Adjacent Non-tumor Liver Tissues Research Articles

Dickkopf-1: As a Diagnostic and Prognostic Serum Marker for Early Hepatocellular Carcinoma

The aim of the present study was to evaluate serum Dickkopf-1 (Dkk-1) as a marker for early detection of hepatocellular carcinoma (HCC), as well as for prognostic prediction of early HCC after hepatic resection. One-hundred and four cases of matched fresh tissue specimens of early HCC and adjacent non-tumorous liver tissue (ANLT) were obtained for RT-PCR, qRT-PCR, western blot and immunohistochemistry assays. Sera were collected from patients with early HCC (n=184), benign liver tumors (n=29), cirrhosis (n=174), non-cirrhotic hepatitis B (n=193), and from healthy individuals (n=202). The levels of Dkk-1 and alpha fetoprotein (AFP) were measured. The Dkk-1 mRNA and protein levels were both upregulated in early HCC. Serum levels of Dkk-1 in patients with early HCC were significantly higher than in the other 4 groups (p<0.001). Dkk-1 had a better sensitivity and accuracy than AFP (p<0.05). More importantly, 73.1% of the patients negative for AFP could be diagnosed with early HCC using Dkk-1. A combination of Dkk-1 and AFP further improved the diagnostic efficacy. Patients with a high serum Dkk-1 level had poorer overall and relapse-free survivals than those with a low Dkk-1 level (p=0.028 and p=0.045, respectively). These results were shown in a testing cohort and confirmed in a validation cohort of patients. Univariable and multivariable Cox regression analyses showed serum Dkk-1 level to be an independent prognostic factor for overall survival. Our data show that Dkk-1 is a diagnostic and prognostic serologic marker for early HCC.

Mitogenic Insulin Receptor-A Is Overexpressed in Human Hepatocellular Carcinoma due to EGFR-Mediated Dysregulation of RNA Splicing Factors

Insulin receptor (IR) exists as two isoforms resulting from the alternative splicing of IR pre-mRNA. IR-B promotes the metabolic effects of insulin, whereas IR-A rather signals proliferative effects. IR-B is predominantly expressed in the adult liver. Here, we show that the alternative splicing of IR pre-mRNA is dysregulated in a panel of 85 human hepatocellular carcinoma (HCC) while being normal in adjacent nontumor liver tissue. An IR-B to IR-A switch is frequently observed in HCC tumors regardless of tumor etiology. Using pharmacologic and siRNA approaches, we show that the autocrine or paracrine activation of the EGF receptor (EGFR)/mitogen-activated protein/extracellular signal-regulated kinase pathway increases the IR-A:IR-B ratio in HCC cell lines, but not in normal hepatocytes, by upregulating the expression of the splicing factors CUGBP1, hnRNPH, hnRNPA1, hnRNPA2B1, and SF2/ASF. In HCC tumors, there is a significant correlation between the expression of IR-A and that of splicing factors. Dysregulation of IR pre-mRNA splicing was confirmed in a chemically induced model of HCC in rat but not in regenerating livers after partial hepatectomy. This study identifies a mechanism responsible for the generation of mitogenic IR-A and provides a novel interplay between IR and EGFR pathways in HCC. Increased expression of IR-A during neoplastic transformation of hepatocytes could mediate some of the adverse effects of hyperinsulinemia on HCC.

Prediction of Disease-free Survival in Hepatocellular Carcinoma by Gene Expression Profiling

Progression of hepatocellular carcinoma (HCC) often leads to vascular invasion and intrahepatic metastasis, which correlate with recurrence after surgical treatment and poor prognosis. The molecular prognostic model that could be applied to the HCC patient population in general is needed for effectively predicting disease-free survival (DFS). A cohort of 286 HCC patients from South Korea and a second cohort of 83 patients from Hong Kong, China, were used as training and validation sets, respectively. RNA extracted from both tumor and adjacent nontumor liver tissues was subjected to microarray gene expression profiling. DFS was the primary clinical end point. Gradient lasso algorithm was used to build prognostic signatures. High-quality gene expression profiles were obtained from 240 tumors and 193 adjacent nontumor liver tissues from the training set. Sets of 30 and 23 gene-based DFS signatures were developed from gene expression profiles of tumor and adjacent nontumor liver, respectively. DFS gene signature of tumor was significantly associated with DFS in an independent validation set of 83 tumors (P = 0.002). DFS gene signature of nontumor liver was not significantly associated with DFS in the validation set (P = 0.827). Multivariate analysis in the validation set showed that DFS gene signature of tumor was an independent predictor of shorter DFS (P = 0.018). We developed and validated survival gene signatures of tumor to successfully predict the length of DFS in HCC patients after surgical resection.

SiRNA‐targeted carboxypeptidase D inhibits hepatocellular carcinoma growth

Carboxypeptidase D (CPD), a membrane-bound metallocarboxypeptidase that functions as a docking receptor for duck hepatitis B virus, is frequently overexpressed in human cancers. We have explored its expression pattern, clinical significance, and biological function of CPD in hepatocellular carcinoma (HCC). CPD expression was markedly elevated in HCCs relative to adjacent non-tumor liver tissues, as determined by quantitative real-time polymerase chain reaction and Western blot analysis. Immunohistochemistry showed that 164 of 400 (41%) HCCs had high expression of CPD. CPD overexpression was significantly associated with serum levels of hepatitis B surface antigen and hepatitis B e antigen, liver cirrhosis, pathological grade, and intrahepatic metastasis. Knockdown of endogenous CPD expression in Huh7 HCC cells by RNA interference reduced cell proliferation, blocked the cell cycle at G1 phase, and increased apoptosis. Many genes implicated in cell-cycle regulation, including P21waf1, P27 Kip1, SKP2, and CDC2, were deregulated by CPD downregulation. Thus CPD is frequently upregulated in HCC, and targeting CPD inhibits HCC cell proliferation through induction of G1 cell-cycle arrest and apoptosis, thereby providing a potential therapeutic target for this malignancy.

Cytohesin-2 as a novel prognostic marker for hepatocellular carcinoma

Cytohesin-2 is overexpressed in human lung cancer and it activates cytoplasmic ErbB receptors. Inhibition of cytohesin-2 by SecinH3 reduces growth of EGFR-dependent lung cancer xenografts and improves the treatment of primarily EGFR-TKI-resistant lung cancers. Cytohesin-2 promotes HepG2 proliferation through the IGF pathway, and VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, vessel permeability and ultimately endothelial proliferation. The purpose of this study was to evaluate the effects of cytohesin-2 in hepatocellular carcinoma (HCC). In the current study, we collected 40 HCC tissues and detected cytohesin-2 mRNA expression in the 40 HCC tissues by using quantitative real-time polymerase chain reaction (qRT-PCR), as well as its protein expression by using immunohistochemistry and western blot analysis. We found that cytohesin-2 was more highly expressed in HCC compared to adjacent non-tumorous liver tissues, and cytohesin-2 expression was significantly increased in specimens with high α-fetoprotein and vascular invasion. Both univariate and multivariate analyses indicated that there is an association between cytohesin-2 expression and overall survival (OS) and disease-free survival (DFS). Moreover, stratified analysis showed that patients in tumor-node-metastasis (TNM) stage I with higher cytohesin-2 levels had shorter OS and DFS than those with lower cytohesin-2 levels. In conclusion, cytohesin-2 may identify low-and high-risk individuals with HCC and may be a valuable indicator for stratifying prognosis of TNM stage I patients. Cytohesin-2 may serve as a novel prognostic biomarker for HCC.

Involvement of microRNA‐224 in cell proliferation, migration, invasion, and anti‐apoptosis in hepatocellular carcinoma

Changes in microRNA (miRNA) expression have been detected in a broad range of biological processes including cancer. Here we determined the role of miRNA dysregulation in hepatocellular carcinoma (HCC). We investigated the expression of nine cancer-related miRNAs in HCC. Among these, miR-224 was the most significantly uprgulated in HCC tissues (n = 18), compared with normal (n = 9) and HCC adjacent non-tumorous liver tissues (n = 18). After leading-in currently reported gene targets from Sanger miRBase, we characterized the expression profiles of target genes of miR-224 using cDNA microarray. The altered expression was subsequently validated by real-time polymerase chain reaction and Western blot. The phenotypic changes by miR-224 expression were identified by cell viability, apoptosis, and in vitro scratch assays. The microarray analysis and miRNA target prediction analysis allowed the identification of significant changes in 68 putative gene targets after overexpression of miR-224. The high-ranking genes CDC42, CDH1, PAK2, BCL-2, and MAPK1 were confirmed as important targets of miR-224 and involvement in hepatocarcinogenesis. Overexpression of miR-224 significantly in Hek293 and Huh7 cells altered the expression levels of CDC42, CDH1, PAK2, and BCL-2 at both mRNA and protein levels. Similar changes in the expression of the same genes were also observed in HCC tissues. Via functional analyses, cell proliferation, migration and anti-apoptosis were proved to be affected by miR-224 expression. The results suggest that miR-224 plays a role in cell proliferation, migration, invasion, and anti-apoptosis in HCC by directly binding to its gene targets, implicating this RNA in HCC development and progression.

Differential Expression of Wnt Pathway Genes in Sporadic Hepatocellular Carcinomas Infected With Hepatitis B Virus Identified With OligoGE Arrays

BackgroundEpidemiological evidence has clearly indicated that chronic infection with the hepatitis B virus (HBV) is the major risk factor for developing hepatocellular carcinoma (HCC). Nonetheless, the mechanisms by which HBV contributes to the pathogenesis of HCC have not been fully elucidated.ObjectivesOur aim was to characterize differential gene expression profiles related to the Wnt signaling pathway between primary tumor and adjacent normal tissues in HCC patients with concomitant HBVinfection .Materials and MethodsAn oligoGEArray® (an oligonucleotide-based gene expression array platform) containing 126 Wnt signaling pathway-related genes was used to compare gene expressions between primary HCC and adjacent non-tumorous liver tissues from 10 patients with HCC. Selected differential genes were identified with real-time RT-PCR and immunohistochemistry (IHC). In particular, the protein of the differential gene DVL3 (disheveled, dsh homolog 3 [Drosophila]) was chosen to investigate whether it is up regulated in primary tumor correlated with the clinic pathological characteristics of HCC patients. For this purpose we examined 56 HCC tissue samples via IHC for the presence of DVL3 protein.ResultsSixteen genes were identified with significant differential expression between HCC and adjacent non-tumorous liver tissue. These genes have been previously associated with the Frizzled signaling pathway, cell cycle, transcription, or protein degradation. All (100%) of the tumor samples results from 56 HCC patients tested were positive for DVL3 via IHC. Based on the intensity of DVL3 immunoreactivity, 25 (44.6%) and 31 (55.4%) of the patients were classified aslow and high-DVL3, respectively, which correlated with tumor stage (P = 0.029).ConclusionsThis study clarified a number of Wnt pathway-related genes which are dysregulated in HBV-associated HCC. These genes may be contributedto the frequent activation of the Wnt signaling pathway. Our results promote the role of the Wnt signaling pathway in HBV-associated HCC.

The clinical and prognostic implications of pluripotent stem cell gene expression in hepatocellular carcinoma

Recently, growing evidence has demonstrated that aberrant expression of pluripotent stem cell-related genes may confer primitive and aggressive traits and be associated with unfavorable clinical outcomes in certain solid cancers. However, the role of pluripotent stem cell gene expression in hepatocellular carcinoma (HCC) remains unexplored. We evaluated the expression of the pluri potent stem cell genes Oct4, Sox2 and Klf4, as well as that of the c-Myc, Nanog and Lin28 genes in HCC samples and corresponding adjacent non-tumor liver samples obtained from 57 patients using quantitative real-time reverse transcription-PCR (qRT-PCR). The results revealed that six pluripotent stem cell gene expression levels were upregulated in the tumor tissues compared with the corresponding adjacent non-tumor liver tissues. In HCC tissues, aberrant expression of Sox2 and Lin28 was associated with a large tumor size (P=0.02 and P=0.03, respectively), while increased expression levels of c-Myc (P=0.01) were correlated with vascular invasion. Moreover, high Klf4 expression levels were associated with aggressive tumor behaviors in terms of vascular invasion (P=0.02) and poor tumor differentiation (P=0.03). Survival analysis revealed that Klf4 expression was independently associated with overall survival [OS; hazard ratio (HR), 8.61; 95% confidential interval (CI), 2.7–27.5; P<0.001] and recurrence-free survival (RFS; HR, 3.96; 95% CI, 1.3–11.6; P=0.01). In conclusion, pluripotent stem cell genes are associated with HCC progression and a poor prognosis. The development of therapeutic strategies, including adjuvant therapy, that take cancer stem cell (CSC)-related markers into consideration is likely to be a key factor in further improvements of the prognosis of HCC patients undergoing curative liver resection.

Oxidative stress-induced 1, N6-ethenodeoxyadenosine adduct formation contributes to hepatocarcinogenesis

Numerous studies have found that oxidative stress-derived 1, N6-ethenodeoxyadenosine (ɛ-dA) can act as a driving force towards hepatocellular carcinoma (HCC) in cancer-prone liver diseases. The aim of the present study was to determine the oxidative stress status and the occurrence of ɛ-dA in HCC and adjacent non-tumor liver tissue, and to clarify whether the occurrence of ɛ-dA is related to liver inflammatory activity, fibrosis and mutant p53 expression. Oxidative stress-related parameters were examined in tumor and (or) non-tumor liver tissues of 32 patients with HCC. ɛ-dA, mutant p53 and proliferating cell nuclear antigen (PCNA) were immunohistochemically investigated in control, HCC and non-tumor liver tissues. The total antioxidant capacity and total superoxide dismutase activity of HCC tissues were lower compared to those of non-tumor tissues (P<0.05 vs. P<0.001). The prevalence of ɛ-dA in HCC was significantly higher compared to control (P<0.0001) and non-tumor liver tissues (P<0.001). A significant correlation between the positive rate of ɛ-dA and mutant p53 was observed (r=0.5162, P<0.01). The positive rate of PCNA in HCC was significantly higher compared to control (P<0.0001) and non-tumor liver tissues (P<0.0001). There was a possible link between the formation of ɛ-dA and chronic inflammation and fibrosis. Therefore, ɛ-dA lesions may gradually accumulate in chronic liver diseases, and partially contribute to mutant p53 overexpression and excessive cell proliferation, making it a potential mechanism in oxidative stress-mediated hepatocarcinogenesis.

MicroRNA-29a-5p Is a Novel Predictor for Early Recurrence of Hepatitis B Virus-Related Hepatocellular Carcinoma after Surgical Resection

It is still difficult to predict the probability of tumor recurrence after resection of hepatocellular carcinoma (HCC). In this study, we set out to identify specific microRNA (miRNA) in microdissected hepatitis B virus (HBV)-related HCC tissue from formalin-fixed paraffin-embedded (FFPE) samples which might be used in predicting early recurrence after HCC resection. Taqman low density arrays were used to detect the 667 miRNA profiles in both the microdissected tumorous and adjacent non-tumorous liver tissues from 20 HCC patients (discovery set) including 10 patients with early tumor recurrence and 10 without early tumor recurrence and to identify the differentially expressed miRNAs related to HCC recurrence. Then quantitative real-time PCR (qRT-PCR) was used to verify the findings in 106 patients (training set), and to develop a predictive assay. The identified miRNAs were further validated in an independent cohort of 112 patients (validation set). Thirty seven miRNAs were identified from 20 HCC patients and validated in 106 HCC patients using qRT-PCR. A significant association was found between miR-29a-5p level in HCC tissues and early tumor recurrence (P = 0.0002). This association was further confirmed in the independent validation set of 112 patients (P = 0.0154). MiR-29a-5p level was significantly associated with both time to tumor recurrence (TTR) (P = 0.0015) and overall survival (OS) (P = 0.0079) in validation set. In the multivariate analyses, miR-29a-5p was identified as an independent factor for TTR, particularly for those patients with early stage of HCC. The sensitivity and specificity of miR-29a-5p for the prediction of early HCC recurrence of BCLC 0/A stage HCC were 74.2% and 68.2%, respectively. These suggest that miR-29a-5p might be a useful marker for the prediction of early tumor recurrence after HCC resection, especially in BCLC 0/A stage HCCs.

SPOCK1 Is Regulated by CHD1L and Blocks Apoptosis and Promotes HCC Cell Invasiveness and Metastasis in Mice

Chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like (CHD1L) is an SNF2-like transcription factor involved in the development of human hepatocellular carcinoma (HCC). Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) is up-regulated by CHD1L; we investigated its role in hepatocellular carcinogenesis. We investigated interactions between SPOCK1 and CHD1L using electrophoretic mobility shift and luciferase reporter assays. Levels of SPOCK1 messenger RNA (mRNA) and protein were measured in samples of HCC and adjacent nontumor liver tissues (135 pairs) and compared using Pearson correlation coefficients. Effects of SPOCK1 overexpression and silencing were determined in HCC cell lines (QGY-7703, PLC-8024, BEL-7402, and QGY-7701). The CHD1L protein bound directly to the promoter region (nt-1662 to +34) of SPOCK1 and activated transcription. Levels of SPOCK1 mRNA and protein were increased in 60% of human HCC samples, compared with nontumor live tissues, and was associated significantly with clinical stage. Levels of SPOCK1 mRNA were increased among tumors that became metastatic, compared with those that did not, and among patients with shorter overall and disease-free survival times. Ectopic expression of SPOCK1 in HCC cells increased proliferation, foci formation, and colony formation in soft agar; these cells also formed larger xenograft tumors, more rapidly, in nude mice than control HCC cells. Silencing SPOCK1 expression with short hairpin RNA had the opposite effects. We found that SPOCK1 prevents apoptosis of HCC cells by activating Akt, to block release of cytochrome c and activation of caspase-9 and caspase-3; these effects were reversed with an Akt inhibitor. HCC cells that overexpressed SPOCK1 expressed higher levels of matrix metallopeptidase 9, were more invasive in Matrigel assays, and formed more metastatic nodules in immunodeficient mice than control HCC cells. CHD1L activates expression of SPOCK1, which activates Akt signaling to block apoptosis and invasion by HCC cells, in culture and in mice. Levels of SPOCK1 increase with progression of human HCC. SPOCK1 might be used as a prognostic factor or therapeutic target.

Decreased levels of nitric oxide production and nitric oxide synthase-2 expression are associated with the development and metastasis of hepatocellular carcinoma

Many studies have demonstrated the function of nitric oxide (NO) or nitric oxide synthase-2 (NOS-2) in cancer as pro-neoplastic or anti-neoplastic effectors, but the role of NO and NOS-2 in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the levels of NO production and NOS-2 expression in HCC and adjacent non-tumor liver tissues and to clarify whether the levels of NO/NOS-2 are related to the clinicopathological features of HCC. The levels of NO production were examined in tumor and adjacent non-tumor liver tissues of 30 patients with HCC. The expression of NOS-2 was detected by real-time polymerase chain reaction (RT-PCR) and immunohistochemical analysis in HCC and/or adjacent non-tumor liver tissues. Mutant p53 and proliferating cell nuclear antigen (PCNA) were also immunohistochemically investigated in liver tissues. The levels of NO in HCC were significantly lower compared to adjacent non-tumor liver tissues (P<0.001). The relative mRNA and protein expression levels of NOS-2 in HCC were also significantly lower compared to adjacent non-tumor liver tissues (P<0.01 for both). We found that the levels of NO in patients suffering from HCC metastasis were lower compared to those without metastasis (P<0.05) and NOS-2 expression was correlated with tumor diameter (P<0.05) and metastasis (P<0.05). In addition, mutant p53 protein was expressed in the majority of HCC samples and the proliferation rate of HCC was significantly higher than that of adjacent non-tumor liver tissues. These data indicate that decreased levels of NO/NOS-2 may partially contribute to overexpression of the mutant p53 protein and excessive proliferation; this may be a potential mechanism in the development and progression of HCC.

A novel quantitative proteomics workflow by isobaric terminal labeling

Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.

Reply to G. Cai et al

We would like to thank Cai et al for their comments on our article. In the study by Gao et al, microRNA (miRNA) was extracted from formalin-fixed, paraffin-embedded dysplastic nodules, hepatocellular carcinomas (HCCs), and the adjacent nontumorous liver tissues. This study showed that miRNA deregulation at the tissue level was an early event and accumulated throughout the various steps of hepatitis B virus–associated hepatocarcinogenesis. In our study, the panel with seven miRNAs (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a, and miR-801) was identified in the plasma of patients with HCC. The discrepancy between the two studies would be primarily a result of the different categories of the study specimens (tissue v plasma). To our knowledge, the expression profiles of plasma miRNA are different from that of tissue miRNA. Plasma circulating miRNA was reported to have diverse origins. It is well known that most hepatitis B virus–related HCCs involve two disease processes: the HCC as well as liver damage as a result of either hepatitis or cirrhosis. Thus, liver damage, tumor immune response, and/or hepatitis may also influence the miRNA profiles in the plasma of patients with HCC. Therefore, the expression profiles of plasma miRNAs in patients with HCC may not trend according to the expression profiles in HCC tissue. For example, studies by Wang et al and Starkey Lewis et al found miR-122 and miR-192 to be associated with liver damage, and the two miRNAs expressed opposite regulations in the tissue and plasma specimens. This indicates that the miRNA profiles in the plasma specimens are more complicated compared with those in pure tumor tissue in patients with HCC. At the tissue level, two studies revealed the differences in miRNA dysregulation in early versus advanced HCCs. Such differences have not been reported in the current plasma studies of miRNA profiles. It would be interesting to further investigate the differences in miRNA spectrum and expression level between early and advanced HCCs in the plasma of patients with HCC.

Suppression of Human Liver Cancer Cell Migration and Invasion via the GABAA Receptor.

ObjectiveTo investigate the roles of the γ-aminobutyric acid (GABA) in the metastasis of hepatocellular carcinoma (HCC) and to explore the potential of a novel therapeutic approach for the treatment of HCC.MethodsThe expression levels of GABA receptor subunit genes in various HCC cell lines and patients‘ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis. Transwell cell migration and invasion assays were carried out for functional analysis. The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer.ResultsThe mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue. GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABAA receptor as a result of the induction of liver cancer cell cytoskeletal reorganization. Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo.ConclusionsThese findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system.

Decreased expression of zinc-alpha2-glycoprotein in hepatocellular carcinoma associates with poor prognosis

BackgroundZinc-alpha2-glycoprotein (AZGP1, ZAG) was recently demonstrated to be an important factor in tumor carcinogenesis. However, AZGP1 expression in hepatocellular carcinoma (HCC) and its significance remain largely unknown.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was applied to determine mRNA level of AZGP1 in 20 paired fresh HCC tissues. Clinical and pathological data of 246 HCC patients were collected. Tissue-microarray-based immunohistochemistry (IHC) was performed to examine AZGP1 expression in HCC samples. Relationship between AZGP1 expression and clinicopathological features was analyzed by Chi-square test, Kaplan-Meier analysis and Cox proportional hazards regression model.ResultsAZGP1 expression was significantly lower in 80.0% (16/20) of tumorous tissues than that in the corresponding adjacent nontumorous liver tissues (P < 0.001). Consistently, IHC data revealed that decreased expression of AZGP1 was present in 80.1% (197/246) of HCC patient tissues (P < 0.001). Furthermore, AZGP1 expression in HCC significantly associated with several clinicopathological parameters, including serum AFP level (P = 0.013), liver cirrhosis (P = 0.002) and tumor differentiation (P = 0.025). Moreover, HCC patients with high AZGP1 expression survived longer, with better overall survival (P = 0.006) and disease-free survival (P = 0.025). In addition, low AZGP1 expression associated with worse relapse-free survival (P = 0.046) and distant metastatic progression-free survival (P = 0.036).ConclusionAZGP1 was downregulated in HCC and could be served as a promising prognostic marker for HCC patients.

Gene expression profiling of liver cancer stem cells by RNA-sequencing.

BackgroundAccumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90+ liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90+ cells sorted from tumor (CD90+CSCs) with parallel non-tumorous liver tissues (CD90+NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis.Methodology/Principal FindingsCD90+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90+CSCs and CD90+NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90+CSCs and CD90+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90+CSCs compared to CD90+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90+CSCs in liver tumor tissues.Conclusions/SignificanceThe identified genes, such as GPC3 that are distinctly expressed in liver CD90+CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells.

Comparison of pathways associated with hepatitis B- and C-infected hepatocellular carcinoma using pathway-based class discrimination method

Molecular signatures causing hepatocellular carcinoma (HCC) from chronic infection of hepatitis B virus (HBV) or hepatitis C virus (HCV) are not clearly known. Using microarray datasets composed of HCV-positive HCC or HBV-positive HCC, pathways that could discriminate tumor tissue from adjacent non-tumor liver tissue were selected by implementing nearest shrunken centroid algorithm. Cancer-related signaling pathways and lipid metabolism-related pathways were predominantly enriched in HCV-positive HCC, whereas functionally diverse pathways including immune-related pathways, cell cycle pathways, and RNA metabolism pathways were mainly enriched in HBV-positive HCC. In addition to differentially involved pathways, signaling pathways such as TGF-β, MAPK, and p53 pathways were commonly significant in both HCCs, suggesting the presence of common hepatocarcinogenesis process. The pathway clustering also verified segregation of pathways into the functional subgroups in both HCCs. This study indicates the functional distinction and similarity on the pathways implicated in the development of HCV- and/or HBV-positive HCC.

Abstract 718: microRNA-29a* is a predictor for early recurrence hepatitis B virus-related hepatocellular carcinoma after surgical resection

Abstract Purpose: Early tumor recurrence after liver resection for hepatocellular carcinoma (HCC) was mainly due to intrahepatic tumor spreading. In this study, we set out to identify specific microRNA (miRNA) which might be used in predicting the possibility of early recurrence after HCC resection. Experimental Design: Taqman low density arrays were used to detect the miRNA expression levels in both the microdissected tumorous and adjacent non-tumorous liver tissues from 20 HCC patients. Quantitative real-time PCR (qRT-PCR) was used to verify the findings in 106 patients, and to develop a predictive assay. The identified miRNAs were further validated in an independent cohort of 112 patients. Results: Thirty seven selected miRNAs were validated in 106 HCC patients using qRT-PCR. A significant association was found between miR-29a* level in HCC tissues and early recurrence (P=0.0002). This association was further confirmed in the independent testing set of 112 patients (P=0.0154), in which miR-29a* level was significantly associated with both time to tumor recurrence (TTR) (P=0.0015) and overall survival (OS) (P=0.0079) of patients. In the univariate and multivariate analyses, miR-29a* was identified as an independent factor for TTR, particularly for those patients with early stage of HCC. The sensitivity and specificity of miR-29a* for the prediction of early HCC recurrence in an independent blinded validation set reached 74.2% and 68.2%, respectively. Conclusion: MiR-29a* might be a useful marker for prediction of early recurrence of HCC, particularly for those with early stage of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 718. doi:1538-7445.AM2012-718

Downregulation of GRIM-19 is associated with hyperactivation of p-STAT3 in hepatocellular carcinoma

Accumulating evidence has implicated that constitutive activation of signal transducer and activator of transcription protein 3 (STAT3) may be a major oncogenic factor involved in hepatocellular carcinoma (HCC) development. Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) has been shown to be a tumor suppressor associated with growth control and suppression of STAT3 activity. The downregulation of GRIM-19 expression has been shown in a number of human tumor types, and it has been correlated with hyperactivation of STAT3. However, the role of GRIM-19 in the pathogenesis of HCC has not been evaluated. The aim of our study was to evaluate GRIM-19 expression levels and investigate their correlation with phosphorylated STAT3 (p-STAT3) levels in HCC. GRIM-19 and p-STAT3 expression levels were analyzed in HCC and adjacent nontumorous liver tissues (ANLT) by immunohistochemistry, western blot analysis, and RT-PCR. GRIM-19 protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. HCC samples exhibited low levels of GRIM-19 and moderate to high levels of p-STAT3 expression. In contrast, ANLT was characterized by high levels of GRIM-19 and low levels of p-STAT3 expression. Downregulation of GRIM-19 was closely correlated with increased histological grade in HCC. GRIM-19 expression is closely correlated with histological grading and p-STAT3 in HCC. Thus, the potential role of GRIM-19 in HCC development may be through these correlations.