HCC Formation Research Articles

FIGNL1 Promotes Hepatocellular Carcinoma Formation via Remodeling ECM-receptor Interaction Pathway Mediated by HMMR.

The development of novel biomarkers is crucial for the treatment of HCC. In this study, we investigated a new molecular therapeutic target for HCC. Fidgetin-like 1 (FIGNL1) has been reported to play a vital role in lung adenocarcinoma. However, the potential function of FIGNL1 in HCC is still unknown. This study aims to investigate the key regulatory mechanisms of FIGNL1 in the formation of HCC. The regulatory effect of FIGNL1 on HCC was studied by lentivirus infection. In vitro, the effects of FIGNL1 on the proliferation, migration and apoptosis of cells were investigated by CCK8, colony formation assay, transwell and flow cytometry. Meanwhile, the regulation of FIGNL1 on HCC formation in vivo was studied by subcutaneous transplanted tumors. In addition, using transcriptome sequencing technology, we further explored the specific molecular mechanism of FIGNL1 regulating the formation of HCC. Functionally, we demonstrated that FIGNL1 knockdown significantly inhibited HCC cell proliferation, migration and promoted cell apoptosis in vitro. Similarly, the knockdown of FIGNL1 meaningfully weakened hepatocarcinogenesis in nude mice. Transcriptome sequencing revealed that FIGNL1 affected the expression of genes involved in extracellular matrix-receptor (ECM-receptor) interaction pathway, such as hyaluronan mediated motility receptor (HMMR). Further validation found that overexpression of HMMR based on knockdown FIGNL1 can rescue the expression abundance of related genes involved in the ECM-receptor interaction pathway. Our study revealed that FIGNL1 could modulate the ECM-receptor interaction pathway through the regulation of HMMR, thus regulating the formation of HCC.

Abstract 3011: The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) stands as the fourth leading cause of global cancer-related mortality due to late-stage detection, tumor heterogeneity, and drug resistance. The pivotal role of epigenetic factors in HCC development and progression necessitates the identification of critical epigenetic regulators. The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3), primarily associated with retinal development, has emerged as a potential tumor suppressor. Earlier studies showed that the loss of NR2E3 leads to epigenetic repression of the estrogen receptor α (ER) through the involvement of Lysine Specific histone Demethylase 1 (LSD1). NR2E3 also forms a transcriptional complex with Sp1, governing the aryl hydrocarbon receptor (AHR) expression, a crucial factor in liver xenobiotic metabolism. In vivo, NR2E3 ablation results in p53 inactivation and severe liver injuries when exposed to liver toxicants, highlighting its role in activating p53. Nonetheless, the precise tumor suppressive and epigenetic role of NR2E3 in HCC remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice show accelerated liver tumor formation and progression accompanied by enhanced activation of the Wnt/β-catenin signaling pathway and inactivation of the p53 signaling pathway. At a cellular level, losing NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates vital genes in the Wnt/β-catenin pathway with enhanced chromatin accessibility. This event is mediated through the increased formation of active transcription complex involving Sp1, β-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. Correspondingly, treatment of p300 inhibitor C646 decreased the expression of critical genes in the Wnt/β-catenin signaling pathway. These findings demonstrate that the loss of NR2E3 promotes Wnt/β-catenin signaling activation at cellular, organismal, and clinical levels. In summary, NR2E3 is a novel tumor suppressor that maintains epigenetic homeostasis, thereby preventing activation of Wnt/β-catenin signaling that promotes HCC formation. Citation Format: Yuet-Kin Leung, Sung-Gwon Lee, Jiang Wang, Shuk-Mei Ho, Nancy Rusch, Chungoo Park, Kyounghyun Kim. The loss of an orphan nuclear receptor NR2E3 augments Wnt/β-Catenin signaling via epigenetic dysregulation that links to the Sp1-β catenin-p300 interactions in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3011.

Abstract 630: The role of AKT pathway in β-catenin mutated hepatocellular carcinoma

Abstract The Wnt/β-catenin and PI3K/AKT pathways are pivotal contributors to the development of the malignant phenotype in cancer. The activated Wnt/β-catenin pathway synergizes with various signaling cascades to propel HCC formation, operating through its downstream effectors. It has been well-established that the mammalian target of rapamycin complex 2 (mTORC2) serves as an upstream regulator of AKT, orchestrating the phosphorylation and activation of AKT. Once activated, AKT phosphorylates and inhibits canonical downstream targets like forkhead box O protein (FOXO) and tuberous sclerosis complex 2 (TSC2). Studies indicated that AKT-mediated phosphorylation of TSC2 activates mTORC1, subsequently suppressing autophagy. However, there are limited data exist on the interplay between Wnt/β-catenin and AKT pathways in HCC. In this study, we sought to explore the role of the AKT pathway in β-catenin activated HCC tumors. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (N90-β-catenin) and c-MET in the livers of mice to establish the HCC model (c-Met/β-catenin model). Notably, the knockout of Rictor, a component of the mTORC2 complex, significantly impeded tumor growth, underscoring the pivotal role of AKT in c-Met/β-catenin tumor development. AKT1 and AKT2 are the two main isoforms expressed in the liver. We determined that both AKT1 and AKT2 isoforms are critical for c-Met/β-catenin tumor growth. Intriguingly, simultaneous knockout of Rictor and activation of mTORC1 in the c-Met/β-catenin model resulted in delayed tumor growth, indicating that mTORC1 is not the sole downstream determinant of AKT signaling during c-Met/β-Catenin tumor development. Furthermore, overexpression of the activated FOXO1 (FOXO1AAA) significantly delayed the tumor growth, which highlighted the importance of FOXO1. Given the limited understanding of FOXO1 in HCC, we extended our investigation on FOXO1 to human HCC cell lines. Overexpression of FOXO1AAA notably delayed tumor growth in vitro. RNAseq analysis of FOXO1AAA-overexpressed human HCC cell lines revealed a significant upregulation of "metabolic pathways", suggesting metabolic reprogramming post FOXO1 activation. This study, for the first time, unveils the role of the AKT pathway in β-catenin-mutated HCC growth and highlights the potential therapeutic value of targeting the AKT pathway in patients with β-catenin-mutated HCC. Citation Format: Xue Wang, Xin Chen. The role of AKT pathway in β-catenin mutated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 630.

Non-alcoholic fatty liver disease and the risk of malignant tumors

The prevalence of non-alcoholic fatty liver disease (NAFLD) and metabolic associated liver disease (MAFLD) is growing world-wide. A new terminology (MAFLD) allows us not only to focus on the “metabolic” genesis of this pathology, but also to take into account other factors affecting damage to hepatocytes, such as alcohol consumption in low doses, viral and toxic hepatitis. Currently, obesity is a pathology, that is growing with MAFLD and causes of various non-communicable diseases. Most deaths in patients with NAFLD/MAFLD are caused, firstly, by adverse cardiovascular events, secondly, by malignant tumors of both the digestive organs (liver, intestine, esophagus, stomach and pancreas) and other localizations (kidney cancer in men, breast cancer in women) and, thirdly, by development of hepatic complications (cirrhosis, hepatocellular carcinoma – HCC). Because of the pandemic growth of MAFLD and its association with cardiovascular diseases and obesity, the question about properly clinical management of patients suffered from comorbid pathology to reduce the risks of deaths is timely and very relevant. This review has been prepared to systematize the available literature dates about association of NAFLD/MAFLD with the malignant tumors. A literature searches were conducted, modern epidemiological dates about the prevalence of NAFLD/MAFLD in the population and their complicated forms were presented. The risk of HCC formation both with and without cirrhosis in NAFLD was assessed. It was found that the severity of liver fibrosis can be useful predictor of the future risk of not only the adverse cardiovascular events, but also the malignant tumors in patients with NAFLD/MAFLD. Possible targets for treatment were discussed, the impact on which is useful for the treatment and prevention of progressive forms of the disease. One of the possible therapeutic molecules is essential phospholipids, which are currently included in the consent documents for the managment of patients with NAFLD.

IMMT promotes hepatocellular carcinoma formation via PI3K/AKT/mTOR pathway

Abstract Objectives Hepatocellular carcinoma (HCC) remains a global challenge. Finding new specific targets has significant clinical value for the treatment of hepatocellular carcinoma. In this study, we discovered a new biomarker targeting HCC. At present, the role of Inner Membrane Mitochondrial Protein (IMMT) in various malignant tumors is receiving increasing attention, but its molecular mechanism of action in the malignant process of HCC has not been fully elucidated. This study aims to investigate the key regulatory mechanisms of IMMT in the formation of HCC. Methods Using TCGA data and clinical HCC samples, we first studied the expression of IMMT in HCC tissues and its correlation with malignant prognosis of patients. The regulatory effect of IMMT on HCC was studied by lentivirus infection. In vitro, the effects of IMMT on the proliferation, migration and apoptosis of cells were investigated by CCK8, colony formation assay, transwell and flow cytometry. Consistently, in vivo experiments, the regulation of tumor growth by IMMT was studied by constructing subcutaneous transplanted tumor and liver carcinoma in situ. In terms of mechanism, we predicted and verified the downstream genes of IMMT with the help of string database. Results We found that IMMT was significantly up-regulated in HCC tissues and was significantly positively correlated with poor prognosis of patients. Functionally, we demonstrated that IMMT knockdown significantly inhibited HCC cell proliferation, migration and promoted cell apoptosis in vitro. Similarly, the knockdown of IMMT also significantly weakened the progression of tumors in vivo. In terms of mechanism, we demonstrate for the first time that IMMT can regulate the progression of HCC by influencing the activation of PI3K-AKT-mTOR pathway. Conclusions Collectively, our research findings elucidate the hitherto unexplored important role of the IMMT/PI3K/AKT/mTOR axis in the formation of HCC, and provide a new biomarker for clinical diagnosis and treatment of HCC.

Abstract 1654: Mouse models of hepatocellular carcinoma: A comprehensive and functional preclinical platform for immunotherapy research

Abstract Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and accounts for over 80% of primary liver cancer worldwide. Early stage HCC can be treated by local ablation, surgical resection or liver transplantation. Systemic pharmacological options are limited (kinase and immune-checkpoint inhibitors). Most cases of liver cancer occur in the setting of chronic liver diseases. Risk factors include chronic Hepatitis B and C, alcohol addiction and metabolic diseases. HCC is a multistep process comprising chronic liver injury, inflammation, fibrosis/cirrhosis and cancer formation. Therefore, providing palliative and curative options remains a high medical need. And with the recent success of immunotherapies in HCC, mouse models that better recapitulate the human disease and antitumor immune response are needed. In order to better evaluate new preventive and curative treatments of liver cancers we developed complementary and integrated strategies to mimic the liver cancer initiation and progression steps in mouse models. These models involve chemotoxic agents, diet-induced disorders and syngeneic or xenogeneic tumor implantation strategies. We established an orthotopic syngeneic model using Hepa1.6 mouse liver hepatoma cells, characterized through liver index, alpha-fetoprotein measurement in serum and liver, and MRI. The response to chemotherapy (sorafenib) and immunotherapy (anti PD-1, anti-TLR) was also assessed and show moderate to high efficiency. Moreover, a panel of xenograft models including Patient-Derived Xenograft models (PDXs) are available to assess new treatment options in human HCC with regards of the genetic mutations and the variety of etiologies seen in human. But as xenograft models are not completely mimicking the human situation of both immune and liver microenvironment, we have recently initiated the development of a double humanized (immune and liver) transgenic mouse as a better host for human tumor engraftment. During the course of HCC formation, the liver undergoes cycles of inflammation, necrosis with regeneration, fibrosis, cell dysplasia and ultimately HCC. Thus, we developed a model induced by a low dose of streptozotocin and high fat diet regimen. In this model, mice develop metabolic syndrome NASH and fibrosis within 12 weeks and HCC within 16 weeks. Of interest, 100% of male mice develop HCC within 16 weeks, in accordance with studies showing that men had a 2-to 7-fold higher risk of developing HCC in human. Treatment of mice with lenvatinib alone or in combination with anti-PD1 increases survival and reduces tumor burden as shown with reduced liver weight/body weight ratio at 16 weeks. Altogether, these results demonstrate the usefulness of this comprehensive platform of preclinical in vivo HCC models to discover and identify novel therapeutic strategies that could circumvent the progression of liver cancers. Citation Format: Olivier Duchamp, Gael Krysa, Loic Morgand, Hugo Quillery, Robin Artus, Maxime Ramelet, Jeremy Odillard, Peggy Provent, Sylvie Maubant, Caroline Mignard, Fabrice Viviani, Samira Benhamouche-Trouillet. Mouse models of hepatocellular carcinoma: A comprehensive and functional preclinical platform for immunotherapy research [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 1654.

The association between metabolic risk factors, nonalcoholic fatty liver disease, and the incidence of liver cancer: a nationwide population-based cohort study.

Liver cancer is a detrimental complication in patients with chronic viral hepatitis and alcoholic or nonalcoholic fatty liver disease (NAFLD). However, metabolic risk factors underlying NAFLD usually cause substantial differences in their clinical outcomes. Recently, several studies have used a novel definition of metabolic dysfunction-associated fatty liver disease (MAFLD) to reassess patients with NAFLD and pointed out the importance of metabolic risk factors. Since patients with NAFLD, MAFLD, or metabolic syndrome (MetS) have different burden of metabolic risk factors, it is crucial to decipher the risk of developing hepatic complications in these populations. Through a longitudinal nationwide cohort study, the risk of liver cancer was investigated in patients with MetS alone, NAFLD alone, overlap NAFLD/MAFLD, and coexisting MetS and NAFLD. The general characteristics, comorbidities, and incidence of liver cancer were also compared. Intriguingly, patients diagnosed with MetS alone did not have a significant risk of developing HCC compared to control individuals, while patients with NAFLD alone, NAFLD/MAFLD, and coexisting NAFLD and MetS exhibited 6.08-, 5.81-, and 15.33-fold risks of developing HCC, respectively. Apart from metabolic risk factors, renal function status and liver cirrhosis were the independent risk factors for the development of HCC among these groups. Our data emphasize that metabolic dysfunction has a significant impact on hepatocarcinogenesis in patients with NAFLD. Moreover, coexisting multiple metabolic risk factors would dampen the risk of developing HCC in patients with NAFLD. Closely tracing HCC formation through laboratory examination or imaging is crucial in these patients.

Beyond the Usual Suspects: Hepatitis E Virus and Its Implications in Hepatocellular Carcinoma.

Simple SummaryCancer is a major threat to global health, accounting worldwide for nearly 10 million fatalities in 2020. Importantly, carcinogenesis caused by so-called oncoviruses accounts for approximately 10% of the global cancer burden. Specifically, hepatitis B virus, hepatitis C virus and to a lesser extent hepatitis D virus infection have been recognized to be mainly responsible for the occurrence of hepatocellular carcinoma (HCC). Recent studies have further drawn attention to a long-neglected hepatotropic virus, namely hepatitis E virus (HEV), in the context of HCC. Here, we summarize current epidemiological, clinical and experimental studies to unravel a putative link between HEV and HCC and provide an outlook for future scientific efforts in HEV-related HCC research.Hepatitis E virus infections are the leading cause of viral hepatitis in humans, contributing to an estimated 3.3 million symptomatic cases and almost 44,000 deaths annually. Recently, HEV infections have been found to result in chronic liver infection and cirrhosis in severely immunocompromised patients, suggesting the possibility of HEV-induced hepatocarcinogenesis. While HEV-associated formation of HCC has rarely been reported, the expansion of HEV’s clinical spectrum and the increasing evidence of chronic HEV infections raise questions about the connection between HEV and HCC. The present review summarizes current clinical evidence of the relationship between HEV and HCC and discusses mechanisms of virus-induced HCC development with regard to HEV pathogenesis. We further elucidate why the development of HEV-induced hepatocellular carcinoma has so rarely been observed and provide an outlook on possible experimental set-ups to study the relationship between HEV and HCC formation.

Combination of curcumin with N-n-butyl haloperidol iodide inhibits hepatocellular carcinoma malignant proliferation by downregulating enhancer of zeste homolog 2 (EZH2) - lncRNA H19 to silence Wnt/β-catenin signaling

BackgroundHepatocellular carcinoma (HCC) is one of the most common cause of cancer-related death worldwide. Curcumin (C) has been extensively investigated in different types of malignancies, including hepatocellular carcinoma, but its physicochemical properties have significantly influenced its clinical use. Several approaches are being explored to enhance curcumin's therapeutic response, including its combination with various drugs. PurposeThis study aimed to evaluate the anti-tumor effect of curcumin (C) in combination with F2 (N-n-butyl haloperidol iodide) on hepatocellular carcinoma and its potential underlying mechanism in vitro and in vivo. MethodsCell proliferation was evaluated by CCK-8 and colony formation assays, and apoptosis was measured by flow cytometry. The migratory and invasive abilities of Hep3B and SMMC-7721 cells were measured by wound-healing and matrigel transwell assays. In order to investigate the molecular pathways, various experiments such as western blotting, qPCR, RNA-seq, immunostaining and transfection were performed. To evaluate the anti-HCC effects in vivo, a xenograft tumor model was used. ResultsOur findings showed that the combination of curcumin (C) & F2 (F2C) strongly inhibited malignant proliferation and migration in SMMC-7721 and Hep3B cells. The F2C treatment downregulates enhancer of zeste homolog 2 (EZH2) transcription and protein expression, which is key epigenetic regulator responsible for HCC development. Moreover, the inhibition of EZH2 by F2C led to Wnt/β-catenin signaling inhibition by decreasing tri-methylation of histone H3 at lysine 27 (H3K27me3) and long non-coding RNA H19 expression. The inhibition of F2C was associated with the suppression of tumorigenicity in xenograft HCC models. ConclusionThese findings suggested that, F2C inhibited HCC formation, migration and its modulatory mechanism seemed to be associated with downregulation of EZH2, silencing Wnt/β-catenin signaling by interacting with H19, suggesting that F2C may be a promising drug in the clinical treatment of HCC.

Circ-CSPP1 knockdown suppresses hepatocellular carcinoma progression through miR-493-5p releasing-mediated HMGB1 downregulation

BackgroundHepatocellular carcinoma (HCC) accounts for over 80% of primary liver cancers and leads to a high death rate. Research on circular RNAs (circRNAs) suggests that circRNAs are promising biomarkers for cancer treatment. This study aimed to explore the function of a novel circRNA (circ-CSPP1) in HCC. MethodsCirc-CSPP1 was obtained from the microarray data downloaded from the Gene Expression Omnibus (GEO) database. The expression of circ-CSPP1, miR-493-5p and high mobility group box 1 (HMGB1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, colony formation ability, migration and invasion were monitored using cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and transwell assay, respectively. The protein levels of CyclinD1, Vimentin, matrix metallopeptidase 9 (MMP-9) and HMGB1 were detected by western blot. Xenograft models were established to investigate the function of circ-CSPP1 in vivo. The association between miR-493-5p and circ-CSPP1 or HMGB1 was predicted by the online tool starBase and ensured by dual-luciferase reporter assay. ResultsThe expression of circ-CSPP1 and HMGB1 was elevated, while the expression of miR-493-5p was declined in HCC tissues and cells. Circ-CSPP1 knockdown not only depleted HCC cell proliferation, formation, migration and invasion in vitro but also inhibited tumor growth in vivo. MiR-493-5p was a target of circ-CSPP1, and HMGB1 was a target of miR-493-5p. Rescue experiments presented that miR-493-5p deficiency reversed the effects of circ-CSPP1 knockdown, and HMGB1 overexpression reversed the effects of miR-493-5p restoration. Circ-CSPP1 sponged miR-493-5p to regulate HMGB1 expression. ConclusionKnockdown of circ-CSPP1 suppressed HCC development both in vitro and in vivo by upregulation of miR-493-5p and downregulation of HMGB1, hinting that circ-CSPP1 participated in HCC pathogenesis.

Overexpression of Mothers Against Decapentaplegic Homolog 7 Activates the Yes-Associated Protein/NOTCH Cascade and Promotes Liver Carcinogenesis in Mice and Humans.

Mothers against decapentaplegic homolog (SMAD) 7 is an antagonist of TGF-β signaling. In the present investigation, we sought to determine the relevance of SMAD7 in liver carcinogenesis using in vitro and in vivo approaches. We found that SMAD7 is up-regulated in a subset of human HCC samples with poor prognosis. Gene set enrichment analysis revealed that SMAD7 expression correlates with activated yes-associated protein (YAP)/NOTCH pathway and cholangiocellular signature genes in HCCs. These findings were substantiated in human HCC cell lines. In vivo, overexpression of Smad7 alone was unable to initiate HCC development, but it significantly accelerated c-Myc/myeloid cell leukemia 1 (MCL1)-induced mouse HCC formation. Consistent with human HCC data, c-Myc/MCL1/Smad7 liver tumors exhibited an increased cholangiocellular gene expression along with Yap/Notch activation and epithelial-mesenchymal transition (EMT). Intriguingly, blocking of the Notch signaling did not affect c-Myc/MCL1/Smad7-induced hepatocarcinogenesis while preventing cholangiocellular signature expression and EMT, whereas ablation of Yap abolished c-Myc/MCL1/Smad7-driven HCC formation. In mice overexpressing a myristoylated/activated form of AKT, coexpression of SMAD7 accelerated carcinogenesis and switched the phenotype from HCC to intrahepatic cholangiocarcinoma (iCCA) lesions. In human iCCA, SMAD7 expression was robustly up-regulated, especially in the most aggressive tumors, and directly correlated with the levels of YAP/NOTCH targets as well as cholangiocellular and EMT markers. The present data indicate that SMAD7 contributes to liver carcinogenesis by activating the YAP/NOTCH signaling cascade and inducing a cholangiocellular and EMT signature.

Synergistic Effect of MC-LR and C-Terminal Truncated HBx on HepG2 Cells and Their Effects on PP2A Mediated Downstream Target of MAPK Signaling Pathway

C-terminally truncated hepatitis B virus (HBV) X (ctHBx) infection and exposure to microcystins-LR (MC-LR) can lead to human hepatitis and liver cancer, but the mechanism associated with their synergistically effects not been fully elucidated. The ctHBx (HBxΔ4 and HBxΔ32) lentivirus were constructed and transfected into the HepG2 cells. Then we investigated the function of MC-LR and ctHBx using the molecular biology approaches, including enzyme-linked immunosorbent assay, clone formation assay, scratch wound testing, transwell assays, carried out flow cytometry respectively to examine cell cycle and apoptosis in each group, and detected the related proteins of HBx, MEK/ERK/JNK/p38 in mitogen-activated protein kinase (MAPK) pathway and the downstream proteins such as cdc2, cdc25C, and p53 by western blotting. We found that the protein phosphorylase 2A (PP2A) enzyme activity in MC-LR and HBxΔ32/HBxΔ4 groups decreased more than in MC-LR and HBx group at the same time point and MC-LR concentration (P < 0.05). Meanwhile the proliferation, migration, invasion and colony formation capability of HepG2 cells were significantly enhanced in MC-LR and ctHBx groups (P < 0.05). In addition the proportion of S stage cells in the MC-LR-treated HBxΔ32/HBxΔ4 groups was significantly greater than that in the untreated groups (P < 0.05). Furthermore, the protein expression of MAPK signaling pathway including phospho-MEK1/2, ERKl/2, p38, and JNK were up-regulated by MC-LR and HBxΔ32, and the expression of cyclin-related proteins, including p53, cdc25C, and cdc2 were also activated (P < 0.05). Taken together, our findings revealed the essential significance of the MC-LR and ctHBx on the PP2A/MAPK/p53, cdc25C and cdc2 axis in the formation and development of HCC and identified MC-LR and ctHBx as potential causal cofactors of hepatocarcinogenesis.

Abstract 5728: Liver progenitor cells induces fibrosis via RAGE signaling upon liver injury

Abstract Liver fibrosis is characterized by hepatic stellate cell activation and extracellular matrix deposition upon persistent injury and inflammation, which can impair hepatic function and its ability to regenerate. The origin and physiological role of facultative liver progenitor cells (LPCs) have been a controversial issue as it was found to be a major player to regenerate the damaged liver, but it is also associated with fibrosis, disease progression or tumor initiation. The receptor for advanced glycation end products (RAGE) signaling axis is often associated with chronic inflammation-associated tissue damage and plays an essential role in modulating the tumor microenvironment. Our previous data suggested that RAGE mediates LPC expansion, onset of liver fibrosis and HCC formation (Pusterla et al. Hepatology. 2013.) Hence, in this study, we seek to delineate the functional role and underlying mechanism of RAGE activity in LPC activation in response to inflammation-associated liver injury. R26TomHnf1β-CreER transgenic mice were crossed with Rage flox/flox (Ragefl/fl) mice to generate tamoxifen-inducible LPC-specific RAGE knockout mice (RAGEΔLPC). They were exposed to a choline-deficient ethionine-supplemented (CDE) diet for three weeks to induce liver damage. Ablation of RAGE in LPCs strongly impairs LPC expanding capacities in CDE diet-treated mice. Strikingly, this is accompanied by reduction of activated hepatic stellate cells and bridging fibrosis. This demonstrated that RAGE signaling in LPCs is a mediator of liver fibrosis in vivo. Necroinflammation is known to be associated with liver fibrosis. To investigate the role of RAGE in LPCs in the context of necroinflammation in vitro, primary LPCs were isolated from CDE-treated Ragefl/fl C57BL/6 mouse. Wildtype and knockout Rage cell lines were established. LPCs were stimulated with supernatants from necrotic hepatocytes followed by whole transcriptome sequencing to identify downstream targets of RAGE-dependent pathways. Stress response, inflammatory and pro-fibrotic pathways were enriched in LPCs upon treatment with necrotic medium. Most interestingly, signaling pathways that regulate organ size, tissue homeostasis and cell survival were found to be RAGE-dependent. Moreover, clusters of stem cell renewal-related genes were deregulated upon ablation of RAGE. In line with the whole transcriptome profile, we demonstrated that ablation of RAGE attenuates LPCs organoid-forming ability, implying that RAGE regulates stemness properties of LPCs. Our recent results demonstrated that RAGE is required for LPCs activation and proliferation, as well as the crosstalk with stellate cells in supporting fibrogenesis. Taken together, our data uncovers a potential mechanistic insight on the role of RAGE in LPCs in association with fibrosis upon chronic liver injury. Citation Format: Wai Ling Macrina Lam, Gisela Gabernet, Tanja Poth, Aurora De Ponti, Anna Saborowski, Sven Nahnsen, Doris Schneller, Peter Angel. Liver progenitor cells induces fibrosis via RAGE signaling upon liver injury [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5728.

Abstract 5972: HLJ1 deficiency enhanced diethylnitrosamine-induced hepatocarcinogenesis in a gene targeting mouse model

Abstract Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related death worldwide. The conventional treatments such as surgical resection and liver transplantation are applicable to only a small proportion of patients and prolongation of survival is restricted. Therefore, it is urgent to develop cancer biomarkers for defining cancer risk, diagnosis, prognosis and even find new therapeutic targets. Human Liver DnaJ-Like Protein (HLJ1), which belongs to DnaJ heat shock protein family (HSP40) member B4 (DNAJB4), has been reported to act as a tumor suppressor in NSCLC, colorectal cancer, melanoma metastasis. However, the precise role of HLJ1 in liver cancer tumorigenesis and the progress of HCC carcinogenesis has not been elucidated yet. Thus, we investigate whether HLJ1 has tumor-suppressive or oncogenic role on DEN-induced liver cancer development in a mouse model. Wild-type (WT) and HLJ1 knockout (HLJ1−/−) mice are injected with 20 mg/kgbw DEN at postnatal day 15. Thereafter, 50 mg/kgbw DEN is administrated weekly to mice between age 4 to 12 weeks. Mice were sacrificed at age 30 and 36 weeks to assess the tumor count and size. In another group, mice are injected with DEN and age day 15 and then 500 ppm PB is administrated in drinking water until mice were sacrifice at age 52 weeks. The liver tissues are frozen and fixed for western blotting and IHC analysis. For cDNA microarray analysis, gene expression microarray samples were from 6-8 weeks wild-type and HLJ1−/− mice livers. When sacrificed at age 30 and 36 weeks, HLJ1−/− mice exhibited higher tumor multiplicity, serum ALT, AST levels, and larger macroscopic tumor nodules than WT mice. In DEN+PB treatment group, higher incidence rate can be observed in HLJ1−/− mice than in WT mice. This indicated HLJ1 protects liver from DEN-induced HCC carcinogenesis and knockout of HLJ1 confers to more severe HCC induction. Furthermore, p-STAT3 expression was higher in normal part of HLJ1−/− mice liver compared to that of WT mice liver, which suggest the role of HLJ1 in regulating p-STAT3 signaling in the early stage of tumor formation. Using IHC staining and western blotting to analyze HLJ1 expression level, we found half of the tumors express higher HLJ1 than adjacent normal part did, which suggested a tumor-promoting role of HLJ1 after tumor initiation and formation. In cDNA microarray analysis, network analysis of genes enriched in HCC map folder revealed involved objects JunB, c-Myc, p21 and Cyclin D1, which was further confirmed by quantitative PCR. p21 and Cyclin D1 were highly expressed in the liver of HLJ1−/− comparing to that of WT mice, which suggested HLJ1-related genes p21 and Cyclin D1 may have roles in HCC formation. Clinical information of 362 liver cancer patients from TCGA database exhibited that higher expression of HLJ1 in tumor part is correlated to lower disease-specific survival rate. It indicated that HLJ1 may be a tumor marker for HCC prognosis and clinical outcome prediction. It also suggested that HLJ1 may play an oncogenic role after tumor formation. In summary, HLJ1 has protective role against DEN-induced hepatocarcinogenesis through down-regulated p-STAT3 and cyclin D1 whereas it could also be an enhancer to promote tumor progression after tumor formation. Hence, HLJ1 could be a target for liver cancer therapy and a prognostic biomarker in the future. Citation Format: Wei-Jia Luo, Min-Hui Chien, Jeremy J. W. Chen, Sung-Liang Yu, Pan-Chyr Yang, Kang-Yi Su. HLJ1 deficiency enhanced diethylnitrosamine-induced hepatocarcinogenesis in a gene targeting mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5972.

Abstract 5638: Liver preclinical models - acute, chronic and cancer models

Abstract Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and accounts for &amp;gt;80% of primary liver cancer worldwide. Early stage HCC can be treated by local ablation, surgical resection or liver transplantation. Systemic pharmacological options are limited and only a few available (kinase and immune-checkpoint inhibitors). Most cases of liver cancer occur in the setting of chronic liver diseases. Risk factors include chronic Hepatitis B and C, alcohol addiction and metabolic diseases. HCC is a multistep process comprising chronic liver injury, inflammation, fibrosis/cirrhosis and cancer formation. Therefore, providing palliative and curative options remains a high medical need. In order to better evaluate new preventive and curative treatments of liver cancers we developed complementary and integrated strategies to mimic the liver cancer initiation and progression steps in mouse models. These models involve chemotoxic agents, diet-induced disorders and implantation strategies. We first established an orthotopic syngeneic model using Hepa1.6 mouse liver hepatoma cells. Hepa1.6-derived tumor growth was characterized through liver index, alpha-fetoprotein measurement in serum and liver, and MRI. The response to chemotherapy (Sorafenib) and immunotherapy (anti PD-1) was also assessed. Moreover, a panel of Patient-Derived Xenograft models (PDXs) are available to assess new treatment options in human HCC with regards of the genetic mutations and the variety of etiologies seen in human. During the course of HCC formation, the liver undergoes cycles of inflammation, necrosis with regeneration, fibrosis, cell dysplasia and ultimately HCC. Thus, we developed two models of non- alcoholic steato-hepatitis (NASH); The first model involves chronic administration of hepatotoxic CCL4 associated with a high-fat and high-fructose diet. Mice develop steatohepatitis and fibrosis within 4 to 8 weeks as assessed with biochemistry parameters (AST, ALT…), gene expression levels of inflammatory and fibrotic genes as well as histological scores and MRI. The effect of obeticholic acid was successfully evaluated in this model. The second model is the STAM model induced by a low dose of Streptozotocin and high fat diet regimen. In this model, mice develop metabolic syndrome (increased body weight gain, hyperglycemia and hyperlipidemia), NASH (steatosis, inflammation, fibrosis) within 12 weeks and HCC within 16 weeks. The responses to standard of care and to immunotherapy together with the characterization of the immune cell populations are currently under investigation. Altogether, these results demonstrate the usefulness of this liver diseases development program to discover and identify new treatments that could circumvent the progression of liver cancer. Citation Format: Olivier Duchamp, Gael Krysa, Robin Artus, Hugo Quillery, Maxime Ramelet, Valerie Boullay, Laure Levenez, Adelaide Ferment, Jeremy Odillard, Anne-Benedict Boullay, Peggy Provent, Edwige Nicodeme, Samira Benhamouche-Trouillet, Anne Lazzari, Loic Morgand. Liver preclinical models - acute, chronic and cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5638.

Functional role of SGK3 in PI3K/Pten driven liver tumor development

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer related deaths worldwide. The PI3K cascade is one of the major signaling pathways underlying HCC development and progression. Activating mutations of PI3K catalytic subunit alpha (PIK3CA) and/or loss of Pten often occur in human HCCs. Serum and glucocorticoid kinase 3 (SGK3) belongs to the SGK family of AGK kinases and functions in parallel to AKT downstream of PI3K. Previous studies have shown that SGK3 may be the major kinase responsible for the oncogenic potential of PIK3CA helical domain mutants, such as PIK3CA(E545K), but not kinase domain mutants, such as PIK3CA(H1047R).MethodsWe investigated the functional contribution of SGK3 in mediating activated PIK3CA mutant or loss of Pten induced HCC development using Sgk3 knockout mice.ResultsWe found that ablation of Sgk3 does not affect PIK3CA(H1047R) or PIK3CA(E545K) induced lipogenesis in the liver. Using PIK3CA(H1047R)/c-Met, PIK3CA(E545K)/c-Met, and sgPten/c-Met murine HCC models, we also demonstrated that deletion of Sgk3 moderately delays PIK3CA(E545K)/c-Met driven HCC, while not affecting PIK3CA(H1047R)/c-Met or sgPten/c-Met HCC formation in mice. Similarly, in human HCC cell lines, silencing of SGK3 reduced PIK3CA(E545K) -but not PIK3CA(H1047R)- induced accelerated tumor cell proliferation.ConclusionAltogether, our data suggest that SGK3 plays a role in transducing helical domain mutant PIK3CA signaling during liver tumor development.

A Potential Antitumor Effect of Dendritic Cells Fused with Cancer Stem Cells in Hepatocellular Carcinoma.

HCC stem cells were reported as posttreatment residual tumor cells that play a pivotal role in tumor relapse. Fusing dendritic cells (DCs) with tumor cells represents an ideal approach to effectively activate the antitumor immunity in vivo. DC/HCC stem cell vaccine provides a potential strategy to generate polyclonal immune response to multiple tumor stem cell antigens including those yet to be unidentified. To assess the potential capacity of DC/HCC stem cell vaccines against HCC, CD90+HepG2 cells were sorted from the HCC cell line HepG2. DC and CD90+HepG2 and DC and HepG2 fused cells were induced by polyethylene glycol (PEG). The influence of fusion cells on proliferation and immunological function transformation of lymphocytes was assessed by FCM and ELISA assay, respectively. The cytotoxicity assay of specific fusion cell-induced CTLs against HepG2 was conducted by CytoTox 96 Non-Radioactive Cytotoxicity Assay kit in vitro. At last, the prevention of HCC formation in vivo was described in a mouse model. The results of FCM analysis showed that the proportion of CD90+HepG2 cells in the spheral CD90+HepG2 enriched by suspension sphere culture was ranging from 98.7% to 99.5%, and 57.1% CD90+HepG2/DC fused cells were successfully constructed. The fusion cells expressed a higher level of costimulatory molecules CD80, CD83, CD86, and MHC-I and MHC-II molecules HLA-ABC and HLA-DR than did immature DCs (P < 0.05). And the functional analysis of fusion cell-induced CTLs also illustrated that CD90+HepG2/DC fusion cells showed a greater capacity to activate proliferation of lymphocytes in vitro (P < 0.05). The CD90+HepG2/DC-activated CTLs had a specific killing ability against CD90+HepG2 cells in vivo. These results suggested that CD90+HepG2/DC fusion cells could efficiently stimulate T lymphocytes to generate specific CTLs targeting CD90+HepG2 cells. It might be a promising strategy of immunotherapy for HCC.

Activity of IL-12/15/18 primed natural killer cells against hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is common, but remains difficult to treat. Natural killer (NK) cells are cells of the innate immune system that have potent anti-cancer activity. Recent work has shown that stimulation with IL-12/15/18 leads to the generation of NK cells with enhanced functional and putative “memory” properties. We have investigated the activity of these NK cells against HCC cell lines in vitro and in a mouse model.MethodsNK cells from healthy donors or individuals with HCC were activated with IL-12/15/18 in vitro and tested for cytotoxic activity against a panel of human HCC cell lines. IL-12/15/18 primed murine NK cells were then infused into a murine model of spontaneously arising HCC to test for anti-tumor activity.ResultsNK cells from patients and healthy controls had similar expression levels of activating and inhibitory NK cell receptors. However, proliferation of NK cells from HCC patients was weaker than healthy controls in response to IL-12/15/18 and IL-2 (p < 0.001 at day 9). In vitro, NK cells from both groups of individuals killed HCC targets to similar levels and this was unrelated to NKG2D expression. In a spontaneous model of HCC, IL-12/15/18 activated NK cells trafficked to the liver and resulted in lower levels of spontaneous HCC formation (p < 0.01).ConclusionCytokine-primed NK cells from patients with HCC have similar levels of activity against HCC cell lines as those from healthy controls. This type of activated NK cell has immunotherapeutic potential against hepatocellular carcinoma.

Oncogenic potential of N-terminal deletion and S45Y mutant \u03b2-catenin in promoting hepatocellular carcinoma development in mice

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with limited treatment options. Mutation of β-catenin is one of the most frequent genetic events along hepatocarcinogenesis. β-catenin mutations can be in the form of point mutation or large N-terminal deletion. Studies suggested that different β-catenin mutations might have distinct oncogenic potential.MethodsWe tested the oncogenic activity of β-cateninS45Y, one of the most frequent point mutations of β-catenin, and ∆N90-β-catenin, a form of β-catenin with a large N-terminal deletion, in promoting HCC development in mice. Thus, we co-expressed β-cateninS45Y or ∆N90-β-catenin together with c-Met into the mouse liver using hydrodynamic injection.ResultsWe found that both β-catenin mutations were able to induce HCC formation in combination with c-Met at the same latency and efficiency. Tumors showed similar histological features and proliferation rates. However, immunohistochemistry showed predominantly nuclear staining of β-catenin in c-Met/∆N90-β-catenin HCC, but membrane immunoreactivity in c-Met/β-cateninS45Y HCC. qRT-PCR analysis demonstrated that both ∆N90-β-catenin and β-cateninS45Y induced the same effectors, although at somewhat different levels. In cultured cells, both ∆N90-β-catenin and β-cateninS45Y were capable of inducing TCF/LEF reporter expression, promoting proliferation, and inhibiting apoptosis.ConclusionsOur study suggests that β-cateninS45Y and ∆N90-β-catenin, in combination with the c-Met proto-oncogene, have similar oncogenic potential. Furthermore, nuclear staining of β-catenin does not always characterize β-catenin activity.

Therapeutic Efficacy of Vitamin D in Experimental c-MET-β-Catenin-Driven Hepatocellular Cancer.

Aberrant activation of β-catenin signaling is frequently observed in hepatocellular cancer. Although Wnt/β-catenin signaling can be targeted by vitamin D, therapeutic use of vitamin D for this purpose is not currently established. We evaluated the therapeutic use of vitamin D or its analogs using a synthetic transgenic mouse of hepatocarcinogenesis induced by mutant β-catenin, and MET overexpression in which 75% of mice develop well-differentiated HCC within 8 weeks in the absence of fibrosis. Vitamin D receptor expression was similar in both tumoral and nontumoral tissue. There was no significant difference in overall survival, or in tumor progression assessed by imaging, biochemical, or tumor cell burden assessments in mice receiving a vitamin D-supplemented diet containing 12.0 IU VD/g (HVD) compared with a standard diet (SD) containing 2.3 IU VD/g. Furthermore, systemic treatment with calcitriol [vitamin D analog 1α,25(OH)₂D₃] or EB1089 (synthetic vitamin D analog) by intraperitoneal injection for 4 weeks prolonged median survival but did not increase overall survival compared with controls. Although tumor formation was delayed in males compared with that in females, there was no difference in overall survival between males and females. In conclusion, although 1α,25(OH)₂D₃ is reported to inhibit β-catenin signaling, as well as proliferation, migration, and differentiation in cancer cells, neither dietary supplementation with vitamin D nor treatment with vitamin D analogs altered the formation or growth of HCC associated with β-catenin activation. These results conclusively demonstrate the lack of utility of targeting vitamin D for therapy of HCC in this setting.