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Background & Aims: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells., Methods: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest., Results: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers., Conclusions: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Metabolic reprogramming is recognized as a cancer hallmark intimately linked to tumor hypoxia, which supports rapid tumor growth and mitigates the consequential oxidative stress. Phosphofructokinase-fructose bisphosphatase (PFKFB) is a family of bidirectional glycolytic enzymes possessing both kinase and phosphatase functions and has emerged as important oncogene in multiple types of cancer. However, its clinical relevance, functional significance, and underlying mechanistic insights in hepatocellular carcinoma (HCC), the primary malignancy that develops in the most important metabolic organ, has never been addressed., Methods: PFKFB4 expression was examined by RNA sequencing in The Cancer Genome Atlas and our in-house HCC cohort. The up-regulation of PFKFB4 expression was confirmed further by quantitative polymerase chain reaction in an expanded hepatitis B virus-associated HCC cohort followed by clinicopathologic correlation analysis. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated PFKFB4 knockout cells were generated for functional characterization in vivo, targeted metabolomic profiling, as well as RNA sequencing analysis to comprehensively examine the impact of PFKFB4 loss in HCC., Results: PFKFB4 expression was up-regulated significantly in HCC and correlated positively with TP53 and TSC2 loss-of-function mutations. In silico transcriptome-based analysis further revealed PFKFB4 functions as a critical hypoxia-inducible gene. Clinically, PFKFB4 up-regulation was associated with more aggressive tumor behavior. Functionally, CRISPR/Cas9-mediated PFKFB4 knockout significantly impaired in vivo HCC development. Targeted metabolomic profiling revealed that PFKFB4 functions as a phosphatase in HCC and its ablation caused an accumulation of metabolites in downstream glycolysis and the pentose phosphate pathway. In addition, PFKFB4 loss induced hypoxia-responsive genes in glycolysis and reactive oxygen species detoxification. Conversely, ectopic PFKFB4 expression conferred sorafenib resistance., Conclusions: PFKFB4 up-regulation supports HCC development and shows therapeutic implications., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is characterized by its high degrees of both inter- and intratumoral heterogeneity. Its complex tumor microenvironment is also crucial in promoting tumor progression. Recent advances in single-cell RNA sequencing provide an important highway to characterize the underlying pathogenesis and heterogeneity of HCC in an unprecedented degree of resolution. This review discusses the up-to-date discoveries from the latest studies of HCC with respect to the strength of single-cell RNA sequencing. We discuss its use in the dissection of the landscape of the intricate HCC ecosystem and highlight the major features at cellular levels, including the malignant cells, different immune cell types, and the various cell-cell interactions, which are crucial for developing effective immunotherapies. Finally, its translational applications will be discussed. Altogether, these explorations may give us some hints at the tumor growth and progression and drug resistance and recurrence, particularly in this era of personalized medicine., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Liver cancer (hepatocellular carcinoma [HCC]) is a fatal cancer worldwide and often is detected at an advanced stage when treatment options are very limited. This drives the development of techniques and platforms for early detection of HCC. In recent years, liquid biopsy has provided a means of noninvasive detection of cancers. By detecting plasma circulating tumor DNA (ctDNA) released from dying cancer cells, the presence of HCC can be detected in a noninvasive manner. In this review, we discuss the molecular characteristics of ctDNA and its various molecular landscapes in HCC. These include the mutational landscape, single-nucleotide variations, copy number variations, methylation landscape, end motif/coordinate preference, hepatitis B virus integration, and mitochondrial DNA mutations. The consistency between the plasma ctDNA and the tumor tissue genomic DNA mutational profile is pivotal for the clinical utility of ctDNA in the clinical management of HCC. With strategic use of genetic information provided from plasma ctDNA profiling and procedure standardization to facilitate implementation in clinical practice, better clinical management would become permissible through more efficient detection and diagnosis of HCC, better prognostication, precision-matched treatment guidance, and more reliable disease monitoring., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)