LANCL1, a cell surface protein, promotes liver tumor initiation through FAM49B-Rac1 axis to suppress oxidative stress.

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.

S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos.

OBJECTIVE: Growing 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. DESIGN: The 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. RESULTS: Functionally, 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. CONCLUSION: Altogether, 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.

Cellular heterogeneity and plasticity in liver cancer.

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.

Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma.

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.

Hepatitis B Virus-Telomerase Reverse Transcriptase Promoter Integration Harnesses Host ELF4, Resulting in Telomerase Reverse Transcriptase Gene Transcription in Hepatocellular Carcinoma.

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.

Cancer stemness in hepatocellular carcinoma: mechanisms and translational potential.

Cancer stemness, referring to the stem-cell-like phenotype of cancer cells, has been recognised to play important roles in different aspects of hepatocarcinogenesis. A number of well-established cell-surface markers already exist for liver cancer stem cells, with potential new markers of liver cancer stem cells being identified. Both genetic and epigenetic factors that affect various signalling pathways are known to contribute to cancer stemness. In addition, the tumour microenvironment-both physical and cellular-is known to play an important role in regulating cancer stemness, and the potential interaction between cancer stem cells and their microenvironment has provided insight into the regulation of the tumour-initiating ability as well as the cellular plasticity of liver CSCs. Potential specific therapeutic targeting of liver cancer stemness is also discussed. With increased knowledge, effective druggable targets might be identified, with the aim of improving treatment outcome by reducing chemoresistance.

APOBEC3B promotes hepatocarcinogenesis and metastasis through novel deaminase-independent activity.

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.

SENP1 promotes hypoxia-induced cancer stemness by HIF-1α deSUMOylation and SENP1/HIF-1α positive feedback loop.

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.

Interplay among extracellular vesicles, cancer stemness and immune regulation in driving hepatocellular carcinoma progression.

The intricate interplay among extracellular vesicles, cancer stemness properties, and the immune system significantly impacts hepatocellular carcinoma (HCC) progression, treatment response, and patient prognosis. Extracellular vesicles (EVs), which are membrane-bound structures, play a pivotal role in conveying proteins, lipids, and nucleic acids between cells, thereby serving as essential mediators of intercellular communication. Since a lot of current research focuses on small extracellular vesicles (sEVs), with diameters ranging from 30 nm to 200 nm, this review emphasizes the role of sEVs in the context of interactions between HCC stemness-bearing cells and the immune cells. sEVs offer promising opportunities for the clinical application of innovative diagnostic and prognostic biomarkers in HCC. By specifically targeting sEVs, novel therapeutics aimed at cancer stemness can be developed. Ongoing investigations into the roles of sEVs in cancer stemness and immune regulation in HCC will broaden our understanding and ultimately pave the way for groundbreaking therapeutic interventions.

Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma.

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.

Sorted-Cell Sequencing on HCC Specimens Reveals EPS8L3 as a Key Player in CD24/CD13/EpCAM-Triple Positive, Stemness-Related HCC Cells.

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.

Multimodal Integrative Genomics and Pathology Analyses in Neoadjuvant Nivolumab Treatment for Intermediate and Locally Advanced Hepatocellular Carcinoma.

Introduction: Immunotherapy has resulted in pathologic responses in hepatocellular carcinoma (HCC), but the benefits and molecular mechanisms of neoadjuvant immune checkpoint blockade are largely unknown. Methods: In this study, we evaluated the efficacy and safety of preoperative nivolumab (anti-PD-1) in patients with intermediate and locally advanced HCC and determined the molecular markers for predicting treatment response. Results: Between July 2020 and November 2021, 20 treatment-naive HCC patients with intermediate and locally advanced tumors received preoperative nivolumab at 3 mg/kg for 3 cycles prior to surgical resection. Nineteen patients underwent surgical resection on trial. Seven (36.8%) of the 19 patients had major pathologic tumor necrosis (≥60%) in the post-nivolumab resection specimens, with 3 having almost complete (>90%) tumor necrosis. The tumor necrosis was hemorrhagic and often accompanied by increased or dense immune cell infiltrate at the border of the tumors. None of the patients developed major adverse reactions contradicting hepatectomy. RNA-sequencing analysis on both pre-nivolumab tumor biopsies and post-nivolumab resected specimens showed that, in cases with major pathologic necrosis, the proportion of CD8 T cells in the HCC tissues predominantly increased after treatment. Moreover, to investigate noninvasive biomarker for nivolumab response, we evaluated the copy number variation (CNV) using target-panel sequencing on plasma cell-free DNA of the patients and derived a CNV-based anti-PD-1 score. The score correlated with the extent of tumor necrosis and was validated in a Korean patient cohort with anti-PD-1 treatment. Conclusion: Neoadjuvant nivolumab demonstrated promising clinical activity in intermediate and locally advanced HCC patients. We also identified useful noninvasive biomarker predicting responsiveness.

Fueling HCC Dynamics: Interplay Between Tumor Microenvironment and Tumor Initiating Cells.

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.

PFKFB4 Drives the Oncogenicity in TP53-Mutated Hepatocellular Carcinoma in a Phosphatase-Dependent Manner.

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.

Single-Cell Transcriptomics of Liver Cancer: Hype or Insights?

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.

Liquid Biopsy Using Cell-Free or Circulating Tumor DNA in the Management of Hepatocellular Carcinoma.

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.

RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-ß2.

Growing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-ß2 mRNA stability by decreasing N6-methyladenosine (m6A) modification. TGF-ß signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-ß2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

Antioxidant supplements promote tumor formation and growth and confer drug resistance in hepatocellular carcinoma by reducing intracellular ROS and induction of TMBIM1.

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.

Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma.

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.

Single-cell transcriptomics reveals the landscape of intra-tumoral heterogeneity and stemness-related subpopulations in liver cancer.

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.