Hepatocellular Carcinoma Transformation Research Articles

CtBP1 Mediates Hypoxia-Induced Sarcomatoid Transformation in Hepatocellular Carcinoma.

BackgroundSarcomatoid hepatocellular carcinoma (sHCC), a highly aggressive subtype of hepatocellular carcinoma (HCC), mostly transforms from classical hepatocellular carcinoma (cHCC). The study intended to explore the role of C-terminal binding protein 1 (CtBP1) in sarcomatoid transformation of hepatocellular carcinoma.MethodsWestern blotting and/or immunohistochemistry were used to confirm the expression of CtBP1 and other proteins in HCC cells, xenografts and clinical tissue samples. CtBP1 shRNA-expressing lentivirus was used to infect HepG2 cells to construct CtBP1 knockdown cells. Cell migration was determined by scratch wound assays and Transwell assays. Immunofluorescence was used to label the a-tubulin cytoskeleton to evaluate cell morphology. HepG2 cells were inoculated subcutaneously in nude mice to construct xenografts and beneath the liver capsule to evaluate in vivo metastasis.ResultsCompared to that in the cHCC area, CtBP1 expression was significantly upregulated in the sHCC area, as shown by immunohistochemistry. HE staining showed that cells in the sHCC area were spindle-shaped, while those in the cHCC area were polygonal. Immunohistochemically, the epithelial markers pancytokeratin (CK) and E-cadherin were partially or completely lost, while the expression of the mesenchymal marker vimentin was upregulated in the sHCC area. Moreover, HepG2, an HCC cell line with high expression of CtBP1, autonomously underwent sarcomatoid transformation, showing a sarcomatoid morphology and phenotype. HIF1a expression was upregulated in epithelial cells adjacent to the sHCC area. Hypoxia upregulated CtBP1 protein expression and induced an EMT phenotype with increased migration and a spindle-shaped morphology in HepG2 cells. Knockdown of CtBP1 partially reversed the EMT phenotype induced by hypoxia. Silencing CtBP1 completely blocked the sarcomatoid transformation of subcutaneous xenografts and decreased lung metastasis in subcapsular xenografts of the liver in nude mice.ConclusionCtBP1 plays a key role in hypoxia-induced EMT and sarcomatoid transformation in HCC and could be a candidate target for the management of sHCC.

New Approach about the Signaling Crosstalk between IQGAPs/ NF- κB/IL-8 and PDCD5/p53/TRAIL Pathways that Modulate Malignant Transformation in Hepatocellular Carcinoma.

Objective:We aimed to investigate the signalling crosstalk of TNF-related apoptosis-inducing ligand, TRAIL death receptors, tumour protein p53, and programmed cell death (PDCD5) with IQGAPs. Also, we targeted the crosstalk between IQGAPs genes with decoy receptors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interleukins -8 (IL-8) and its receptor genes in a designed model of hepatocellular carcinoma induced in male Balb/c mice. Methods:The presence of HCC was confirmed by histological and morphological alterations. In parallel to the incidence of hepatic cancer, we found lung, heart, and kidney cancer after treatment with DEN. Results:Our results show that the expression of mRNA of IQGAP1, TRAIL decoy receptors, NF-κB, and IL-8 genes was elevated in hepatocellular carcinoma, as compared to normal liver tissue, while their expression was further up-regulated by increasing the dose of diethylnitrosamine. The expression of IQGAP2, TRAIL death receptors, p53, and PDCD5 was significantly down-regulated in HCC (p˂0.05). For confirmation of gene expression, protein levels of both IQGAP1 and P53 were measured by western blot analysis, which showed that diethylnitrosamine enhanced protein expression of IQGAP1 and diminished that of p53. Conclusion:IQGAPs have a direct signaling relationship with p53, IL-8, and TRAIL family. This interaction is recognized as a key signalling pathway for hepatocellular carcinogenesis.

Aerobic exercise suppresses hepatocellular carcinoma by downregulating dynamin-related protein 1 through PI3K/AKT pathway

ObjectiveExercise, as a common non-drug intervention, is one of several lifestyle choices known to reduce the risk of cancer. Mitochondrial division has been reported to play a key role in the occurrence and transformation of hepatocellular carcinoma (HCC). This study investigated whether exercise could regulate the occurrence and development of HCC through mitosis. MethodsBioinformatics technology was used to analyze the expression level of dynamin-related protein 1 (DRP1), a key protein of mitochondrial division. The effects of DRP1 and DRP1 inhibitor (mdivi-1) on the proliferation and migration of liver cancer cells BEL-7402 were observed using cell counting kit-8, plate colony formation, transwell cell migration, and scratch experiments. Enzyme-linked immunosorbent assay, Western blot and real-time polymerase chain reaction were used to detect the expression of DRP1 and its downstream phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. A treadmill exercise intervention was tested in a nude mouse human liver cancer subcutaneous tumor model expressing different levels of DRP1. The size and weight of subcutaneous tumors in mice were detected before and after exercise. ResultsThe expression of DRP1 in liver cancer tissues was significantly upregulated compared with normal liver tissues (P < 0.001). The proliferation rate and the migration of BEL-7402 cells in the DRP1 over-expression group were higher than that in the control group. The mdivi-1 group showed an inhibitory effect on the proliferation and migration of BEL-7402 cells at 50 μmol/L. Aerobic exercise was able to inhibit the expression of DRP1 and decrease the size and weight of subcutaneous tumors. Moreover, the expression of phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) decreased in the exercise group. However, exercise could not change p-PI3K and p-AKT levels after knocking down DRP1 or using mdivi-1 on subcutaneous tumor. ConclusionAerobic exercise can suppress the development of tumors partially by regulating DRP1 through PI3K/AKT pathway.

Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma

A subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.

Cinobufagin Triggers Defects in Spindle Formation and Cap-Dependent Translation in Liver Cancer Cells by Inhibiting the AURKA-mTOR-eIF4E Axis.

Cinobufagin is a Na+/K+-ATPase (NKA) inhibitor with excellent anticancer effects to prolong the survival of patients. The purpose of the present study was to clarify the underlying mechanism of the anticancer effects of cinobufagin using overexpression or inhibition of aurora kinase A (AURKA) signaling. First, high expression of Na+/K+-ATPase alpha 1 subunit (ATP1A1) and AURAK resulted in increased malignant transformation in hepatocellular carcinoma (HCC) patients using the cancer genome atlas (TCGA) data and tissue samples. After treatment with cinobufagin, we successfully screened 202, 249, and 335 changing expression proteins in Huh-7 cells under normal, overexpression, and inhibition of AURKA using tandem mass tags (TMT)-labeled quantitative proteomics coupled to 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these molecules were closely associated with chromosome segregation, DNA damage, and regulation of translation processes. We further confirmed that cinobufagin induced DNA damage and chromosome segregation disorders and suppresses translational processing in oncogenes by decreasing the expression of AURKA, mechanistic target of rapamycin kinase (mTOR), p-mTOR, p-extracellular regulated protein kinases (ERK), eukaryotic translation initiation factor 4E (eIF4E), and p-eIF4E, while increasing the expression of p-eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (S65, T37, T46, T45) and increasing the interaction between eIF4 and 4E-BP1. Our results suggested that cinobufagin performed an antitumor effects in liver cancer cells by inhibiting the AURKA-mTOR-eIF4E axis.

Biocompatible co-loading vehicles for delivering both nanoplatin cores and siRNA to treat hepatocellular carcinoma.

Bmi-1 is a gene related to malignant transformation in hepatocellular carcinoma (HCC). The liver cancer cells developed the ability to tolerate CDDP treatment with the elevation of Bmi-1. Bmi-1 is also an oncogene promoting malignance of tumor and an anti-cancer target in many studies. Herein, a biocompatible nanocarrier was designed in the study to deliver a chemotherapeutical agent CDDP and Bmi-1 siRNA to kill cancer cells and silence drug resistance related gene simultaneously. Calciumphosphate (CaP) was applied to coat both nanoplatin cores and siRNA as a shell for the purpose of delivering cargos to the cytosol of the tumor cells. Nanoplatin and siRNA co-loaded CaP nanoparticles (NPSC) enhanced cell uptake of CDDP and showed elevated drug accumulation in tumor. NPSC achieved considerable anti-cancer efficacy and counter-regulated drug tolerance, therefore, warranted a further investigation as a novel therapeutic nanosystem to improve cancer therapy.

The Down-Regulation of SUZ12 Accelerates the Migration and Invasion of Liver Cancer Cells via Activating ERK1/2 Pathway.

The suppressor of zest 12 (SUZ12), an essential subunit of the transcription polycomb repressive complex 2 (PRC2), has been found to be involved in HBV X-induced oncogenic transformation in hepatocellular carcinoma (HCC). However, the specific function of SUZ12 has not yet been determined in the pathogenesis of migration and invasion of HBV-associated HCC. Here, our results showed that SUZ12 was significantly down-regulated in HBV-related HCC tissues compared with adjacent non-tumor tissues by immunohistochemical and Western blot assays. The 5-years survival rate was worse in patients with low expression level of SUZ12. SUZ12 silencing increased the migration and invasion of HCC cells, and its overexpression impaired HCC cells migration and invasion. Knockdown of SUZ12 activated ERK1/2 pathway and increased MMP9 (matrix metallopeptidase 9) and MMP2 (matrix metallopeptidase 2) expression, whereas SUZ12 overexpression had opposite effects. Specific ERK1/2 inhibitor (SCH772984) significantly decreased HCC cells migration and invasion caused by SUZ12 shRNA. Thus, the liver cancer-down-regulated SUZ12 accelerated the invasion and metastasis of HCC cells. These effects might be associated with deregulation of SUZ12 activating ERK1/2, MMP2 and MMP9 in HCC cells.

Sarcomatoid hepatocellular carcinoma (SHC): a case report

BackgroundSarcomatoid hepatocellular carcinoma (SHC) is a rare malignant hepatic tumor. Recurrent interventional therapies such as transcatheter arterial chemo-embolization (TACE), radiofrequency ablation (RFA), and percutaneous ethanol injection have been reported previously utilized in a majority of SHC cases. To date, the exact pathogenic mechanisms underlying sarcomatoid transformation of hepatocellular carcinoma (HCC) remain unknown.Case presentationIn this study, we report a 68-year-old female SHC patient admitted to our hospital due to discrete abdominal distention for more than 20 days. Abdominal computed tomography (CT) with tri-phase enhancement revealed portal vein tumor thrombi (PVTT) and a left hepatic lobe lesion measuring 110.0 mm × 160.0 mm. The patient subsequently underwent liver resection, after which pathological examination revealed proliferation of spindle-shaped SHC cells. A sarcomatoid, T4 stage carcinoma was eventually diagnosed. Forty-seven days after the operation, tri-phase enhanced CT detected extensive lesions in the liver, spleen, peritoneum, omentum majus, and mesentery, indicating SHC recurrence and metastases. Combination chemotherapy with pirarubicin and cisplatin was initiated for 1 cycle, but terminated due to resultant severe myelosuppression and medication intolerance. The patient was lost to therapy after 3 months of follow-up.ConclusionsThis case is unique because of hepatitis C virus infection. We should consider the possibility of this disease in patients with atypical clinical presentation.

Multifocal β-catenin activated hepatocellular adenomas (HCA) with malignant transformation in hepatocellular carcinoma (HCC) after abuse of anabolic steroids

Multifocal β-catenin activated hepatocellular adenomas (HCA) with malignant transformation in hepatocellular carcinoma (HCC) after abuse of anabolic steroids

Molecular classification of hepatocellular adenomas.

Hepatocellular adenomas (HCAs) are benign tumors developed in normal liver most frequently in women before menopause. HCAs lead to diagnostic pitfalls and several difficulties to assess the risk of malignant transformation in these young patients. Recent advances in basic knowledge have revealed a molecular classification related to risk factors, pathological features, and risk of transformation in hepatocellular carcinoma. Three major molecular pathways have been identified altered in specific HCA subgroups that are defined by either (1) inactivation of hepatocyte nuclear factor 1A (HNF1A) transcription factor, (2) activation of the WNT/β-catenin by CTNNB1 mutations, or (3) activation of the IL6/STAT3 pathway by somatic mutation of IL6ST, GNAS, or STAT3. Here, we will review the different molecular classes of HCA.

Molecular characterization of hepatocellular adenomas developed in patients with glycogen storage disease type I

Hepatocellular adenomas (HCA) are benign liver tumors mainly related to oral contraception and classified into 4 molecular subgroups: inflammatory (IHCA), HNF1A-inactivated (H-HCA), β-catenin-activated (bHCA) or unclassified (UHCA). Glycogen storage disease type I (GSD) is a rare hereditary metabolic disease that predisposes to HCA development. The aim of our study was to characterize the molecular profile of GSD-associated HCA. We characterized a series of 25 HCAs developed in 15 patients with GSD by gene expression and DNA sequence of HNF1A, CTNNB1, IL6ST, GNAS, and STAT3 genes. Moreover, we searched for glycolysis, gluconeogenesis, and fatty acid synthesis alterations in GSD non-tumor livers and compared our results to those observed in a series of sporadic H-HCA and various non-GSD liver samples. GSD adenomas were classified as IHCA (52%) mutated for IL6ST or GNAS, bHCA (28%) or UHCA (20%). In contrast, no HNF1A inactivation was observed, showing a different molecular subtype distribution in GSD-associated HCA from that observed in sporadic HCA (p = 0.0008). In non-tumor GSD liver samples, we identified glycolysis and fatty acid synthesis activation with gluconeogenesis repression. Interestingly, this gene expression profile was similar to that observed in sporadic H-HCA. Our study showed a particular molecular profile in GSD-related HCA characterized by a lack of HNF1A inactivation. This exclusion could be explained by similar metabolic defects observed with HNF1A inactivation and glucose-6-phosphatase deficiency. Inversely, the high frequency of β-catenin mutations could be related to the increased frequency of malignant transformation in hepatocellular carcinoma.

Role of the Hypoxia-Related Gene, JMJD1A, in Hepatocellular Carcinoma: Clinical Impact on Recurrence after Hepatic Resection

Intratumoral hypoxia affects every major aspect of cancer biology, but the relationship between hypoxia-induced genes and hepatocellular carcinoma has not been fully investigated. From a previously ranked microarray of hypoxia-inducible genes related to hepatocellular carcinoma, we focused on a histone H3 lysine 9 demethylase, known as Jumonji domain containing 1A. One function of this demethylase is to amplify hypoxia-inducible gene expression. We hypothesized that the demethylase would be a significant marker of hepatocellular carcinoma. We examined Jumonji domain containing 1A expression in 110 hepatocellular carcinoma samples with quantitative real-time polymerase chain reaction and immunohistochemistry. We performed a small interfering RNA suppression analysis to determine the biological roles of the demethylase in proliferation, invasion, and the expression of epithelial-mesenchymal transition-related genes. The level of Jumonji domain containing 1A in cancer tissues was higher than in normal tissues (P < 0.0001). Protein expression was significantly related to gene expression (P < 0.0001). Samples with high Jumonji domain containing 1A expression (n = 47) had higher recurrence rates (P = 0.0006) than those with low expression. Multivariate Cox regression analysis revealed that Jumonji domain containing 1A expression was an independent predictor of recurrence (P = 0.0016), but was not significantly associated with any clinicopathological characteristics. Moreover, suppression of Jumonji domain containing 1A expression in hepatocellular carcinoma cell lines under hypoxic conditions reduced cell growth inhibition, reduced invasion ability, and arrested epithelial-mesenchymal transitions. Jumonji domain containing 1A is a useful prognostic marker and may ameliorate malignant transformation in hepatocellular carcinoma.

Revisiting the Pathology of Resected Benign Hepatocellular Nodules Using New Immunohistochemical Markers

In this review, the authors focus on the use of immunohistochemistry to characterize the different types and subtypes of benign hepatocellular nodules. They describe the classical and currently accepted features leading to the easy and formal diagnosis of focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA). In addition, they report some atypical features and difficulties in the interpretation of section staining analyses, which represent important parameters for pathologists. A significant contribution of molecular biology to the characterization of FNH has been to reclassify some cases of FNH as inflammatory HCA. Furthermore, the pattern of overexpression of glutamine synthetase (GS), a target gene of β-catenin has been successfully used to identify FNH by immunohistochemistry. Molecular approaches have demonstrated that HCA is a heterogeneous entity. Genotype classification of HCA has allowed the identification of three subtypes: HNF1A-mutated HCA (H-HCA) in 35% of cases, β-catenin-mutated HCA (b-HCA) in 10%, and inflammatory HCA (IHCA) in 55%. Following molecular data, the diagnosis of H-HCA relies on the lack of liver fatty acid binding protein (LFABP) immunostaining. The diagnosis of b-HCA is straightforward when GS is strongly and diffusely expressed by lesional hepatocytes, and is accompanied by nuclear β-catenin immunoreactivity. In IHCA, serum amyloid protein and C- reactive protein are strongly and usually diffusely expressed by tumoral hepatocytes with a sharp limit with the surrounding nontumoral liver. IHCA can also be β-catenin activated (10%). Due to the strong association of b-HCA with hepatocellular carcinoma transformation, the identification of this HCA subtype is extremely important.

Decreased Expression of UK114 Is Related to the Differentiation Status of Human Hepatocellular Carcinoma

Previous studies have identified that the expression of UK114 is tissue specific and the protein has been found to be most abundant in liver and kidney. However, the expression of UK114 in human hepatocellular carcinoma and its relationship to differentiation and transformation of hepatocellular carcinoma have not been studied. In this study, the expression of UK114 in human hepatocellular carcinoma was examined by Northern and Western blot analyses. We found that UK114 was significantly down-regulated in most of hepatocellular carcinoma tissues compared with adjacent nontumor tissues (72.7%) at both mRNA and protein levels. We looked into the possibility that this decreased expression of UK114 in the hepatocellular carcinoma tissues may play a role in the differentiation or tumorigenicity of hepatocellular carcinoma. Immunohistochemical staining showed that the reduced expression of UK114 in hepatocellular carcinoma tissues was correlated with the tumor differentiation status as graded by the Edmondson-Steiner classification. On the other hand, overexpression of UK114 was not able to suppress the proliferation of human hepatoma cells and tumorigenicity in nude mice. These results suggest that UK114 does not seem to act as a tumor suppressor gene; however, it may useful as a biomarker that will assist in the grading of the differentiation status of hepatocellular carcinoma samples.

Could the cytokeratin molecule be modulated during tumor transformation in hepatocellular carcinoma?

The stability of cytokeratin during tumor transformation in hepatocellular carcinoma was studied. We applied biochemical methodology to look into the switching of cytokeratin molecules in tumor transformation. First, by centrifugation the cytokeratin molecules were extracted from both liver and hepatoma tissues. The extracts were then soaked with cyanogen bromide-activated Sepharose 4B beads previously coated by monoclonal anti-cytokeratin antibody. The bound molecules were then released from the resin with salt. Second, the isolated molecules of both were treated with lysosomal enzyme and analyzed on two-dimensional gels. The results demonstrated that there was a modulation in cytokeratin molecules, and the hepatoma cytokeratin was generated from the hepatocyte cytokeratin.