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Background: Deregulating cellular metabolism is one of the prominent hallmarks of malignancy, with a critical role in tumor survival and growth. However, the role of reprogramming aspartate metabolism in hepatocellular carcinoma (HCC) are largely unknown. Methods: The multi-omics data of HCC patients were downloaded from public databases. Univariate and multivariate stepwise Cox regression were used to establish an aspartate metabolism-related gene signature (AMGS) in HCC. The Kaplan–Meier and receiver operating characteristic curve analyses were performed to evaluate the predictive ability for overall survival (OS) in HCC patients. Gene set enrichment analysis and immune infiltration analysis were operated to determine the potential mechanisms underlying the AMGS. Single-cell RNA sequencing (scRNA-seq) data of liver cancer stem cells were visualized by t-SNE algorithm. In vivo and in vitro experiments were implemented to investigate the biological function of CAD in HCC. In addition, a nomogram based on the AMGS and clinicopathologic characteristics was constructed by univariate and multivariate Cox regression analyses. Results: Patients in the high-AMGS subgroup exerted advanced tumor status and poor prognosis. Mechanistically, the high-AMGS subgroup patients had significantly enhanced proliferation and stemness-related pathways, increased infiltration of regulatory T cells and upregulated expression levels of suppressive immune checkpoints in the tumor immune microenvironment. Notably, scRNA-seq data revealed CAD, one of the aspartate metabolism-related gene, is significantly upregulated in liver cancer stem cells. Silencing CAD inhibited proliferative capacity and stemness properties of HCC cells in vitro and in vivo. Finally, a novel nomogram based on the AMGS showed an accurate prediction in HCC patients. Conclusions: The AMGS represents a promising prognostic value for HCC patients, providing a perspective for finding novel biomarkers and therapeutic targets for HCC. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a prevalent malignant cancer worldwide, characterized by high morbidity and mortality rates. Alpha-fetoprotein (AFP) is a glycoprotein synthesized by the liver and yolk sac during fetal development. However, the serum levels of AFP exhibit a significant correlation with the onset and progression of HCC in adults. Extensive research has demonstrated that the tumor microenvironment (TME) plays a crucial role in the malignant transformation of HCC, and AFP is a key factor in the TME, promoting HCC development. The objective of this review was to analyze the existing knowledge regarding the role of AFP in the TME. Specifically, this review focused on the effect of AFP on various cells in the TME, tumor immune evasion, and clinical application of AFP in the diagnosis and treatment of HCC. These findings offer valuable insights into the clinical treatment of HCC. [ABSTRACT FROM AUTHOR]

The treatment of advanced hepatocellular carcinoma (HCC) is limited, and immunotherapy is the current research focus of multi-disciplinary collaborative comprehensive treatment of HCC. Herein, we constructed a bio-responsive Au-miR-183 inhibitor (Au@miR-183i) delivery system targeting liver cancer stem cells (LCSCs), and adopted the strategy of combining αPD-L1 immunotherapy. The multifunctional Au@miR-183i nanocomplexes (NCs), which self-assemble based on the tumor microenvironment, consume NADPH and H2O2, leading to redox homeostasis disturbance, ROS accumulation, regulation of the LCSC niche, and induction of stemness regression. Moreover, self-assembled Au@miR-183i NCs specifically target the delivery of miR-183i to LCSCs, triggering the immunogenic cell death (ICD) effect, promoting the maturation of dendritic cells, inducing infiltration of CD8+ T cells, and facilitating the transformation of 'cold' tumors into 'hot' tumors. More importantly, consistent with the results in vitro, Au@miR-183i NCs demonstrated effective tumor targeting and strong ICD induction in vivo, assisted in enhancing αPD-L1 immunotherapy, and activated a robust systemic anti-tumor immune response in tumor-bearing mouse models. Overall, we provide a simple and universal therapeutic strategy by constructing a multifunctional bio-responsive Au@miR-183i NCs delivery system with LCSC targeting capability. Furthermore, nanocomplex-based ICD inducers have great promise in enhancing anti-tumor immunity and the PD-1/PD-L1 blocking efficacy in HCC, which provides a theoretical basis for effectively eliminating LCSCs and achieving a high-efficiency synergistic treatment strategy for HCC. [Display omitted] • Self-assembled Au@miR-183i NCs target LCSCs for effective delivery of miR-183i. • Au@miR-183i NCs lead to redox homeostasis disturbance inducing stemness regression. • Au@miR-183i NCs exhibit potent ICD-inducing ability. • Au@miR-183i NCs activate anti-tumor immunity and effectively enhances αPD-L1 immunotherapy. [ABSTRACT FROM AUTHOR]

The progression of hepatocellular carcinoma (HCC) is influenced by disrupted metabolic processes, presenting challenges in prognostic outcomes. Hepatocellular carcinoma (HCC), a leading cause of cancer-related mortality, is closely associated with metabolic reprogramming and stem cell-like properties in liver cancer stem cells (LCSCs). This study explored the potential molecular mechanisms by which tLyP-1-modified extracellular vesicles (EVs) delivering CTCF shRNA (tLyp-1-EV-shCTCF) regulate mitochondrial DNA methylation-induced glycolytic metabolic reprogramming and LCSC self-renewal. Through a series of methods, including Western blot, nanoparticle tracking analysis, and immunofluorescence, we demonstrated the successful delivery and internalization of tLyp-1-EV in HCC cells. Our results identified SALL3 as a critical factor underexpressed in HCC and LCSCs, while CTCF was overexpressed. Overexpression of SALL3 inhibited LCSC self-renewal and immune evasion by blocking the CTCF-DNMT3A interaction, thus repressing DNMT3A methyltransferase activity and subsequent mitochondrial DNA methylation-mediated glycolytic metabolic reprogramming. In vivo experiments further supported these findings, showing that tLyp-1-EV-shCTCF treatment significantly reduced tumor growth by upregulating SALL3 expression, thereby inhibiting glycolytic metabolic reprogramming and enhancing the immune response against HCC cells. This study provides novel insights into the role of SALL3 and mitochondrial DNA methylation in HCC progression, offering potential therapeutic targets for combating HCC and its stem cell-like properties.

Abstract Hepatocellular carcinoma (HCC), one of the most prevalent forms of cancer worldwide, presents a significant global healthcare challenge. Cancer stem cells (CSCs), which can influence neighboring non-CSCs, are believed to play a crucial role in tumor growth and resistance to treatment, but the specific mechanisms and mediators are not fully understood. Regulation of the CSC state is considered an ideal therapeutic strategy both in the early stages of tumor formation and within established tumors. Exosomes have emerged as key players in intercellular communication, similar to classical hormone signaling, and are essential for facilitating communication between cells in liver cancer. Here, by coupling immunomagnetic bead sorting and exosomal sequencing, we found that exosome-derived circRNAs enriched in liver cancer CSCs were the key subsets with stemness characteristics and ultimately promoted HCC development. Of interest, we found that circ-ZEB1 and circ-AFAP1 are strongly correlated with liver cancer stemness and a poor prognosis, and can regulate the epithelial-mesenchymal transition (EMT) process. Our novel exosome-derived circRNAs play a vital role as key components of various intercellular crosstalk and communication systems in malignant transmission. This finding not only provides valuable support for utilizing plasma exosomal circRNAs as clinical prognostic indicators for HCC patients but also highlights a new research direction in exploring the signaling between liver CSCs and the messenger molecules contained within exosomes.

Hepatocellular carcinoma (HCC), one of the most prevalent forms of cancer worldwide, presents a significant global healthcare challenge. Cancer stem cells (CSCs), which can influence neighboring non-CSCs, are believed to play a crucial role in tumor growth and resistance to treatment, but the specific mechanisms and mediators are not fully understood. Regulation of the CSC state is considered an ideal therapeutic strategy both in the early stages of tumor formation and within established tumors. Exosomes have emerged as key players in intercellular communication, similar to classical hormone signaling, and are essential for facilitating communication between cells in liver cancer. Here, by coupling immunomagnetic bead sorting and exosomal sequencing, we found that exosome-derived circRNAs enriched in liver cancer CSCs were the key subsets with stemness characteristics and ultimately promoted HCC development. Of interest, we found that circ-ZEB1 and circ-AFAP1 are strongly correlated with liver cancer stemness and a poor prognosis, and can regulate the epithelial-mesenchymal transition (EMT) process. Our novel exosome-derived circRNAs play a vital role as key components of various intercellular crosstalk and communication systems in malignant transmission. This finding not only provides valuable support for utilizing plasma exosomal circRNAs as clinical prognostic indicators for HCC patients but also highlights a new research direction in exploring the signaling between liver CSCs and the messenger molecules contained within exosomes. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNA (lncRNA) HOTAIR acts importantly in liver cancer development, but its effect on radioresistance remains poorly understood. Here, our study probed into the possible impact of HOTAIR in radioresistance in liver cancer stem cells (LCSCs) and to elucidate its molecular basis. Methods: Following sorting of stem and non-stem liver cancer cells, LCSCs were identified and subjected to RNA-seq analysis for selecting differentially expressed genes. Expression of HOTAIR was determined in liver cancer tissues and CSCs. The stemness, proliferation, apoptosis and radioresistance of LCSCs were then detected in response to altered expression of HOTAIR-LSD1-JMJD6-BRD4. Results: Ectopic HOTAIR expression was found to promote radioresistance of LCSCs by maintaining its stemness. Mechanistic investigations indicated that HOTAIR recruited LSD1 to the MAPK1 promoter region and reduced the level of H3K9me2 in the promoter region, thus elevating ERK2 (MAPK1) expression. JMJD6–BRD4 complex promoted HOTAIR transcription by forming a complex and positively regulated ERK2 (MAPK1) expression, maintaining the stemness of LCSCs, and ultimately promoting their radioresistance in vitro and in vivo. Conclusion: Collectively, our work highlights the promoting effect of the JMJD6–BRD4 complex on the radioresistance of LCSCs through a HOTAIR-dependent mechanism. [ABSTRACT FROM AUTHOR]

Objective To observe the effects of amarogentinon liver cancer stem cells (LCSCs) after insufficient thermal ablation and its mechanism. Methods A insufficient thermal ablation model of HepG2 cells was established by water bath method.The percentage of CD133-positive LCSCs and the mRNA and protein levels of CD133 were detected by flow cytometry, qRT-PCR and Western blot.The insufficient thermal ablation model of HepG2 cells was treated with variable doses of amarogentin for 24 h; the percentage of CD133-positive LCSCs, the proliferation and apoptosis of liver cancer cells, and the mRNA and protein levels of CD133, TBC1D15, and p53were detected by flow cytometry, qRT-PCR and Western blot. Results The percentage of CD133-positive HepG2 cells and the mRNA and protein levels of CD133 and TBC1D15in the insufficient thermal ablation model were significantly higher than those in the normal HepG2 cells.Amarogentin then markedly decreased the percentage of CD133-positive LCSCs, the proliferation rate of HepG2 cells, and the mRNA and protein levels of CD133 and TBC1D15 in the insufficient thermal ablationresidual model (all P < 0.05);inversely, the apoptosis rate of HepG2 cells and the phosphorylated levels of p53 in the insufficient thermal ablation model were significantly increased (all P < 0.05). Conclusion Amarogentin could reduce the proportion of LCSCs after insufficient thermal ablation, inhibit the proliferation, and promote the apoptosis of LCSCs, which maybe associated with increasing the phosphorylation of p53 and inhibiting the expression of TBC1D15.

Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, is associated with a high degree of malignancy and poor prognosis. Patients with early HCC may benefit from surgical resection to remove tumor tissue and a margin of healthy tissue surrounding it. Unfortunately, most patients with HCC are diagnosed at an advanced or distant stage, at which point resection is not feasible. Systemic therapy is now routinely prescribed to patients with advanced HCC; however, drug resistance has become a major obstacle to the treatment of HCC and exploring purported mechanisms promoting drug resistance remains a challenge. Here, we focus on the determinants of drug resistance from the perspective of non-coding RNAs (ncRNAs), liver cancer stem cells (LCSCs), autophagy, epithelial-mesenchymal transition (EMT), exosomes, ferroptosis, and the tumor microenvironment (TME), with the aim to provide new insights into HCC treatment.

Objective To observe the effects of amarogentinon liver cancer stem cells (LCSCs) after insufficient thermal ablation and its mechanism. Methods A insufficient thermal ablation model of HepG2 cells was established by water bath method. The percentage of CD133-positive LCSCs and the mRNA and protein levels of CD133 were detected by flow cytometry, qRT-PCR and Western blot.The insufficient thermal ablation model of HepG2 cells was treated with variable doses of amarogentin for 24 h; the percentage of CD133-positive LCSCs,the proliferation and apoptosis of liver cancer cells,and the mRNA and protein levels of CD133, TBC1D15, and p53were detected by flow cytometry, qRT-PCR and Western blot. Results The percentage of CD133-positive HepG2 cells and the mRNA and protein levels of CD133 and TBC1D15in the insufficient thermal ablation model were significantly higher than those in the normal HepG2 cells. Amarogentin then markedly decreased the percentage of CD133-positive LCSCs, the proliferation rate of HepG2 cells, and the mRNA and protein levels of CD133 and TBC1D15 in the insufficient thermal ablationresidual model (all P<0.05); inversely, the apoptosis rate of HepG2 cells and the phosphorylated levels of p53 in the insufficient thermal ablation model were significantly increased (all P<0.05). Conclusion Amarogentin could reduce the proportion of LCSCs after insufficient thermal ablation, inhibit the proliferation, and promote the apoptosis of LCSCs, which maybe associated with increasing the phosphorylation of p53 and inhibiting the expression of TBC1D15. [ABSTRACT FROM AUTHOR]

Initiation and progression of liver cancer are driven by a subpopulation of tumor cells—the cancer stem cells (CSCs). Thus, targeted therapy of CSCs holds great promise for providing important new impetus to tumor biology and clinical oncology. CD133 is one of the important surface markers of liver CSCs. Cell-penetrating peptide (CPP) is defined as the peptide sequence capable of transporting various molecules efficiently into cells across membranes. In this study, paclitaxel-loaded nanoparticles (NPs) are modified with anti-CD133 antibody and CPP (Polyarginine) to construct multifunctional drug delivery system for targeting liver CSCs and efficient endocytosis. NPs were prepared by emulsification–solvent evaporation method and modified by carbodiimide hydrochloride and hydroxysuccinimide (EDC/NHS). The physicochemical characteristics of NPs (i.e., encapsulation efficiency, particle size distribution, morphology, release in vitro) were determined. Cellular uptake of NPs was investigated. Using the hepatocellular carcinoma (HCC) cell lines (HepG2 and Huh7), cell survival assay was performed to assess anti-tumor activity in vitro of NPs. Particle size distribution of NPs was between 50 and 200 nm. Drug-loaded NPs showed spherical morphology and higher encapsulation efficiency. The release behavior of paclitaxel from the polymer NPs showed an immediate burst effect. The accumulation of NPs modified with the ligands depended on passive and active targeting. Drug-loaded NPs caused cytotoxicity on HepG2 and Huh7 cells. NPs modified with both ligands maximized the selectivity and therapeutic effect for liver CSCs. This study provided a new idea for HCC therapy in the future. [ABSTRACT FROM AUTHOR]

Abstract Hepatocellular carcinoma (HCC) is the most common type of liver malignant tumor, with high recurrence and mortality rates. Mesenchymal stem cells (MSCs) are multipotent cells that can be recruited into the tumor microenvironment (TME). What is known, TME plays a vital part in tumor progression. In recent years, accumulating studies have found that MSCs have a dual role of promotion and inhibition in the occurrence and development of HCC. In this review, we analyzed the role of MSCs in TME and summarized the relationship between MSCs and liver cancer stem cells, the molecular signaling pathway mechanisms of MSCs promoting and inhibiting HCC, and the latest research progress of MSCs in the treatment of HCC.

•.CAP@CD133-D/X-Lip was constructed. • CAP@CD133-D/X-Lip inhibits metastasis and recurrence of HCC. Hepatocellular carcinoma (HCC) is a prevalent global malignancy, presenting significant challenges in developing safe and effective treatments to prevent recurrence. This is mainly attributed to the inability of traditional treatments to eliminate liver cancer stem cells (LCSCs). LCSCs exhibit resistance to standard therapies, such as radiotherapy and chemotherapy, playing a noticeable role in drug resistance, metastasis, and recurrence of HCC. Their "stemness" is maintained by cancer-associated fibroblasts (CAFs), especially dense CAFs that form a barrier, hindering drug delivery. These two components interact to drive the progression of HCC. Regarding this unique tumor microenvironment, a novel nanoliposome (CAP@CD133-D/X-Lip) was developed. This nanoliposome could initially target CAFs by leveraging fibroblast activating protein (FAP) overexpression on their surface, activating CAP-degrading peptides upon reaching CAFs. Subsequent size reduction and exposure of CD133 aptamers enable secondary targeting of LCSCs, leading to the release of the antitumor drug doxorubicin hydrochloride (DOX) and the LCSCs' inhibitor XAV-939 in deep tumor regions. The efficacy of CAP@CD133-D/X-Lip was validated in vitro and in vivo , demonstrating its ability to target therapeutic drugs and disrupt the 'CAFs-LCSCs' communication, thereby slowing HCC progression and enhancing treatment outcomes. This approach holds significant promise for the clinical management of HCC. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are reported to play essential roles in chemoresistance and metastasis. Pathways regulating CSC self‐renewal and proliferation, such as Hedgehog, Notch, Wnt/β‐catenin, TGF‐β, and Myc, may be potential therapeutic targets. Here, a functional screening from the focused library with 365 compounds is performed by a step‐by‐step strategy. Among these candidate molecules, phenyl‐2‐pyrimidinyl ketone 4‐allyl‐3‐amino selenourea (CU27) is chosen for further identification because it proves to be the most effective compound over others on CSC inhibition. Through ingenuity pathway analysis, it is shown CU27 may inhibit CSC through a well‐known stemness‐related transcription factor c‐Myc. Gene set enrichment analysis, dual‐luciferase reporter assays, expression levels of typical c‐Myc targets, molecular docking, surface plasmon resonance, immunoprecipitation, and chromatin immunoprecipitation are conducted. These results together suggest CU27 binds c‐Myc bHLH/LZ domains, inhibits c‐Myc‐Max complex formation, and prevents its occupancy on target gene promoters. In mouse models, CU27 significantly sensitizes sorafenib‐resistant tumor to sorafenib, reduces the primary tumor size, and inhibits CSC generation, showing a dramatic anti‐metastasis potential. Taken together, CU27 exerts inhibitory effects on CSC and CSC‐associated traits in hepatocellular carcinoma (HCC) via c‐Myc transcription activity inhibition. CU27 may be a promising therapeutic to treat sorafenib‐resistant HCC. [ABSTRACT FROM AUTHOR]

Lactate plays a critical role as an energy substrate, metabolite, and signaling molecule in hepatocellular carcinoma (HCC). Intracellular lactate-derived protein lysine lactylation (Kla) is identified as a contributor to the progression of HCC. Liver cancer stem cells (LCSCs) are believed to be the root cause of phenotypic and functional heterogeneity in HCC. However, the impact of Kla on the biological processes of LCSCs remains poorly understood. Here enhanced glycolytic metabolism, lactate accumulation, and elevated levels of lactylation are observed in LCSCs compared to HCC cells. H3K56la was found to be closely associated with tumourigenesis and stemness of LCSCs. Notably, a comprehensive examination of the lactylome and proteome of LCSCs and HCC cells identified the ALDOA K230/322 lactylation, which plays a critical role in promoting the stemness of LCSCs. Furthermore, this study demonstrated the tight binding between aldolase A (ALDOA) and dead box deconjugate enzyme 17 (DDX17), which is attenuated by ALDOA lactylation, ultimately enhancing the regulatory function of DDX17 in maintaining the stemness of LCSCs. This investigation highlights the significance of Kla in modulating the stemness of LCSCs and its impact on the progression of HCC. Targeting lactylation in LCSCs may offer a promising therapeutic approach for treating HCC., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Targeting liver cancer stem cells (LCSCs) is a promising strategy for hepatocellular carcinoma (HCC) therapy. Target selection and corresponding inhibitor screening are of vital importance for eliminating the stemness of LCSCs. Peptide-based agents are hopeful but have long been hindered for in vivo application. Herein, we selected a clinically significant target MUC13 and screened out a suitable peptide for preparation of an albumin-based MUC13 peptide nanomedicine, P3@HSA, which suppressed liver cancer stem cells via JNK-ERK signaling pathway-mediated autophagy inhibition. The selected target MUC13 was highly expressed in LCSCs and associated with the prognosis of liver cancer patients. Encouraged by this observation, we screened the corresponding peptide-based inhibitor P3 for further evaluation. P3 could interact with albumin through the intrinsic hydrophobic force and formed the nanomedicine P3@HSA. The prepared nanomedicine could inhibit LCSCs through JNK-ERK signaling pathway-mediated autophagy inhibition and exert potent antitumor effect both in vitro and in vivo. Together, this study provides a promising peptide-based nanomedicine for high-performance HCC treatment.

Abstract Background Accumulating evidence demonstrates that tRFs (tRNA-derived small RNA fragments) and tiRNAs (tRNA-derived stress-induced RNA), an emerging category of regulatory RNA molecules derived from transfer RNAs (tRNAs), are dysregulated in in various human cancer types and play crucial roles. However, their roles and mechanisms in hepatocellular carcinoma (HCC) and liver cancer stem cells (LCSCs) are still unknown. Methods The expression of glycine tRNA-derived fragment (Gly-tRF) was measured by qRT-PCR. Flow cytometric analysis and sphere formation assays were used to determine the properties of LCSCs. Transwell assays and scratch wound assays were performed to detect HCC cell migration. Western blotting was conducted to evaluate the abundance change of Epithelial-mesenchymal transition (EMT)-related proteins. Dual luciferase reporter assays and signalling pathway analysis were performed to explore the underlying mechanism of Gly-tRF functions. Results Gly-tRF was highly expressed in HCC cell lines and tumour tissues. Gly-tRF mimic increased the LCSC subpopulation proportion and LCSC-like cell properties. Gly-tRF mimic promoted HCC cell migration and EMT. Loss of Gly-tRF inhibited HCC cell migration and EMT. Mechanistically, Gly-tRF decreased the level of NDFIP2 mRNA by binding to the NDFIP2 mRNA 3′ UTR. Importantly, overexpression of NDFIP2 weakened the promotive effects of Gly-tRF on LCSC-like cell sphere formation and HCC cell migration. Signalling pathway analysis showed that Gly-tRF increased the abundance of phosphorylated AKT. Conclusions Gly-tRF enhances LCSC-like cell properties and promotes EMT by targeting NDFIP2 and activating the AKT signalling pathway. Gly-tRF plays tumor-promoting role in HCC and may lead to a potential therapeutic target for HCC.

Circular RNA (CircRNA) is a newly identified special class of non-coding RNA (ncRNA) that plays an important regulatory role in the progression of certain diseases. Herein, our results indicate that CircMEG3 is downregulated expression and negatively correlated with the expression of telomerase-related gene Cbf5 in human liver cancer. Moreover, CircMEG3 inhibits the growth of human liver cancer stem cells in vivo and in vitro. CircMEG3 inhibits the expression of m6A methyltransferase METTL3 dependent on HULC. Moreover, CircMEG3 inhibits the expression of Cbf5, a component of telomere synthetase H/ACA ribonucleoprotein (RNP; catalyst RNA pseudouracil modification) through METTL3 dependent on HULC. Thereby, CircMEG3 inhibits telomerase activity and shortens telomere lifespan dependent on HULC and Cbf5 in human liver cancer stem cell. Strikingly, increased Cbf5 abrogates the ability of CircMEG3 to inhibit malignant differentiation of human liver cancer stem cells. In summary, these observations provide important basic information for finding effective liver cancer therapeutic targets.

Objective: This study aims to explore the effects of miR-342-3p on liver cancer stem cells (LCSC) and related mechanism. Methods: LCSC were sorted using immunomagnetic beads and flow cytometry was used to determine CD133+ and CD133− sorted cells. The self-renewal ability and growth ability of LCSC were measured by tumor spheroid formation assay and soft agar colony formation assay. Protein and mRNA expressions of CD44, ALDH1, Bmi1, Sox2 and Oct4 were detected by western blot and quantitative PCR. The relationship between miR-342-3p and HDAC7 was analyzed by dual-luciferase assay. The acetylation level of H3 protein was measured by acetyl Lysine antibody. Results: miR-342-3p overexpression in LCSC lead to lower tumor volume, reduced tumor spheroid formation and agar colony formation rates, as well as lower mRNA and protein expressions of CD44, ALDH1, Bmi1, Sox2, and Oct4. Dual-luciferase reporter assay confirmed HDAC7 as a target gene of miR-342-3p. Inhibition of HDAC7 or overexpression of PTEN suppressed the carcinogenicity and stemness of LCSC. PTEN expression was increased in sh-HDAC7 group and decreased in pcDNA3.1-HDAC7 group. HDAC7 promoted H3 deacetylation and inhibited PTEN expression. Overexpression of HDAC7 or silencing of PTEN could reverse the inhibitory effect of overexpression of miR-342-3p on LCSC carcinogenicity and cell stemness. Conclusion: MiR-342-3p inhibited LCSC oncogenicity and cell stemness by promoting PTEN and inhibiting HDAC7. [ABSTRACT FROM AUTHOR]

The progression of hepatocellular carcinoma (HCC) is influenced by disrupted metabolic processes, presenting challenges in prognostic outcomes. Hepatocellular carcinoma (HCC), a leading cause of cancer-related mortality, is closely associated with metabolic reprogramming and stem cell-like properties in liver cancer stem cells (LCSCs). This study explored the potential molecular mechanisms by which tLyP-1-modified extracellular vesicles (EVs) delivering CTCF shRNA (tLyp-1-EV-shCTCF) regulate mitochondrial DNA methylation-induced glycolytic metabolic reprogramming and LCSC self-renewal. Through a series of methods, including Western blot, nanoparticle tracking analysis, and immunofluorescence, we demonstrated the successful delivery and internalization of tLyp-1-EV in HCC cells. Our results identified SALL3 as a critical factor underexpressed in HCC and LCSCs, while CTCF was overexpressed. Overexpression of SALL3 inhibited LCSC self-renewal and immune evasion by blocking the CTCF-DNMT3A interaction, thus repressing DNMT3A methyltransferase activity and subsequent mitochondrial DNA methylation-mediated glycolytic metabolic reprogramming. In vivo experiments further supported these findings, showing that tLyp-1-EV-shCTCF treatment significantly reduced tumor growth by upregulating SALL3 expression, thereby inhibiting glycolytic metabolic reprogramming and enhancing the immune response against HCC cells. This study provides novel insights into the role of SALL3 and mitochondrial DNA methylation in HCC progression, offering potential therapeutic targets for combating HCC and its stem cell-like properties., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Matrix stiffening is one of the major risk factors for hepatocellular carcinoma (HCC) and drives tumor progression. The extracellular matrix (ECM) stiffness of HCC displays mechanical heterogeneity, with stiffness increasing from the core to the invasive frontier. The distribution of liver cancer stem cells (CSCs) is related to this mechanical property. However, it is not sufficiently understood how heterogeneous matrix stiffness regulates the stemness of CSCs. In this study, we developed an adjustable gelatin/alginate hydrogel to investigate the effect of various matrix stiffnesses on CSC stemness under three-dimensional culture conditions. Gelatin/alginate hydrogel with the stiffness of soft (5 kPa), medium (16 kPa), and stiff (81 kPa) were prepared by altering the concentration of calcium ions. It was found that a stiffer matrix promoted stemness-associated gene expression, reduced drug sensitivity, enhanced sphere-forming and clonogenic ability, and tumorigenic potential. Mechanistically, matrix stiffening facilitates CSC stemness by increasing Yes-associated protein (YAP) activity and inhibiting Bcl-2 modifying factor (BMF) expression. Knockdown of YAP or overexpression of BMF significantly attenuated matrix stiffening-induced stemness, suggesting the involvement of YAP and BMF in this process. Together, our results unravel the regulatory mechanism of heterogeneous matrix stiffness on CSC stemness and also provide a novel therapeutic strategy for eradicating CSCs and improving the efficiency of HCC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The aim of this study was to elucidate the mechanism of action of the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) on the phenotype of the liver cancer stem cells (LCSCs). To gain insight into the mechanism of action of the IGF2BP1 on LCSCs, the IGF2BP1 shRNA sequences were transfected into hepatocellular carcinoma (HCC) cells. The LCSC phenotypes were measured by stemness gene expressions, spheroid formations, percentages of the CD133+ cells, colony formations, and tumorigenesis in vivo. Next, we screened for possible molecular mechanisms from the Cancer Genome Atlas (TCGA) database, and a methylated RNA immunoprecipitation-quantitative polymerase chain reaction (MeRIP-qPCR) was used to adjust the binding of IGF2BP1 to the target gene, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase (MGAT5). The MeRIP-qPCR was used to detect the binding of IGF2BP1 and MGAT5 through N6 methyladenosine (m6A) modification. Furthermore, we adjusted the attenuation of the mRNA of the MGAT5 using quantitative real-time PCR (qRT-PCR). The IGF2BP1 was upregulated in the LCSCs. Furthermore, the IGF2BP1 promoted self-renewal and chemoresistance in human LCSCs and tumorigenesis in mice and it enhanced the expression of stemness genes in the LCSCs compared with the HCC cells. Further exploration indicated that the IGF2BP1 binds directly to the MGAT5 and inhibits its mRNA attenuation, suggesting that the IGF2BP1 impacts MGAT5 mRNA stability through m6A modification. Thus, it can be concluded that the IGF2BP1 facilitated the LCSC phenotypes by promoting the MGAT5 mRNA stability through the upregulation of m6A modification of the MGAT5 mRNA. [ABSTRACT FROM AUTHOR]

Liver cancer stem cells (L‐CSCs) are considered to be an important therapeutic target for hepatocellular carcinoma (HCC). This study provides a new in vitro long‐term culture model for a specific subpopulation of L‐CSCs enriched by cell surface markers. We combined CD13, CD133 and EpCAM to selectively enrich L‐CSCs, which we then cultured in modified chemically defined medium. The enriched L‐CSCs exhibited enhanced proliferation, self‐renewal and long‐term clonal maintenance ability as compared with non‐CSCs. Compared with wild‐type hepatocellular carcinoma, the expression of stemness surface markers, oncogenes, drug resistance and tumorigenicity in enriched L‐CSCs was significantly increased. In summary, the subpopulation of L‐CSCs still maintains cancer stem cell‐related phenotypes after 14 days of culture.

Abstract Background Cancer stem cells are the main reason of relapse, metastasis and resistance to anti-cancer therapies of Hepatocellular carcinoma (HCC). Mesenchymal stem cells (MSCs) are an important part of the tumor microenvironment. MSCs have been demonstrated to be involved in drug resistance in tumor. How MSCs contribute to radiotherapy resistance of HCC is still indistinct. Methods Flow cytometry analysis was performed to isolate CD133+ cells from HCC cell lines Huh7 and PLC. The stemness of Huh7-CD133 and PLC-CD133 those were co-cultured with IR-MSCs were investigated by Colony formation assay. Tumor formation in nude mice was used to explore the tumorigenicity of CD133+ cancer cells. The activating Wnt/β-catenin signaling pathway in CSCs were also detected by RT-PCR and Western blotting. Results We report that irradiated MSCs (IR-MSCs) could increase the ratio of CD133+ cells in hepatocellular carcinoma cells. IR-MSCs could promote stemness maintenance of HCC stem cells. After co-cultured with IR-MSCs, liver cancer stem cells (CSCs) presented increased colony formation ability and tumor formation ability. We also found IR-MSCs promoted Wnt expression of CSCs. Reverse suppression experiment showed that when Wnt inhibitor was added into the culture medium, the effect of IR-MSCs on stemness maintenance was counteracted. Conclusions These data showed that IR-MSCs could support stemness maintenance of CSCs by activating Wnt/β-catenin signaling pathway.

Abstract Background Liver cancer stem cells (LCSCs) contribute to hepatocellular carcinoma (HCC) development, metastasis, and drug resistance. MSI2 and Notch1 signaling are involved in the maintenance of CSCs. However, it is unknown whether MSI2 and Notch1 are involved in the maintenance of CD44v6+ LCSCs. Therefore, we investigated the clinical significance and function of MSI2 and its relationship with Notch1 signaling in the maintenance of stemness properties in CD44v6+ LCSCs. Methods The expression of MSI2 and CD44v6 were detected by fresh specimens and a HCC tissue microarray. The tissue microarray containing 82 HCC samples was used to analyze the correlation between CD44v6 and MSI2. CD44v6+/− cells were isolated using microbeads sorting. We explored the roles of MSI2 and Notch1 signaling in CD44v6+ LCSCs by sphere formation assay, transwell assay, clone formation assay in vitro, and xenograft tumor models in vivo. A Notch RT2 PCR Array, Co-immunoprecipitation, and RNA-immunoprecipitation were used to further investigate the molecular mechanism of MSI2 in activating Notch1 signaling. Results Here, we found MSI2 expression was positively correlated with high CD44v6 expression in HCC tissues, and further correlated with tumor differentiation. CD44v6+ cells isolated from HCC cell lines exhibited increased self-renewal, proliferation, migration and invasion, resistance to Sorafenib and tumorigenic capacity. Both MSI2 and Notch1 signaling were elevated in sorted CD44v6+ cells than CD44v6- cells and played essential roles in the maintenance of stemness of CD44v6+ LCSCs. Mechanically, MSI2 directly bound to Lunatic fringe (LFNG) mRNA and protein, resulting in Notch1 activation. Conclusions Our results demonstrated that MSI2 maintained the stemness of CD44v6+ LCSCs by activating Notch1 signaling through the interaction with LFNG, which could be a potential molecular target for stem cell-targeted therapy for liver cancer.

Hepatocellular carcinoma (HCC) is a prevalent malignant cancer worldwide, characterized by high morbidity and mortality rates. Alpha-fetoprotein (AFP) is a glycoprotein synthesized by the liver and yolk sac during fetal development. However, the serum levels of AFP exhibit a significant correlation with the onset and progression of HCC in adults. Extensive research has demonstrated that the tumor microenvironment (TME) plays a crucial role in the malignant transformation of HCC, and AFP is a key factor in the TME, promoting HCC development. The objective of this review was to analyze the existing knowledge regarding the role of AFP in the TME. Specifically, this review focused on the effect of AFP on various cells in the TME, tumor immune evasion, and clinical application of AFP in the diagnosis and treatment of HCC. These findings offer valuable insights into the clinical treatment of 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 © 2024 Lu, Lin and Li.)

Background: Accumulating evidence demonstrates that tRFs (tRNA-derived small RNA fragments) and tiRNAs (tRNA-derived stress-induced RNA), an emerging category of regulatory RNA molecules derived from transfer RNAs (tRNAs), are dysregulated in in various human cancer types and play crucial roles. However, their roles and mechanisms in hepatocellular carcinoma (HCC) and liver cancer stem cells (LCSCs) are still unknown. Methods: The expression of glycine tRNA-derived fragment (Gly-tRF) was measured by qRT-PCR. Flow cytometric analysis and sphere formation assays were used to determine the properties of LCSCs. Transwell assays and scratch wound assays were performed to detect HCC cell migration. Western blotting was conducted to evaluate the abundance change of Epithelial-mesenchymal transition (EMT)-related proteins. Dual luciferase reporter assays and signalling pathway analysis were performed to explore the underlying mechanism of Gly-tRF functions. Results: Gly-tRF was highly expressed in HCC cell lines and tumour tissues. Gly-tRF mimic increased the LCSC subpopulation proportion and LCSC-like cell properties. Gly-tRF mimic promoted HCC cell migration and EMT. Loss of Gly-tRF inhibited HCC cell migration and EMT. Mechanistically, Gly-tRF decreased the level of NDFIP2 mRNA by binding to the NDFIP2 mRNA 3′ UTR. Importantly, overexpression of NDFIP2 weakened the promotive effects of Gly-tRF on LCSC-like cell sphere formation and HCC cell migration. Signalling pathway analysis showed that Gly-tRF increased the abundance of phosphorylated AKT. Conclusions: Gly-tRF enhances LCSC-like cell properties and promotes EMT by targeting NDFIP2 and activating the AKT signalling pathway. Gly-tRF plays tumor-promoting role in HCC and may lead to a potential therapeutic target for HCC. [ABSTRACT FROM AUTHOR]

BACKGROUND: The malignant transformation of cancer originating from stem cells is increasingly recognized. Liver cancer stem cells have the potential of continuous self-renewal and multi-directional differentiation, which can promote the occurrence and development of liver cancer, postoperative recurrence and the production of drug resistance. OBJECTIVE: To explore the effect of Fuhe preparation formula on proliferation of CD133+ liver cancer stem cells and stemness transcription factor expression. METHODS: CD133+ HepG2 liver cancer stem cells were selected by immunomagnetic bead method. CD133+ HepG2 cells were treated with serum containing 2%, 4%, 8%, 10%, 12% and 16% Fuhe preparation formula respectively. The inhibitory rate of CD133+ HepG2 cells was determined by CCK-8 method, and the optimal volume fraction of serum containing drugs was selected. The CD133+ HepG2 liver cancer stem cells were divided into dimethyl sulfoxide group, application and preparation group, serum control group and blank control group. Cells in the dimethyl sulfoxide group were intervened with 0.05% dimethyl sulfoxide. Cells in the application and preparation group were intervened with 16% application and preparation containing serum. Cells in the serum control group were intervened with 16% normal rat serum. After 6 days of intervention, the percentage of CD133+ HepG2 liver cancer stem cells was detected by flow cytometry. The mRNA and protein levels of transcription factors SOX2, NANOG, and OCT4 were detected by RT-PCR and western blot assay. RESULTS AND CONCLUSION: (1) The serum containing application and preparation formula inhibited the proliferation of CD133+ HepG2 cells, which was related to the time and concentration. The inhibitory effect of 16% application and preparation formula was the most obvious in 72 hours (P < 0.05). (2) Compared with the serum control and blank control groups, the percentage of CD133+ HepG2 liver cancer stem cells was decreased in the application and preparation and dimethyl sulfoxide groups (P < 0.05), and the effect was more obvious in the application and preparation group. (3) Compared with the serum control and blank control groups, SOX2, NANOG, OCT4 mRNA and protein expression levels were downregulated in the application and preparation and dimethyl sulfoxide groups (P < 0.05), and the effect was more obvious in the application and preparation group. (4) These findings conclude that the serum containing application and preparation formula can effectively reduce the expression levels of CD133, a marker on the surface of liver cancer stem cells, and transcription factors SOX2, NANOG and OCT4, and can reduce the malignant degree of liver cancer stem cells. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) originates from fully differentiated hepatocytes, but the decisive events for converting hepatocytes to the cells of origin for HCC are still unclear. Liver cancer stem cells (LCSCs) cause HCC but are not bona fide cells of origin. Here, the expressions of POU2F2 and IL‐31 are identified in macroscopically normal livers of diethylnitrosamine‐challenged mice. An autoregulatory circuit formed by mutual induction between POU2F2 and IL‐31 drives hepatocytes to progress to LCSCs by acquiring stemness, as well as stimulates them to in vivo grow and malignantly progress. The development of the autoregulatory circuit is a decisive event for converting hepatocytes into the cells of origin, since hepatocytes expressing the circuit have acquired tumorigenic potential before progressing to LCSCs. Nonetheless, acquiring stemness is still required for the cells of origin to initiate hepatocarcinogenesis. The circuit also occurs in human cirrhotic tissues, partially elucidating how premalignant lesions progress to HCC. [ABSTRACT FROM AUTHOR]

The survival of cancer stem cells is usually limited to a specific tumor microenvironment, and this microenvironment plays a vital role in the development of tumors. The mechanical properties of the microenvironment differ in different regions of solid tumors. However, in solid tumors, whether the distribution of cancer stem cells relates to the mechanical microenvironment of different regions is still unclear. In this study, we undertook a biophysical and biochemical assessment of the changes in the mechanical properties of liver tissue during the progression of liver cancer and explored the distribution of liver cancer stem cells in liver cancer tissues. Our analysis confirmed previous observations that the stiffness of liver tissue gradually increased with the progress of fibrosis. In liver cancer tissues, we found obvious mechanical heterogeneity: the core of the tumor was soft, the invasive front tissue was the hardest, and the para-cancer tissue was in an intermediate state. Interestingly, the greatest number of liver cancer stem cells was found in the invasive front part of the tumor. We finally established that stroma stiffness correlated with the number of liver cancer stem cells. These findings indicate that the distribution of liver cancer stem cells correlates with the mechanical heterogeneity of liver cancer tissue. This result provides a theoretical basis for the development of targeted therapies against the mechanical microenvironment of liver cancer stem cells. [ABSTRACT FROM AUTHOR]

Background: Liver cancer stem cells, characterized by self-renewal and initiating cancer cells, were decisive drivers of progression and therapeutic resistance in hepatocellular carcinoma (HCC). However, a comprehensive understanding of HCC stemness has not been identified.Methods: RNA sequencing information, corresponding clinical annotation, and mutation data of HCC were downloaded from The Cancer Genome Atlas-LIHC project. Two stemness indices, mRNA expression-based stemness index (mRNAsi) and epigenetically regulated-mRNAsi, were used to comprehensively analyze HCC stemness. Estimation of Stromal and Immune Cells in Malignant Tumors using Expression Data and single-sample gene-set enrichment analysis algorithm were performed to characterize the context of tumor immune microenvironment (TIME). Next, differentially expressed gene (DEG) analysis and weighted gene co-expression network analysis (WGCNA) were employed to identify significant mRNAsi-related modules with hub genes. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment pathways were analyzed to functionally annotate these key genes. The least absolute shrinkage and selection operator (LASSO) Cox regression analysis was performed to establish a prognostic signature. Kaplan–Meier survival curves and receiver operating characteristic (ROC) analysis were applied for prognostic value validation. Seven algorithms (XCELL, TIMER, QUANTISEQ, MCPcounter, EPIC, CIBERSORT, and CIBERSORT-ABS) were utilized to draw the landscape of TIME. Finally, the mutation data were analyzed by employing “maftools” package.Results: mRNAsi was significantly elevated in HCC samples. mRNAsi escalated as tumor grade increased, with poor prognosis presenting the higher stemness index. The stemness-related (greenyellow) modules with 175 hub genes were screened based on DEGs and WGCNA. A prognostic signature was established using LASSO analysis of prognostic hub genes to classify samples into two risk subgroups, which exhibited good prognostic performance. Additionally, prognostic risk-clinical nomogram was drawn to estimate risk quantitatively. Moreover, risk score was significantly associated with contexture of TIME and immunotherapeutic targets. Finally, potential interaction between risk score with tumor mutation burden (TMB) was elucidated.Conclusion: This work comprehensively elucidated that stemness characteristics served as a crucial player in clinical outcome, complexity of TIME, and immunotherapeutic prediction from both mRNAsi and mRNA level. Quantitative identification of stemness characteristics in individual tumor will contribute into predicting clinical outcome, mapping landscape of TIME further optimizing precision immunotherapy.

Abstract Hepatocellular carcinoma (HCC) originates from fully differentiated hepatocytes, but the decisive events for converting hepatocytes to the cells of origin for HCC are still unclear. Liver cancer stem cells (LCSCs) cause HCC but are not bona fide cells of origin. Here, the expressions of POU2F2 and IL‐31 are identified in macroscopically normal livers of diethylnitrosamine‐challenged mice. An autoregulatory circuit formed by mutual induction between POU2F2 and IL‐31 drives hepatocytes to progress to LCSCs by acquiring stemness, as well as stimulates them to in vivo grow and malignantly progress. The development of the autoregulatory circuit is a decisive event for converting hepatocytes into the cells of origin, since hepatocytes expressing the circuit have acquired tumorigenic potential before progressing to LCSCs. Nonetheless, acquiring stemness is still required for the cells of origin to initiate hepatocarcinogenesis. The circuit also occurs in human cirrhotic tissues, partially elucidating how premalignant lesions progress to HCC.

Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and one of the leading causes of cancer-related deaths worldwide. A growing body of evidence supports the hypothesis that HCC is driven by a population of cells called liver cancer stem cells (LCSCs). LCSCs have been proposed to contribute to malignant HCC progression, including promoting tumor occurrence and growth, mediating tumor metastasis, and treatment resistance, but the regulatory mechanism of LCSCs in HCC remains unclear. Understanding the signaling pathways responsible for LCSC maintenance and survival may provide opportunities to improve patient outcomes. Here, we review the current literature about the origin of LCSCs and the niche composition, describe the current evidence of signaling pathways that mediate LCSC stemness, then highlight several mechanisms that modulate LCSC properties in HCC progression, and finally, summarize the new developments in therapeutic strategies targeting LCSCs markers and regulatory pathways.

The combination of chemotherapy and phototherapy has emerged as a promising therapeutic approach for enhancing the efficacy of cancer treatment and mitigating drug resistance. Salinomycin (SAL), a polyether antibiotic, exhibits potent cytotoxicity against chemotherapy-resistant cancer cells. IR780 iodide, a novel photosensitive reagent with excellent near-infrared (NIR) light absorption and photothermal conversion abilities, is suitable for use in photothermal therapy for cancers. However, both SAL and IR780 exhibit hydrophobic properties that limit their clinical applicability. Upconversion nanoparticles (UCNPs) are an emerging class of fluorescent probe materials capable of emitting high-energy photons upon excitation by low-energy NIR light. The UCNPs not only function as nanocarriers for drug delivery but also serve as light transducers to activate photosensitizers for deep-tissue photodynamic therapy. Here, to enhance the targeting and bioavailability of hydrophobic drugs in liver cancer stem cells (LCSCs), we employ distearoyl phosphorethanolamine-polyethylene glycol (DSPE-PEG) to encapsulate SAL and IR780 on the surface of UCNPs. Cell viability was evaluated using the CCK-8 assay. Cell migration was assessed by the Transwell Boyden Chamber. The activation of the mitogen-activated protein kinase (MAPK) signaling pathway was measured via western blot. The results demonstrated successful loading of both IR780 and SAL onto the UCNPs, and the SAL and IR780-loaded UCNPs (UISP) exhibited a robust photothermal effect under NIR light irradiation. The UISP effectively inhibited the viability of HCCLM3 and LCSCs. Under NIR light irradiation, the UISP further suppressed HCCLM3 viability but had no impact on LCSC viability; however, it could further inhibit LCSC migration. Meanwhile, under NIR light irradiation, the UISP persistently activated the MAPK pathway more significantly in LCSCs. These findings suggest that exposure to NIR light results in persistent activation of the MAPK pathway by UISP, thereby influencing the biological behavior of LCSCs and enhancing their therapeutic efficacy against liver cancer. • SAL and IR780-loaded UCNPs (UISP) inhibited cell viability in HCCLM3 and LCSCs. • Under NIR light irradiation, UISP further inhibited HCCLM3 viability. • Under NIR light irradiation, UISP had no effect on LCSC viability. • Under NIR light irradiation, UISP could further inhibit LCSC migration. • UISP activated MAPK signal more persistently in LCSCs under NIR light irradiation. [ABSTRACT FROM AUTHOR]

Abstract Background Application of dendritic cells (DC) for cancer immunotherapy involves tumor-associated immunogenic antigens for effective therapeutic strategies. The present study investigated whether DC co-cultured with autologous cytokine-induced killer cells (CIK) could induce a more specific immune response against liver cancer stem cells (LCSC) generated from human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Methods Human DC and CIK were generated from peripheral blood mononuclear cells (PBMCs) taken from consenting liver cancer patients. Flow cytometry was used to determine the phenotypes of DC and CIK, and cell proliferation. The tumor growth and anti-tumor activity of these cells were further evaluated using a nude mouse tumor model. Results We demonstrated that DC and CIK significantly enhanced the apoptosis ratio, depending on DC-CIK cell numbers, by increasing caspase-3 protein expression and reducing proliferating cell nuclear antigen (PCNA) protein expression against LCSC. The in vivo data indicated that DC-CIK exhibited significant LCSC cell-induced tumor growth inhibition in nude mice, which was most significant with LCSC antigen loaded DCs. Conclusions The results showed, that DC-CIK cells could inhibit HCC and LCSC growths in vitro and in vivo and the most successful DC triggering of cell cytotoxic activity could be achieved by their LCSC antigen loading.

It is generally believed that the existence of cancer stem cells (CSCs) is related to tumor recurrence and metastasis of hepatocellular carcinoma (HCC). Neuropilin1 (NRP1) is involved in numerous pathophysiological processes of tumor progression, however, whether NRP1 is involved in the regulation of liver CSCs and metastasis of HCC is still unknown. In the present study, we examined the effect of NRP1 on the population of liver CSCs and the metastasis mechanism of HCC. In NRP1 small hairpin RNA (shRNA)-transduced HCC cells, liver CSCs surface markers (CD133+/EpCAM+/CD13+/CD44+) expressing cells, which imply the CSCs population, were decreased. Transwell assay and nude mouse liver orthotopic transplantation model confirmed that NRP1 knockdown inhibited HCC cells' migration and lung metastasis. Our data showed that the expression of NRP1 was upregulated in 5 independent cohorts of HCC patients, consequently, high levels of NRP1 correlated with recurrence and poor prognosis in HCC. Mechanism research showed that NRP1 promotes cell spreading through the epithelial-mesenchymal transition (EMT) signaling pathway. In summary, NRP1 enhanced the population of liver CSCs and migration of HCC via EMT, indicating that NRP1 might be a novel target for HCC treatments. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies around the world. The self-renewal, proliferation, differentiation, and tumorigenic potential of liver cancer stem cells (LCSCs) may account for the high recurrence rate and the refractory feature of HCC. Despite extensive researches, the underlying regulatory mechanism of LCSCs has not been fully disclosed. Long nonprotein coding RNAs (lncRNAs) may exert an essential role in regulating various biological functions of LCSCs, such as maintaining the stemness of cancer stem cells (CSCs) and promoting tumor development. Therefore, it is highly critical to determine which lncRNAs can control LCSCs functions and understand how LCSCs are regulated by lncRNAs. Herein, we summarized lncRNAs and the main signaling pathways involved in the regulation of LCSCs found in recent years. Moreover, we shed light on the existence of the network system of lncRNAs and LCSCs, which may provide valuable clues on targeting LCSCs. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide, with high morbidity, relapse, metastasis and mortality rates. Although liver surgical resection, transplantation, chemotherapy, radiotherapy and some molecular targeted therapeutics may prolong the survival of HCC patients to a certain degree, the curative effect is still poor, primarily because of tumor recurrence and the drug resistance of HCC cells. Liver cancer stem cells (LCSCs), also known as liver tumor-initiating cells, represent one small subset of cancer cells that are responsible for disease recurrence, drug resistance and death. Therefore, understanding the regulatory mechanism of LCSCs in HCC is of vital importance. Thus, new studies that present gene regulation strategies to control LCSC differentiation and replication are under development. In this review, we provide an update on the latest advances in experimental studies on non-coding RNAs (ncRNAs), oncogenes and oncoproteins. All the articles addressed the crosstalk between different ncRNAs, oncogenes and oncoproteins, as well as their upstream and downstream products targeting LCSCs. In this review, we summarize three pathways, the Wnt/β-catenin signaling pathway, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, and interleukin 6/Janus kinase 2/signal transducer and activator of transcription 3 (IL6/JAK2/STAT3) signaling pathway, and their targeting gene, c-Myc. Furthermore, we conclude that octamer 4 (OCT4) and Nanog are two important functional genes that play a pivotal role in LCSC regulation and HCC prognosis.

Liver cancer stem cells (L‐CSCs) are considered to be an important therapeutic target for hepatocellular carcinoma (HCC). This study provides a new in vitro long‐term culture model for a specific subpopulation of L‐CSCs enriched by cell surface markers. We combined CD13, CD133 and EpCAM to selectively enrich L‐CSCs, which we then cultured in modified chemically defined medium. The enriched L‐CSCs exhibited enhanced proliferation, self‐renewal and long‐term clonal maintenance ability as compared with non‐CSCs. Compared with wild‐type hepatocellular carcinoma, the expression of stemness surface markers, oncogenes, drug resistance and tumorigenicity in enriched L‐CSCs was significantly increased. In summary, the subpopulation of L‐CSCs still maintains cancer stem cell‐related phenotypes after 14 days of culture. [ABSTRACT FROM AUTHOR]

Background: Cancer stem cells are the main reason of relapse, metastasis and resistance to anti-cancer therapies of Hepatocellular carcinoma (HCC). Mesenchymal stem cells (MSCs) are an important part of the tumor microenvironment. MSCs have been demonstrated to be involved in drug resistance in tumor. How MSCs contribute to radiotherapy resistance of HCC is still indistinct. Methods: Flow cytometry analysis was performed to isolate CD133+ cells from HCC cell lines Huh7 and PLC. The stemness of Huh7-CD133 and PLC-CD133 those were co-cultured with IR-MSCs were investigated by Colony formation assay. Tumor formation in nude mice was used to explore the tumorigenicity of CD133+ cancer cells. The activating Wnt/β-catenin signaling pathway in CSCs were also detected by RT-PCR and Western blotting. Results: We report that irradiated MSCs (IR-MSCs) could increase the ratio of CD133+ cells in hepatocellular carcinoma cells. IR-MSCs could promote stemness maintenance of HCC stem cells. After co-cultured with IR-MSCs, liver cancer stem cells (CSCs) presented increased colony formation ability and tumor formation ability. We also found IR-MSCs promoted Wnt expression of CSCs. Reverse suppression experiment showed that when Wnt inhibitor was added into the culture medium, the effect of IR-MSCs on stemness maintenance was counteracted. Conclusions: These data showed that IR-MSCs could support stemness maintenance of CSCs by activating Wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Objective To observe the expression of long non-coding RNA KCNQl OTl in liver cancer tissues and liver cancer cell lines and its regulatory effect on self-renewal ability of liver cancer stem cells, and to explore its mechanism. Methods The expression levels of KCNQl OTl in the liver cancer tissues and adjacent tissues, liver cancer cell lines and human immortalized normal liver epithelial cells were detected by RT-PCR. CD13 + CD133 + liver cancer stem cells were screened out by Huh7, and were divided into groups A, B and C. Cells in the group B were transfected with KCNQlOTl shRNA to inhibit the expression of KCNQl OTl, and group C with YAPl overexpression vector after transfection with KC NQl OTl shRNA. Sphere forming ability of liver cancer stem cells in each group was observed by sphere formation assay, col ony formation ability of liver cancer stem cells in each group was observed by colony formation assay, and proliferation ability of liver cancer stem cells in each group was observed by CCK8 assay. In addition, the expression levels of Hippo-YAP path way target genes ( Yapl, Birc5 and Foxml) in the liver cancer stem cells of each group were determined by RT-PCR. Re sults The relative expression level of KCNQlOTl in the liver cancer tissues was higher than that in the adjacent tissues (P < 0. 05), and the relative expression levels of KCNQl OTl in Huh7, SMMC7721 and Hep3B cells were higher than that in ID2 cells (all P < 0. 05). The sphere formation rate, the number of colony formation, and the OD values at 24, 48, 72, and 96 h of the group B were lower than those of groups A and C (all P < 0. 05). The relative expression levels of Y apl, Birc5 and Foxml mRNA in the liver cancer stem cells of group B were lower than those of groups A and C (all P < 0. 05). Conclusion KCNQl OTl is highly expressed in the liver cancer tissues and liver cancer cell lines; inhibiting KCNQl OTl expres sion can reduce the self-renewal ability of liver cancer stem cells, while high expression of YAPl can antagonize this process; KCNQlOTl may affect the self-renewal ability of liver cancer stem cells by activating Hippo-YAP pathway. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) represents a major cause of cancer death, but the molecular mechanism for its development has not yet been well characterized. Long noncoding RNAs (lncRNAs) are involved in a wide range of biological processes via their roles as oncogenes or tumor suppressor genes. The present study aimed to elucidate the role of LINC00324 in HCC through its interaction with Fas ligand (FasL). Initially, microarray‐based gene expression profiling of HCC was employed to identify differentially expressed genes. Next, the expression of LINC00324 in HCC tissues and liver cancer stem cell (LCSC) lines was examined using RT‐qPCR. Then, the interaction among LINC00324, PU box binding protein (PU.1) and FasL was identified with RIP, ChIP and dual‐luciferase reporter gene assays. The effect of LINC00324 on viability, proliferation, migration, invasion, and apoptosis as well as the tumorigenesis of transfected cells was examined with gain‐ and loss‐of‐function experiments. LINC00324 and FasL were highly expressed in HCC. LINC00324 regulated FasL expression via interaction with PU.1. Silencing of LINC00324 or FasL suppressed expression of stemness‐related genes, cell viability, proliferation, migration, invasion, self‐renewal, and tumorigenesis, but enhanced cell apoptosis. Taken together, LINC00324 promotes the expression of FasL through the recruitment of PU.1, which ultimately maintains the biological properties of LCSCs, thus, highlighting LINC00324 as a promising therapeutic candidate for HCC. [ABSTRACT FROM AUTHOR]

Objective: This study is performed to explore the role of transcription factor FOXM1 in promoting the self‐renewal and proliferation of liver cancer stem cells (LCSCs) by regulating the expression of acetaldehyde dehydrogenase‐2 (ALDH2). Methods: CD133+CD24+ LCSCs were sorted and identified. A series of experiments were carried out to determine the proliferation, colony formation rate, migration, invasion, and apoptosis of LCSCs after interfering with FOXM1. Proliferation‐, epithelial–mesenchymal transition (EMT)‐, apoptosis‐, and stemness‐related factors were then detected by western blot analysis. Tumor xenograft in nude mice was used to figure out the role of FOXM1 in tumorigenesis in vivo by regulating ALDH2 expression. Luciferase activity assay was conducted to determine whether FOXM1 could target ALDH2 promoter region and thereby affecting ALDH2 expression. Results: The sorted CD133+CD24+ Huh‐7 cells had the characteristic of stem cells. FOXM1 was highly expressed in CD133+CD24+Huh‐7 cells. Silencing FOXM1 inhibited the proliferation and colony formation of LCSCs and decreased the expression of proliferating cell nuclear antigen and Ki‐67 protein; inhibited the migration, invasion, and EMT of LCSCs while promoting the apoptosis of LCSCs, as well as promoted the expression of Bax and cleaved‐caspase‐3, and inhibited the expression of Bcl‐2. Silencing FOXM1 inhibited the expression of Nanog, Oct4, and Sox2 in LCSCs by decreasing the expression of ALDH2. in vivo experiment, silencing FOXM1 suppressed tumorigenesis of LCSCs by decreasing the expression of ALDH2. Conclusion: Our study provides evidence that silencing FOXM1 inhibits stemness of LCSCs by decreasing the expression of ALDH2, and represses the proliferation, migration, invasion, and tumorigenesis while inducing the apoptosis of LCSCs. [ABSTRACT FROM AUTHOR]

Background: Liver cancer stem cells (LCSCs) contribute to hepatocellular carcinoma (HCC) development, metastasis, and drug resistance. MSI2 and Notch1 signaling are involved in the maintenance of CSCs. However, it is unknown whether MSI2 and Notch1 are involved in the maintenance of CD44v6+ LCSCs. Therefore, we investigated the clinical significance and function of MSI2 and its relationship with Notch1 signaling in the maintenance of stemness properties in CD44v6+ LCSCs. Methods: The expression of MSI2 and CD44v6 were detected by fresh specimens and a HCC tissue microarray. The tissue microarray containing 82 HCC samples was used to analyze the correlation between CD44v6 and MSI2. CD44v6+/− cells were isolated using microbeads sorting. We explored the roles of MSI2 and Notch1 signaling in CD44v6+ LCSCs by sphere formation assay, transwell assay, clone formation assay in vitro, and xenograft tumor models in vivo. A Notch RT2 PCR Array, Co-immunoprecipitation, and RNA-immunoprecipitation were used to further investigate the molecular mechanism of MSI2 in activating Notch1 signaling. Results: Here, we found MSI2 expression was positively correlated with high CD44v6 expression in HCC tissues, and further correlated with tumor differentiation. CD44v6+ cells isolated from HCC cell lines exhibited increased self-renewal, proliferation, migration and invasion, resistance to Sorafenib and tumorigenic capacity. Both MSI2 and Notch1 signaling were elevated in sorted CD44v6+ cells than CD44v6- cells and played essential roles in the maintenance of stemness of CD44v6+ LCSCs. Mechanically, MSI2 directly bound to Lunatic fringe (LFNG) mRNA and protein, resulting in Notch1 activation. Conclusions: Our results demonstrated that MSI2 maintained the stemness of CD44v6+ LCSCs by activating Notch1 signaling through the interaction with LFNG, which could be a potential molecular target for stem cell-targeted therapy for liver cancer. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, is associated with a high degree of malignancy and poor prognosis. Patients with early HCC may benefit from surgical resection to remove tumor tissue and a margin of healthy tissue surrounding it. Unfortunately, most patients with HCC are diagnosed at an advanced or distant stage, at which point resection is not feasible. Systemic therapy is now routinely prescribed to patients with advanced HCC; however, drug resistance has become a major obstacle to the treatment of HCC and exploring purported mechanisms promoting drug resistance remains a challenge. Here, we focus on the determinants of drug resistance from the perspective of non-coding RNAs (ncRNAs), liver cancer stem cells (LCSCs), autophagy, epithelial-mesenchymal transition (EMT), exosomes, ferroptosis, and the tumor microenvironment (TME), with the aim to provide new insights into HCC treatment. [Display omitted] • A review of drug resistance in HCC was conducted. • Drug resistance in HCC related to LCSCs, autophagy, EMT, exosomes, ferroptosis, and the TME are focused on ncRNAs. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser897, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.

Abstract Shear stress-activated cellular signalling is involved in cellular migration, proliferation and differentiation. However, it remains unclear about the effects of shear stress on the differentiation of liver cancer stem cells (LCSCs). In this study, using a parallel plated flow chamber system, we found that low-shear stress loading (2 dyne/cm2) for 48 h could significantly inhibit the sphere-forming ability, increase the chemotherapeutic drug sensitivity, downregulate cancer stem cell marker expression and suppress the in vivo tumorigenicity potential of LCSCs. Moreover, using an atomic force microscope (AFM), we found that shear stress increased the Young's modulus of LCSCs. These results indicate LCSC's differentiation after shear stress exposure. In addition, low-shear stress loading suppressed the β-catenin expression in LCSCs. More importantly, pharmacological activation of Wnt/β-catenin signalling restored the shear stress-suppressed β-catenin expression in LCSCs, and abolished the shear stress-altered sphere-forming ability, chemotherapeutic drug sensitivity, cancer stem cell marker expression, Young's modulus and in vivo tumorigenicity potential of LCSCs. Our results suggest that low shear stress could induce LCSC differentiation via the Wnt/β-catenin signalling pathways, providing a new insight into the role of shear stress in cancer cell biological behaviors that might contribute to the development of new therapeutic strategies for liver cancer treatment. Highlights • Low-shear stress loading suppresses the stemness of Liver cancer stem cells (LCSCs). • Low-shear stress loading inhibits the wnt/β-catenin signalling pathway in LCSCs. • Low-shear stress loading weakens the tumorigenicity of LCSCs in vivo. • Low-shear stress induces LCSC differentiation through the Wnt/β-catenin signalling pathway. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of inhibition of K.LF8 expression on the proportion of CD133+ phenotype cell subsets in the hepatocellular carcinoma stern cells, tumor formation in vivo and lung metastasis in the nude mice. Methods Liver cancer stem cell subsets (LCSCs) with CD133+ phenotype in the lung metastases of liver cancer patients were isolated and cultured. LCSCs were infected with the recombinant lentiviral plasmid (sh-KLF8) interfered by RNA and its negative control empty vector plasmid (sh-control) , respectively. CD133+ phenotype liver cancer stem cell line (LCSC-ssh-KLF8) and control cell line (LCSCssh-eontrol) with stably knocking out K.LF8 gene were constructed, then we constructed the liver cancer stem cell xenograft model and lung metastasis model. The qRT-PCR was used to detect the expression of K.LF8 in the two cell lines, and the ratio of CD133+ phenotypic cells in the two cell lines was detected by flow cytometry. We injected LCSCssh-KLF8 and LCSCssh-eontrol to construct the nude mouse xenograft model of liver cancer stem cells, and observed the effect of knocking out of K.LF8 gene on the tumorigenic ability of liver cancer stem cells in nude mice. We injected LCSCssh-KLF8 and LCSCssh-eontrol into the nude mice through the tail vein to construct the nude mouse lung metastasis model, and observed the effect of knocking out of K.LF8 gene on the lung metastasis ability of liver cancer stem cells in nude mice. K.LF8 protein was detected by immunohistoehemistry in the lung metastases formed by LCSCssh-KLF8 and LCSCssh-eontrol. Results Compared with LCSCssh-eontrol, the relative expression level of K.LF8 mR-JNA in LCSCssh-KLF8 significantly decreased (P<0.05) , and the proportion of CD133+ phenotype stem cell subsets significantly decreased, and the tumor weight and number of lung metastases in vivo significantly decreased in the nude mice, and the positive staining of K.LF8 protein in the lung metastases was much less (all P < 0. 05). Conclusion Inhibiting JVLF8 gene can reduce the proportion of CD133+ phenotype stem cell subsets, tumor formation in vivo, and lung metastasis ability, and therefore KCFS may be the key molecule that inhibits the development and progression of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]