ARID3A Promotes Tumor Progression by Activating TNF-α/NF-κB Signaling in Dual-Phenotype Hepatocellular Carcinoma

Transcriptional regulators in hepatocarcinogenesis – Key integrators of malignant transformation

BackgroundAberrant alternative splicing (AS) contributes to cancer stemness and progression in hepatocellular carcinoma (HCC). However, the regulatory roles of long noncoding RNAs (lncRNAs) in linking AS dysregulation to tumor stemness remain elusive.MethodsWe performed integrated bulk and single-cell RNA-Seq analyses combined with functional assays to identify key lncRNAs associated with splicing regulation and cancer stemness in HCC. Mechanistic studies were conducted to elucidate the molecular interplay between lncRNAs, splicing factors, and transcriptional regulators. Drug sensitivity assays were used to evaluate therapeutic potential.ResultsGlobal analysis revealed increased splicing regulator activity during hepatocellular carcinoma (HCC) progression, which correlated with poor prognosis. This splicing dysregulation led us to identify 28 lncRNAs that connect aberrant splicing with cancer stemness. Among these, RAB30-DT was significantly overexpressed in malignant epithelial cells and associated with advanced tumor stage, stemness features, genomic instability, and poor patient prognosis. Functional assays demonstrated that RAB30-DT promotes proliferation, migration, invasion, colony and sphere formation in vitro, and tumor growth in vivo. Mechanistically, RAB30-DT is transcriptionally activated by CREB1 and directly binds and stabilizes the splicing kinase SRPK1, facilitating its nuclear localization. This interaction broadly reshapes the AS landscape, including splicing of the cell cycle regulator CDCA7, to drive tumor stemness and malignancy. Importantly, pharmacological disruption of the CREB1–RAB30-DT–SRPK1 axis sensitizes HCC cells to targeted therapies.ConclusionsOur study reveals a novel lncRNA-mediated signaling axis that integrates transcriptional regulation and splicing reprogramming to sustain cancer stemness and progression in HCC. Targeting this axis offers promising therapeutic opportunities for HCC treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-025-03546-w.

Background: Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer but accounts for 10% of breast cancer-related deaths. Cancer stem cells (CSCs; tumor stemness) play a key role in tumor dormancy, progression, and treatment resistance, yet mechanisms that drive CSCs remain poorly defined. We recently identified that NDRG1 promotes tumor growth and progression in IBC mouse models, and that its depletion inhibits AKT phosphorylation. We hypothesized that NDRG1 is a key regulator of tumor stemness in IBC via activating AKT signaling. Methods: CSCs were assessed using surrogate markers including CD44+/CD24-, mammospheres, and in vivo limiting dilution assay experiments. To identify which AKT isoforms (AKT1, AKT2, or AKT3) or upstream kinases (SGK1, GSK3β) mediate NDRG1-induced CSCs, NDRG1-depleted cells were transfected with NDRG1 WT, AKT plasmids or SGK1/GSK3β phospho-site mutants or siRNA for SGK1/GSK3β. Results: NDRG1 depletion significantly reduced CD44+/CD24- subpopulation and mammosphere formation (p<0.001). Limiting dilution experiments demonstrated a significant reduction in tumor incidence and stemness frequency in mice transplanted with NDRG1 knockdown cells (p= 1 x 10-12). Each of the three AKT isoforms partially rescued tumor stemness in the NDRG1 knockdown IBC cells [CD44+/CD24-: NDRG1 KD vs AKT1 OE, p=0.008; vs AKT2 OE, p=0.005; vs AKT3 OE, p=0.001)]. Overexpression of known inactive SGK1 phospho-site mutants of NDRG1 restored tumor stemness in NDRG1-depleted cells (p≤0.005). While silencing GSK3β reduced CSCs in IBC cells SGK1 did not affect this subpopulation, indicating that the tumor stemness effect of NDRG1 is independent of SGK1 and its kinase activity and is regulated by GSK3β. Conclusions: Our findings underscore the critical role of NDRG1 as a regulator of IBC tumor stemness. We observed all three isoforms of AKT restored CSCs in NDRG1-depleted breast cancer cells, highlighting the significance of the NDRG1-AKT axis in governing stemness and tumor progression in IBC. Citation Format: Emilly Schlee Villodre, Xiaoding Hu, Wendy A. Woodward, Stefan Pusch, Debu Tripathy, Bisrat G. Debeb. NDRG1-AKT signaling promotes tumor stemness in inflammatory breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-23.

Proteomic and single-cell landscape reveals novel pathogenic mechanisms of HBV-infected intrahepatic cholangiocarcinoma

Global cancer incidence and mortality increases dramatically word wild, epically in China, even though significant progress has been made against cancer. Cancer will account for the first most common cause of disease-related death in China. Abundant evidences demonstrated both genetic and environmental factors might contribute to human cancer initiation and progression. In our lab, we focus on role of gain-of-function mutant SHP-2 (genetic factor) and heavy mental contamination or alcohol drinking (environmental factors) on tumor progression. Here, we reported environmental factors like heavy mental arsenic and alcohol consumption promote digestive tumor and breast cancer malignant behaviors. The first, clinic data showed that an increasing arsenic concentration in III/IV TNM stage gastric cancer patient, then an enhanced cancer cell malignant behaviors (proliferation/ motility/ metastasis, etc) were found in gastric cancer or colon cancer cell lines chronic treated with 5-15 nm arsenic in vitro and in vivo. EMT might contribute to this promotion process induced by arsenic. The second: a positive correlation between alcohol consumption and advanced Tumor-Node-Metastasis (TNM) stages, higher vessel invasion and poorer prognosis in Hepatocellular carcinoma (HCC) were found. In vitro and in vivo experiments further indicated that alcohol promoted the progression and migration/invasion of HCC, breast cancer and lewis lung carcinoma (LLC). Specifically, we found that alcohol enhanced the migration/invasion of tumor cells and increased tumor angiogenesis. Consistently, higher expression of VEGF, MCP-1, activated NF-κB and more infiltrated M2 macrophage was observed in tumor tissues of alcohol-drinkers. Alcohol induced the accumulation of intracellular reactive oxygen species (ROS) and the activation of NF-κB signaling in HepG2 cells. Conversely, blockage of alcohol-mediated ROS accumulation and NF-κB signaling inhibited alcohol-induced expression of VEGF and MCP-1, the tumor growth, angiogenesis and metastasis. These results suggested that chronic moderate alcohol consumption may promote the progression and metastasis of tumor; the oncogenic effect may be at least partially mediated by the ROS accumulation and NF-ĸB-dependent VEGF and MCP-1 up-regulation. Even more, an increasing cancer stem cell population was found in tumor cell lines under low dose alcohol induction. Our results make us presume chronic stress induced by environmental factors like alcohol, heavy mental, etc might activate cell signal and enhance tumor progression in a universal way. Those studies will be benefited to tumor prevention. This study was supported by a grant from the National Natural Science Foundation of China (codes: 30873046, 30973424, and 81072663). Note: This abstract was not presented at the meeting. Citation Format: Siying Wang. Environmental factors (heavy metal, alcohol) enhance tumor malignant behaviors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1248. doi:10.1158/1538-7445.AM2015-1248

Wnt/β-catenin signaling in malignant mammary tumor progression and metastasis formation & Mechanisms of evasive resistance to sorafenib in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, it has a poor prognosis due to its highly invasive and metastatic nature. Consequently, identifying effective prognostic markers and potential therapeutic targets has been extensively investigated. METTL5, an 18S rRNA methyltransferase, is abnormally high in HCC. But its biological function and prognostic significance in HCC remain largely unelucidated. This study aimed to investigate the role of METTL5 in HCC progression, and elucidate its possible molecular mechanisms in HCC via transcriptome sequencing, providing new insights for identifying new HCC prognostic markers and therapeutic targets. The METTL5 expression in HCC and paracancerous tissues was analyzed using HCC immunohistochemical microarrays and bioinformatic retrieval methods to correlate METTL5 with clinicopathological features and survival prognosis. We constructed a METTL5 knockdown hepatocellular carcinoma cell line model and an animal model to determine the effect of METTL5 on hepatocellular carcinoma progression. Subsequently, RNA sequencing was performed to analyze the molecular mechanism of METTL5 in HCC based on the sequencing results, and relevant experiments were performed to verify it. We found that METTL5 expression was elevated in hepatocellular carcinoma tissues and correlated with poor patient prognosis, and in the analysis of clinicopathological features showed a correlation with TNM staging. In hepatocellular carcinoma cell lines with knockdown of METTL5, the malignant biological behavior was significantly reduced both invitro and invivo. Based on the sequencing results as well as the results of GO functional enrichment analysis and KEGG pathway enrichment analysis, we found that METTL5 could promote the generation and release of neutrophil extracellular capture network (NETs) and might further accelerate the progression of HCC. The m6A methyltransferase METTL5 is overexpressed in hepatocellular carcinoma (HCC) and correlates with poor prognosis. METTL5 accelerates malignant progression of HCC by promoting generation and release of the neutrophil extracellular traps (NETs) network, providing new insights for clinical biomarkers and immunotherapeutic targets in HCC prognosis.

BackgroundTransarterial chemoembolization (TACE) refractoriness is a significant challenge in treating intermediate to advanced-stage hepatocellular carcinoma (HCC). A few studies suggest that liver cancer stem cells (LCSCs) may be associated with TACE refractoriness. This study aims to explore the potential correlation between TACE refractoriness and HCC stemness, highlighting its clinical significance.MethodsThis research encompassed the analysis of diverse HCC datasets, including RNA-sequencing, microarray, single-cell RNA-sequencing, and clinical cohorts. We identified common genes between TACE refractoriness and tumor stemness (TSGs). Unsupervised clustering was employed to classify HCC patients into different clusters based on TSGs (TRS clusters). The study explored the differences in clinical prognosis, biological characteristics, genomic variations, immune landscapes, and treatment responses among the TRS clusters.ResultsPatients with TACE-refractoriness demonstrated significantly higher tumor stemness. Our study identified 33 TSGs and established two TRS clusters, including C1 and C2. C1 was associated with TACE refractoriness, elevated tumor stemness, and poorer prognosis. Genomic alterations were found to be significantly different between the TRS clusters. The C1 exhibited signs of immunosuppression and lower activity of immune effector cells, while the C2 had a more robust immune response and higher level of immune cell presence. Single-cell RNA-seq revealed distinct cell type characteristics in each subtypes, with the C1 showing a higher proportion of stem cells and malignant cells.ConclusionOur findings establish a connection between TACE refractoriness and tumor stemness in HCC, proposing a novel subtype classification to guide personalized treatment. Insights gained may facilitate overcoming TACE refractoriness and the development of innovative therapies.

CD168+ macrophages promote hepatocellular carcinoma tumor stemness and progression through TOP2A/β-catenin/YAP1 axis

How should patients with hepatocellular carcinoma recurrence after liver transplantation be treated?

MYH9 has dual functions in tumors. However, its role in inducing tumor stemness in hepatocellular carcinoma (HCC) is not yet determined. Here, we found that MYH9 is an effective promoter of tumor stemness that facilitates hepatocellular carcinoma pathogenesis. Importantly, targeting MYH9 remarkably improved the survival of hepatocellular carcinoma-bearing mice and promoted sorafenib sensitivity of hepatocellular carcinoma cells in vivo. Mechanistic analysis suggested that MYH9 interacted with GSK3β and reduced its protein expression by ubiquitin-mediated degradation, which therefore dysregulated the β-catenin destruction complex and induced the downstream tumor stemness phenotype, epithelial–mesenchymal transition, and c-Jun signaling in HCC. C-Jun transcriptionally stimulated MYH9 expression and formed an MYH9/GSK3β/β-catenin/c-Jun feedback loop. X protein is a hepatitis B virus (HBV)-encoded key oncogenic protein that promotes HCC pathogenesis. Interestingly, we observed that HBV X protein (HBX) interacted with MYH9 and induced its expression by modulating GSK3β/β-catenin/c-Jun signaling. Targeting MYH9 blocked HBX-induced GSK3β ubiquitination to activate the β-catenin destruction complex and suppressed cancer stemness and EMT. Based on TCGA database analysis, MYH9 was found to be elevated and conferred poor prognosis for hepatocellular carcinoma patients. In clinical samples, high MYH9 expression levels predicted poor prognosis of hepatocellular carcinoma patients. These findings identify the suppression of MYH9 as an alternative approach for the effective eradication of CSC properties to inhibit cancer migration, invasion, growth, and sorafenib resistance in HCC patients. Our study demonstrated that MYH9 is a crucial therapeutic target in HCC.

This study aimed to investigate the expression levels of the MKNK2 gene in pan-cancer, its prognostic significance, and its relationship with the tumor immune microenvironment, as well as to assess its potential as an immunological and prognostic biomarker. The research utilized data from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Cancer Cell Line Encyclopedia (CCLE), including clinical and mutational information. Bioinformatic tools were employed to analyze the association of MKNK2 with carcinogenesis, including its links to prognosis, immune cell infiltration, tumor immune microenvironment, gene mutation, and the stemness of various tumor cells. A variety of statistical software and analytical tools were applied, including R software, SPSS 27.0, TIMER, CIBERSORT algorithm, and EPIC algorithm. The study found that MKNK2 is abnormally expressed in pan-cancer and is associated with a poor prognosis. The levels of MKNK2 are highly related to immune cell infiltration and tumor stemness. Notably, in liver hepatocellular carcinoma, glioblastoma multiforme, low-grade gliomas, and acute myeloid leukemia, MKNK2 expression shows a strong correlation with clinical outcomes and immune infiltration. Furthermore, the expression of MKNK2 shows significant correlations with immune cell infiltration, immune checkpoints, tumor mutational burden (TMB), microsatellite instability (MSI), and stemness scores across various cancers. The abnormal expression of MKNK2 is associated with tumor progression, immune checkpoint genes, immune cell infiltration, microsatellite instability (MSI), tumor mutational burden (TMB), and stemness in a variety of tumors, especially in glioblastoma multiforme low-grade gliomas (GBMLGG). Therefore, MKNK2 may serve as a potent prognostic physiological marker and provide new avenues for the development of tumor mechanisms and therapeutic strategies targeting MKNK2 to enhance the efficacy of immunotherapy.

Purpose: CK19 has been recognized as an important molecular marker for poor prognosis of Hepatocellular carcinoma (HCC), however, its molecular determinants and clinical implications is still remains a largely unmet challenge. We sought to identify molecular features and develop a CK19-based molecular classification model to provide new strategies for precise treatment of this deadly HCC. Experimental Design: Tumor and matched non-tumor liver specimens were collected from 116 patients who underwent partial hepatectomy for primary or recurrent HCC in Guangxi region. HCC patients were classified into two CK19 positive subtypes and one CK19 negative subtype based on CK19 related transcriptomic profiling. The tumor heterogeneity of three subtypes were further characterized by multiomics technology including whole-exome sequencing, bulk and single-cell RNA sequencing, whole genome methylation , and imaging mass cytometry (IMC). Subtype scoring were validated using the TCGA database and in vitro cell line. Results: We innovatively constructed an accurate classification scoring system for CK19 related HCC, which divided CK19 positive HCC into “Hepatic stem cell”(K1) and “Hepatic progenitor cell”(K2) two distinct subtypes with respective pathway and clinicopathological phenotypes . K1 is characterized by poor prognosis, and activation of Myc and proliferation pathway. Meanwhile, down-regulation of p53 pathway and low differentiation are also unique characteristics of K1, Compared with K1, K2 showed enrichment of pro-oncogene Ras pathway and high expression of inflammatory signals and immune cells. Moreover, these two CK19 positive subtypes have different cell lineage gene expression and they may come from different cell lineage types. K1 may come from CK19 negative HCC by epigenetic regulation but K2 may come from hepatic progenitor cells activated by inflammatory stimulation. Specifically, we identified K1 as a new poor prognosis stem cell subtype of CK19 positive HCC. K1 highly expressed tumor stem cell markers and displayed very low expression of androgen receptor AR, as well as activation of neural related signal pathway, which is ineffective for postoperative TACE treatment. Conclusion: These data suggest that lineage plasticity may contribute to heterogeneity of CK19 positive HCC, which in turn carry clinical implications regarding new treatment strategies for treatment of HCC with highly recurrence. Citation Format: Qiuyan Wang, Qinchen Lu, Yaobang Wang, Zhenxing Wang, Xi Wang, Yuting Tao, Bangde Xiang, Herry Yang. CK19 based molecular classification defined three distinct hepatocellular carcinoma subtypes with different cell lineage features and therapeutic vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2240.

N4-acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N-acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10-mediated ac4C in HCC progression and provide a promising therapeutic approach. The ac4C levels were evaluated by dot blot and ultra-performance liquid chromatography-tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10-ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull-down, mass spectrometry, and site-specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10-ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high-throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10-mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. Our study identified a novel oncogenic epi-transcriptome axis involving NAT10-ac4C/eEF2-HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

Hepatocellular carcinoma (HCC) is an aggressive malignant tumor with a poor prognosis. Reactive oxygen species (ROS) play an important role in tumors; however, the role of ROS-related genes is still unclear in HCC. Therefore, we analyzed the role of ROS-related genes in HCC via bioinformatics methods. Firstly, a prognosis model was constructed using LASSO Cox regression and multivariate analyses. We also investigated the potential function of the ROS-related genes and the correlation with immune infiltration, tumor stemness, and drug sensitivity. ICGC database was used for validation. Secondly, we further analyzed the role of 11 ROS-related genes in HCC. As a member of ROS gene family, the role of STK25 has remained unclear in HCC. We explored the biological function of STK25 using in vitro experiments. The present study was the first to construct a ROS-related prognostic model in HCC. The correlation of ROS-related genes with immune infiltration, tumor stemness, and drug sensitivity was dissected. Furthermore, we demonstrated that STK25 knockdown could increase the proliferation, migration, and invasion capacity of HCC cells.