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Extracellular vesicles (EVs) play a crucial role in triggering tumour-aggressive behaviours. However, the energetic process by which tumour cells produce EVs remains poorly understood. Here, we demonstrate the involvement of β-hexosaminidase B (HEXB) in mediating EV release in response to oxidative stress, thereby promoting the development of hepatocellular carcinoma (HCC). Mechanistically, reactive oxygen species (ROS) stimulate the nuclear translocation of transcription factor EB (TFEB), leading to the upregulation of both HEXB and its antisense lncRNA HEXB-AS. HEXB-AS can bind HEXB to form a protein/RNA complex, which elevates the protein stability of HEXB. The stabilized HEXB interacts with lysosome-associated membrane glycoprotein 1 (LAMP1), disrupting lysosome-multivesicular body (MVB) fusion, which protects EVs from degradation. Knockdown of HEXB efficiently inhibits EV release and curbs HCC growth both in vitro and in vivo. Moreover, targeting HEXB by M-31850 significantly inhibits HCC growth, especially when combined with GW4869, an inhibitor of exosome release. Our results underscore the critical role of HEXB as a modulator that promotes EV release during HCC development., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Loss of E-cadherin (ECAD) is required in tumor metastasis. Protein degradation of ECAD in response to oxidative stress is found in metastasis of hepatocellular carcinoma (HCC) and is independent of transcriptional repression as usually known. Mechanistically, protein kinase A (PKA) senses oxidative stress by redox modification in its β catalytic subunit (PRKACB) at Cys200 and Cys344. The activation of PKA kinase activity subsequently induces RNF25 phosphorylation at Ser450 to initiate RNF25-catalyzed degradation of ECAD. Functionally, RNF25 repression induces ECAD protein expression and inhibits HCC metastasis in vitro and in vivo. Altogether, these results indicate that RNF25 is a critical regulator of ECAD protein turnover, and PKA is a necessary redox sensor to enable this process. This study provides some mechanistic insight into how oxidative stress-induced ECAD degradation promotes tumor metastasis of HCC., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

N6-methyladenosine (m 6 A) modification orchestrates cancer formation and progression by affecting the tumor microenvironment (TME). For hepatocellular carcinoma (HCC), immune evasion and angiogenesis are characteristic features of its TME. The role of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), as an m 6 A reader, in regulating HCC TME are not fully understood. Herein, it is discovered that trimethylated histone H3 lysine 4 and H3 lysine 27 acetylation modification in the promoter region of YTHDF2 enhanced its expression in HCC, and upregulated YTHDF2 in HCC predicted a worse prognosis. Animal experiments demonstrated that Ythdf2 depletion inhibited spontaneous HCC formation, while its overexpression promoted xenografted HCC progression. Mechanistically, YTHDF2 recognized the m 6 A modification in the 5'-untranslational region of ETS variant transcription factor 5 (ETV5) mRNA and recruited eukaryotic translation initiation factor 3 subunit B to facilitate its translation. Elevated ETV5 expression induced the transcription of programmed death ligand-1 and vascular endothelial growth factor A, thereby promoting HCC immune evasion and angiogenesis. Targeting YTHDF2 via small interference RNA-containing aptamer/liposomes successfully both inhibited HCC immune evasion and angiogenesis. Together, this findings reveal the potential application of YTHDF2 in HCC prognosis and targeted treatment., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background and Aims: The incidence and mortality of hepatocellular carcinoma (HCC) are increasing. It is urgent to develop more effective HCC biomarkers for diagnosis and treatment. This project intends to verify the expression of enhancer of zeste 1 polycomb repressive complex 2 subunit (EZH1) and its mechanism in HCC., Methods: This study integrates global microarray and high-throughput sequencing datasets, combined with internal immunohistochemistry, to analyze the expression and prognostic value of EZH1 in HCC. Functional enrichment analysis was conducted to investigate transcriptional targets, which were achieved by intersecting HCC over-expressed genes, EZH1 co-expressed genes and putative transcriptional targets. The relationship between EZH1 and anticancer drugs was detected by drug sensitivity analysis., Results: In this study, 84 datasets from 40 platforms (3,926 HCC samples and 3,428 non-cancerous liver tissues) were included to show the high expression of EZH1 in HCC. Immunohistochemistry with 159 HCC samples and 62 non-HCC samples confirmed the high expression level. HCC patients with high EZH1 expression had worse survival prognoses. Gene ontology and Reactome analysis revealed that metabolism-related pathways, including autophagy, are critical for HCC. Interestingly, as one of the EZH1 potential transcriptional targets, autophagy-related 7 (ATG7) appeared in the above pathways. ATG7 was positively correlated with EZH1, upregulated in HCC, and mediated poor prognosis. Upregulation of EZH1 was found to be in contact with HCC anti-tumor drug resistance., Conclusions: The upregulation of EZH1 expression can promote the occurrence of HCC and lead to poor clinical progression and drug resistance; these effects may be mediated by regulating ATG7.

Background: Liver cancer is a malignant tumor commonly seen in infants and young children. Serabelisib is a novel and effective phosphoinositide-3-kinase (PI3Kα) inhibitor, currently in trials for solid malignancy treatment, such as bladder cancer. However, it is unclear whether serabelisib affects liver cancer., Objectives: To explore the effects of serabelisib on the proliferation, apoptosis, invasion, and metastasis of HepG2 and HuH-6 cells, and elucidate the relevant molecular mechanisms., Material and Methods: The HepG2 cells were treated with 2 μM, 4 μM or 8 μM serabelisib, while the 0-μM group was used as a control. A plate clone formation assay was utilized to measure colony formation ability in each group, a 5-ethynyl-2'-deoxyuridine (EdU) assay examined cell proliferation, a Cell Counting Kit-8 (CCK-8) assay assessed cell viability, flow cytometry measured the cell cycle and apoptosis, JC-1 staining determined mitochondrial membrane potential, and transmission electron microscopy evaluated cell morphology. In addition, gene and protein expression levels of apoptosis markers, epithelial-mesenchymal transition (EMT), Gasdermin D (GSDMD), and the PI3K/protein kinase B (AKT) signaling pathway were measured., Results: After serabelisib intervention, HepG2 and HuH-6 cells formed fewer colonies, proliferated more slowly, and had reduced viability. The number of HepG2 and HuH-6 cells in the G2 and S phases decreased, apoptosis and the number of apoptotic bodies increased, and mitochondrial membrane potential decreased. Serabelisib treatment also reduced the migration and invasion capacity of the cells. Furthermore, genes and proteins of the PI3K/AKT signaling pathway were downregulated, while those that promote apoptosis and pyroptosis or inhibit EMT were upregulated., Conclusions: Serabelisib inhibited the PI3K/AKT signaling pathway, thereby inhibiting EMT and promoting apoptosis and pyroptosis in HepG2 and HuH-6 cells.

The centromere proteins (CENPs), a critical mitosis-related protein complexes, are involved in the kinetochore assembly and chromosome segregation. In this study, we identified that CENPA was significantly up-regulated in HCC and highly expressed CENPA correlated with poor prognosis for HCC patients. Knockdown of CENPA inhibited HCC cell proliferation and tumor growth in vitro and in vivo . Mechanistically, CENPA transcriptionally activated and cooperated with YY1 to drive the expression of cyclin D1 (CCND1) and neuropilin 2 (NRP2). Moreover, we identified that CENPA can be lactylated at lysine 124 (K124). The lactylation of CENPA at K124 promotes CENPA activation, leading to enhanced expression of its target genes. In summary, CENPA function as a transcriptional regulator to promote HCC via cooperating with YY1. Targeting the CENPA-YY1-CCND1/NRP2 axis may provide candidate therapeutic targets for HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: The role of circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT) is not fully understood., Methods: In this retrospective analysis, we included 316 HCC patients who underwent hepatectomy and preoperative CTC detection. We selected 41 pairs of matched HCC patients with and without PVTT using propensity score matching (PSM) analysis. We compared the preoperative CTC counts in patients from both the full cohort and the PSM model. We also analyzed their associations with disease-free survival (DFS) and overall survival (OS)., Results: Before and after PSM analysis, the preoperative CTC counts in the HCC with PVTT group were substantially higher than in the HCC without PVTT group. In both the full cohort of patients and the PSM model, patients with CTC ≥ 2 had significantly shorter OS and DFS than patients with CTC < 2. The outcomes of HCC patients with PVTT could be well differentiated by preoperative CTC levels. HCC patients with CTC ≥ 2 had noticeably shorter OS (9.9 months vs. 24.6 months, P = 0.0003) and DFS (6.0 months vs. 12.3 months, P = 0.0041) than those with CTC < 2. Moreover, preoperative CTC ≥ 2 remained an independent predictor in all groups' multivariate analysis., Conclusion: We discovered a link between preoperative CTC counts and the occurrence of PVTT and confirmed the prognostic significance of preoperative CTC in HCC patients with PVTT. These findings suggest that preoperative CTC counts have the potential to assist in identifying patients with HCC and PVTT who may benefit from surgery., (© 2023. The Author(s).)

Bone is the second leading metastatic site for hepatocellular carcinoma (HCC). Patients with HCC and bone metastasis suffer poor quality of life and reduced survival time. Extracellular vesicles (EVs) are widely involved in HCC formation and metastasis. However, the communication between primary HCC and bone lesions mediated by EVs remains unclear and the possible effect of bone metastasis on the progression of HCC remains largely unknown. Here, bone-metastasized HCC-derived EVs (BM-EVs) are found to localize to orthotropic HCC cells and promote HCC progression. Mechanistically, miR-3190-5p (miR-3190) is upregulated in intracellular HCC cells isolated from bone lesions as well as in their derived EVs. miR-3190 in BM-EVs is transferred into orthotopic tumor cells and enhances their metastatic capacity by downregulating AlkB homolog 5 (ALKBH5) expression. Decreased level of ALKBH5 exacerbates the prometastatic characteristics of HCC by modulating gene expression in N6-methyladenosine-dependent and -independent ways. Finally, antagomir-miR-3190-loaded liposomes with HCC affinity successfully suppress HCC progression in mice treated with BM-EVs. These findings reveal that BM-EVs initiate prometastatic cascades in orthotopic HCC by transferring ALKBH5-targeting miR-3190 and miR-3190 is serving as a promising therapeutic target for inhibiting the progression of HCC in patients with bone metastasis., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The single-nucleotide polymorphism (SNP) of p53, in particular the codon 72 variants, has recently been implicated as a critical regulator in tumor progression. However, the underlying mechanism remains elusive. Here we found that cancer cells carrying codon 72-Pro variant of p53 showed impaired metastatic potential upon serine supplementation. Proteome-wide mapping of p53-interacting proteins uncovered a specific interaction of the codon 72 proline variant (but not p53 72R ) with phosphoserine aminotransferase 1 (PSAT1). Interestingly, p53 72P -PSAT1 interaction resulted in dissociation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) that otherwise bound to p53 72P , leading to subsequent nuclear translocation of PGC-1α and activation of oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle. Depletion of PSAT1 restored p53 72P -PGC-1α interaction and impeded the OXPHOS and TCA function, resulting in mitochondrial dysfunction and metastasis suppression. Notably, pharmacological targeting the PSAT1-p53 72P interaction by aminooxyacetic acid (AOA) crippled the growth of liver cancer cells carrying the p53 72P variant in both in vitro and patient-derived xenograft models. Moreover, AOA plus regorafenib, an FDA-proved drug for hepatocellular carcinoma and colorectal cancer, achieved a better anti-tumor effect on tumors carrying the p53 72P variant. Therefore, our findings identified a gain of function of the p53 72P variant on mitochondrial function and provided a promising precision strategy to treat tumors vulnerable to p53 72P -PSAT1 perturbation., (© 2022. The Author(s).)

Metformin, a well-known antidiabetic drug, has been repurposed for cancer treatment; however, recently observed drug resistance and tumor metastasis have questioned its further application. Here, we found that long-term metformin exposure led to metabolic adaptation of hepatocellular carcinoma (HCC) cells, which was characterized by an obvious epithelial-mesenchymal transition (EMT) phenotype and compensatory elevation of oxidative phosphorylation (OXPHOS). TOMM34, a translocase of the outer mitochondrial membrane, was upregulated to promote tumor metastasis in response to metformin-induced metabolic stress. Mechanistically, TOMM34 interacted with ATP5B to preserve F 1 F O -ATPase activity, which conferred mitochondrial OXPHOS and ATP production. This metabolic preference for OXPHOS suggested a large requirement of energy supply by cancer cells to survive and spread in response to therapeutic stress. Notably, disturbing the interaction between TOMM34 and ATP5B using Gboxin, a specific OXPHOS inhibitor, increased sensitivity to metformin and suppressed tumor progression both in vitro and in vivo. Overall, this study demonstrates a molecular link of the TOMM34/ATP5B-ATP synthesis axis during metformin adaptation and provides promising therapeutic targets for metformin sensitization in cancer treatment., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: MicroRNAs (miRNAs) play crucial roles in the development of hepatocellular carcinoma (HCC). Hsa-microRNA-27b-3p (hsa-miR-27b) is involved in the formation and progression of various cancers, but its role and clinical value in HCC remain unclear., Methods: The expression of hsa-miR-27b in HCC was examined by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) assays of clinical samples. Cell Counting Kit-8 assays (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, Transwell assays, filamentous actin (F-actin) staining and western blot analyses were used to determine the effects of hsa-miR-27b on HCC cells in vitro. Subcutaneous xenograft and lung metastatic animal experiments were conducted to verify the role of hsa-miR-27b in HCC in vivo. In silico prediction, qRT-PCR, western blot, anti-Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) and dual luciferase reporter assays were applied to identify the target genes of hsa-miR-27b. To detect the impacts of hsa-miR-27b on nuclear factor kappa B (NF-кB) signalling cascades mediated by transforming growth factor-activated kinase-binding protein 3 (TAB3), we performed qRT-PCR, western blot assays, immunofluorescence staining, immunohistochemistry (IHC) and dual-luciferase reporter assays. Recombinant oncolytic adenovirus (Onco Ad ) overexpressing hsa-miR-27b was constructed to detect their therapeutic value in HCC., Results: The expression of hsa-miR-27b was lower in HCC than in adjacent non-tumourous tissues (ANTs), and the reduced expression of hsa-miR-27b was associated with worse outcomes in patients with HCC. Hsa-miR-27b significantly inhibited the proliferation, migration, invasion, subcutaneous tumour growth and lung metastasis of HCC cells. The suppression of hsa-miR-27b promoted the nuclear translocation of NF-κB by upregulating TAB3 expression. TAB3 was highly expressed in HCC compared with ANTs and was negatively correlated with the expression of hsa-miR-27b. The impaired cell proliferation, migration and invasion by hsa-miR-27b overexpression were recovered by ectopic expression of TAB3. Recombinant Onco Ad with overexpression of hsa-miR-27b induced anti-tumour activity compared with that induced by negative control (NC) Onco Ad in vivo and in vitro., Conclusions: By targeting TAB3, hsa-miR-27b acted as a tumour suppressor by inactivating the NF-кB pathway in HCC in vitro and in vivo, indicating its therapeutic value against HCC., (© 2022. The Author(s).)

The tripartite motif (TRIM) protein family is a highly conserved group of E3 ligases with 77 members known in the human, most of which consist of a RING-finger domain, one or two B-box domains, and a coiled-coil domain. Generally, TRIM proteins function as E3 ligases to facilitate specific proteasomal degradation of target proteins. In addition, E3 ligase independent functions of TRIM protein were also reported. In hepatocellular carcinoma, expressions of TRIM proteins are both regulated by genetic and epigenetic mechanisms. TRIM proteins regulate multiple biological activities and signaling cascades. And TRIM proteins influence hallmarks of HCC. This review systematically demonstrates the versatile roles of TRIM proteins in HCC and helps us better understand the molecular mechanism of the development and progression of HCC., (© 2022. The Author(s).)

Patients with hepatocellular carcinoma (HCC) and bone metastasis (BM) suffer from greatly reduced life quality and a dismal prognosis. However, BM in HCC has long been overlooked possibly due to its relatively low prevalence in previous decades. To date, no consensus or guidelines have been reached or formulated for the prevention and management of HCC BM. Our narrative review manifests the increasing incidence of HCC BM to sound the alarm for additional attention. The risk factors, diagnosis, prognosis, and therapeutic approaches of HCC BM are detailed to provide a panoramic view of this disease to clinicians and specialists. We further delineate an informative cancer bone metastatic cascade based on evidence from recent studies and point out the main factors responsible for the tumor-associated disruption of bone homeostasis and the formation of skeletal cancer lesions. We also present the advances in the pathological and molecular mechanisms of HCC BM to shed light on translational opportunities. Dilemmas and challenges in the treatment and investigation of HCC BM are outlined and discussed to encourage further endeavors in the exploration of underlying pathogenic and molecular mechanisms, as well as the development of novel effective therapies for HCC patients with BM., (© 2022. Higher Education Press.)

Recent evidence suggests that small nucleolar RNAs (snoRNAs) are involved in the progression of various cancers, but their precise roles in hepatocellular carcinoma (HCC) remain largely unclear. Here, we report that SNORD17 promotes the progression of HCC through a positive feedback loop with p53. HCC-related microarray datasets from the Gene Expression Omnibus (GEO) database and clinical HCC samples were used to identify clinically relevant snoRNAs in HCC. SNORD17 was found upregulated in HCC tissues compared with normal liver tissues, and the higher expression of SNORD17 predicted poor outcomes in patients with HCC, especially in those with wild-type p53. SNORD17 promoted the growth and tumorigenicity of HCC cells in vitro and in vivo by inhibiting p53-mediated cell cycle arrest and apoptosis. Mechanistically, SNORD17 anchored nucleophosmin 1 (NPM1) and MYB binding protein 1a (MYBBP1A) in the nucleolus by binding them simultaneously. Loss of SNORD17 promoted the translocation of NPM1 and MYBBP1A into the nucleoplasm, leading to NPM1/MDM2-mediated stability and MYBBP1A/p300-mediated activation of p53. Interestingly, p300-mediated acetylation of p53 inhibited SNORD17 expression by binding to the promoter of SNORD17 in turn, forming a positive feedback loop between SNORD17 and p53. Administration of SNORD17 antisense oligonucleotides (ASOs) significantly suppressed the growth of xenograft tumors in mice. In summary, this study suggests that SNORD17 drives cancer progression by constitutively inhibiting p53 signaling in HCC and may represent a potential therapeutic target for HCC., (© 2022. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Background and Aims: Bone is the second most frequent site of metastasis for HCC, which leads to an extremely poor prognosis. HCC bone metastasis is typically osteolytic, involving the activation of osteoclasts. Long noncoding RNA H19 plays an important role in the pathogenesis of human cancers. Nonetheless, the mechanism underlying the participation of H19 in HCC bone metastasis remains unclear., Approach and Results: The current study established a mouse HCC bone metastasis model by using serial intracardiac injection and cell isolation to obtain cells with distinct bone metastasis ability. H19 was highly expressed in these cells and in clinical HCC bone metastasis specimens. Both osteoclastogenesis in vitro and HCC bone metastasis in vivo were promoted by H19 overexpression, whereas these processes were suppressed by H19 knockdown. H19 overexpression attenuated p38 phosphorylation and further down-regulated the expression of osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor. However, up-regulated OPG expression as well as suppressed osteoclastogenesis caused by H19 knockdown were recovered by p38 interference, indicating that p38 mitogen-activated protein kinase (MAPK)-OPG contributed to H19-promoted HCC bone metastasis. Furthermore, we demonstrated that H19 inhibited the expression of OPG by binding with protein phosphatase 1 catalytic subunit alpha (PPP1CA), which dephosphorylates p38. SB-203580-mediated inactivation of p38MAPK reversed the down-regulation of HCC bone metastasis caused by H19 knockdown in vivo. Additionally, H19 enhanced cell migration and invasion by up-regulating zinc finger E-box binding homeobox 1 through the sequestration of microRNA (miR) 200b-3p., Conclusions: H19 plays a critical role in HCC bone metastasis by reducing OPG expression, which is mediated by the PPP1CA-induced inactivation of the p38MAPK pathway; and H19 also functions as a sponge for miR-200b-3p., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: Tumor metastasis is a major factor of high recurrence and mortality in hepatocellular carcinoma (HCC), but its underlying mechanism remains elusive. We report that PDZ and LIM domain protein 1 (PDLIM1) is significantly down-regulated in metastatic human HCC tissues, which predicts unfavorable prognosis, suggesting that PDLIM1 may play an important inhibitory role during HCC metastasis., Approach and Results: Functional studies indicate that PDLIM1 knockdown induces epithelial-to-mesenchymal transition (EMT) of HCC cells, elevates their invasive capacity, and promotes metastasis in vitro and in vivo, whereas overexpression of PDLIM1 exhibits opposite phenotypes. Mechanistically, PDLIM1 competitively binds to the cytoskeleton cross-linking protein alpha-actinin 4 (ACTN4), leading to the disassociation of ACTN4 from F-actin, thus preventing F-actin overgrowth. In contrast, loss of PDLIM1 induces excessive F-actin formation, resulting in dephosphorylation of large tumor suppressor kinase 1 and activation of Yes-associated protein, thereby promoting HCC metastasis. Moreover, Asn145 (N145) of PDLIM1 is critical for its interaction with ACTN4, and N145A mutation abolishes its regulatory function in Hippo signaling and HCC metastasis., Conclusions: Our findings indicate that PDLIM1 suppresses HCC metastasis by modulating Hippo signaling, suggesting that PDLIM1 may be a potential prognostic marker for metastatic HCC., (© 2020 by the American Association for the Study of Liver Diseases.)

Hepatocellular carcinoma (HCC) is the most frequent subtype of primary liver cancer and one of the leading causes of cancer-related death worldwide. However, the molecular mechanisms underlying HCC pathogenesis have not been fully understood. Emerging evidences have recently suggested the crucial role of long noncoding RNAs (lncRNAs) in the tumorigenesis and progression of HCC. Various HCC-related lncRNAs have been shown to possess aberrant expression and participate in cancerous phenotypes (e.g. persistent proliferation, evading apoptosis, accelerated vessel formation and gain of invasive capability) through their binding with DNA, RNA or proteins, or encoding small peptides. Thus, a deeper understanding of lncRNA dysregulation would provide new insights into HCC pathogenesis and novel tools for the early diagnosis and treatment of HCC. In this review, we summarize the dysregulation of lncRNAs expression in HCC and their tumor suppressive or oncogenic roles during HCC tumorigenesis. Moreover, we discuss the diagnostic and therapeutic potentials of lncRNAs in HCC.

The landscape of cellular plasticity and sources with relevant niche signals in hepatocellular carcinoma is still obscure. Transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel, is involved in a variety of malignancies and overexpressed in hepatocellular carcinoma (HCC). We have investigated the role of TRPV1 in HCC from different angles by various experimental techniques, such as in vivo and in vitro experiments, and by bioinformatics analysis of data from genetic models induced by diethylnitrosamine (DEN), mice samples and human HCC samples. We find that TRPV1 knockout promotes to hepatocarcinogenesis and deconstructs the portal triad adjacent to tumor border that is contributed by originations of tumor initiating cells and biliary cells. Epithelial to mesenchymal transition (EMT) is involved and transcription factors Ovol2 and Zeb1 coordinated with Sox 10 drive gene expression in the event which is also confirmed by the expression of these proteins in human HCC samples. Treatment with TRPV1 agonist Capsaicin inhibits the growth of HCC cells in xenograft models. Our findings demonstrate that TRPV1 is a potential therapeutic target in human HCC and exerts effects on cellular plasticity with modulation of Ovol2, Zeb1 and Sox10., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background & Aims: Hepatocellular carcinoma (HCC) is a common cancer worldwide and remains a major clinical challenge. Ketoconazole, a traditional antifungal agent, has attracted considerable attention as a therapeutic option for cancer treatment. However, its mechanism of action is still not clearly defined. We aimed to evaluate the effect of ketoconazole on HCC and investigate the underlying mechanisms., Methods: We examined the antitumor effect of ketoconazole on HCC cells, cell line-derived xenografts, and a patient-derived xenograft (PDX) model. Ketoconazole-induced mitophagy was quantified by immunofluorescence, immunoblotting and transmission electron microscopy analysis. We used mitophagy inhibitors to study the role of mitophagy on HCC cell death induced by ketoconazole. The role of cyclooxygenase-2 (COX-2 [encoded by PTGS2]) on ketoconazole-induced mitophagy was evaluated using gain- and loss-of-function methods. The synergistic effect of ketoconazole with sorafenib on HCC was measured in vivo and in vitro., Results: Ketoconazole stimulated apoptosis in HCC cells by triggering mitophagy in vitro and in vivo. Mechanistically, ketoconazole downregulated COX-2, which led to PINK1 accumulation and subsequent mitochondrial translocation of Parkin (PRKN), and thereby promoted mitophagy-mediated mitochondrial dysfunction. Inhibiting mitophagy alleviated ketoconazole-induced mitochondrial dysfunction and apoptosis, supporting a causal role for mitophagy in the antitumor effect of ketoconazole. In the HCC PDX model, ketoconazole demonstrated a marked antitumor effect characterized by COX-2 downregulation, mitophagy activation, and apoptosis induction. Moreover, ketoconazole acted synergistically with sorafenib to suppress HCC xenograft growth in vivo., Conclusion: Our results demonstrate a novel link between ketoconazole and mitophagy machinery, providing preclinical proof of concept for the use of ketoconazole in HCC treatment., Lay Summary: Hepatocellular carcinoma (HCC) is a common malignancy worldwide and remains a major clinical challenge. Our study reveals that ketoconazole, a broad-spectrum antifungal agent, activates PINK1/Parkin-mediated mitophagy by downregulating COX-2, consequently resulting in the acceleration of apoptosis and thereby inhibiting the growth of HCC. Furthermore, ketoconazole acts synergistically with sorafenib in the suppression of HCC growth in vitro and in vivo., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

18β-Glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicine, Glycyrrhrzae Radix et Rhizoma. Here, we explored the effects of GA on hepatocellular carcinoma (HCC) in vitro and in vivo and the underlying molecular mechanisms. We confirmed that GA suppressed proliferation of various HCC cell lines. Treatment of GA caused G0/G1 arrest, apoptosis and autophagy in HCC cells. GA-induced apoptosis and autophagy were mainly due to the unfolded protein response. We compared the roles of the ATF4/CHOP and IRE1α/XBP1s UPR pathways, which were both induced by GA. The ATF4/CHOP cascade induced autophagy and was indispensable for the induction of apoptosis in GA-treated HCC cells. In contrast, the IRE1α/XBP1s cascade protected HCC cells from apoptosis in vitro and in vivo induced by GA. Despite this, activation of autophagy protected HCC cells from apoptosis induced by GA. We concluded that pharmacological inhibition of autophagy or IRE1α may be of benefit to enhance the antitumor activity of GA.

Liver cancer is a major global health problem being the sixth most common cancer and the third cause of cancer-related death, with hepatocellular carcinoma (HCC) representing more than 90% of primary liver cancers. Mounting evidence suggests that, compared with their normal counterparts, many types of cancer cell have increased levels of ROS. Therefore, cancer cells need to combat high levels of ROS, especially at early stages of tumor development. Recent studies have revealed that ROS-mediated regulation of redox-sensitive proteins (redox sensors) is involved in the pathogenesis and/or progression of many human diseases, including cancer. Unraveling the altered functions of redox sensors and the underlying mechanisms in hepatocarcinogenesis is critical for the development of novel cancer therapeutics. For this reason, redox proteomics has been developed for the high-throughput screening of redox sensors, which will benefit the development of novel therapeutic strategies for the treatment of HCC. In this review, we will briefly introduce several novel redox proteomics techniques that are currently available to study various oxidative modifications in hepatocarcinogenesis and summarize the most important discoveries in the study of redox processes related to the development and progression of HCC., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Numb, an endocytic adaptor, is a known cell fate determinant that participates in asymmetric cell division. The present study aimed to explore the potential roles of Numb in hepatocarcinogenesis. Numb expression was investigated in hepatocellular carcinomas (HCC) with reverse transcription‑quantitative polymerase chain reaction and immunohistochemical examination; its association with the prognosis of HCC patients was analyzed. In addition, the effects of Numb deletion on proliferation of HCC cells and its relevant molecules were evaluated in Huh7 and HepG2 cells. Numb overexpression was observed in 62% of adjacent non‑tumor tissues and 46% of tumor tissues. Overexpression of Numb in HCC was associated with histological grade, portal vein invasion and the number of tumors (P=0.001, 0.022 and 0.034 respectively). Multivariate analysis revealed that Numb expression was an independent prognostic indicator of HCC patients. Methylation of the Numb promoter contributed to hepatocarcinogenesis. In vitro assays demonstrated that Numb silencing resulted in inhibition of cell proliferation, induction of apoptosis, downregulation of cyclin‑dependent protein kinase 4 (CDK4) and S‑phase kinase‑associated protein 2 (SKP2), and upregulation of Bcl‑2 homologous antagonist/killer (BAK) and cyclin‑dependent kinase inhibitor 1 (p21). The present study suggests that downregulation of Numb inhibits colony formation and cell proliferation, induces apoptosis of HCC cells and independently predicts the poor prognosis of HCC patients. Thus, Numb has a potential role in the development and progression of HCC.

Background: This study was conducted to determine DEPDC1 expression in hepatocelluar carcinomas (HCCs) and to reveal its potential role in diagnosis and prognosis of affected patients., Materials and Methods: DEPDC1 expression at the mRNA level was detected by quantitative real-time PCR (qRT-PCR) in 205 cases of HCC and paired adjacent normal liver tissues, and by semi-quantitative RT-PCR in 20 cases. Survival curves were obtained by using Kaplan-Meier method and Log-rank test. Independent predictors associated with regard to disease free survival (DFS) and overall survival (OS) were identified using the Cox proportional hazard model., Results: High DEPDC1 mRNA levels were detected in 144 out of 205 cases (70.24%) of HCC, significantly associated with clinicopathological parameters, including tumor size (≥4cm), alpha-fetoprotein (≥100ng/ml), B-C of BCLC stage and recurrence. Kaplan-Meier survival analysis revealed that HCC patients with high DEPDC1 expression had poor OS and DFS. Multivariate analysis demonstrated that high DEPDC1 expression was an independent predictor of OS (HR=1.651; 95% 95%CI, 1.041- 2.617; p=0.033) and DFS (HR=1.583; 95%CI, 1.01- 2.483; p=0.045)., Conclusions: Our results indicate DEPDC1 might be a novel diagnostic marker and an independent prognostic predictor for HCC patients.

Background: This study was conducted to investigate whether apparent diffusion coefficient (ADC) measurements by dividing the liver into left and right hepatic lobes may be utilized to improve the accuracy of differential diagnosis of benign and malignant focal liver lesions., Materials and Methods: A total of 269 consecutive patients with 429 focal liver lesions were examined by 3-T magnetic resonance imaging that included diffusion-weighted imaging. For 58 patients with focal liver lesions of the same etiology in left and right hepatic lobes, ADCs of normal liver parenchyma and focal liver lesions were calculated and compared using the paired t-test. For all 269 patients, ADC cutoffs for focal liver lesions and diagnostic accuracy in the left hepatic lobe, right hepatic lobe and whole liver were evaluated by receiver operating characteristic curve analysis., Results: For the group of 58 patients, mean ADCs of normal liver parenchyma and focal liver lesions in the left hepatic lobe were significantly higher than those in the right hepatic lobe. For differentiating malignant lesions from benign lesions in all patients, the sensitivity and specificity were 92.6% and 92.0% in the left hepatic lobe, 94.4% and 94.4% in the right hepatic lobe, and 90.4% and 94.7% in the whole liver, respectively. The area under the curve of the right hepatic lobe, but not the left hepatic lobe, was higher than that of the whole liver., Conclusions: ADCs of normal liver parenchyma and focal liver lesions in the left hepatic lobe were significantly higher than those in the right hepatic lobe. Optimal ADC cutoff for focal liver lesions in the right hepatic lobe, but not in the left hepatic lobe, had higher diagnostic accuracy compared with that in the whole liver.

Unlabelled: MicroRNA 370 (miR-370) is located within the DLK1/DIO3 imprinting region on human chromosome 14, which has been identified as a cancer-associated genomic region. However, the role of miR-370 in malignances remains controversial. Here, we report that miR-370 was repressed in human hepatocellular carcinoma (HCC) tissues and hepatoma cell lines. Using gain-of-function and loss-of-function experiments, we demonstrated that miR-370 inhibited the malignant phenotype of HCC cells in vitro. Overexpression of miR-370 inhibited growth and metastasis of HCC cells in vivo. Moreover, the RNA-binding protein, LIN28A, was identified as a direct functional target of miR-370, which, in turn, blocked the biogenesis of miR-370 by binding to its precursor. LIN28A also mediated the suppressive effects of miR-370 on migration and invasion of HCC cells by post-transcriptionally regulating RelA/p65, which is an important effector of the canonical nuclear factor kappa B (NF-κB) pathway. Interleukin-6 (IL-6), a well-known NF-κB downstream inflammatory molecule, reduced miR-370 but increased LIN28A levels in HCC. Furthermore, miR-370 levels were inversely correlated with LIN28A and IL-6 messenger RNA (mRNA) levels, whereas LIN28A mRNA expression was positively correlated with IL-6 expression in human HCC samples. Interestingly, reduction of miR-370 expression was associated with the development of HCC in rats, as well as with aggressive tumor behavior and short survival in HCC patients., Conclusions: These data demonstrate the involvement of a novel regulatory circuit consisting of miR-370, LIN28A, RelA/p65 and IL-6 in HCC progression. Manipulating this feedback loop may have beneficial effect in HCC treatment., (© 2013 by the American Association for the Study of Liver Diseases.)

Peptide vaccine based on tumor-associated antigen (TAA), which usually belongs to self-antigen with poor immunogenicity, has been considered as an attractive option for treatment of malignant tumors. The ideal TAA epitopes should have stable affinity to major histocompatibility complex (MHC) molecules and elicit strong anti-tumor immune response. Although point-mutation technology of TAA peptide may increase the binding capability to MHC molecules, some previous studies have revealed that part of the variant peptides results in lymphocyte not to effectively cross-recognize and kill the target tumor expressed wild-type TAA. Here, we designed a novel HLA-A2-restricted mutated TAA Survivin epitope nonapeptide Sur79L2 (KLSSGCAFL) that showed higher binding ability compared to wild-type peptide Sur79 (KHSSGCAFL) in T2-binding assays. To investigate whether Sur79L2 can induce Survivin-specific anti-hepatocellular carcinoma (HCC) response, we stimulated tumor-associated lymphocytes from a HCC patient with Sur79L2 in vitro. IFN-γ release and cytotoxicity assays showed Sur79L2 could effectively cross-recognize and lysis T2 cell plus peptide Sur79 and HCC cell lines (expression of wild-type Survivin antigen) in an HLA-A2-restricted manner. In contrast, peptide Sur95 (ELTLGEFLKL) that has been reported as a very promising anti-tumor epitope in a variety of tumors except HCC were not able to generate detectable cytotoxic immune responses against HCC in this study. Our results suggest that point-mutated peptide Sur79L2 is a new HLA-A2-restricted CTL epitope and may be useful for the immunotherapy for patients with HCC.

This study was aimed to evaluate the clinical value of plasma methylation analysis of a panel of four genes (APC, GSTP1, RASSF1A, and SFRP1), which was identified by our previous work, for the noninvasive diagnosis of hepatocellular carcinoma (HCC). The methylation status of these four genes in 150 plasma samples from 72 patients with HCC, 37 benign live diseases and 41 normal controls was detected with methylation-sensitive restriction enzymes-based quantitative PCR (MSRE-qPCR) method. The plasma methylation levels of APC, GSTP1, RASSF1A, and SFRP1 were significantly higher in HCCs than those in normal or benign controls (P<0.05). Although the area under the receiver-operation characteristic curve (AUC-ROC) for individual gene was moderate (range, from 0.800 to 0.881), the combination analysis of these four genes resulted in an increased AUC of 0.933 with 92.7% sensitivity, 81.9% specificity, 90.5% positive predictive value (PPV), and 87.2% negative predictive value (NPV) in discriminating HCC from normal control. The combination analysis also indicated an increased AUC of 0.877 when compared with individual gene (from 0.666 to 0.850) in discriminating HCC from benign control, and the consultant sensitivity, specificity, PPV, and NPV was 84.7%, 81.1%, 89.7%, and 73.2%, respectively. Patients with elevated plasma methylation levels of APC or RASSF1A showed poorer overall survival than those with low levels (P<0.05). Cox multivariate analysis demonstrated methylated RASSF1A in plasma to be an independent prognostic factor for overall survival (hazard ratio=3.262, 95% CI: 1.476-7.209, P=0.003). These data showed that quantitative analysis of multiple methylated genes in plasma may be a promising tool for noninvasive diagnosis of HCC; and methylated plasma RASSF1A appears to be a prognostic marker of HCC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

DNA methylation is a promising biomarker for cancer. This study was aimed at investigating the methylation levels of multiple genes in hepatocellular carcinoma (HCC) and to identify a combination of methylation markers that would be useful for the diagnosis of HCC. The methylation status of a panel of nine tumor-associated genes (APC, GSTP1, RASSF1A, CDKN2A, SFRP1, RUNX3, SOCS1, Hint1, and HIC-1) in 8 normal liver tissues and 47 paired HCCs and non-tumorous tissues (NTs) was determined using a modified methylation-sensitive, restriction enzyme-based quantitative PCR (MSRE-qPCR) method. The methylation levels of six genes (APC, CDKN2A, GSTP1, RASSF1A, SFRP1 and RUNX3) were significantly higher in HCCs than in adjacent NTs (P<0.05). Although the AUC (area under the curve) for each individual gene was low to moderate (range: 0.576 to 0.835) according to receiver operator characteristic (ROC) curve analysis, the combination analysis of these six genes resulted in an increase of AUC of 0.954 with 85.1% sensitivity, 89.4% specificity, 88.9% positive predictive value, and 85.7% negative predictive value in discriminating HCC tissues from NT tissues. These results indicate that the analysis of a combination of these six methylated genes may be a promising method for the risk assessment and diagnosis of HCC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Transient receptor potential vanilloid 2 (TRPV2), one of the members of TRP (transient receptor potential) superfamily of ion channels, has been suggested to contribute to pain associated with inflammation or neuropathy. To investigate its role in hepatocarcinogenesis, we examined the expression of TRPV2 in human hepatocellular carcinoma (HCC) samples and analyzed the association of TRPV2 expression with its clinical significance. TRPV2 expression in 55 HCC patients was examined by immunohistochemistry, and the correlation between TRPV2 levels and clinicopathologic parameters was analyzed. Thirteen paired HCC specimens and their nontumor counterparts were investigated by quantitative real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. Quantitative RT-PCR and Western blotting analysis revealed that expression of TRPV2 at both the mRNA and protein levels were increased in cirrhotic livers compared with chronic hepatitis, whereas that also occurred in moderately and well-differentiated tumors compared with that of poorly differentiated tumors. Immunohistochemistry of the 55 HCC samples showed that the expression of TRPV2 increased when going from normal liver or chronic hepatitis to cirrhosis. Increased TRPV2 expression was observed in tissues of liver cirrhosis (31/37, 83.8%). In HCC, increased expression of TRPV2 was identified in 16/55 (29%) cases. Clinicopathologic assessment suggested a significant association between TRPV2 expression and portal vein invasion and histopathologic differentiation (P = 0.036 and 0.001, respectively). Our data suggest that TRPV2 plays a role in human hepatocarcinogenesis and might be a prognostic marker of patients with HCC., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Purpose: The relationship between aflatoxin B1 (AFB1) exposure and hepatocellular carcinoma (HCC) has been previously demonstrated and supported with strong epidemiological evidence. However, the role of genetic polymorphism of X-ray cross-complementing group 3 (XRCC3) codon 241 (namely: Thr241Met), which may be involved in the repair of DNA double-strand breaks caused by carcinogens such as AFB1, been less well elaborated., Methods: We conducted a case-control study including 491 cases and 862 controls to evaluate the associations between this polymorphism and HCC risk for Guangxi population by means of polymerase chain reaction-restriction fragment length polymorphism analysis., Results: We found that individuals with the XRCC3 genotypes with codon 241 Met (namely XRCC3-TM or XRCC3-MM) had an increased risk of HCC than those with the homozygote of XRCC3 codon 241 Thr alleles (namely XRCC3-TT, adjusted odds ratios 2.22 and 7.19; 95% confidence intervals 1.72-2.88 and 4.52-11.42, respectively). The risk of HCC, moreover, did appear to differ more significantly among individuals featuring high-level AFB1-DNA adducts, whose adjusted odds ratios (95% confidence intervals) were 11.59 (5.73-23.47) and 37.54 (16.32-86.32), respectively., Conclusions: These findings support the hypothesis that the XRCC3 Thr241Met polymorphism may be associated with the risk of AFB1-related HCC among the Guangxi population.

Objective: To evaluate the efficacy of hepatitis B vaccination on hepatitis B prevention and on hepatocellular carcinoma., Methods: Birth cohort study, cross-sectional seroepidemiological survey, and surveillance of hepatitis B (HBV) and hepatocellular carcinoma were used to evaluate the efficacy of hepatitis B vaccination., Results: During the 14 years after hepatitis B vaccination, the HBsAg positive rates were found to be 0.7% - 2.9%, with an average of 1.5%, and the protective rates were 83.5% - 96.6%. Hepatitis B virus infection rates of children immunized with hepatitis B vaccine were 1.1% - 5.1%, with an average of 2.2% and the protective rates of 93.5% - 98.4%. 15 years after hepatitis B vaccination, the incidence of hepatitis B dropped from 3.27/10 000 to 0.17/10 000, a 94.8% decrease, in the group of 0 - 19 year-olds., Conclusion: The universal infant hepatitis B vaccination has proved to be effective in reducing the incidence rate of acute hepatitis B as well as the mortality of hepatocellular carcinoma.