Search

Timothy Kwok,1,2 Anita Wong,3 Grace Chan,4 YY Shiu,3 Ko-Chuen Lam,2 Daniel Young,2 Daniel WH Ho,2 Florence Ho21Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, People's Republic of China; 2Jockey Club Centre for Positive Ageing, Shatin, Hong Kong, People's Republic of China; 3The Hong Kong Chinese Women's Club Madam Wong Chan Sook Ying Memorial Care and Attention Home for the Aged, Hong Kong, People's Republic of China; 4The Hong Kong Council of Social Service, Hong Kong, People's Republic of ChinaAbstract: In Hong Kong, the evidence for cognitive-training programs in fighting against memory complaints is lacking. This study aimed to evaluate the effectiveness of the Active Mind cognitive-training program in improving the cognitive function and quality of life (QoL) for local community-dwelling Chinese older adults. A total of 200 subjects were recruited from 20 different district elderly community centers (DECCs). Centers were randomly assigned into either the intervention group or control group. The intervention group underwent eight 1-hour sessions of cognitive training, while the control group were included in the usual group activities provided by the DECCs. Standardized neuropsychological tests (the Chinese version of Mattis Dementia Rating Scale [CDRS] and the Cantonese version of the Mini-Mental State Examination) and the QoL questionnaire SF12 were used to assess participants' cognitive function and QoL before and after the trial. A total of 176 subjects completed the study. The intervention group showed greater improvement in the cognitive function measured by total CDRS score (treatment: 12.24 ± 11.57 vs control: 4.37 ± 7.99; P < 0.001) and QoL measured by total SF12 score (treatment: 7.82 ± 13.19 vs control: 3.18 ± 11.61; P = 0.014). Subjects with lower education level were associated with better cognitive response to the cognitive-training program. The current findings indicated that the Active Mind cognitive-training program was effective in improving the cognitive function and QoL for community-dwelling Chinese older adults in Hong Kong.Keywords: cognitive training, cognitive stimulation, memory, older adults, effectiveness

Xue Bai,1,2 Daniel WH Ho,2 Karen Fung,3 Lily Tang,3 Moon He,3 Kim Wan Young,4 Florence Ho,2 Timothy Kwok2,5 1Department of Applied Social Sciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong; 2Jockey Club Centre for Positive Ageing, Shatin, Hong Kong; 3Hong Chi Association, Hong Kong; 4Department of Social Work, Hong Kong Baptist University, Hong Kong; 5Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, Hong Kong Objective: This study examines the effectiveness of a life story work program (LSWp) in older adults with mild-to-moderate levels of intellectual disability (ID). Methods: Using a quasiexperimental design, this study assigned 60 older adults who were between 50–90 years old with mild-to-moderate levels of ID to receive either the LSWp (intervention group, N=32) or usual activities (control group, N=28) during a period of 6 months. Evaluation was made based on the outcomes assessed by the Mood Interest and Pleasure Questionnaire, the Vineland Adaptive Behavior Scales, and the Personal Well-being Index – ID. Results and conclusion: LSWp shows potential for improving the quality of life and preventing the loss of interest and pleasure in older adults with ID. It also shows promise in enhancing their socialization skills. Patients with better communication abilities seemed to benefit more from the LSWp. Keywords: life story work, life story book, intellectual disabilities, older adults, effectiveness

Timothy Kwok,1,2 Ping Chung Leung,3 Yun Kwok Wing,4 Isaac Ip,2 Bel Wong,2 Daniel Wai Hung Ho,2 Wai Ming Wong,3 Florence Ho2 1Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong; 2Jockey Club Centre for Positive Ageing, Hong Kong; 3Institute of Chinese Medicine, 4Department of Psychiatry, The Chinese University of Hong Kong, Hong Kong Purpose: Elderly with dementia are often afflicted with sleep problems. Recent studies have suggested that acupuncture may be a feasible alternative to traditional sleep medicine for treating sleep disturbance. This study investigated the effectiveness of acupuncture on sleep quality of elderly with dementia. Patients and methods: Nineteen elders with dementia were followed through a control period and an acupuncture treatment period, each lasting 6 weeks. Outcome measures were subjects' sleep quality and cognitive function. Sleep parameters were recorded by wrist actigraphy. Cognitive function was assessed by the Alzheimer's Disease Assessment Scale – Cognitive (ADAS-Cog). Pretests and posttests were conducted immediately before and after the control and treatment periods. Changes in the outcome measures between control and treatment periods were compared. Results: Wilcoxon signed rank tests revealed that the subjects gained significantly more resting time and total sleep time in the treatment period than in the control period (P < 0.05). A nonsignificant trend for improvement in sleep efficiency was observed. Improvement in cognitive function was not statistically significant. A total of 86% of the subjects completed the treatment regime. Conclusion: Results reveal that acupuncture was effective in improving some domains of sleep quality of elderly with dementia, and the subjects showed acceptance towards the intervention. Strengths and limitations of the present study as well as suggestions for further studies were considered. Keywords: acupuncture, sleep disturbance, patients with dementia

Aggressive features of hepatocellular carcinoma (HCC) are highly related to liver tumor-initiating cells (TICs), which are heterogeneous and plastic. In this issue of Cancer Cell, Yang et al. reveal the ability of CD49f-high TICs in shaping the tumor immunosuppressive microenvironment in HCC., Competing Interests: Declaration of interests The authors declare no completing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Aims: HCC is the most common type of primary liver cancer and is a common malignancy worldwide. About half of all new liver cancers worldwide each year occur in China, including Hong Kong, due to a high prevalence of HBV infection. HBV DNA integrates into the human genome, disrupting the endogenous tumor suppressors/regulatory genes or enhancing the activity of proto-oncogenes. It would be useful to examine the different NGS-based databases to provide a more unbiased and comprehensive survey of HBV integration., Approach and Results: We aimed to take advantage of publicly available data sets of different regional cohorts to determine the disparity landscapes of integration events among sample cohorts, tissue types, chromosomal positions, individual host, and viral genes, as well as genic locations. By comparing HCC tumors with non tumorous livers, the landscape of HBV integration was delineated in gene-independent and gene-dependent manners. Moreover, we performed mechanistic investigations on how HBV-TERT integration led to TERT activation and derived a score to predict patients' prognostication according to their clonal disparity landscape of HBV integration., Conclusions: Our study uncovered the different levels of clonal enrichment of HBV integration and identified mechanistic insights and prognostic biomarkers. This strengthens our understanding of HBV-associated hepatocarcinogenesis., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Mitochondria generate energy to support cells. They are important organelles that engage in key biological pathways. The dysfunction of mitochondria can be linked to hepatocarcinogenesis, which has been actively explored in recent years. To investigate the mitochondrial dysfunction caused by genetic variations, target-panel sequencing is a flexible and promising strategy. However, the copy number of mitochondria generally exceeds nuclear DNA, which raises a concern that uneven target enrichment of mitochondrial DNA (mtDNA) and nuclear DNA (ncDNA) in target-panel sequencing would lead to an undesirably biased representation of them. To resolve this issue, we evaluated the optimal pooling of mtDNA probes and ncDNA probes by a series of dilutions of mtDNA probes in both genomic DNA (gDNA) and cell-free DNA (cfDNA) samples. The evaluation was based on read count, average sequencing depth and coverage of targeted regions. We determined that an mtDNA:ncDNA probe ratio of around 1:10 would offer a good balance of sequencing performance and cost effectiveness. Moreover, we estimated the median physiological mtDNA:ncDNA copy ratio as 38.1 and 2.9 in cfDNA and gDNA samples of non-liver cancer subjects, respectively, whereas they were 20.0 and 2.1 in the liver cancer patients. Taken together, this study revealed the appropriate pooling strategy of mtDNA probes and ncDNA probes in target-panel sequencing and suggested the normal range of physiological variation of the mtDNA:ncDNA copy ratio in non-liver cancer individuals. This can serve as a useful reference for future target-panel sequencing investigations of the mitochondrial genome in liver cancer.

We developed a cell atlas named LiverSCA on human liver cancer single-cell RNA sequencing data. It has a user-friendly web interface and comprehensive functionalities aiming to help researchers to make easy access to cellular and molecular landscapes of the tumor microenvironment in liver cancer. LiverSCA includes a complete analytical pipeline that allow mechanistic exploration on a wide variety of functionalities, such as cell clustering, cell annotation, identification of differentially expressed genes, functional enrichment analysis, analysis of cellular crosstalk, and pseudo-time trajectory analysis. Notably, our intuitive web interface allows users, particularly wet-lab researchers, to easily explore and undertake data discovery, without the need to handle any of the raw data., (© 2024 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Objective: Fat mass and obesity-associated protein (FTO), an eraser of N 6 -methyadenosine (m6A), plays oncogenic roles in various cancers. However, its role in hepatocellular carcinoma (HCC) is unclear. Furthermore, small extracellular vesicles (sEVs, or exosomes) are critical mediators of tumourigenesis and metastasis, but the relationship between FTO-mediated m6A modification and sEVs in HCC is unknown., Design: The functions and mechanisms of FTO and glycoprotein non-metastatic melanoma protein B (GPNMB) in HCC progression were investigated in vitro and in vivo. Neutralising antibody of syndecan-4 (SDC4) was used to assess the significance of sEV-GPNMB. FTO inhibitor CS2 was used to examine the effects on anti-PD-1 and sorafenib treatment., Results: FTO expression was upregulated in patient HCC tumours. Functionally, FTO promoted HCC cell proliferation, migration and invasion in vitro, and tumour growth and metastasis in vivo. FTO knockdown enhanced the activation and recruitment of tumour-infiltrating CD8 + T cells. Furthermore, we identified GPNMB to be a downstream target of FTO, which reduced the m6A abundance of GPNMB, hence, stabilising it from degradation by YTH N 6 -methyladenosine RNA binding protein F2. Of note, GPNMB was packaged into sEVs derived from HCC cells and bound to the surface receptor SDC4 of CD8 + T cells, resulting in the inhibition of CD8 + T cell activation. A potential FTO inhibitor, CS2, suppresses the oncogenic functions of HCC cells and enhances the sensitivity of anti-PD-1 and sorafenib treatment., Conclusion: Targeting the FTO/m6A/GPNMB axis could significantly suppress tumour growth and metastasis, and enhance immune activation, highlighting the potential of targeting FTO signalling with effective inhibitors for HCC therapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Aims: HCC is an aggressive cancer with a poor clinical outcome. Understanding the mechanisms that drive tumor initiation is important for improving treatment strategy. This study aimed to identify functional cell membrane proteins that promote HCC tumor initiation., Approach and Results: Tailor-made siRNA library screening was performed for all membrane protein-encoding genes that are upregulated in human HCC (n = 134), with sphere formation as a surrogate readout for tumor initiation. Upon confirmation of membranous localization by immunofluorescence and tumor initiation ability by limiting dilution assay in vivo, LanC-like protein-1 (LANCL1) was selected for further characterization. LANCL1 suppressed intracellular reactive oxygen species (ROS) and promoted tumorigenicity both in vitro and in vivo. Mechanistically, with mass spectrometry, FAM49B was identified as a downstream binding partner of LANCL1. LANCL1 stabilized FAM49B by blocking the interaction of FAM49B with the specific E3 ubiquitin ligase TRIM21, thus protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppressed the Rac1-NADPH oxidase-driven ROS production, but this suppression of ROS was independent of the glutathione transferase function of LANCL1. Clinically, HCCs with high co-expression of LANCL1 and FAM49B were associated with more advanced tumor stage, poorer overall survival, and disease-free survival. In addition, anti-LANCL1 antibodies targeting the extracellular N-terminal domain were able to suppress the self-renewal ability, as demonstrated by the sphere formation ability of HCC cells., Conclusions: Our data showed that LANCL1 is a cell surface protein and a key contributor to HCC initiation. Targeting the LANCL1-FAM49B-Rac1-NADPH oxidase-ROS signaling axis may be a promising therapeutic strategy for HCC., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of in vitro assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple in vivo preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. Results: Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both in vitro and in vivo validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. Conclusions: Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background & Aims: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells., Methods: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest., Results: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers., Conclusions: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Understanding the mechanisms of HCC progression and metastasis is crucial to improve early diagnosis and treatment. This study aimed to identify key molecular targets involved in HCC metastasis., Approach and Results: Using whole-transcriptome sequencing of patients' HCCs, we identified and validated midline 1 interacting protein 1 (MID1IP1) as one of the most significantly upregulated genes in metastatic HCCs, suggesting its potential role in HCC metastasis. Clinicopathological correlation demonstrated that MID1IP1 upregulation significantly correlated with more aggressive tumor phenotypes and poorer patient overall survival rates. Functionally, overexpression of MID1IP1 significantly promoted the migratory and invasive abilities and enhanced the sphere-forming ability and expression of cancer stemness-related genes of HCC cells, whereas its stable knockdown abrogated these effects. Perturbation of MID1IP1 led to significant tumor shrinkage and reduced pulmonary metastases in an orthotopic liver injection mouse model and reduced pulmonary metastases in a tail-vein injection model in vivo . Mechanistically, SP1 transcriptional factor was found to be an upstream driver of MID1IP1 transcription. Furthermore, transcriptomic sequencing on MID1IP1-overexpressing HCC cells identified FOS-like 1 (FRA1) as a critical downstream mediator of MID1IP1. MID1IP1 upregulated FRA1 to subsequently promote its transcriptional activity and extracellular matrix degradation activity of matrix metalloproteinase MMP9, while knockdown of FRA1 effectively abolished the MID1IP1-induced migratory and invasive abilities., Conclusions: Our study identified MID1IP1 as a regulator in promoting FRA1-mediated-MMP9 signaling and demonstrated its role in HCC metastasis. Targeting MID1IP1-mediated FRA1 pathway may serve as a potential therapeutic strategy against HCC progression., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Primary liver cancer (PLC), consisting mainly of hepatocellular carcinoma and intrahepatic cholangiocarcinoma, is one of the major causes of cancer-related mortality worldwide. The curative therapy for PLC is surgical resection and liver transplantation, but most PLCs are inoperable at diagnosis. Even after surgery, there is a high rate of tumor recurrence. There is an unmet clinical need to discover more effective treatment options for advanced PLCs. Pre-clinical mouse models in PLC research have played a critical role in identifying key oncogenic drivers and signaling pathways in hepatocarcinogenesis. Furthermore, recent advances in single-cell RNA sequencing (scRNA-seq) have provided an unprecedented degree of resolution in such characterization. In this review, we will summarize the recent studies that utilized pre-clinical mouse models with the combination of scRNA-seq to provide an understanding of different aspects of PLC. We will focus particularly on the potentially actionable targets regarding the cellular and molecular components. We anticipate that the findings in mouse models could complement those in patients. With more defined etiological background, mouse models may provide valuable insights.

Background & Aims: Metabolic reprogramming is recognized as a cancer hallmark intimately linked to tumor hypoxia, which supports rapid tumor growth and mitigates the consequential oxidative stress. Phosphofructokinase-fructose bisphosphatase (PFKFB) is a family of bidirectional glycolytic enzymes possessing both kinase and phosphatase functions and has emerged as important oncogene in multiple types of cancer. However, its clinical relevance, functional significance, and underlying mechanistic insights in hepatocellular carcinoma (HCC), the primary malignancy that develops in the most important metabolic organ, has never been addressed., Methods: PFKFB4 expression was examined by RNA sequencing in The Cancer Genome Atlas and our in-house HCC cohort. The up-regulation of PFKFB4 expression was confirmed further by quantitative polymerase chain reaction in an expanded hepatitis B virus-associated HCC cohort followed by clinicopathologic correlation analysis. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated PFKFB4 knockout cells were generated for functional characterization in vivo, targeted metabolomic profiling, as well as RNA sequencing analysis to comprehensively examine the impact of PFKFB4 loss in HCC., Results: PFKFB4 expression was up-regulated significantly in HCC and correlated positively with TP53 and TSC2 loss-of-function mutations. In silico transcriptome-based analysis further revealed PFKFB4 functions as a critical hypoxia-inducible gene. Clinically, PFKFB4 up-regulation was associated with more aggressive tumor behavior. Functionally, CRISPR/Cas9-mediated PFKFB4 knockout significantly impaired in vivo HCC development. Targeted metabolomic profiling revealed that PFKFB4 functions as a phosphatase in HCC and its ablation caused an accumulation of metabolites in downstream glycolysis and the pentose phosphate pathway. In addition, PFKFB4 loss induced hypoxia-responsive genes in glycolysis and reactive oxygen species detoxification. Conversely, ectopic PFKFB4 expression conferred sorafenib resistance., Conclusions: PFKFB4 up-regulation supports HCC development and shows therapeutic implications., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The highly proliferative nature of hepatocellular carcinoma (HCC) frequently results in a hypoxic intratumoural microenvironment, which creates a therapeutic challenge owing to a lack of mechanistic understanding of the phenomenon. We aimed to identify critical drivers of HCC development and progression in the hypoxic microenvironment., Methods: We performed integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia and identified the Ephrin-A3/Eph receptor A2 (EphA2) axis as a clinically relevant and hypoxia-inducible signalling axis in HCC. The functional significance and mechanistic consequences of the Ephrin-A3/EphA2 axis were examined in EFNA3- and EPHA2- knockdown/overexpressing HCC cells. The potential downstream pathways were investigated by transcriptome sequencing, quantitative reverse-transcription PCR, western blotting analysis and metabolomics., Results: EFNA3 was frequently upregulated in HCC and its overexpression was associated with more aggressive tumour behaviours. HIF-1α directly and positively regulated EFNA3 expression under hypoxia. EFNA3 functionally contributed to self-renewal, proliferation and migration in HCC cells. EphA2 was identified as a key functional downstream mediator of EFNA3. Functional characterisation of the Ephrin-A3/EphA2 forward-signalling axis demonstrated a promotion of self-renewal ability and tumour initiation. Mechanistically, the Ephrin-A3/EphA2 axis promoted the maturation of SREBP1 and expression of its transcriptional target, ACLY, was significantly associated with the expression of EFNA3 and hypoxia markers in clinical cohorts. The metabolic signature of EPHA2 and ACLY stable knockdown HCC cells demonstrated significant overlap in fatty acid, cholesterol and tricarboxylic acid cycle metabolite profiles. ACLY was confirmed to mediate the self-renewal function of the Ephrin-A3/EphA2 axis., Conclusions: Our findings revealed the novel role of the Ephrin-A3/EphA2 axis as a hypoxia-sensitive modulator of HCC cell metabolism and a key contributor to HCC initiation and progression., Lay Summary: Hepatocellular carcinoma (HCC) is a fast-growing tumour; hence, areas of the tumour often have insufficient vasculature and become hypoxic. The presence of hypoxia within tumours has been shown to negatively impact on the survival of patients with tumours, including HCC. Herein, we identified the Ephrin-A3/EphA2 axis as a key functional driver of tumour initiation and progression in response to hypoxia. Additionally, we showed that SREBP1-ACLY-mediated metabolic rewiring was an important downstream effector that induced cancer stemness in response to Ephrin-A3/EphA2 forward-signalling., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Lines of evidence implicate CENPF and FOXM1 may have novel co-operative roles in driving hepatocellular carcinoma (HCC)., Objective: We investigated the clinicopathological correlation, functional characterization, molecular mechanism and translational significance of CENPF and FOXM1., Methods: We carried out integrative studies investigating functional synergism of CENPF and FOXM1 in HCC and its metastasis. Human HCC samples, HCC cell lines and mouse model were used in the studies. Stable knockdown, q-PCR, Western blotting, whole-transcriptomic sequencing (RNA-seq), as well as cell and mouse assays were performed., Results: Upon clinicopathological correlation, we found that co-overexpression of CENPF and FOXM1 in human HCCs was associated with more aggressive tumor behavior including presence of venous invasion, tumor microsatellite formation, and absence of tumor encapsulation. Moreover, co-silencing FOXM1 and CENPF using shRNA approach in HCC cell lines resulted in significantly reduced cell proliferation. Furthermore, our RNA-seq and differential gene expression analysis delineated that CENPF and FOXM1 co-regulated a specific set of target genes in various metabolic processes and oncogenic signaling pathways. Among them, POLD1, which encodes the catalytic subunit of DNA polymerase δ, was ranked as the top downstream target co-regulated by CENPF and FOXM1. POLD1 expression was positively correlated with that of FOXM1 and CENPF in HCCs. In addition, POLD1 expression was significantly upregulated in HCC tumors. Functionally, in vivo orthotopic injection model showed that stable knockdown of POLD1 in HCC cells suppressed tumor incidence and tumorigenicity and had a trend of diminished lung metastasis., Conclusion: Taken together, our data suggest that CENPF and FOXM1 could synergistically support hepatocarcinogenesis via the regulation of POLD1. CENPF and FOXM1 may represent new vulnerabilities to novel drug-based therapy in HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ho, Lam, Chan and Ng.)

Background and Aims: Ras-like (Ral) small guanosine triphosphatases (GTPases), RalA and RalB, are proto-oncogenes directly downstream of Ras and cycle between the active guanosine triphosphate-bound and inactive guanosine diphosphate-bound forms. RalGTPase-activating protein (RalGAP) complex exerts a negative regulation. Currently, the role of Ral up-regulation in cancers remains unclear. We aimed to examine the clinical significance, functional implications, and underlying mechanisms of RalA signaling in HCC., Approach and Results: Our in-house and The Cancer Genome Atlas RNA sequencing data and quantitative PCR data revealed significant up-regulation of RalA in patients' HCCs. Up-regulation of RalA was associated with more aggressive tumor behavior and poorer prognosis. Consistently, knockdown of RalA in HCC cells attenuated cell proliferation and migration in vitro and tumorigenicity and metastasis in vivo. We found that RalA up-regulation was driven by copy number gain and uncovered that SP1 and ETS proto-oncogene 2 transcription factor cotranscriptionally drove RalA expression. On the other hand, RalGAPA2 knockdown increased the RalA activity and promoted intrahepatic and extrahepatic metastasis in vivo. Consistently, we observed significant RalGAPA2 down-regulation in patients' HCCs. Intriguingly, HCC tumors showing simultaneous down-regulation of RalGAPA2 and up-regulation of RalA displayed a significant association with more aggressive tumor behavior in terms of more frequent venous invasion, more advanced tumor stage, and poorer overall survival. Of note, Ral inhibition by a Ral-specific inhibitor RBC8 suppressed the oncogenic functions in a dose-dependent manner and sensitized HCC cells to sorafenib treatment, with an underlying enhanced inhibition of mammalian target of rapamycin signaling., Conclusions: Our results provide biological insight that dysregulation of RalA signaling through dual regulatory mechanisms supports its oncogenic functions in HCC. Targeting RalA may serve as a potential alternative therapeutic approach alone or in combination with currently available therapy., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Hepatocarcinogenesis involves complex genetic and cellular dysregulations which drive the formation of hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, with extensive heterogeneity. In contrast to the broad spectrum of molecularly driven therapies available for defined patient groups in certain cancer types, unfortunately the treatment options for HCC are highly limited. The lack of representative molecular and cellular signatures in the heterogeneous HCC tumors that can effectively guide the choice of the most appropriate treatment among the patients unavoidably limits the treatment outcome. Advancement and wide availability of the next-generation sequencing technologies have empowered us to examine and capture not only the detailed genetic alterations of the HCC cells but also the precise composition of different cell types within the tumor microenvironment and their interactions with the HCC cells at an unprecedented level. The information generated has provided new insight and better defined the inter-patient intertumoral heterogeneity, intra-patient intratumoral heterogeneity as well as the plasticity of HCC cells. These collectively provide a robust scientific basis in guiding the development and use of targeted therapy and immunotherapy. To complement, liquid biopsy coupled with high-sensitivity sequencing could potentially be adopted as a more practical and safer approach to detect and reflect the tumor heterogeneity in HCC patients in guiding the choice of treatment and monitoring disease progression., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hepatocellular carcinoma (HCC) is characterized by its high degrees of both inter- and intratumoral heterogeneity. Its complex tumor microenvironment is also crucial in promoting tumor progression. Recent advances in single-cell RNA sequencing provide an important highway to characterize the underlying pathogenesis and heterogeneity of HCC in an unprecedented degree of resolution. This review discusses the up-to-date discoveries from the latest studies of HCC with respect to the strength of single-cell RNA sequencing. We discuss its use in the dissection of the landscape of the intricate HCC ecosystem and highlight the major features at cellular levels, including the malignant cells, different immune cell types, and the various cell-cell interactions, which are crucial for developing effective immunotherapies. Finally, its translational applications will be discussed. Altogether, these explorations may give us some hints at the tumor growth and progression and drug resistance and recurrence, particularly in this era of personalized medicine., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Liver cancer (hepatocellular carcinoma [HCC]) is a fatal cancer worldwide and often is detected at an advanced stage when treatment options are very limited. This drives the development of techniques and platforms for early detection of HCC. In recent years, liquid biopsy has provided a means of noninvasive detection of cancers. By detecting plasma circulating tumor DNA (ctDNA) released from dying cancer cells, the presence of HCC can be detected in a noninvasive manner. In this review, we discuss the molecular characteristics of ctDNA and its various molecular landscapes in HCC. These include the mutational landscape, single-nucleotide variations, copy number variations, methylation landscape, end motif/coordinate preference, hepatitis B virus integration, and mitochondrial DNA mutations. The consistency between the plasma ctDNA and the tumor tissue genomic DNA mutational profile is pivotal for the clinical utility of ctDNA in the clinical management of HCC. With strategic use of genetic information provided from plasma ctDNA profiling and procedure standardization to facilitate implementation in clinical practice, better clinical management would become permissible through more efficient detection and diagnosis of HCC, better prognostication, precision-matched treatment guidance, and more reliable disease monitoring., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The application of single-cell RNA sequencing to delineate tissue heterogeneity and complexity has become increasingly popular. Given its tremendous resolution and high-dimensional capacity for in-depth investigation, single-cell RNA sequencing offers an unprecedented research power. Although some popular software packages are available for single-cell RNA sequencing data analysis and visualization, it is still a big challenge for their usage, as they provide only a command-line interface and require significant level of bioinformatics skills. Methods: We have developed scAnalyzeR, which is a single-cell RNA sequencing analysis pipeline with an interactive and user-friendly graphical interface for analyzing and visualizing single-cell RNA sequencing data. It accepts single-cell RNA sequencing data from various technology platforms and different model organisms (human and mouse) and allows flexibility in input file format. It provides functionalities for data preprocessing, quality control, basic summary statistics, dimension reduction, unsupervised clustering, differential gene expression, gene set enrichment analysis, correlation analysis, pseudotime cell trajectory inference, and various visualization plots. It also provides default parameters for easy usage and allows a wide range of flexibility and optimization by accepting user-defined options. It has been developed as a docker image that can be run in any docker-supported environment including Linux, Mac, and Windows, without installing any dependencies. Results: We compared the performance of scAnalyzeR with 2 other graphical tools that are popular for analyzing single-cell RNA sequencing data. The comparison was based on the comprehensiveness of functionalities, ease of usage and flexibility, and execution time. In general, scAnalyzeR outperformed the other tested counterparts in various aspects, demonstrating its superior overall performance. To illustrate the usefulness of scAnalyzeR in cancer research, we have analyzed the in-house liver cancer single-cell RNA sequencing dataset. Liver cancer tumor cells were revealed to have multiple subpopulations with distinctive gene expression signatures. Conclusion: scAnalyzeR has comprehensive functionalities and demonstrated usability. We anticipate more functionalities to be adopted in the future development.

Background: Controversy over the benefits of antioxidants supplements in cancers persists for long. Using hepatocellular carcinoma (HCC) as a model, we investigated the effects of exogenous antioxidants N-acetylcysteine (NAC) and glutathione (GSH) on tumor formation and growth., Methods: Multiple mouse models, including diethylnitrosamine (DEN)-induced and Trp53KO/C-MycOE-induced HCC models, mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection were used. In vitro assays including ROS assay, colony formation, sphere formation, proliferation, migration and invasion, apoptosis, cell cycle assays were conducted. Western blot was performed for protein expression and RNA-sequencing to identify potential gene targets., Results: In these multiple different mouse and cell line models, we observed that NAC and GSH promoted HCC tumor formation and growth, accompanied with significant reduction of intracellular reactive oxygen species (ROS) levels. Moreover, NAC and GSH promoted cancer stemness, and abrogated the tumor-suppressive effects of Sorafenib both in vitro and in vivo. Exogenous supplementation of NAC or GSH reduced the expression of NRF2 and GCLC, suggesting the NRF2/GCLC-related antioxidant production pathway might be desensitized. Using transcriptomic analysis to identify potential gene targets, we found that TMBIM1 was significantly upregulated upon NAC and GSH treatment. Both TCGA and in-house RNA-sequence databases showed that TMBIM1 was overexpressed in HCC tumors. Stable knockdown of TMBIM1 increased the intracellular ROS; it also abolished the promoting effects of the antioxidants in HCC cells. On the other hand, BSO and SSA, inhibitors targeting NAC and GSH metabolism respectively, partially abrogated the pro-oncogenic effects induced by NAC and GSH in vitro and in vivo., Conclusions: Our data implicate that exogenous antioxidants NAC and GSH, by reducing the intracellular ROS levels and inducing TMBIM expression, promoted HCC formation and tumor growth, and counteracted the therapeutic effect of Sorafenib. Our study provides scientific insight regarding the use of exogenous antioxidant supplements in cancers., (© 2021. The Author(s).)

Rab GTPases are major mediators that ensure the proper spatiotemporal regulation of intracellular trafficking. Functional impairment and altered expression of Rab proteins have been revealed in various human cancers. There is an emerging evidence about the role of Rab proteins in the biogenesis of extracellular vesicles (EVs). In hepatocellular carcinoma (HCC), using RNA sequencing comparing expression profiles of adjacent non-tumorous tissues and HCC, Rab20 is identified to be the most frequently downregulated Rab member in HCC. Functionally, restoration of Rab20 in metastatic HCC cells results in the release of EVs with a diminished activity to promote cell growth, motility and metastasis. Conversely, EVs released from normal liver cells with Rab20 knockdown loses suppressive effect on HCC cell growth and motility. Proteomic profiling revealed the level of triosephosphate isomerase 1 (TPI1), a glycolytic enzyme, in EVs to be positively associated with Rab20 expression of the releasing cells. TPI1 targeted to be expressed in EVs released by Rab20 knockdown cells compromises the oncogenic activity of EVs. Besides, EVs released by TPI1 knockdown cells recapitulates the promoting effect of EVs derived from HCC cells with Rab20 underexpression. Aerobic glycolysis is beneficial to the survival and proliferation of tumour cells. Here, we observed that the enhanced cell growth and motility are driven by the enhanced aerobic glycolysis induced by EVs with reduced TPI1. The addition of glycolytic inhibitor blocks the promoting effect of EVs with reduced TPI1. Taken together, our study provides a mechanistic link among tumour cell-derived EVs and glucose metabolism in HCC with Rab20 deregulation., Competing Interests: The authors report no conflict of interest., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Interaction between tumor cells and immune cells in the tumor microenvironment is important in cancer development. Immune cells interact with the tumor cells to shape this process. Here, we use single-cell RNA sequencing analysis to delineate the immune landscape and tumor heterogeneity in a cohort of patients with HBV-associated human hepatocellular carcinoma (HCC). We found that tumor-associated macrophages suppress tumor T cell infiltration and TIGIT-NECTIN2 interaction regulates the immunosuppressive environment. The cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplifies the overall cancer-promoting immunocellular landscape. Furthermore, the heterogeneity of global molecular profiles reveals co-existence of intra-tumoral and inter-tumoral heterogeneity, but is more apparent in the latter. This analysis of the immunosuppressive landscape and intercellular interactions provides mechanistic information for the design of efficacious immune-oncology treatments in hepatocellular carcinoma.

Background & Aims: Mutational profiling of patient tumors has suggested that hepatocellular carcinoma (HCC) development is mainly driven by loss-of-function mutations in tumor suppressor genes. p90 ribosomal S6 kinase 2 (RSK2) functions as a direct downstream kinase of ERK1/2 and elevated RSK2 expression has been reported to support oncogenic functions in some cancers. We investigated if RSK2 was also dysregulated by inactivating mutations in cancers including HCC., Methods: We performed exome sequencing and targeted DNA sequencing on HBV-associated HCCs to examine recurrent RSK2 mutations. The functional significance and mechanistic consequences of RSK2 mutations were examined in natural RSK2-null HCC cells, and RSK2-knockout HCC cells. The potential downstream pathways underlying RSK2 mutations were investigated by RNA sequencing, qRT-PCR and mass spectrometry., Results: We detected recurrent somatic RSK2 mutations at a rate of 6.3% in our HCC cohorts and revealed that, among many cancer types, HCC was the cancer most commonly harboring RSK2 mutations. The RSK2 mutations were inactivating and associated with a more aggressive tumor phenotype. We found that, functionally, restoring RSK2 expression in natural RSK2-null HBV-positive Hep3B cells suppressed proliferation and migration in vitro and tumorigenicity in vivo. Mechanistically, RSK2-inactivating mutations attenuated a SOS1/2-dependent negative feedback loop, leading to the activation of MAPK signaling. Of note, this RSK2 mutation-mediated MAPK upregulation rendered HCC cells more sensitive to sorafenib, a first-line multi-kinase inhibitor for advanced HCC. Furthermore, such activation of MAPK signaling enhanced cholesterol biosynthesis-related gene expression in HCC cells., Conclusions: Our findings reveal the mechanistic and functional significance of RSK2-inactivating mutations in HCC. These inactivating mutations may serve as an alternative route to activate MAPK signaling and cholesterol metabolism in HCC., Lay Summary: In this study, we identified and functionally characterized RSK2-inactivating mutations in human hepatocellular carcinoma and demonstrated their association with aggressive tumor behavior. Mutations in RSK2 drive signaling pathways with known oncogenic potential, leading to enhanced cholesterol biosynthesis and potentially sensitizing tumors to sorafenib treatment., Competing Interests: Conflict of interest The authors declare no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Hepatitis B virus (HBV) integrations are common in hepatocellular carcinoma (HCC). In particular, alterations of the telomerase reverse transcriptase (TERT) gene by HBV integrations are frequent; however, the molecular mechanism and functional consequence underlying TERT HBV integration are unclear., Approach and Results: We adopted a targeted sequencing strategy to survey HBV integrations in human HBV-associated HCCs (n = 95). HBV integration at the TERT promoter was frequent (35.8%, n = 34/95) in HCC tumors and was associated with increased TERT mRNA expression and more aggressive tumor behavior. To investigate the functional importance of various integrated HBV components, we employed different luciferase reporter constructs and found that HBV enhancer I (EnhI) was the key viral component leading to TERT activation on integration at the TERT promoter. In addition, the orientation of the HBV integration at the TERT promoter further modulated the degree of TERT transcription activation in HCC cell lines and patients' HCCs. Furthermore, we performed array-based small interfering RNA library functional screening to interrogate the potential major transcription factors that physically interacted with HBV and investigated the cis-activation of host TERT gene transcription on viral integration. We identified a molecular mechanism of TERT activation through the E74 like ETS transcription factor 4 (ELF4), which normally could drive HBV gene transcription. ELF4 bound to the chimeric HBV EnhI at the TERT promoter, resulting in telomerase activation. Stable knockdown of ELF4 significantly reduced the TERT expression and sphere-forming ability in HCC cells., Conclusions: Our results reveal a cis-activating mechanism harnessing host ELF4 and HBV integrated at the TERT promoter and uncover how TERT HBV-integrated HCCs may achieve TERT activation in hepatocarcinogenesis., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Accumulating evidence illustrates the significance of cell plasticity in the molecular biology of liver cancer. Reprogramming of mature parenchymal cells to a less differentiated state by key molecular targets contributes to the pathogenesis of hepatocellular carcinoma (HCC). Hereby, we investigated the role of GATA6, a transcription factor implicated in hepatocyte lineage specification, in HCC. Our results demonstrated a lower expression of GATA6 in HCC tissues compared to the corresponding nontumoral liver tissues. Moreover, GATA6 underexpression, as observed in about 50% cases in our clinical cohort, was associated with a poorer degree of tumor cell differentiation and worse disease-free survival outcome. In vitro, silencing of GATA6 in HCC cells augmented cell migration and invasion abilities of HCC cells by activating epithelial-mesenchymal transition. Self-renewal was also enhanced in vitro. Consistently, in vivo tumorigenicity and self-renewal was promoted upon GATA6 knockdown. Notably, suppression of GATA6 converts HCC cells to a metabolic phenotype recapitulating stem-cell state. Expression of glycolytic markers was elevated in GATA6-knockdown clones accompanied by increased glucose uptake; while overexpression of GATA6 resulted in opposite effects. Further to this, we identified that GATA6 bound to the promoter region of PKM gene and regulated PKM2 transcription. Taken together, downregulation of GATA6 directs HCC cells to glycolytic metabolism and fosters tumorigenicity, self-renewal and metastasis. GATA6 is a transcriptional regulator and a genetic switch that converts the phenotypic reprogramming of HCC cells. It is a potential prognostic biomarker and therapeutic target for liver cancer., (© 2019 UICC.)

Hepatocellular carcinoma (HCC) is heterogeneous, rendering its current curative treatments ineffective. The emergence of single-cell genomics represents a powerful strategy in delineating the complex molecular landscapes of cancers. In this study, we demonstrated the feasibility and merit of using single-cell RNA sequencing to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare cell subpopulations of significance. Exploration of the inter-relationship among liver cancer stem cell markers showed two distinct major cell populations according to EPCAM expression, and the EPCAM + cells had upregulated expression of multiple oncogenes. We also identified a CD24 + /CD44 + -enriched cell subpopulation within the EPCAM + cells which had specific signature genes and might indicate a novel stemness-related cell subclone in HCC. Notably, knockdown of signature gene CTSE for CD24 + /CD44 + cells significantly reduced self-renewal ability on HCC cells in vitro and the stemness-related role of CTSE was further confirmed by in vivo tumorigenicity assays in nude mice. In summary, single-cell genomics is a useful tool to delineate HCC intratumoral heterogeneity at better resolution. It can identify rare but important cell subpopulations, and may guide better precision medicine in the long run., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. Increasing evidence shows that epigenetic alterations play an important role in human carcinogenesis. Deregulation of DNA methylation and histone modifications have recently been characterized in HCC, but the significance of chromatin remodeling in liver carcinogenesis remains to be explored. In this study, by systematically analyzing the expression of chromatin remodeling genes in human HCCs, we found that helicase, lymphoid-specific (HELLS), an SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC. Overexpression of HELLS correlated with more aggressive clinicopathological features and poorer patient prognosis compared to patients with lower HELLS expression. We further showed that up-regulation of HELLS in HCC was conferred by hyperactivation of transcription factor specificity protein 1 (SP1). To investigate the functions of HELLS in HCC, we generated both gain-of-function and loss-of-function models by the CRISPR activation system, lentiviral short hairpin RNA, and the CRISPR/Cas9 genome editing system. We demonstrated that overexpression of HELLS augmented HCC cell proliferation and migration. In contrast, depletion of HELLS reduced HCC growth and metastasis both in vitro and in vivo. Moreover, inactivation of HELLS led to metabolic reprogramming and reversed the Warburg effect in HCC cells. Mechanistically, by integrating analysis of RNA sequencing and micrococcal nuclease sequencing, we revealed that overexpression of HELLS increased nucleosome occupancy, which obstructed the accessibility of enhancers and hindered formation of the nucleosome-free region (NFR) at the transcription start site. Though this mechanism, up-regulation of HELLS mediated epigenetic silencing of multiple tumor suppressor genes including E-cadherin, FBP1, IGFBP3, XAF1 and CREB3L3 in HCC. Conclusion: Our data reveal that HELLS is a key epigenetic driver of HCC; by altering the nucleosome occupancy at the NFR and enhancer, HELLS epigenetically suppresses multiple tumor suppressor genes to promote HCC progression., (© 2019 by the American Association for the Study of Liver Diseases.)

Cytidine deaminase APOBEC3B (A3B) is known to play important roles in creating de novo genomic C-to-T mutations in cancers and contribute to induction of genomic instability. Our study evaluated the roles of A3B in the progression and metastasis of human hepatocellular carcinoma (HCC). Using whole-transcriptome and whole-exome sequencing, and quantitative PCR, we found that A3B was overexpressed in human HCCs and A3B expression was significantly correlated with the proportion of genomic C-to-A and G-to-T mutations. Upon clinicopathological correlation, higher A3B expression was associated with more aggressive tumor behavior. Wild-type A3B (wt-A3B) overexpression in HCC cells promoted cell proliferation, and cell migratory and invasive abilities in vitro, and tumorigenicity and metastasis in vivo. On the other hand, knockdown of A3B suppressed cell proliferation, migratory, and invasive abilities of HCC cells with high endogenous A3B level. However, to our surprise, overexpression of A3B deaminase-dead double mutant (E68A/E255Q) led to similar results as wt-A3B in HCC. Furthermore, overexpression of wt-A3B and mutant A3B both enhanced cell cycle progression in HCC cells. Altogether, our data demonstrated a novel deaminase-independent role of A3B in contributing to HCC tumorigenesis and metastasis., (© 2018 Wiley Periodicals, Inc.)

Purpose: The study examined the relationship between verbal short-term memory (STM) and language impairment in Cantonese speakers after stroke. It is hypothesised that Cantonese speakers with left-hemisphere (LH) stroke would perform worse than those with right hemisphere (RH) stroke and normal controls. Specific linguistic factors of Cantonese might affect results in the tasks., Method: Fifteen participants with LH stroke, 10 with RH stroke and 25 healthy controls were tested with auditory-verbal immediate serial recall (ISR) tasks and auditory linguistic tasks. All stroke participants were assessed with the Cantonese version of Western Aphasia Battery (CAB)., Result: The LH group performed significantly worse than the RH and healthy control groups in the auditory verbal ISR and auditory linguistic tasks. There were significant lexicality, frequency and imageability effects in most tasks. Auditory discrimination and word comprehension tasks, but not the auditory word recognition task had correlations with ISR tasks., Conclusion: Verbal STM and language performance of Cantonese-speakers with history of LH stroke were inferior to RH stroke and healthy controls. The effects of lexicality, word frequency and imageability on verbal STM memory performance were found. Cantonese tones have effects on performance in auditory word recognition task, similar to onset, nucleus and rime.

Identification and characterization of functional molecular targets conferring stemness properties in hepatocellular carcinoma (HCC) offers crucial insights to overcome the major hurdles of tumor recurrence, metastasis and chemoresistance in clinical management. In the current study, we investigated the significance of Cripto-1 in contributing to HCC stemness. Cripto-1 was upregulated in the sorafenib-resistant clones derived from HCC cell lines and patient-derived xenograft that we previously developed, suggesting an association between Cripto-1 and stemness. By in vitro experiments, Cripto-1 fostered cell proliferation, migration, and invasion. It also enhanced self-renewal ability and conferred chemoresistance of HCC cells. Consistently, silencing of Cripto-1 suppressed in vivo tumorigenicity on serial transplantation. On the downstream signaling mechanism, expression of major components of Wnt/β-catenin pathway β-catenin, AXIN2, and C-MYC, accompanied by β-catenin activity was reduced upon Cripto-1 knockdown. The suppressive effects on stemness properties with Cripto-1 knockdown in vitro and in vivo were partially rescued by forced expression of constitutively active β-catenin. Further elucidation revealed the binding of Cripto-1 to Frizzled-7 (FZD7), low-density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled-3 (DVL3) of the Wnt/β-catenin pathway and stabilized DVL3 protein. Analyses with clinical samples validated Cripto-1 overexpression in HCC tissues, as well as a positive correlation between Cripto-1 and AXIN2 expressions. High Cripto-1 level in tumor was associated with poorer disease-free survival of HCC patients. Taken together, Cripto-1 binds to FZD7/LRP6 and DVL3, stabilizes DVL3 expression and activates the Wnt/β-catenin signaling cascade to confer stemness in HCC. Our study findings substantiated the role of Cripto-1 in determining stemness phenotypes of HCC and mechanistically in modulating the Wnt/β-catenin signaling cascade, one of the most frequently deregulated pathways in liver cancer.

Epigenetic alterations have contributed greatly to human carcinogenesis. Conventional epigenetic studies have predominantly focused on DNA methylation, histone modifications, and chromatin remodeling. Recently, diverse and reversible chemical modifications of RNAs have emerged as a new layer of epigenetic regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of eukaryotic messenger RNA (mRNA) and is important for the regulation of mRNA stability, splicing, and translation. Using transcriptome sequencing, we discovered that methyltransferase-like 3 (METTL3), a major RNA N6-adenosine methyltransferase, was significantly up-regulated in human hepatocellular carcinoma (HCC) and multiple solid tumors. Clinically, overexpression of METTL3 is associated with poor prognosis of patients with HCC. Functionally, we proved that knockdown of METTL3 drastically reduced HCC cell proliferation, migration, and colony formation in vitro. Knockout of METTL3 remarkably suppressed HCC tumorigenicity and lung metastasis in vivo. On the other hand, using the CRISPR/dCas9-VP64 activation system, we demonstrated that overexpression of METTL3 significantly promoted HCC growth both in vitro and in vivo. Through transcriptome sequencing, m6A sequencing, and m6A methylated RNA immuno-precipitation quantitative reverse-transcription polymerase chain reaction, we identified suppressor of cytokine signaling 2 (SOCS2) as a target of METTL3-mediated m6A modification. Knockdown of METTL3 substantially abolished SOCS2 mRNA m6A modification and augmented SOCS2 mRNA expression. We also showed that m6A-mediated SOCS2 mRNA degradation relied on the m6A reader protein YTHDF2-dependent pathway., Conclusion: METTL3 is frequently up-regulated in human HCC and contributes to HCC progression. METTL3 represses SOCS2 expression in HCC through an m6A-YTHDF2-dependent mechanism. Our findings suggest an important mechanism of epigenetic alteration in liver carcinogenesis. (Hepatology 2018;67:2254-2270)., (© 2017 by the American Association for the Study of Liver Diseases.)

Objective: We investigated the effect and mechanism of hypoxic microenvironment and hypoxia-inducible factors (HIFs) on hepatocellular carcinoma (HCC) cancer stemness., Design: HCC cancer stemness was analysed by self-renewal ability, chemoresistance, expression of stemness-related genes and cancer stem cell (CSC) marker-positive cell population. Specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) mRNA level was examined with quantitative PCR in human paired HCCs. Immunoprecipitation was used to examine the binding of proteins and chromatin immunoprecipitation assay to detect the binding of HIFs with hypoxia response element sequence. In vivo characterisation was performed in immunocompromised mice and stem cell frequency was analysed., Results: We showed that hypoxia enhanced the stemness of HCC cells and hepatocarcinogenesis through enhancing HIF-1α deSUMOylation by SENP1 and increasing stabilisation and transcriptional activity of HIF-1α. Furthermore, we demonstrated that SENP1 is a direct target of HIF-1/2α and a previously unrecognised positive feedback loop exists between SENP1 and HIF-1α., Conclusions: Taken together, our findings suggest the significance of this positive feedback loop between HIF-1α and SENP1 in contributing to the increased cancer stemness in HCC and hepatocarcinogenesis under hypoxia. Drugs that specifically target SENP1 may offer a potential novel therapeutic approach for HCC., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The hypoxic microenvironment is well-characterized in hepatocellular carcinoma (HCC). Delineation of hypoxia-responsive events is an integral part to understand the pathogenesis of HCC. We studied the functional role and clinical relevance of Stanniocalcin 1 (STC1), a hypoxia-induced molecular target, in HCC. In our clinical cohort, STC1 transcript was up-regulated in HCC tumor tissues. Moreover, STC1 protein was detected in the sera of HCC patients. A higher serum STC1 level was correlated with larger tumor size and poorer 5-year disease-free survival. Functionally, recombinant STC1 protein (rhSTC1) promoted cell migration and cell invasion in vitro; and the effect was abolished by co-treatment of anti-STC1 neutralizing antibody. By in vivo mouse model, silencing of STC1 in HCC cells downregulated secretory STC1 level and suppressed lung metastasis. Furthermore, we found that rhSTC1 activated the JNK pathway, as evidenced by altered expression of the key molecular targets pJNK and p-c-Jun. The functional effects conferred by rhSTC1 were abrogated by co-treatment of JNK inhibitor. In summary, secretory STC1 enhances metastatic potential of HCC via JNK signaling. It potentially serves as a prognostic serum biomarker and a therapeutic target for HCC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Dishevelled-3 (Dvl3) is regarded as a binding hub with many different interacting partners. However, its regulation and mechanism on cancer stemness remain to be explored. In this study, we showed that Dvl3 was significantly overexpressed in human hepatocellular carcinomas (HCCs) and promoted cancer stemness both in vitro and in vivo. We found that the non-phosphorylated (NP)-Dvl3 was more stable than the phosphorylated form, more active in activating β-catenin transcriptional activity, and more potent in enhancing self-renewal ability in HCC cells. Mechanistically, we confirmed that the homeodomain-interacting protein kinase-2 (HIPK2) and E3 ubiquitin ligase ITCH were able to physically bind to Dvl3 protein. Knockdown of HIPK2 and the protein phosphatase regulatory unit C-alpha (PP1Cα) resulted in sustained Dvl3 phosphorylation and hence decrease in the NP form of Dvl3. On the other hand, knockdown of E3 ubiquitin ligase ITCH reduced the phosphorylation-induced degradation and stabilized the phosphorylated Dvl3 protein. Furthermore, the NP-Dvl3 enhanced the LGR5 promoter activity to upregulate LGR5 expression, which was associated with increased cancer stemness in HCC. Our findings established that HIPK2/PP1Cα/ITCH axis sustains the de-phosphorylation of Dvl3. This post-translational modification of Dvl3 in turn maintains LGR5 expression and enhances the cancer stemness properties in HCC.

This paper investigates the leader-following consensus for multiagent systems with general linear dynamics by means of event-triggered scheme (ETS). We propose three types of schemes, namely, distributed ETS (distributed-ETS), centralized ETS (centralized-ETS), and clustered ETS (clustered-ETS) for different network topologies. All these schemes guarantee that all followers can track the leader eventually. It should be emphasized that all event-triggered protocols in this paper depend on local information and their executions are distributed. Moreover, it is shown that such event-triggered mechanism can significantly reduce the frequency of control's update. Further, positive inner-event time intervals are assured for those cases of distributed-ETS, centralized-ETS, and clustered-ETS. In addition, two methods are proposed to avoid continuous communication between agents for event detection. Finally, numerical examples are provided to illustrate the effectiveness of the ETSs.

The pathogenesis of hepatocellular carcinoma (HCC) is a multistep process involving the progressive accumulation of molecular alterations pinpointing different molecular and cellular events. The next-generation sequencing technology is facilitating the global and systematic evaluation of molecular landscapes in HCC. There is emerging evidence supporting the importance of cancer metabolism and tumor microenvironment in providing a favorable and supportive niche to expedite HCC development. Moreover, recent studies have identified distinct surface markers of cancer stem cell (CSC) in HCC, and they also put forward the profound involvement of altered signaling pathways and epigenetic modifications in CSCs, in addition to the concomitant drug resistance and metastasis. Taken together, multiple key genetic and non-genetic factors, as well as liver CSCs, result in the development and progression of HCC.

This study reports the validation of the Hong Kong version of Oxford Cognitive Screen (HK-OCS). Seventy Cantonese-speaking healthy individuals participated to establish normative data and 46 chronic stroke survivors were assessed using the HK-OCS, Albert's Test of Visual Neglect, short test of gestural production, and Hong Kong version of the following assessments: Western Aphasia Battery, MMSE, MoCA, Modified Barthel Index, and Lawton Instrumental Activities of Daily Living scale. The validity of the HK-OCS was appraised by the difference between the two participant groups. Neurologically unimpaired individuals performed significantly better than stroke survivors on the HK-OCS. Positive and significant correlations found between cognitive subtests in the HK-OCS and related assessments indicated good concurrent validity. Excellent intra-rater and inter-rater reliabilities, fair test-retest reliability, and acceptable internal consistency suggested that the HK-OCS had good reliability. Specific HK-OCS subtests including semantics, episodic memory, number writing, and orientation were the best predictors of functional outcomes.

In this paper, we study the distributed constrained optimization problem where the objective function is the sum of local convex cost functions of distributed nodes in a network, subject to a global inequality constraint. To solve this problem, we propose a consensus-based distributed regularized primal-dual subgradient method. In contrast to the existing methods, most of which require projecting the estimates onto the constraint set at every iteration, only one projection at the last iteration is needed for our proposed method. We establish the convergence of the method by showing that it achieves an O ( K (-1/4) ) convergence rate for general distributed constrained optimization, where K is the iteration counter. Finally, a numerical example is provided to validate the convergence of the propose method.

Background & Aims: Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs., Methods: Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs., Results: Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions., Conclusions: A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact., Lay Summary: HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

In this article, we develop some approaches, which enable us to more accurately and analytically identify the essential patterns that guarantee the almost sure stability of discrete-time systems with random switches. We allow for the case that the elements in the switching connection matrix even obey some unbounded and continuous-valued distributions. In addition to the almost sure stability, we further investigate the almost sure synchronization in complex dynamical networks consisting of randomly connected nodes. Numerical examples illustrate that a chaotic dynamics in the synchronization manifold is preserved when statistical parameters enter some almost sure synchronization region established by the developed approach. Moreover, some delicate configurations are considered on probability space for ensuring synchronization in networks whose nodes are described by nonlinear maps. Both theoretical and numerical results on synchronization are presented by setting only a few random connections in each switch duration. More interestingly, we analytically find it possible to achieve almost sure synchronization in the randomly switching complex networks even with very large population sizes, which cannot be easily realized in non-switching but deterministically connected networks.

This paper studies the problem of minimizing a sum of (possible nonsmooth) convex functions that are corresponding to multiple interacting nodes, subject to a convex state constraint set. Time-varying directed network is considered here. Two types of computational constraints are investigated in this paper: one where the information of gradients is not available and the other where the projection steps can only be calculated approximately. We devise a distributed zeroth-order method, the implementation of which needs only functional evaluations and approximate projection. In particular, we show that the proposed method generates expected function value sequences that converge to the optimal value, provided that the projection errors decrease at appropriate rates.

Unlabelled: Epigenetic deregulation plays an important role in liver carcinogenesis. Using transcriptome sequencing, we examined the expression of 591 epigenetic regulators in hepatitis B-associated human hepatocellular carcinoma (HCC). We found that aberrant expression of epigenetic regulators was a common event in HCC. We further identified SETDB1 (SET domain, bifurcated 1), an H3K9-specific histone methyltransferase, as the most significantly up-regulated epigenetic regulator in human HCCs. Up-regulation of SETDB1 was significantly associated with HCC disease progression, cancer aggressiveness, and poorer prognosis of HCC patients. Functionally, we showed that knockdown of SETDB1 reduced HCC cell proliferation in vitro and suppressed orthotopic tumorigenicity in vivo. Inactivation of SETDB1 also impeded HCC cell migration and abolished lung metastasis in nude mice. Interestingly, SETDB1 protein was consistently up-regulated in all metastatic foci found in different organs, suggesting that SETDB1 was essential for HCC metastatic progression. Mechanistically, we showed that the frequent up-regulation of SETDB1 in human HCC was attributed to the recurrent SETDB1 gene copy gain at chromosome 1q21. In addition, hyperactivation of specificity protein 1 transcription factor in HCC enhanced SETDB1 expression at the transcriptional level. Furthermore, we identified miR-29 as a negative regulator of SETDB1. Down-regulation of miR-29 expression in human HCC contributed to SETDB1 up-regulation by relieving its post-transcriptional regulation., Conclusion: SETDB1 is an oncogene that is frequently up-regulated in human HCCs; the multiplicity of SETDB1 activating mechanisms at the chromosomal, transcriptional, and posttranscriptional levels together facilitates SETDB1 up-regulation in human HCC., (© 2015 by the American Association for the Study of Liver Diseases.)

Multiagent systems (MASs) are ubiquitous in our real world. There is an increasing attention focusing on the consensus (or synchronization) problem of MASs over the past decade. Although there are numerous results reported on the convergence of a discrete-time MAS based on the infinite products of matrices, few results are on the convergence rate. Because of the switching topology, the traditional eigenvalue analysis and the Lyapunov function methods are both invalid for the convergence rate analysis of an MAS with a switching topology. Therefore, the estimation of the convergence rate for a discrete-time MAS with time-varying delays remains a difficult problem. To overcome the essential difficulty of switching topology, this paper aims at developing a contractive-set approach to analyze the convergence rate of a discrete-time MAS in the presence of time-varying delays and generalized coupling coefficients. Using the proposed approach, we obtain an upper bound of the convergence rate under the condition of joint connectivity. In particular, the proposed method neither requires the nonnegative property of the coupling coefficients nor the basic assumption of a uniform lower bound for all positive coupling coefficients, which have been widely applied in the existing works on this topic. As an application of the main results, we will show that the classical Vicsek model with time delays can realize synchronization if the initial topology is connected.

This paper studies the stability and Hopf bifurcation in a class of high-dimension neural network involving the discrete and distributed delays under a new framework. By introducing some virtual neurons to the original system, the impact of distributed delay can be described in a simplified way via an equivalent new model. This paper extends the existing works on neural networks to high-dimension cases, which is much closer to complex and real neural networks. Here, we first analyze the Hopf bifurcation in this special class of high dimensional model with weak delay kernel from two aspects: one is induced by the time delay, the other is induced by a rate parameter, to reveal the roles of discrete and distributed delays on stability and bifurcation. Sufficient conditions for keeping the original system to be stable, and undergoing the Hopf bifurcation are obtained. Besides, this new framework can also apply to deal with the case of the strong delay kernel and corresponding analysis for different dynamical behaviors is provided. Finally, the simulation results are presented to justify the validity of our theoretical analysis.