Hepatocellular Carcinoma Stem Research Articles

LARP4B promotes hepatocellular carcinoma progression and impairs sorafenib efficacy by activating SPINK1-mediated EGFR pathway.

La-related proteins (LARPs) regulate gene expression by binding to RNAs and exhibit critical effects on disease progression, including tumors. However, the role of LARP4B and its underlying mechanisms in the progression of hepatocellular carcinoma (HCC) remain largely unclear. In this study, we found that LARP4B expression is upregulated and correlates with poor prognosis in patients with HCC. Gain- and loss-of-function assays showed that LARP4B promotes stemness, proliferation, metastasis, and angiogenesis in vitro and in vivo. Furthermore, LARP4B inhibition enhances the antitumor effects of sorafenib and blocks the metastasis-enhancing effects of low sorafenib concentrations in HCC. Mechanistically, LARP4B expression is upregulated by METTL3-mediated N6-methyladenosine (m6A)-IGF2BP3-dependent modification in HCC. RNA- and RNA immunoprecipitation (RIP)- sequencing uncovered that LARP4B upregulates SPINK1 by binding to SPINK1 mRNA via the La motif and maintaining mRNA stability. LARP4B activates the SPINK1-mediated EGFR signaling pathway, which supports stemness, progression and sorafenib resistance in HCC. Additionally, a positive feedback loop with the LARP4B/SPINK1/p-AKT/C/EBP-β axis is responsible for the sorafenib-therapeutic benefit of LARP4B depletion. Overall, this study demonstrated that LARP4B facilitates HCC progression, and LARP4B inhibition provides benefits to sorafenib treatment in HCC, suggesting that LARP4B might be a potential therapeutic target for HCC.

Hepatitis B Virus-mediated m6A demethylation increases hepatocellular carcinoma stemness and immune escape.

Hepatitis B viral (HBV) persistent infection plays a significant role in hepatocellular carcinoma (HCC) tumorigenesis. Many studies have revealed the pivotal roles of N6-methyladenosine (m6A) in multiple cancers, while the regulatory mechanism in stemness maintenance of HBV persistent infection-related HCC remains elusive. Here, we demonstrated that the level of m6A modification was downregulated by HBV in HBV-positive HCC, through enhanced stability of ALKBH5 mRNA. More specifically, we also identified that ALKBH5 mRNA was functionally required for the stemness maintenance and self-renewal in the HBV-positive HCC, but dispensable in HBV-negative HCC. Mechanistically, ALKBH5 demethylated the m6A modification in the 3'UTR region of the oncogenic gene SNAI2 to prevent the recognition of YTHDF2 therewith stabilize SNAI2 transcripts, contributing to cancer stem cell traits in HBV-positive HCC. Moreover, the expression of SNAI2 reversed the suppression of stemness properties by knocking down ALKBH5. Additionally, ALKBH5/SNAI2 axis accelerates tumor immune evasion through activated ligand of immune checkpoint CD155. Our study unveiled that the ALKBH5 induces m6A demethylation of the SNAI2 as a key regulator in HBV-related HCC, and identifies the function of ALKBH5/SNAI2/YTHDF2 axis in promoting the stem-like cells phenotype and immune escape during HBV infection. Implications: HBV promotes hepatocellular carcinoma stemness maintenance through elevate m6A modification of SNAI2 in an ALKBH5-YTHDF2 dependent manner and increases the expression of the ligand of immune checkpoint CD155.

Abstract 5447: Microtubule-associated protein MAP2 promotes drug resistance and cancer stemness in hepatocellular carcinoma through integrin dysregulation

Abstract Integrins are adhesion molecules that mediate mechanical and chemical signal transduction pathways to support cell survival and proliferation. Dysregulated integrin signaling empowers tumor cells to drive oncogenic stemness functions, including tumor initiation, epithelial plasticity, metastatic reactivation, and resistance to therapies. However, the interplay between cancer stemness and integrin signaling in hepatocellular carcinoma (HCC) remains poorly understood. HCC cells marked by CD133 represent an important functional subset of HCC tumors, displaying a dedifferentiated status with stem cell traits. In this study, transcriptome profiling reveals specific downregulation of integrin α family genes and integrin signaling in ‘HCC’ CD133+ cells isolated from NRAS+AKT-driven HCC, but not epithelial-specific ‘normal’ CD133+ cells isolated from regenerating liver. Of note, one of the most differentially upregulated genes identified in CD133+ HCC cell profiling, microtubule-associated protein MAP2, demonstrates the ability to suppress integrin expression. MAP2 overexpression is frequently observed in HCC and correlates with aggressive clinical and stemness features, including survival, tumor stage, and stemness signatures. Epigenetic modifications by H3K27Ac contribute to MAP2 upregulation. Functionally, MAP2 promotes cancer stemness and cell invasion, and confers resistance to the targeted therapy sorafenib. Mechanistically, the inhibition of collagen-binding integrin α subunits by MAP2 subsequently ablates integrin β1-mediated cell adhesion and FAK signaling. Estramustine Phosphate (EMP), previously reported to inhibit the interaction of MAP2 with actin filaments, attains a synergistic effect in suppressing tumor initiation and growth of HCC cell lines, HCC patient-derived organoids and NRAS+AKT HCC mouse model when used in combination with sorafenib. In summary, MAP2 inhibition may represent a potential novel therapy for HCC by targeting its cancer stemness roots and altering integrin signaling. Ongoing work is focused on the study of MAP2 regulation of integrin signaling and cell behaviours in the maintenance of a more stemness state in HCC. Citation Format: Ut Kei Lou, Ka-Hei Lam, Huajian Yu, Jia Jian Loh, Ki-Fong Man, Lei Zhou, Yuan Gao, Tin-Lok Wong, Cheng-Han Yu, Stephanie Ma. Microtubule-associated protein MAP2 promotes drug resistance and cancer stemness in hepatocellular carcinoma through integrin dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5447.

Abstract 5437: Chemoproteomics-enabled discovery of covalent inhibitors targeted at AGPAT4: Unravelling tumor lineage plasticity to overcome drug resistance in hepatocellular carcinoma

Abstract The liver is a unique organ that is responsible for many metabolic functions. During hepatocellular carcinoma (HCC) development, these metabolic machineries are extensively reprogrammed to support the insatiable nutrient requirements of HCC. Tumor lineage plasticity, a recognized hallmark of cancer, is a phenomenon in which tumor cells co-opt developmental pathways to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. Cancer cells can reprogram their metabolic pathways to match their increased metabolic needs for cancer cell survival under adverse conditions. Identifying novel metabolic targets related to stemness can offer promising strategies for targeting cancer stemness roots and hence to kill and control their growth. Through pathway enrichment analysis of genes that linked metabolism and stemness, we identified an aberrant glycerophospholipid metabolism signature, with AGPAT4 ranking as the top-hit. AGPAT4 upregulation in HCC is strongly correlated with aggressive clinical features, including survival, metastasis, and stemness signatures. AGPAT4 expression peaked during early liver progenitor development, decreased during hepatocyte maturation and progressively increased from well-differentiated to poorly differentiated HCCs. Enrichment of AGPAT4 in HCC is mediated by promoter binding of SOX9 to drive AGPAT4 transcriptional activity. AGPAT4 inhibition can mitigate tumor initiation, self-renewal, metastasis, and sorafenib resistance. Mechanistic studies revealed an AGPAT4-mediated phosphatidic acid production axis that promotes HCC through the regulation of mTOR signaling. Inhibition of Agpat4 by AAV8 shRNA reduced tumorigenicity and stemness, and sensitized HCC tumors to sorafenib. AGPAT4 overexpression can predict sorafenib response in clinical settings. Through chemoproteomics screening of a cysteine-reacting compound library using activity-based protein profiling, a cysteine-reacting compound with high binding affinity and selectivity towards AGPAT4 was identified and found to work synergistically with sorafenib to suppress HCC, as demonstrated in HCC patient-derived tumor xenograft (PDTX) models. Toxicity analysis through histological examination of organs, body weight measurements, and biochemical tests revealed minimal toxicity associated with the covalent inhibitor. In conclusion, AGPAT4 is a novel metabolic driver of oncogenic stemness, dedifferentiation, and metastasis in HCC. AGPAT4-induced tumor lineage plasticity may represent an Achilles heel for HCC treatment, and inhibition of AGPAT4 may widen the therapeutic window for sorafenib treatment in the clinic. Citation Format: Kai-Yu Ng, Tin-Yan Koo, Tsz-Lok Fong, Ya Gao, Tin-Lok Wong, Yuan Gao, Jing-Ping Yun, Xin-Yuan Guan, Ming Liu, Clive YS Chung, Stephanie Ma. Chemoproteomics-enabled discovery of covalent inhibitors targeted at AGPAT4: Unravelling tumor lineage plasticity to overcome drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5437.

Abstract 7065: AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer, with emerging evidence highlighting the role of cancer stem cells (CSCs) in its progression. AMBRA1 has been implicated to exert both tumor-promoting and tumor-suppressive roles through regulating cell cycle and mitophagy in various solid tumors. However, the functional roles of AMBRA1 in modulating cancer stemness and tumorigenesis in HCC remain elusive. Initial analysis of The Cancer Genome Atlas data revealed a significant inverse correlation between AMBRA1 expression and stemness indices (TSS) in HCC patients, with low AMBRA1 levels associated with an upregulated oxidative phosphorylation (OXPHOS) gene signature. To elucidate the functional consequences of AMBRA1 modulation in liver tumor development and stemness maintenance, we exploited both hydrodynamic mouse models and human HCC cell lines. Our findings indicate that hepatic AMBRA1 deletion enhances cancer stemness and tumorigenesis both in vivo and in vitro. Moreover, HCC cells with reduced AMBRA1 expression demonstrated heightened mitochondrial activity, characterized by increased oxygen consumption, respiration, mitochondrial membrane potential, and intracellular ATP levels. Apart from OXPHOS, we also observed a concomitant up-regulation and enrichment of MYC targets in TSS-high and AMBRA1-low HCC patients, pointing towards a deregulated c-MYC signaling to promote cancer stemness under AMBRA1 regulation in HCC. Collectively, our results demonstrate that AMBRA1 loss enhances mitochondrial function to support hepatic tumorigenesis in HCC. Citation Format: Yan Liu, Yunong Xie, Yimiao He, Stephanie Ma, Man Tong. AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7065.

Metadherin promotes stem cell phenotypes and correlated with immune infiltration in hepatocellular carcinoma.

Metadherin (MTDH) is a key oncogene in most cancer types, including hepatocellular carcinoma (HCC). Notably, MTDH does not affect the stemness pheno-type or immune infiltration of HCC. To explore the role of MTDH on stemness and immune infiltration in HCC. MTDH expression in HCC tissues was detected using TCGA and GEO databases. Immunohistochemistry was used to analyze the tissue samples. MTDH was stably knocked down or overexpressed by lentiviral transfection in the two HCC cell lines. The invasion and migration abilities of HCC cells were evaluated using Matrigel invasion and wound healing assays. Next, we obtained liver cancer stem cells from the spheroids by culturing them in a serum-free medium. Gene expression was determined by western blotting and quantitative reverse transcri-ption PCR. Flow cytometry, immunofluorescence, and tumor sphere formation assays were used to characterize stem-like cells. The effects of MTDH inhibition on tumor growth were evaluated in vivo. The correlation of MTDH with immune cells, immunomodulators, and chemokines was analyzed using ssGSEA and TISIDB databases. HCC tissues expressed higher levels of MTDH than normal liver tissues. High MTDH expression was associated with a poor prognosis. HCC cells overexpressing MTDH exhibited stronger invasion and migration abilities, exhibited a stem cell-like phenotype, and formed spheres; however, MTDH inhibition attenuated these effects. MTDH inhibition suppressed HCC progression and CD133 expression in vivo. MTDH was positively correlated with immature dendritic, T helper 2 cells, central memory CD8+ T, memory B, activated dendritic, natural killer (NK) T, NK, activated CD4+ T, and central memory CD4+ T cells. MTDH was negatively correlated with activated CD8+ T cells, eosinophils, activated B cells, monocytes, macrophages, and mast cells. A positive correlation was observed between the MTDH level and CXCL2 expression, whereas a negative correlation was observed between the MTDH level and CX3CL1 and CXCL12 expression. High levels of MTDH expression in patients with HCC are associated with poor prognosis, promoting tumor stemness, immune infiltration, and HCC progression.

Extracellular vesicles and cancer stemness in hepatocellular carcinoma - is there a link?

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy, with high recurrence rates and notorious resistance to conventional chemotherapy. Cancer stemness refers to the stem-cell-like phenotype of cancer cells and has been recognized to play important roles in different aspects of hepatocarcinogenesis. Small extracellular vesicles (sEVs) are small membranous particles secreted by cells that can transfer bioactive molecules, such as nucleic acids, proteins, lipids, and metabolites, to neighboring or distant cells. Recent studies have highlighted the role of sEVs in modulating different aspects of the cancer stemness properties of HCC. Furthermore, sEVs derived from diverse cellular sources, such as cancer cells, stromal cells, and immune cells, contribute to the maintenance of the cancer stemness phenotype in HCC. Through cargo transfer, specific signaling pathways are activated within the recipient cells, thus promoting the stemness properties. Additionally, sEVs can govern the secretion of growth factors from non-cancer cells to further maintain their stemness features. Clinically, plasma sEVs may hold promise as potential biomarkers for HCC diagnosis and treatment prediction. Understanding the underlying mechanisms by which sEVs promote cancer stemness in HCC is crucial, as targeting sEV-mediated communication may offer novel strategies in treatment and improve patient outcome.

An integrated investigation of sulfotransferases (SULTs) in hepatocellular carcinoma and identification of the role of SULT2A1 on stemness.

The cytosolic sulfotransferases (SULTs) are phase II conjugating enzymes, which are widely expressed in the liver and mainly mediate the sulfation of numerous xenobiotics and endogenous compounds. However, the role of various SULTs genes has not been reported in hepatocellular carcinoma (HCC). This study aims to analyze the expression and potential functional roles of SULTs genes in HCC and to identify the role of SULT2A1 in HCC stemness as well as the possible mechanism. We found that all of the 12 SULTs genes were differentially expressed in HCC. Moreover, clinicopathological features and survival rates were also investigated. Multivariate regression analysis showed that SULT2A1 and SULT1C2 could be used as independent prognostic factors in HCC. SULT1C4, SULT1E1, and SULT2A1 were significantly associated with immune infiltration. SULT2A1 deficiency in HCC promoted chemotherapy resistance and stemness maintenance. Mechanistically, silencing of SULT2A1 activated the AKT signaling pathway, on the one hand, promoted the expression of downstream stemness gene c-Myc, on the other hand, facilitated the NRF2 expression to reduce the accumulation of ROS, and jointly increased HCC stemness. Moreover, knockdown NR1I3 was involved in the transcriptional regulation of SULT2A1 in stemness maintenance. In addition, SULT2A1 knockdown HCC cells promoted the proliferation and activation of hepatic stellate cells (HSCs), thereby exerting a potential stroma remodeling effect. Our study revealed the expression and role of SULTs genes in HCC and identified the contribution of SULT2A1 to the initiation and progression of HCC.

Interferon-α induces differentiation of cancer stem cells and immunosuppression in hepatocellular carcinoma by upregulating CXCL8 secretion

Interferon-alpha (IFN-α) is widely used in the clinical treatment of patients with chronic hepatitis B and hepatocellular carcinoma (HCC). However, high levels of CXCL8 are associated with resistance to IFN-α therapy and poorer prognosis in advanced cancers. In this study, we investigated whether IFN-α could directly induce the production of CXCL8 in HCC cells and whether CXCL8 could antagonize the antitumor activity of IFN-α. We found that IFN-α not only upregulated the expression of the inducible genes CXCL9, CXCL10, CXCL11 and PD-L1, but also significantly stimulated CXCL8 secretion in HCC cells. Mechanically, IFN-α induces CXCL8 expression by activating the AKT and JNK pathways. In addition, our results demonstrate that IFN-α exposure significantly increases the differentiation of HCC stem cells, but this effect is reversed by the addition of the CXCL8 receptor CXCR1/2 inhibitor Reparixin and STAT3 inhibitor Stattic. Besides, our study reveals that the cytokine CXCL8 secreted by IFN-α-induced HCC cells inhibits T-cell function. Conversely, inhibition of CXCL8 promotes TNF-α and IFN-γ secretion by T cells. Finally, liver cancer patients who received anti-PD-1/PD-L1 immunotherapy with high CXCL8 expression had a lower immunotherapy efficacy. Overall, our findings clarify that IFN-α triggers immunosuppression and cancer stem cell differentiation in hepatocellular carcinoma by upregulating CXCL8 secretion. This discovery provides a novel approach to enhance the effectiveness of HCC treatment in the future.

LAPTM4B-mediated hepatocellular carcinoma stem cell proliferation and MDSC migration: implications for HCC progression and sensitivity to PD-L1 monoclonal antibody therapy

In hepatocellular carcinoma (HCC), immunotherapy is vital for advanced-stage patients. However, diverse individual responses and tumor heterogeneity have resulted in heterogenous treatment outcomes. Our mechanistic investigations identified LAPTM4B as a crucial gene regulated by ETV1 (a transcription factor), especially in liver cancer stem cells (LCSCs). The influence of LAPTM4B on LCSCs is mediated via the Wnt1/c-Myc/β-catenin pathway. CXCL8 secretion by LAPTM4B drove myeloid-derived suppressor cell (MDSC) migration, inducing unfavorable patient prognosis. LAPTM4B affected PD-L1 receptor expression in tumor microenvironment and enhanced tumor suppression induced by PD-L1 monoclonal antibodies in HCC patients. LAPTM4B up-regulation is correlated with adverse outcomes in HCC patients, sensitizing them to PD-L1 monoclonal antibody therapy.

USP40 promotes hepatocellular carcinoma cell proliferation, migration and stemness by deubiquitinating and stabilizing Claudin1

BackgroundHepatocellular carcinoma (HCC) is a prevalent malignant tumor that poses a major threat to people’s lives and health. Previous studies have found that multiple deubiquitinating enzymes are involved in the pathogenesis of HCC. The purpose of this work was to elucidate the function and mechanism of the deubiquitinating enzyme USP40 in HCC progression.MethodsThe expression of USP40 in human HCC tissues and HCC cell lines was investigated using RT-qPCR, western blotting and immunohistochemistry (IHC). Both in vitro and in vivo experiments were conducted to determine the crucial role of USP40 in HCC progression. The interaction between USP40 and Claudin1 was identified by immunofluorescence, co-immunoprecipitation and ubiquitination assays.ResultsWe discovered that USP40 is elevated in HCC tissues and predicts poor prognosis in HCC patients. USP40 knockdown inhibits HCC cell proliferation, migration and stemness, whereas USP40 overexpression shows the opposite impact. Furthermore, we confirmed that Claudin1 is a downstream gene of USP40. Mechanistically, USP40 interacts with Claudin1 and inhibits its polyubiquitination to stabilize Claudin1 protein.ConclusionsOur study reveals that USP40 enhances HCC malignant development by deubiquitinating and stabilizing Claudin1, suggesting that targeting USP40 may be a novel approach for HCC therapy.

Ubiquitination-specific protease 7 enhances stemness of hepatocellular carcinoma by stabilizing basic transcription factor 3.

This study aims to explore the molecular regulation mechanism of ubiquitination-specific protease 7 (USP7) in facilitating the stemness properties of hepatocellular carcinoma (HCC). Gain-of-function and loss-of-function assays were conducted in SK-Hep1 and HepG2 cells transfected with USP7 overexpression/knockdown plasmids and USP7 inhibitor P22077. The proliferation, migration, invasion, and self-renewal capacity of hepatocellular carcinoma cells were detected by CCK-8, colony formation, Transwell, scratch, and tumor sphere formation, respectively. MS was performed to identify the potential substrate of USP7 following P22077 treatment. Co-IP assay was used to verify the interaction between USP7 and basic transcription factor 3 (BTF3) in HCC cells. The overexpression of USP7 could promote the proliferation, migration, invasion, and colony formation capacity of SK-Hep1 and HepG2 cells. Additionally, ectopic UPS7 enhanced the epithelial-mesenchymal transition (EMT) and stem-like characteristics of the HCC cells. In contrast, USP7 depletion by knockdown of USP7 or administrating inhibitor P22077 significantly inhibited these malignant phenotypes of SK-Hep1 and HepG2 cells. Following MS analysis, BTF3 was identified as a potential substrate for USP7. USP7 could interact with BTF3 and upregulate its protein level, while USP7 depletion significantly upregulated the ubiquitination levels. Overexpression of BTF3 partially rescue the inhibitory effects of USP7 depletion on the malignant phenotypes and stemness properties of SK-Hep1 and HepG2 cells. USP7 can promote the stemness and malignant phenotype of HCC by stabilizing BTF3.

N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression.

Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.

GCF2 mediates nicotine-induced cancer stemness and progression in hepatocellular carcinoma

Cigarette smoking is one of the most impactful behavior-related risk factors for multiple cancers including hepatocellular carcinoma (HCC). Nicotine, as the principal component of tobacco, is not only responsible for smoking addiction but also a carcinogen; nevertheless, the underlying mechanisms remain unclear. Here we report that nicotine enhances HCC cancer stemness and malignant progression by upregulating the expression of GC-rich binding factor 2 (GCF2), a gene that was revealed to be upregulated in HCC and whose upregulation predicts poor prognosis, and subsequently activating the Wnt/ꞵ-catenin/SOX2 signaling pathway. We found that nicotine significantly increased GCF2 expression and that silencing of GCF2 reduced nicotine-induced cancer stemness and progression. Mechanistically, nicotine could stabilize the protein level of GCF2, and then GCF2 could robustly activate its downstream Wnt/β-catenin signaling pathway. Taken together, our results thus suggest that GCF2 is a potential target for a therapeutic strategy against nicotine-promoted HCC.

Study on the Mechanism of Notch Pathway Mediates the Role of Lenvatinib-resistant Hepatocellular Carcinoma Based on Organoids.

The emergence of treatment resistance has hindered the efficacy of targeted therapies used to treat patients with hepatocellular carcinoma (HCC). This study aimed to explore the mechanism of organoids constructed from lenvatinib-resistant HCC cells. Hep3B cell and human HCC organoids were cultured and identified using hematoxylin and eosin staining and Immunohistochemistry. Lenvatinib-sensitive/ resistant Hep3B cells were constructed using lenvatinib (0, 0.1, 1, and 10 μM) and lenvatinib (0, 1, 10, and 100 μM). qRT-PCR and flow cytometry were utilized to determine HCC stem cell markers CD44, CD90, and CD133 expressions. Transcriptome sequencing was performed on organoids.-Western blot evaluated Notch pathwayrelated proteins (NOTCH1 and Jagged) expressions. Furthermore, DAPT, an inhibitor of the Notch pathway, was used to investigate the effects of lenvatinib on resistance or stemness in organoids and human HCC tissues. The organoids were successfully cultivated. With the increase of lenvatinib concentration, sensitive cell organoids were markedly degraded and ATP activity was gradually decreased, while there was no significant change in ATP activity of resistant cell organoids. CD44 expressions were elevated after lenvatinib treatment compared with the control group. KEGG showed that lenvatinib treatment of organoids constructed from Hep3B cells mainly activated the Notch pathway. Compared with the control group, NOTCH1 and Jagged expressions elevated, and ATP activity decreased after lenvatinib treatment. However, ATP activity was notably decreased after DAPT treatment. Moreover, DAPT inhibited lenvatinib resistance and the increase in the expressions of CD44 caused by lenvatinib. Besides, 100 μM lenvatinib significantly inhibited the growth and ATP activity of human HCC organoids, and DAPT increased the inhibitory effect of lenvatinib. Lenvatinib regulated resistance and stemness in organoids via the Notch pathway.

Transcription Factor E2F1 Enhances Hepatocellular Carcinoma Cell Proliferation and Stemness by Activating GINS1.

Present studies report that high expression of GINS complex subunit 1 (GINS1) is notably pertinent to poor survival for hepatocellular carcinoma (HCC), but it remains unclear how GINS1 affects the progression of HCC. This study aims at investigating the mechanism by which GINS1 affects HCC cell proliferation and stemness. We performed bioinformatics analysis for determining GINS1 expression in HCC tissues, as well as the HCC patients' survival rate with different expression levels of GINS1. E2F transcription factor 1 (E2F1) was predicted as the upstream transcription factor of GINS1, and the binding relation between the two was verified by chromatin immunoprecipitation and dual-luciferase reporter assays. Quantitative real-time polymerase chain reaction was adopted to evaluate the expression of GINS1 and E2F1. The protein expression levels of GINS1, E2F1, and cell stemness-related genes (SOX-2, NANOG, OCT4, and CD133) were detected by Western blot. Afterward, the proliferative capacity and stemness of HCC tumor cells were determined through colony formation, cell counting kit-8, and sphere formation assays. Our study found the high expression of GINS1 and E2F1 in HCC, and overexpressed GINS1 markedly enhanced the sphere formation and proliferation of HCC cells, while silencing GINS1 led to the opposite results. Besides, E2F1 promoted the transcription of GINS1 by working as an upstream transcription factor. The results of the rescue experiment suggested that overexpressed E2F1 could offset the suppressive effect of GINS1 silencing on HCC cell stemness and proliferation. We demonstrated that the transcription factor E2F1 accelerated cell proliferation and stemness in HCC by activating GINS1 transcription. The results can provide new insight into the GINS1-related regulatory mechanism in HCC, which suggest that it may be an effective way for HCC treatment by targeting the E2F1/GINS1 axis.

Comprehensive analysis of IGF2BP3 with expression features, prognosis, immune modulation and stemness in hepatocellular carcinoma and pan-cancer.

Insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) is a critical m6A reader. It encodes proteins that contain several KH domains, which are important in RNA binding, RNA synthesis and metabolism. Lots of researches have studied the malignant potential of m6A readers in tumors. However, the biological functional analysis of IGF2BP3 in hepatocellular carcinoma (HCC) and pan-cancer is not comprehensive. In this study, we used a bioinformatics approach to comprehensively analyze the significance of IGF2BP3 in HCC through analyzing its expression, mutation, prognosis, protein-protein interaction (PPI) network, functional enrichment, and the correlation with ferroptosis, stemness as well as immune modulation in HCC. IGF2BP3 presented a negative correlation with the ferroptosis molecule NFE2L2, and a positive correlation with the ferroptosis molecule SLC1A5 as well as the immune checkpoint HAVCR2. In addition, we also analyzed IGF2BP3 expression, prognosis and immune modulation in pan-cancer, revealing the prognostic value of IGF2BP3 in a variety of tumors. Finally, we verified the biological functions of IGF2BP3 in HCC through various experiments. The data showed that IGF2BP3 may enhance the proliferation, colony formation and invasion capacities of HCC cells, and IGF2BP3 is mainly positively correlated with the expression level of stemness marker SOX2. In conclusion, IGF2BP3 had a potential to be a new perspective biomarker in forecasting the immune response, ferroptosis, stemness and prognosis of HCC or even pan-cancer.

Interventing mitochondrial PD-L1 suppressed IFN-γ-induced cancer stemness in hepatocellular carcinoma by sensitizing sorafenib-induced ferroptosis

Evidence recently showed that pleiotropic cytokine interferon-gamma (IFN-γ) in the tumor microenvironment (TME) plays a positive role in hepatocellular carcinoma (HCC) progression through the regulation of liver cancer stem cells (LCSCs) in HCC. The present study explored the role and potential mechanism of mitochondrial programmed cell death-ligand 1 (PD-L1) and its regulation of ferroptosis in modulating the cancer stemness of LCSCs. It was shown that mimicking TME IFN-γ exposure increased the LCSCs ratio and cancer stemness phenotypes in HCC cells. IFN-γ exposure inhibited sorafenib (Sora)-induced ferroptosis by enhancing glutathione peroxidase 4 (GPX4) expression as well reactive oxygen species (ROS) and lipid peroxidation (LPO) generation in LCSCs. Furthermore, IFN-γ exposure upregulated PD-L1 expression and its mitochondrial translocation, inducing dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and correlating with glycolytic metabolism reprogramming in LCSCs. The genetic intervention of PD-L1 promoted ferroptosis-dependent anti-tumor effects of Sora, reduced glycolytic metabolism reprogramming, and inhibited cancer stemness of HCC in vitro and in vivo. Our results revealed a novel mechanism that IFN-γ exposure-induced mitochondrial translocation of PD-L1 enhanced glycolytic reprogramming to mediate the GPX4-dependent ferroptosis resistance and cancer stemness in LCSCs. This study provided new insights into the role of mitochondrial PD-L1-Drp1-GPX4 signal axis in regulating IFN-γ exposure-associated cancer stemness in LCSCs and verified that PD-L1-targeted intervention in combination with Sora might achieve promising synergistic anti-HCC effects.

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.

Integration of single-cell RNA-seq and bulk RNA-seq to construct liver hepatocellular carcinoma stem cell signatures to explore their impact on patient prognosis and treatment.

Liver hepatocellular carcinoma (LIHC) is a prevalent form of primary liver cancer. Research has demonstrated the contribution of tumor stem cells in facilitating tumor recurrence, metastasis, and treatment resistance. Despite this, there remains a lack of established cancer stem cells (CSCs)-associated genes signatures for effectively predicting the prognosis and guiding the treatment strategies for patients diagnosed with LIHC. The single-cell RNA sequencing (scRNA-seq) and bulk RNA transcriptome data were obtained based on public datasets and computerized firstly using CytoTRACE package and One Class Linear Regression (OCLR) algorithm to evaluate stemness level, respectively. Then, we explored the association of stemness indicators (CytoTRACE score and stemness index, mRNAsi) with survival outcomes and clinical characteristics by combining clinical information and survival analyses. Subsequently, weighted co-expression network analysis (WGCNA) and Cox were applied to assess mRNAsi-related genes in bulk LIHC data and construct a prognostic model for LIHC patients. Single-sample gene-set enrichment analysis (ssGSEA), Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) and Tumor Immune Estimation Resource (TIMER) analysis were employed for immune infiltration assessment. Finally, the potential immunotherapeutic response was predicted by the Tumor Immune Dysfunction and Exclusion (TIDE), and the tumor mutation burden (TMB). Additionally, pRRophetic package was applied to evaluate the sensitivity of high and low-risk groups to common chemotherapeutic drugs. A total of four genes (including STIP1, H2AFZ, BRIX1, and TUBB) associated with stemness score (CytoTRACE score and mRNAsi) were identified and constructed a risk model that could predict prognosis in LIHC patients. It was observed that high stemness cells occurred predominantly in the late stages of LIHC and that poor overall survival in LIHC patients was also associated with high mRNAsi scores. In addition, pathway analysis confirmed the biological uniqueness of the two risk groups. Personalized treatment predictions suggest that patients with a low risk benefited more from immunotherapy, while those with a high risk group may be conducive to chemotherapeutic drugs. The current study developed a novel prognostic risk signature with genes related to CSCs, which provides novel ideas for the diagnosis, prognosis and treatment of LIHC.