumorigenesis. To address this issue, human tissue array and TCGA analysis were conducted in HCCs to find overexpression of UBE2M in HCCs. In addition, a differential profile was confirmed in UBE2M-depleted HepG2 cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells via binding to MDM2 and ribosomal protein L11 by immunoprecipitation and immunofluorescence. These findings provide evidence that UBE2M is critically involved in liver cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11.Though UBE2M, an E2 NEDD8-conjugating enzyme, is overexpressed in HepG2, Hep3B, Huh7 and PLC/PRF5 HCCs with poor prognosis by human tissue array and TCGA analysis, its underlying oncogenic mechanism remains unclear. Herein, UBE2M depletion suppressed viability and proliferation and induced cell cycle arrest and apoptosis via cleavages of PARP and caspase 3 and upregulation of p53, Bax and PUMA in HepG2, Huh7 and Hep3B cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells. Interestingly, UBE2M binds to MDM2 or ribosomal protein L11, but not p53 in HepG2 cells, despite crosstalk between p53 and UBE2M. Consistently, the colocalization between UBE2M and MDM2 was observed by immunofluorescence. Notably, L11 was required in p53 activation by UBE2M depletion. Furthermore, UBE2M depletion retarded the growth of HepG2 cells in athymic nude mice along with elevated p53. Overall, these findings suggest that UBE2M promotes cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11 in HCCs.

The Potential of Serum Exosomal hsa_circ_0028861 as the Novel Diagnostic Biomarker of HBV-Derived Hepatocellular Cancer.

Hepatitis B virus (HBV)-derived hepatocellular cancer (HCC) is a serious threat to human health, especially in China. There is no highly sensitive and specific HCC biomarker at present, which makes it difficult to detect HCC at the early stage. Serum exosomal circular RNAs (circRNAs) have been reported as novel diagnostic and prognostic biomarkers of cancers. In the present study, we aimed to explore the diagnostic performance of serum exosomal circRNAs for HBV-derived HCC screening. At first, many circRNAs were found to be differentially expressed in the serum exosomes of HCC individuals by microarray analysis. The validation of dysregulated circRNAs by qRT-PCR revealed that serum exosomal hsa_circ_0028861 was decreased in HCC compared to chronic HBV and cirrhosis. Then, hsa_circ_0028861 was identified as a novel biomarker for HCC diagnosis with an area under the ROC curve (AUC) of 0.79 for discriminating HCC from chronic HBV and cirrhosis individuals. Hsa_circ_0028861 was capable of detecting small (AUC = 0.81), early-stage (AUC = 0.82) and AFP-negative [AFP (−)] (AUC = 0.78) tumors as well. The combination of hsa_circ_0028861 and AFP exhibited better diagnostic ability (AUC = 0.86 for discriminating HCC from chronic HBV and cirrhosis). Moreover, bioinformatics prediction suggested that hsa_circ_0028861 might influence HCC progression by regulating its targeted microRNAs (miRNAs) and downstream tumor-related signaling pathways. Collectively, our study reveals a novel diagnostic tool for HBV-derived HCC.

Abstract 2544: TGF-β receptors 1 and 2 are functional biomarkers that stratify risk of hepatocellular cancer (HCC). Artificial intelligence based validation at three centers

Abstract Background: Hepatocellular carcinoma (HCC) is the fastest rising cancer in the USA and is the fourth leading cause of cancer deaths globally. A critical unmet need to reduce high mortality associated with advanced HCC is the ability to identify high-risk individuals for cost-effective, targeted screening, early detection, and prevention strategies. Human genomics and animal models have revealed etiological patterns and multiple genes and signaling pathways such as TGF-β, WNT, VEGF to be associated with the initiation and progression of HCC. We used integrated functional approaches (combining bioinformatics analysis, in vivo mouse models, and in vitro biological and biochemical methods) and identified potential biomarkers for HCC. We examined and validated 10 functional biomarkers for HCC risk prediction. We further harnessed machine learning tools and Artificial Intelligence (AI)-based technology to validate manual analyses of Immunohistochemical (IHC) labeled slides across different centers. Methods: >280 liver tissue samples were collected from patients with HCC and cirrhosis. Immunohistochemistry was performed against 10 functional biomarkers (TGFBR1, TGFBR2, SPTBN1, HMGA2, RSPO3, ITIH4, EPCAM, PLK1, SIRT6, FANCD2). Artificial intelligence-based deep learning technology by AIFORIA was used for the unbiased, intensive, and rapid validation of IHC. Results: The most promising results were for TGFBR1 and TGFBR2, on >80 HCC and cirrhotic samples, IHC labeling revealed: • Both TGFBR1 (p<0.001) and TGFBR2 (p<0.02) showed considerably reduced expression levels in tumor tissue within HCC samples, compared to non-tumorous adjacent tissue and cirrhotic samples. • The results were validated with another set of 70 patient samples at two other centers. Results obtained at George Washington University (GW), University of Maryland (UMD), and the University of Hawaii (UH) for TGFBR2 were consistent. For TGFBR1, GW and UMD showed reproducible results while results at UH lacked statistical significance. • AI-based analysis for TGFBR2 (p<0.002) validated the results from manual analysis ensuring the consistency of results across different sites. • Of the other biomarkers, PLK1 also displayed potentially substantial alterations with increased labeling in tumor tissue as compared to non-tumor tissue. Conclusions: TGFBR2 is a promising functional predictive HCC tissue biomarker. • Tissue biomarkers are important as they provide spatial and contextual information. • Computerized analysis is a powerful tool to capture microscopic patterns in visual data that are not normally assessed in manual analysis such as the pattern of immune cells in cirrhosis and HCC. • AI adds consistency and confidence to results obtained at different sites. Citation Format: Sobia Zaidi, Kirty Shetty, Herbert Yu, Linda Wong, Shuyun Rao, Wilma Jogunoori, Richard Amdur, Shulin Li, Patricia latham, Bao-Ngoc Nguyen, Lopa Mishra. TGF-β receptors 1 and 2 are functional biomarkers that stratify risk of hepatocellular cancer (HCC). Artificial intelligence based validation at three centers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2544.

Abstract 89: A TGF-beta Pathway-SREBP1 axis controls liver diseases from nonalcoholic steatohepatitis to hepatocellular carcinoma

Abstract Background/Aims: The global epidemic of obesity has led to an alarming rise in nonalcoholic steatohepatitis (NASH) and hepatocellular cancer (HCC). In addition to abnormal fat accumulation and liver injury, fibrosis is a key predictive factor for progression and transformation. Yet, our understanding of how a major fibrosis pathway, transforming growth factor β (TGF-β), and how members, SMAD3 with its adaptor SPTBN 1 -contribute to the progression of NASH-driven HCC remains unclear. Here, we sought to better understand the role of TGF-β1/SMAD3/SPTBN1 in regulating the switch between NASH and HCC. Methods: We generated liver-specific SPTBN1 knockout mice (Albumin Cre+Sptbn1loxp/loxp, LKO). LKO and controls were given a high-fat diet (HFD) or DEN; Phenotypic analyses and mechanistic insight were obtained through RNA-seq, mass spectrometry, structure modeling, cell fractionation, and imaging-immunofluorescence, immunohistochemistry, and interactions studies were performed in human HCC cell lines (Huh7, Hep3B). Results: Strikingly, LKO did not develop HFD induced obesity, NASH, and cancer, that occurred in wild type, Flox control, NASH, and DEN/HCC models. Compared to controls, LKO liver tissues displayed 2-7-fold decreases in NASH associated pro-fibrotic genes (Col1a1, Col1a2) and lipid metabolism genes (CD36, Slc27a1, Plin4, Plin2) (p<0.05), and over 4-fold increases in Fgf21 mRNA levels (LKO vs control: 3.67±1.9 vs 0.84±0.16). Analysis of key regulators of lipogenesis revealed decreased SREBP1 (0.33±0.11 VS 1.35±0.22, p=0.006) and targets as well as PPARG (3.15±1.24 VS 19.03±4.98, p=0.02) in LKO compared to controls. Functionally, Sptbn1 MEF cells show marked reduction in SREBP1 reporter activity compared to WT MEFs (LDLR-luc: 0.25±0.04 vs 1.0±0.08, p<0.05) with decreased nuclear SREBP1 labeling in LKO tissues. Depletion of Sptbn1 leads to impaired TGF-β1/SMAD3 signaling and decreased TNF-α induced ER stress/caspase-3 activated SREBP1. N-terminal SPTBN1 (D50-T975) binds SREBP1 (Q295-K374) and induces its nuclear translocation. HFD -driven caspase-3 activation and SREBP1-SPTBN1 cleavage and binding occur independently of insulin induced gene (INSIG) and SREBP cleavage-activating protein (SCAP). We observe raised levels of SPTBN1 together with Caspase-3, and lipogenic pathways (e.g., FASN, SCD1) in human NASH/HCC progression. Additional human HCC TCGA analysis revealed poor prognosis in patients with high SREBP1 activity. Finally, siRNA targeting Sptbn1 in vivo protects mice from HFD-induced NASH and reduces NASH-associated transcriptional changes in a human 3D-culture NASH model. Conclusions: We uncovered a surprising and promising role of SPTBN1 siRNA in inhibiting NASH and HCC, through modulation of its binding partners, SMAD3 and SREBP1. These data provide new insights into the switch in obesity driven liver cancer as well as new therapeutic targets. Citation Format: Shuyun Rao, Zhanhuai Wang, Kazufumi Ohshiro, Sobia Zaidi, Xiaochun Yang, Patricia Latham, Wilma Jogunoori, Xianyan Xiang, Inhee Chung, Kirti Shetty, Michele Vacca, Antonio Vidal-Puig, Lopa Mishra. A TGF-beta Pathway-SREBP1 axis controls liver diseases from nonalcoholic steatohepatitis to hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 89.

Abstract 1984: Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer

Abstract Background: Cancer stem cells that escape suppression by key pathways such as TGF-β represent one of the mechanisms for poor prognosis in hepatocellular cancer (HCC). Our mouse models with disruption of TGF-β signaling (β2SP+/-/Smad3+/-) develop HCC and phenocopy an epigenetically human cancer stem cell disorder, providing clues towards the role of TGF-β in the switch in stem cell transformation to HCC. An examination of HCC genomics revealed a surprising and strong correlation between TGF-β and Sirtuins (SIRTs) expression. SIRTs are also implicated in cancer stem cell and HCC suppression. Here we hypothesized that the TGF-β pathway harnesses Sirtuin members to maintain stem cell homeostasis, which may mediate the progression of HCC. Methods: Overall Survival (OS) analysis was carried out by using early-stage HCC (TCGA, Firehose Legacy, stage N1, n=186) from cBioportal. ChIP assay was performed in HepG2 cells using anti-Smad3 and normal rabbit IgG. Biotinylated SIRT6 promoter with/without SMAD binding sites was used as probes to pull down the DNA-interacting proteins in HepG2 cells treated with/without TGF-β for 2h. Results: Analysis of TCGA HCC dataset (cBioportal, stage N1, n=186) indicated reduced OS for HCC patients with decreased SIRT6 together with loss of SPTBN1 in the early stage (Median survival months: 40.37 vs 83.18, p=0.0417). • Chip-qPCR and the DNA-protein pulldown assay revealed that Smad3 positively upregulates the SIRT6 mRNA expression (Smad3 vs IgG, SBE1: 3.27±0.53 vs 0.99±0.01, p<0.05; SBE2: 3.32±0.74 vs 1.12±0.3, p<0.05) through direct binding to the SIRT6 promoter in HepG2 cells after TGF-β treatment. • Co-IP analysis revealed directly binding of SIRT6 with β2sp and Smad3. • Overexpression of SIRT6 represses Smad3 and β2sp expression (p<0.01) and results in inhibition of the TGF-β target genes (p<0.05) such as MMP9 (68.2% inhibited), HMGA2 (53.4% inhibited), and PAI-1 (35.4% inhibited); whereas knockdown of SIRT6 increases β2sp expression in HepG2 cells (3 fold, p<0.05). • Similar to mice with disruption of SIRT6 (SIRT6-/-) that develop liver inflammation, steatosis with progression to tumorigenesis, mice with TGF-β signaling defect (Smad3+/-Sptbn1+/-) also develop fatty liver with progression to tumorigenesis under HFD. Interestingly, we found that SIRT6 was significantly downregulated in MEF cells from mice with loss of TGF-β signaling (Smad3+/-Sptbn1+/-). Conclusions: Our findings suggest that the TGF-β signaling participates in positively regulating the expression of SIRT6. In turn, SIRT6 acts as a negative regulator of TGF-β signaling through de-acetylating Smad3 and β2sp. SIRT6 and TGF-β signaling exert integrative yet complex roles in inhibiting hepatic steatosis, inflammation, and HCC that when disrupted lead to poor prognosis in HCC. The data provide new combined driving roles for the two pathways in suppressing NASH and HCC. Citation Format: Xiyan Xiang, Sobia Zaidi, Shuyun Rao, Kazufumi Ohshiro, Zhanhuai Wang, Wilma Jogunoori, Chuxia Deng, Patricia Latham, Lopa Mishra. Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1984.

Natural Killer Cells and T Cells in Hepatocellular Carcinoma and Viral Hepatitis: Current Status and Perspectives for Future Immunotherapeutic Approaches.

Natural killer (NK) cells account for 25–50% of the total number of hepatic lymphocytes, which implicates that NK cells play an important role in liver immunity. The frequencies of both circulating and tumor infiltrating NK cells are positively correlated with survival benefit in hepatocellular cancer (HCC) and have prognostic implications, which suggests that functional impairment in NK cells and HCC progression are strongly associated. In HCC, T cell exhaustion is accompanied by the interaction between immune checkpoint ligands and their receptors on tumor cells and antigen presenting cells (APC). Immune checkpoint inhibitors (ICIs) have been shown to interfere with this interaction and have altered the therapeutic landscape of multiple cancer types including HCC. Immunotherapy with check-point inhibitors, aimed at rescuing T-cells from exhaustion, has been applied as first-line therapy for HCC. NK cells are the first line effectors in viral hepatitis and play an important role by directly eliminating virus infected cells or by activating antigen specific T cells through IFN-γ production. Furthermore, chimeric antigen receptor (CAR)-engineered NK cells and T cells offer unique opportunities to create CAR-NK with multiple specificities learning from the experience gained with CAR-T cells with potentially less adverse effects. This review focus on the abnormalities of NK cells, T cells, and their functional impairment in patients with chronic viral hepatitis, which contributes to progression to hepatic malignancy. Furthermore, we discuss and summarize recent advances in the NK cell and T cell based immunotherapeutic approaches in HCC.

Ferroptosis is involved in the progression of hepatocellular carcinoma through the circ0097009/miR-1261/SLC7A11 axis.

BackgroundCircular RNAs (circRNAs) are a class of non-coding RNAs that have been demonstrated to play important roles in tumorigenesis. However, how circRNAs regulate the progression of hepatocellular cancer (HCC) remains unclear.MethodsIn the present study, circRNA microarray analyses were performed with HCC tissues to identify circRNAs that are differentially expressed. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was conducted on HCC cell lines and tissues, and circ0097009 was found to be significantly upregulated. The functions of circ0097009 in HCC were investigated by a series of experiments, including cell proliferation, invasion, and mouse xenograft assays. Additionally, luciferase assays and RNA immunoprecipitation (RIP) assays were used to explore the interactions of circ0097009, microRNA-1261 (miR-1261), and solute carrier family 7 member 11 (SLC7A11) in HCC.ResultsMicroarray analysis and qRT-PCR verified that circRNA, circ0097009, was significantly upregulated in HCC tissues and cell lines. Knockdown of circ0097009 inhibited the proliferation and invasion of HCC cells. Luciferase reporter assays showed that circ0097009 and SLC7A11 directly bound to miR-1261. Subsequent experiments showed that circ0097009 and SLC7A11 reciprocally regulated their expression via miR-1261 sponging by circ0097009.ConclusionsCirc0097009 acts as a competing endogenous RNA to regulate the expression of SLC7A11, a key regulator of cancer cell ferroptosis, by sponging miR-1261 in HCC. Circ0097009 may be used as a diagnostic biomarker for HCC and as a potential target for HCC therapy.