ObjectiveGrowing evidence indicates that tumour cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) are critical mediators of tumourigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown.DesignThe functions and mechanisms of S100A10 and EV-S100A10 in HCC progression were investigated in vitro and in vivo. Neutralising antibody (NA) to S100A10 was used to evaluate the significance of EV-S100A10.ResultsFunctionally, S100A10 promoted HCC initiation, self-renewal, chemoresistance and metastasis in vitro and in vivo. Of significance, we found that S100A10 was secreted by HCC cells into EVs both in vitro and in the plasma of patients with HCC. S100A10-enriched EVs enhanced the stemness and metastatic ability of HCC cells, upregulated epidermal growth factor receptor (EGFR), AKT and ERK signalling, and promoted epithelial–mesenchymal transition. EV-S100A10 also functioned as a chemoattractant in HCC cell motility. Of significance, S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αⅤ. Importantly, blockage of EV-S100A10 with S100A10-NA significantly abrogated these enhancing effects.ConclusionAltogether, our results uncovered that S100A10 promotes HCC progression significantly via its transfer in EVs and regulating the protein cargoes of EVs. EV-S100A10 may be a potential therapeutic target and biomarker for HCC progression.

Liver tumor initiating cells (LTICs) are a subpopulation of liver cancer cells that play important roles in tumor initia-tion, recurrence, metastasis, and chemo-drug resistance. LTICs and their surrounding tumor microenvironment have been under intense research. In this review, we discuss LTIC plasticity, tumor microenvironment components, and the dynamic interaction between LTICs and the tumor micro -environment and their underlying molecular mechanisms. We also highlight potential therapeutic targets and strate-gies related to their mutual interactions. Liver cancer (hepatocellular carcinoma) is a common cancer worldwide. It is an aggressive cancer, with high rates of tumor relapse and metastasis, high chemoresistance, and poor prognosis. Liver tumor-initiating cells (LTICs) are a distinctive subset of liver cancer cells with self-renewal and differentiation capacities that contribute to intratumoral heterogeneity, tumor recurrence, metastasis, and chemo-drug resistance. LTICs, marked by different TIC markers, have high plasticity and use diverse signaling pathways to promote tumorigenesis and tumor progression. LTICs are nurtured in the tumor microenvironment (TME), where noncellular and cellular components participate to build an immunosuppressive and tumor-promoting niche. As a result, the TME has emerged as a promising anticancer therapeutic target, as exemplified by some successful applications of tumor immunotherapy. In this review, we discuss the plasticity of LTICs in terms of cellular differentiation, epithelial-mesenchymal transition, and cellular metabolism. We also discuss the various components of the TME, including its noncellular and cellular components. Thereafter, we discuss the mutual interactions between TME and LTICs, including recently reported molecular mechanisms. Lastly, we summarize and describe new ideas concerning novel approaches and strategies for liver cancer therapy.

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.

Background & Aims: The highly proliferative nature of hepato-cellular carcinoma (HCC) frequently results in a hypoxic intra-tumoural 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 tran-scriptomic 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-1a 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 theEphrin-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 wasan important downstream effector that induced cancer stemnessin response to Ephrin-A3/EphA2 forward-signalling. (C) 2022 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.

Abstract RalA is a Ras-related small GTP binding protein A; however, its functional roles and regulatory mechanisms in hepatocellular carcinoma (HCC) are unclear. In this study, using the RNA-Seq data from TCGA database and our in-house HKU database, we observed that RalA expression was significantly up-regulated in human HCCs (P=0.001). This RalA over-expression was validated in a separate cohort of our HCC patients. Upon clinicopathological correlation of RalA in HCC, we found that over-expression of RalA was associated with more aggressive features of HCC patients, with more frequent tumor microsatellite formation (P=0.001), venous invasion (P=0.005) and absence of tumor encapsulation (P=0.005). The over-expression also correlated with poorer overall survival of HCC patients (P<0.001). Functionally, we established RalA stable knockdown (KD) in three HCC cell lines (Hep3B, BEL7402 and MHCC-97L-Luc) and demonstrated that KD of RalA significantly inhibited cell proliferation, colony formation, migration and invasion in vitro. Conversely, ectopic expression of RalA, by transfecting the RalA dominant active form G23V construct into HCC cells, promoted HCC metastasis using transwell invasion assays. Also, with immunofluorescence assay, RalA mainly located in the cytoplasm and cell membrane. Over-expression of RalA changed the HCC cell morphology from polygonal to spindled shape, suggestive of epithelial-mesenchymal transition. We conducted in vivo study using the orthotopic liver injection model of RalA stable-KD MHCC-97L cells in nude mice, and observed that KD of RalA suppressed HCC tumorigenicity and reduced lung metastasis (P<0.001). Moreover, KD of RalA also reduced sphere formation ability of HCC cells, as well as suppressed the expression of stemness markers, including CD24, NANOG, NOTCH1, NESTIN, and EpCAM, using qPCR. For the liver cancer stem cell surface markers, the expression level of CD24 was significantly decreased in RalA KD Hep3B and BEL7402 cells by flow cytometry. Additionally, with RNA-Seq analysis of Hep3B and BEL7402 shRalA clones, 12 genes were found to be significantly altered (among them 5 were up-regulated and 7 were down-regulated) for both cell lines, when compared with in-house cohort as well as TCGA cohort. Further analysis showed that RalA expression level was positively and negatively correlated with a small group of genes. Taken together, our findings have shown that RalA is significantly up-regulated in human HCCs and its over-expression enhances HCC metastasis and cancer stemness. Further investigation of the interplay between RalA and its downstream targets will derive novel mechanistic insight regarding the oncogenic role of RalA in HCC. Citation Format: Luqing Zhao, Lo-Kong Chan, Daniel Wai-Hung Ho, Goofy Yu-Man Tsui, Macrina Wai-Ling Lam, Charles Shing Kam, Karen Man-Fong Sze, Vanilla Xin Zhang, Abdullah Husain, Joyce Man-Fong Lee, Irene Oi-Lin Ng. RalA is frequently up-regulated in human hepatocellular carcinoma and promotes metastasis and cancer stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 91.

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.