A Minimal Subset of Seven Genes Associated with Tumor Hepatocyte Differentiation Predicts a Poor Prognosis in Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the frequently diagnosed cancers worldwide and has emerged as the main cause of cancer-related death with a very poor prognosis, which is closely related to cancer stem cell (CSC), a subset of cells known to be a root of tumor recurrence, metastasis, and chemoresistance in HCC. However, the characteristics of CSCs in hepatocellular carcinoma (HCC) are still elusive. Our previous in vitro hepatocyte differentiation model revealed that the expression of anti-silencing function 1B histone chaperone (ASF1B) was remarkably upregulated in the stem cell-like stages. In HCC patients, ASF1B was highly expressed in cancer tissues compared with normal counterparts and was positively correlated with poor survival. Functional assays indicated that ASF1B could considerably promote the self-renewal, growth, and metastasis of HCC both in vitro and in vivo. Mechanistically, ASF1B could directly bind to histone H3 Variant H3.3 and enhance its incorporation into the promoter regions of certain stemness- and metastasis-associated genes including SOX9, KLF4, and TBX3, contributing to their augmented transcriptional and protein expression levels. H3.3 knock-down decreased the expression of these stemness and metastasis-associated genes, eliminating the stemness and oncogenic properties of ASF1B. Overall, our study revealed that ASF1B could facilitate HCC stemness and progression via H3.3-dependent transcriptional reprogramming. ASF1B might be a novel diagnostic and prognostic biomarker for HCC patients. Citation Format: Xiaona Fang, Beilei Liu, Shan Liu, Yu Zhang, Lanqi Gong, Qian Yan, Xinyuan Guan. ASF1B potentiates stem cell traits and tumor progression in hepatocellular carcinoma via histone H3.3-dependent transcriptional reprogramming [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 6065.

EFNA3 is a prognostic biomarker for the overall survival of patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Uncontrolled angiogenesis plays a critical role in hepatocellular tumor growth and metastasis. In this study, we aimed to investigate the effects of circular RNA hsa_circ_0000519 and the potential involvement of microRNA (miR)-1296 and E2F transcription factor 7 (E2F7) in HCC development. Hsa_circ_0000519 was highly expressed in HCC cells and hepatocellular tumor tissues, and correlated with poor prognosis of HCC patients. Knockdown of hsa_circ_0000519 significantly reduced HCC cell viability, suppressed cell proliferation, and induced cell cycle arrest in G0/G1. Downregulation of hsa_circ_0000519 also inhibited formation of capillary-like endothelial structures in vitro and impeded microvessel formation in mice bearing HCC tumors. The migration and invasive capacities of HCC cells were markedly reduced by hsa_circ_0000519 knockdown. Hsa_circ_0000519 possessed a binding site for microRNA (miR)-1296. Upregulation of hsa_circ_0000519 significantly decreased the miR-1296 expression in both HCC cells and mouse xenografts. Furthermore, E2F7 was a target of miR-1296. Hsa_circ_0000519 positively regulated E2F7 via acting as a miR-1296 sponge. Upregulation of E2F7 abolished the inhibitory effects of hsa_circ_0000519 knockdown on HCC cell proliferation and angiogenesis. In conclusion, hsa_circ_0000519 promoted tumor progression and angiogenesis in HCC through the miR-1296/E2F7 axis. These data suggest the potential clinical application of hsa_circ_0000519 in HCC treatment.

Background: A recent study reported the loss of gluconeogenic capacity in hepatocellular carcinoma (HCC). Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in some cancers. However, the role of FBP1 in altered glucose metabolism in HCC was unclear. Therefore, the objective of this study was to examine the function and clinical significance of FBP1 expression in HCC. Materials and Methods: First, three independent cohorts totaling 594 cases of HCC (118 real-time RT-PCR data from our institution, 242 expression array data from GSE14520, and 234 RNA-sequencing data from The Cancer Genome Atlas (TCGA)) were analyzed to address clinical significance. Data from methylation arrays, SNP arrays, and whole-exome sequencing were also analyzed to investigate the regulation of FBP1 expression in the TCGA cohort. Second, we analyzed mRNA expression, promoter methylation, and DNA copy number profiles of 967 human cancer cell lines, including 27 liver cancer, in the Cancer Cell Line Encyclopedia. Third, we established HCC cell lines stably expressing FBP1 or empty vector control. We performed sphere formation assay and xenograft studies to evaluate the role of FBP1 on HCC progression. Furthermore, in order to assess the effect of FBP1 on altered glucose metabolism, isotopomer distribution analysis was performed using [U-13C] glucose. Finally, to validate the effects of FBP1 expression on survival, risk of recurrence, and glucose metabolism, we performed gene set enrichment analysis (GSEA). Results: Lower FBP1 expression associated with advanced tumor stage, poor overall survival (OS), and poor recurrence-free survival (RFS) in three independent HCC cohorts. For either OS or RFS in each cohort, this prognostic impact persisted, even after adjusting for tumor stage. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by GSEA. Conclusion: Our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Citation Format: Hiroaki Wakiyama, Hidenari Hirata, Keishi Sugimachi, Takaaki Masuda, Naoki Hayashi, Yohsuke Kuroda, Shuhei Ito, Hidetoshi Eguchi, Kotaro Terashima, Katsumi Sakamoto, Masakazu Hirakawa, Hiroshi Honda, Koshi Mimori. Loss of fructose-1,6-bisphosphatase expression induces altering glucose metabolism and tumor progression in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4411. doi:10.1158/1538-7445.AM2017-4411

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated deaths worldwide. Although recent studies have proposed different biomarkers for HCC progression and therapy resistance, a better understanding of the molecular mechanisms underlying HCC progression and recurrence, as well as the identification of molecular markers with a higher diagnostic accuracy, are necessary for the development of more effective clinical management strategies. Here, we aimed to identify novel players in HCC progression. SPRYD4 mRNA and protein expression analyses were carried out on a normal liver-derived cell line (HL-7702) and four HCC-derived cell lines (HepG2, SMMC7721, Huh-7, BEL-7402) using qRT-PCR and Western blotting, respectively. Cell proliferation Cell Counting Kit-8 (CCK-8) assays, protein expression analyses for apoptosis markers using Western blotting, and Caspase-Glo 3/7 apoptosis assays were carried out onthe four HCC-derived cell lines. Expression comparison, functional annotation, gene set enrichment, correlation and survival analyses were carried out on patient data retrieved fromthe NCBI Gene module,the NCBI GEO database andthe TCGA database. Through a meta-analysis we found that the expression of SPRYD4was downregulated in primary HCC tissues compared to non-tumor tissues. We also found that the expression of SPRYD4 was downregulated in HCC-derived cells compared to normal liver-derived cells. Subsequently, we found that the expression of SPRYD4 was inversely correlated with a gene signature associated with HCC cell proliferation. Exogenous SPRYD4 expression was found to inhibit HCC cell proliferation by inducing apoptotic cell death. We also found that SPRYD4 expressionwas associated with a good prognosis and that its expression became downregulated when HCCs progressed towards more aggressive stages and higher grades. Finally, we found that SPRYD4 expression may serve as a biomarker for a good overall and relapse-free survival in HCC patients. Our data indicate that a decreased SPRYD4 expression may serve as an independent predictor for a poor prognosis in patients with HCC and that increased SPRYD4 expression may reduce HCC growth and progression through the induction of apoptotic cell death, thereby providing a potential therapeutic target.

Circular RNAs (circRNAs) could sponge micro-RNAs (miRNAs) to regulate tumor progression of hepatocellular carcinoma (HCC). Hsa_circ_104566 contributes to papillary thyroid carcinoma progression. However, the tumorigenic mechanism of hsa_circ_104566 on HCC remains enigmatic. The role of hsa_circ_104566 on HCC was therefore evaluated in this study. First, the high expression of hsa_circ_104566 was found in HCC tissues, which was significantly associated with poor prognosis in HCC patients. Second, Hsa_circ_104566 promoted HCC progression by decreasing apoptosis and E-cadherin, while increasing cell viability, proliferation, migration, invasion, and N-cadherin. On the other hand, HCC progression was suppressed by knockdown of hsa_circ_104566. Hsa_circ_104566 could target miR-338-3p, and its expression was negatively correlated with miR-338-3p in HCC patients. Moreover, miR-338-3p suppressed protein expression of Forkhead box protein 1 (FOXP1) and had a negative correlation with FOXP1 in HCC patients. Functional assay further indicated that the promotion of HCC progression by hsa_circ_104566 was reversed by miR-338-3p, and miR-338-3p inhibitor could counteract the effect of hsa_circ_104566 knockdown on the suppression of HCC progression. In vivo assay indicated that hsa_circ_104566 knockdown suppressed HCC tumor growth and metastasis. In conclusion, hsa_circ_104566 sponged miR-338-3p to promote HCC progression, providing a potential therapeutic target for cancer intervention.

IntroductionHCC is the leading cause of cancer related mortality world wide. Emerging evidence suggests aberrant activation of an embryological trans-differentiation programme termed epithelial-mesenchymal transition (EMT) is critical in promoting metastasis...

Percutaneous radiofrequency ablation of tumor feeding artery before target tumor ablation may reduce local tumor progression in hepatocellular carcinoma

Introduction: Tumour immune suppression has been identified as an emerging hallmark of cancer that facilitates cancer progression, including hepatocellular carcinoma (HCC). Studies suggest that cancer cells can remain undetected and proliferate by evading local immune surveillance. A major pathway in tryptophan metabolism, the kynurenine pathway (KP), has been suggested to be one of the key mechanisms mediating tumoral immune evasion. In a pro-inflammatory tumour microenvironment, the KP is highly activated which can suppress activation of tumour-targeting immune cells and enhance cancer cell growth. To elucidate the mechanisms underlying KP activation and its contribution to tumour progression in HCC, this study aims to characterize the KP profile and tumour secretome, including cytokines and proteins in HCC patients, to examine the relationship between changes in plasma KP activity and the inflammation-associated secretome. Methods: We have acquired plasma samples collected from patients with HCC (n=30), cirrhosis only (n=30), and healthy control (n=30). The KP metabolomic analysis was carried out using high pressure liquid chromatography (HPLC), Ultra-HPLC and Gas-chromatography Mass-spectrometry. The cytokine and proteomics analysis was performed using flow cytometry and data-independent acquisition mass-spectrometry respectively. Results/Discussion: Our findings demonstrate that the KP is highly activated in HCC, as inferred by the elevated enzymatic activities of indoleamine-2, 3-dioxygenase/tryptophan-2, 3-dioxygenase and kynurenine 3-monooxygenase in HCC patients. Activation of these KP enzymes implies that HCC patients have a suppressed immune profile to facilitate the progression of cancer. Cytokine profiling revealed a more inflamed systemic profile in HCC patients compared to controls and cirrhotic patients. Given that the KP is induced by inflammation, this provides a potential explanation for the elevated KP activity measured in HCC patients. Proteomics analysis further identified differentially expressed proteins linked to inflammation and immune system activation, highlighting that the immune system plays a major role in HCC pathophysiology, potentially driving inflammation and the observed KP profile in HCC patients. Conclusion: Collectively, these results suggest that KP activation may potentially be one of the mechanisms used by HCC tumour to create an immune-tolerant environment, facilitating tumour progression and development. Citation Format: Shivani Krishnamurthy, Bavani Gunasegaran, Sharron Chow, Vincent Lam, Ken Liu, Fiona Guan, Joo-Shik Shin, Avik Majumdar, Geoffrey McCaughan, Seong Beom Ahn, Benjamin Heng. Hiding in plain sight: Understanding the role of tryptophan metabolism in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4161.

Chapter 2: Diagnosis and surveillance

RASA1 (p120RasGAP), encodes Ras GTPase-activating protein 1 and, is a potent tumor suppressor gene that is frequently inactivated in several human cancer types. However, its precise role in hepatocellular carcinoma (HCC) has been blurred. We hypothesized that RASA1 plays a crucial role in tumor pathogenesis and progression of HCC. RASA1 expression levels were analyzed in 226 cases of HCC by immunohistochemistry. It was found that 38.68% (41/106) of the high-grade HCC samples and 54.17% (65/120) of the low-grade HCC samples expressed RASA1 protein. The difference between RASA1 expression in high-grade and low-grade HCC was statistically significant (p=0.02). Additionally, RASA1 high expression was inversely associated with larger tumor size (p<0.001). Although RASA1 is known as a tumor suppressor, its role in overall survival (OS) in HCC is unclear. Kaplan-Meier survival analysis showed that patients with low level of RASA1 expression correlated with a significantly poorer survival compared to those with high level of RASA1 expression. These data support that RASA1 could serve as an independent prognostic marker for HCC patients.

Myeloid-derived suppressor cells (MDSCs) play a pivotal role in immunosuppression and tumor progression in hepatocellular carcinoma (HCC). While various treatments like surgical resection, ablation, and radiotherapy have been studied for their effects on circulating MDSC frequencies in HCC patients, the findings remain inconclusive. Transarterial Chemoembolization (TACE) stands as the standard care for unresectable HCC, with Microparticle TACE (mTACE) gaining prominence for its capacity to induce significant tumor necrosis. However, the immunological ramifications of such pathological outcomes are scarcely reported. This study aims to elucidate the alterations in MDSC subtypes, specifically monocytic MDSCs (mMDSCs) and early-stage MDSCs (eMDSCs), post-mTACE and to investigate their clinical correlations in HCC patients. A cohort comprising 75 HCC patients, 16 liver cirrhosis patients, and 20 healthy controls (HC) was studied. Peripheral blood samples were collected and analyzed for MDSC subtypes. The study also explored the associations between MDSC frequencies and various clinical parameters in HCC patients. The frequency of mMDSCs was significantly elevated in the HCC group compared to liver cirrhosis and HC. Importantly, mMDSC levels were strongly correlated with aggressive clinical features of HCC, including tumor size, vascular invasion, and distant metastasis. Post-mTACE, a marked reduction in mMDSC frequencies was observed, while eMDSC levels remained stable. Our findings underscore the critical role of mMDSCs in HCC pathogenesis and their potential as a therapeutic target. The study also highlights the efficacy of mTACE in modulating the immunosuppressive tumor microenvironment, thereby opening new avenues for combinatorial immunotherapeutic strategies in HCC management.

We previously identified JWA as a novel microtubule-associated protein (MAP), which is implicated in carcinogenesis and tumor progression. The aims of the present study were to investigate the biological action and the prognostic significance of JWA in hepatocellular carcinoma (HCC). Quantitative real-time PCR and Western blot were used to detect JWA mRNA and protein expression, respectively, in stepwise metastatic HCC cell lines and HCC tissues. Short hairpin RNA was used to inhibit JWA expression in HCC cells. The effects of JWA depletion on cell migration, invasion, adhesion and in vivo metastasis were investigated. Immunohistochemistry of JWA was conducted in microarrays with tissue from 314 HCC patients who had undergone surgical resection. Prognostic significance was assessed using the Kaplan-Meier method and log-rank tests. The result showed JWA expression was decreased in the highly metastatic HCC cell lines and HCC tissues. Depletion of JWA caused a notable increase in cell migration, invasion and adhesion in vitro and metastasis in vivo. Furthermore, there was an inverse correlation between JWA expression and FAK expression and phosphorylation, RhoA activation and matrix metalloproteinase-2 (MMP-2) activity in HCC cells. More notably, multivariate analysis revealed that a low level of JWA expression was an independent prognosticator for both recurrence-free and overall survival for HCC patients after surgical resection, especially for AFP-normal HCC patients. Taken together, our data demonstrate that JWA plays a crucial role in HCC progression and suggest JWA may be a potential prognostic biomarker and therapeutic target for HCC.

Year in review: Liver cancer research in 2022: tumor microenvironment takes the central stage.

How should patients with hepatocellular carcinoma recurrence after liver transplantation be treated?