Human HCC Tissues Research Articles

Up-regulation and subcellular localization of hnRNP A2/B1 in the development of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the world's leading causes of death among cancer patients. It is important to find a new biomarker that diagnoses HCC and monitors its treatment. In our previous work, we screened a single-chain antibody (scFv) N14, which could specifically recognize human HepG2 HCC cells but not human non-cancerous liver LO2 cells. However, the antigen it recognized in the cells remained unknown.MethodsRecombinant scFv N14 antibody was expressed as an active antibody. Using this antibody with a combination of immunological and proteomic approaches, we identified the antigen of scFv N14 antibody as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). The expression of hnRNP A2/B1 in HCC cells was then investigated by semi-quantitative RT-PCR and immunohistochemistry.ResultsWe found that the up-regulation of hnRNP A2/B1 was measured at both transcriptional and translational levels in rat HCC cells but not in rat hepatic cells. We also found that in various human hepatic tissues, hnRNP A2/B1 was highly expressed in both human hepatitis virus positive liver tissues and human HCC tissues but not in normal liver tissues. Interestingly, we observed that the localization of hnRNP A2/B1 in HCC cells was altered during the development of HCC. In human hepatitis virus infected tissues hnRNP A2/B1 resides exclusively in the nuclei of hepatocytes. However, when the HCC progressed from a well differentiated to a poorly differentiated stage, hnRNP A2/B1 was increasingly localized in the cytoplasm. In contrast, the HCC tissues with hnRNP A2/B1 highly expressed in the nucleus decreased.ConclusionsThis work is the first to show that hnRNP A2/B1 is the antigen specifically recognized by the scFv N14 antibody in HCC cells. The over-expression of hnRNP A2/B1 was confirmed in cultured human and rat HCC cell lines, human virus related hepatitis liver tissues and human HCC tissues. The increased localization of hnRNP A2/B1 in the cytoplasm of HCC cells was revealed during the dedifferentiation of hepatocellular carcinoma. Therefore, we suggest that the increased expression and cytoplasmic localization of hnRNP A2/B1 can be used as a diagnostic biomarker to assess the risk of human liver cancer.

Abstract 4067: A T-cell factor-4 motif regulates the phenotype of hepatocellular carcinoma cells

Abstract T-cell factor (TCF) family proteins play a crucial role in regulating the activation of Wnt/β-catenin responsive target genes. Among the family proteins, TCF-4 is predominantly expressed in human HCC tissues. We have recently cloned 14 TCF-4 isoforms from human hepatoma cell lines, and naming them in alphabetical order from A to M. A TCF-4J and K pair has been identified based on the presence (K) or absence (J) of the SxxSS motif at the beginning of exon 9. This motif may modulate transcriptional activity of TCF-4 through phosphorylation/dephosphorylation of serine residues (Pukrop et al., Cancer Res 2001). However, cell-based functional analysis of this motif has yet to be performed. The AIM of this study was to investigate whether TCF-4J and K have distinct cellular phenotypes due to SxxSS motif expression. Methods: The human hepatoma cell line Huh-7 was used in this study. The cells were transfected with TCF-4J and K expression plasmids, and protein levels in cell lysates and nuclear extracts were examined by Western blot analysis. TCF-4K mutants with substitution of serine (S) to alanine (A) were generated by site-directed mutagenesis. Results: Protein expression of all cloned TCF-4 isoforms was confirmed by Western blot analysis. Expression levels of cyclin D1, which is a direct target of TCF-4, was increased in both cell lysates and the nuclear extracts of the TCF-4K, but not in TCF-4J-transfected cells. The TCF-4K isoform with a S269A mutation revealed a decrease in the expression level of cyclin D1, while the mutant with either a S272A or S273A substitution did not, which suggests that phosphorylation of serine 269 was critical to up-regulating cyclin D1 gene expression. Conclusion: The findings from functional analysis following mutagenesis of the SxxSS motif suggest that serine phosphorylation of this motif resulted in the altered target genes expression, including cyclin D1. Therefore, TCF-4 isoforms regulate the phenotype of HCC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4067.

Blockade of Wnt-1 signaling leads to anti-tumor effects in hepatocellular carcinoma cells

BackgroundHepatocellular carcinoma (HCC) is an aggressive cancer, and is the third leading cause of cancer death worldwide. Standard therapy is ineffective partly because HCC is intrinsically resistant to conventional chemotherapy. Its poor prognosis and limited treatment options make it critical to develop novel and selective chemotherapeutic agents. Since the Wnt/β-catenin pathway is essential in HCC carcinogenesis, we studied the inhibition of Wnt-1-mediated signaling as a potential molecular target in HCC.ResultsWe demonstrated that Wnt-1 is highly expressed in human hepatoma cell lines and a subgroup of human HCC tissues compared to paired adjacent non-tumor tissues. An anti-Wnt-1 antibody dose-dependently decreased viability and proliferation of Huh7 and Hep40 cells over-expressing Wnt-1 and harboring wild type β-catenin, but did not affect normal hepatocytes with undetectable Wnt-1 expression. Apoptosis was also observed in Huh7 and Hep40 cells after treatment with anti-Wnt-1 antibody. In these two cell lines, the anti-Wnt-1 antibody decreased β-catenin/Tcf4 transcriptional activities, which were associated with down-regulation of the endogenous β-catenin/Tcf4 target genes c-Myc, cyclin D1, and survivin. Intratumoral injection of anti-Wnt-1 antibody suppressed in vivo tumor growth in a Huh7 xenograft model, which was also associated with apoptosis and reduced c-Myc, cyclin D1, and survivin expressions.ConclusionOur results suggest that Wnt-1 is a survival factor for HCC cells, and that the blockade of Wnt-1-mediated signaling may offer a potential pathway-specific therapeutic strategy for the treatment of a subgroup of HCC that over-expresses Wnt-1.

Expression and biological significance of c-FLIP in human hepatocellular carcinomas

Backgroundc-FLIP can be considered as a tumor-progression factor in regard to its anti-apoptotic functions. In the present study, we intended to investigate the expression of c-FLIP in human HCC tissues, and its relation with drug-induced cell apoptosis through the specific inhibition of c-FLIP expression by siRNA in 7721 cells.Methodsc-FLIP expression was quantified immunohistochemically in HCC tissues(eighty-six cases), and corresponding noncancerous tissues (fifty-seven cases). Patients with HCC were followed up for cancer recurrence. Then, the c-FLIP gene was silenced with specific siRNA in 7721 HCC cells. c-FLIP expression was detected by RT-PCR, Western Blot and immunocytochemical staining. The cellular viability and cell apoptosis were assayed in vitro with cells treated with doxorubicin.ResultsPositive immunostaining was detected for c-FLIP in 83.72% (72/86) human HCC tissues, 14.81% (4/27) hepatic cirrhosis, 11.11% (2/18) hepatic hemangioma tissues, and absent in normal hepatic tissues. The overexpression(more than 50%) of c-FLIP in HCC adversely affected the recurrence-free survival. Through c-FLIP gene silencing with siRNA, the expressions of c-FLIP mRNA and protein were remarkably down-regulated in 7721 HCC cells. And doxorubicin showed apparent inhibition on cell proliferations, and induced more apoptosis.ConclusionThese results indicate that c-FLIP is frequently expressed in human HCCs, and its overexpression implied a lesser probability of recurrence-free survival. The specific silencing of c-FLIP gene can apparently up-regulate drug-induced HCC cell apoptosis, and may have therapeutic potential for the treatment of human HCC.

800 TGF-β Pathway Members ELF and SMAD3 Are Key Tumor Suppressors in Human Beckwith-Wiedemann Syndrome

The Smad3/4 adaptor protein ELF is emerging as a potent regulator of tumorigenesis by its ability to affect TGF-β tumor suppressor function (Science, 2003, 299:574-577, Science, 2005, 310:68-71). However, to date, the role of the TGF-β pathway at specific stages in gastrointestinal (GI) tumor development such as metaplasia, dysplasia and carcinoma remains unclear. A surprising and dramatic discovery by us is that elf+/and elf+/-/Smad3+/mice develop multiple GI cancers, with a phenotype strongly suggestive of Beckwith Wiedemann syndrome (BWS), a hereditary human cancer syndrome with no known causal mutations, and associated with loss of imprinting (LOI) at multiple loci on chromosome 11p15 including IGF2. Methods and Results: 1. Elf+/and Elf+/-/Smad3+/mice develop visceromegaly, multiple liver lobes, abnormal ear folds, and 70% of the mice develop multiple tumors that include metastatic pancreatic carcinoma, hepatocellular carcinoma, and small bowl adenocarcinoma. 2. A dramatic decrease in elf mRNA (200-2000 fold) was observed in elf+/-/Smad3+/tumor tissues without significant decrease of Smad3 or p53 compared to normal tissues. 3.We observed a loss of the 200 Kda ELF protein expression 4 out of 11 human cancer cell lines (HCC, gastric and pancreatic) by Western Blotting and in 19/20 human HCC and gastric cancer tissues by IHC labeling using a N-terminal peptide specific anti-ELF Ab. 4.We identified a missense mutation Arg 928 to Ile (G→A) in elf exon15 in 2 of the human GI cancer cell lines with loss of ELF expression. 5. Importantly we observed a loss of “C” to “U” RNA editing at elf Exon 15 in GI cancer cell lines with loss of ELF expression. 6. All 4/4 Human BWS patient tissues displayed a marked decrease in ELF RNA (by 50%), and a complete loss of ELF protein expression (western blot and IHC). Smad3 RNA and protein expression were not significantly altered. 7. In all 4 BWS tissues, we observed a loss of “C” to “U” RNA editing at elf Exon 15 with loss of ELF expression. 8 Array, proteomic analyses and IHC revealed a markedly high expression (about 4-6 fold) of IGF2, β-catenin, CDK4, and hTERT in Elf+/and Elf+/-/Smad3+/tissues. Conclusions: 1.The TGF-β pathway through elf+/and elf+/-/Smad3+/phenotype provides a novel and strongly compelling model for Beckwith-Wiedemann (BWS) a human cancer overgrowth syndrome. 2. Alterations of ELF/ SMAD3 expression at the mRNA level are the major genetic disposition of human BWS. 3. In BWS patient samples, aberrant elf RNA editing may be a predominant mechanism for loss of ELF protein expression observed in BWS.

Expression profiling reveals dysregulation of cellular cytoskeletal genes in HBx–induced hepatocarcinogenesis

The molecular mechanisms underlying hepatitis B virus encoded HBx protein -mediated tumorigenesis are not fully understood. In order to gain a better view of the effects of HBx on transcriptional regulation and hepatocarcinogenesis, the expression profiles of liver and tumor tissues from 6 and 18 month-old p21-HBx transgenic and control mice were monitored using oligo microarrays. Data analysis demonstrated that 42 genes were de-regulated in both 6 and 18 month-old HBx transgenic mouse tissues. Gene ontology assisted analysis classified these genes into functionally related clusters that encode proteins related to metabolism, signal transduction, transcription regulation and stress responses. Among them, cytoskeletal genes, including microtubule genes tubulin-β2 (Tubb2), tubulin-β3 (Tubb3) and tubulin-β6 (Tubb6) , intermediate filament genes periplakin, keratin 8 (K8) and keratin 18 (K18) and actin-γ1 (Actg1) , were closely clustered and up-regulated in liver tissues. These results were validated by semi-quantitative RT-PCR in both mouse and human HCC tissues. The up-regulation of K8 and K18 was only detected in p21-HBx but not p21-HBsAg liver tissues, suggesting that the global change in the expression of cellular cytoskeletal genes was correlated with the expression of HBx transgene. These findings propose for the first time that systemic dysregulation of cellular cytoskeletal genes is involved in HBx–induced hepatocarcinogenesis.

Decreased hepatocyte membrane potential differences and GABAa-β3 expression in human hepatocellular carcinoma

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and gamma-aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (-19.8+/-1.3 versus -25.9+/-3.2 mV, respectively [P<0.05]), and GABAA-beta3 expression was down-regulated (GABAA-beta3 mRNA and protein expression in HCC; 5,693+/-1,385 and 0.29+/-0.11 versus 11,046+/-4,979 copies/100 mg RNA and 0.62+/-0.16 optical density in adjacent tumor tissues, respectively [P=0.002 and P<0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-beta3 receptor expression (Huh-7 cells) were transfected with GABAA-beta3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n=16-17 group). Tumors developed after a mean (+/-SD) of 51+/-6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70+/-12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-beta3 cDNA-transfected cells (P<0.005). The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-beta3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-beta3 receptor expression results in attenuated in vivo tumor growth in nude mice.

Chemokine CCL20 enhances the growth of HuH7 cells via phosphorylation of p44/42 MAPK in vitro

CCR6 is the receptor of chemokine CCL20. In the present study, we demonstrated that the surface expression of CCR6 was enhanced on the human HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially on HuH7 cells, but not on HLE or HLF cells. These HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially the HuH7 cells secreted a significant amount of CCL20 spontaneously, whereas HLE or HLF did not. Stimulation by CCL20 up-regulated the mRNA expression of CCR6 in HuH7 cells and significantly enhanced the growth of HuH7 cells. CCL20-stimulated growth of HuH7 cells was abrogated by the inhibition of downstream signal transduction pathway mediated by p44/42 MAPK, but not by p38 MAPK or SAPK/JNK. CCR6 expression in human HCC tissues was confirmed by RT-PCR. These results indicate that the growth of a proportion of human HCC cells may be mediated by CCL20–CCR6 axis, like HuH7 cells, in an autocrine or paracrine manner.

Identification of genes differentially expressed in human hepatocellular carcinoma by a modified suppression subtractive hybridization method.

To identify differentially expressed genes in human HCC in China, we applied a modified SSH method for cDNA subtraction. Such modification has made the method more effective for subtraction. We have obtained 36 and 24 differentially expressed cDNA fragments after modified SSH from 4 paired samples of human HCC and non-HCC tissues, respectively. Reverse Northern blotting analysis was performed to further identify the genes differentially expressed in the HCC and non-HCC tissue samples. There were 25 genes really overexpressed in HCC, and their corresponding encoding molecules may reflect the events of cell accelerated metabolism, proliferation, angiogenesis, anti-apoptosis, tumorigenesis (TLH107, TFH9) and the potential for metastasis. Of the 25 genes overexpressed in HCC, 5 were novel and their full-length cDNAs were cloned. These 5 novel genes are functionally associated with the occurrence and development of HCC according to the Database analysis. In the paired non-HCC tissues, there were 15 genes lowly or not expressed in HCC, and their encoding proteins function as tumor suppressors (TFA3, TFA11), acute-phase reactive proteins, and the blood plasma proteins that are mainly or exclusively synthesized in the liver. The distinct profiles of the differentially expressed genes in HCC and the paired non-HCC tissues have partially reflected the genetic alterations during HCC tumorigenesis. The novel HCC-specific gene TLH6 and the CT antigen encoding gene TLH107 may have diagnostic and therapeutic potentials in HCC and/or other solid cancers.

Structure analysis and expressions of a novel tetratransmembrane protein, lysosoma-associated protein transmembrane 4 beta associated with hepatocellular carcinoma.

To analyze the structure and expressions of the protein encoded by an HCC-associated novel gene, lysosome-associated protein transmembrane 4 beta (LAPTM4B). Primary structure and fundamental characteristics of LAPTM4B protein were analysed with bioinformatics. Expressions of LAPTM4B in HCC tissues and various cell lines were detected using polyclonal antibodies and Western blot. LAPTM4B encoded two isoforms of proteins with molecular masses 35-ku and 24-ku, respectively. The expression level of LAPTM4B-35 protein in HCC tissues was dramatically upregulated and related to the differentiation status of HCC tissues, and it was also high in some cancer cell lines. Computer analysis showed LAPTM4B was an integral membrane protein with four transmembrane domains. LAPTM4B showed relatively high homology to LAPTM4A and LAPTM5 in various species. LAPTM4B gene encoded two isoforms of tetratransmembrane proteins, LAPTM4B-35 and LAPTM4B-24. The expression of LAPTM4B-35 protein is upregulated and associated with poor differentiation in human HCC tissues, and also at high levels in some cancer cell lines. LAPTM4B is an original and conserved protein.

Cloning and expression of MXR7 gene in human HCC tissue.

AIM:To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS:The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using (32)P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS:Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively. The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC <5cm was 90% (9/10) and 83.3% (5/6), respectively.CONCLUSION:MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.