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.
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.
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.
PFKFB4 up-regulation supports HCC development and shows therapeutic implications.
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In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including ...aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5‐Aza‐dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor‐2 (TFPI‐2), a Kunitz‐type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI‐2 was frequently silenced in human HCC and HCC cell lines. TFPI‐2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI‐2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI‐2 messenger RNA expression. In addition, TFPI‐2 expression in HCC cell lines can be robustly restored by combined treatment with 5‐Aza‐dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI‐2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI‐2 significantly suppressed the proliferation and invasiveness of HCC cells. Conclusion: Our findings suggest that TFPI‐2 is a candidate tumor suppressor gene in human HCC. (HEPATOLOGY 2007;45:1129–1138.)
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.
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
assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple
preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection.
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
and
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.
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.
Hepatitis B virus (HBV) is a major risk factor of chronic liver disease and hepatocellular carcinoma (HCC). Random integration of HBV DNA into the host genome is frequent in HCC leading to truncation ...of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). C-terminally truncated HBx (HBx-ΔC) has been implicated in playing a pro-oncogenic role in hepatocarcinogenesis. However, the mechanism whereby HBx-ΔC1 contributes to hepatocarcinogenesis remains unclear. In this study, we investigated the functional role of HBx-ΔC1 in regulating liver cancer stem cell (CSC) properties. Using Tet-on inducible system, we found that HBx-ΔC1 enhanced CSC properties including self-renewal, tumorigenicity, chemoresistance, migration and expression of liver CSC markers, when compared with the full-length HBx counterpart and vector control. Interestingly, HBx-ΔC1 conferred resistance in HCC cells towards sorafenib treatment through suppression of apoptotic cascade. In addition, HBx-ΔC1 upregulated a panel of stemness genes, in which Nanog was found to be among the most significant one in both trasnfected cell lines. Consistently, Nanog was upregulated in human HCC samples which had HBx-ΔC1 expression. Furthermore, the induction of CSC properties by HBx-ΔC1 was via the Stat3/Nanog pathway, as administration of Stat3 inhibitor abolished the HBx-ΔC1-induced self-renewing capacity. In conclusion, our data suggest that HBx-ΔC1 enhances liver CSCs properties through Stat3/Nanog cascade, and provide a new insight for the therapeutic intervention for HBV-related HCC.
MicroRNAs (miRNAs), an important class of small non-coding RNAs, regulate gene expression at the post-transcriptional level. miRNAs are involved in a wide range of biological processes and implicated ...in different diseases, including cancers. In this study, miRNA profiling and qRT-PCR validation revealed that miR-142-3p and miR-142-5p were significantly downregulated in hepatocellular carcinoma (HCC) and their expression levels decreased as the disease progressed. The ectopic expression of miR-142 significantly reduced HCC cell migration and invasion. Overexpression of either miR-142-3p or miR-142-5p suppressed HCC cell migration, and overexpression of both synergistically inhibited cell migration, which indicated that miR-142-3p and miR-142-5p may cooperatively regulate cell movement. miR-142-3p and miR-142-5p, which are mature miRNAs derived from the 3′- and 5′-strands of the precursor miR-142, target distinct pools of genes because of their different seed sequences. Pathway enrichment analysis showed a strong association of the putative gene targets of miR-142-3p and miR-142-5p with several cell motility-associated pathways, including those regulating actin cytoskeleton, adherens junctions, and focal adhesion. Importantly, a number of the putative gene targets were also significantly upregulated in human HCC cells. Moreover, overexpression of miR-142 significantly abrogated stress fiber formation in HCC cells and led to cell shrinkage. This study shows that mature miR-142 pairs collaboratively regulate different components of distinct signaling cascades and therefore affects the motility of HCC cells.
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.
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.
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.
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.
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Transarterial chemoembolization (TACE) is a commonly used treatment modality in hepatocellular carcinoma (HCC). The ability to identify patients who will respond to TACE represents an important ...clinical need, and tumor gene expression patterns may be associated with TACE response. We investigated whether tumor transcriptome is associated with TACE response in patients with HCC. We analyzed transcriptome data of treatment-naïve tumor tissues from a Chinese cohort of 191 HCC patients, including 105 patients who underwent TACE following resection with curative intent. We then developed a gene signature, TACE Navigator, which was associated with improved survival in patients that received either adjuvant or post-relapse TACE. To validate our findings, we applied our signature in a blinded manner to three independent cohorts comprising an additional 130 patients with diverse ethnic backgrounds enrolled in three different hospitals who received either adjuvant TACE or palliative TACE. TACE Navigator stratified patients into Responders and Non-Responders which was associated with improved survival following TACE in our test cohort (Responders: 67 months vs Non-Responders: 39.5 months, p<0.0001). In addition, multivariable Cox model demonstrates that TACE Navigator was independently associated with survival (HR: 9.31, 95% CI: 3.46-25.0, p<0.001). In our validation cohorts, the association between TACE Navigator and survival remained robust in both Asian patients who received adjuvant TACE (Hong Kong: 60 months vs 25.6 months p=0.007; Shandong: 61.3 months vs 32.1 months, p=0.027) and European patients who received TACE as primary therapy (Mainz: 60 months vs 41.5 months, p=0.041). These results indicate that a TACE-specific molecular classifier is robust in predicting TACE response. This gene signature can be used to identify patients who will have the greatest survival benefit after TACE treatment and enable personalized treatment modalities for patients with HCC.
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.
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