Rui-Xia Sun

Inhibition of Growth and Metastasis of Human Hepatocellular Carcinoma by Antisense Oligonucleotide Targeting Signal Transducer and Activator of Transcription 3

Purpose: Hepatocellular carcinoma (HCC) is an aggressive malignancy and is a devastating clinical complication of chronic liver disease. Therapeutic options are limited mainly because the genetic and biochemical understanding of this disease remains fragmented. We intended to study the role of signal transducer and activator of transcription 3 (STAT3) aberrant signaling in HCC malignancy, and the therapeutic potential of inhibition of STAT3 expression for HCC. Experimental Design: A 2′-O-methoxyethylribose–modified phosphorothioate antisense oligonucleotide (ASO) was used to knock down STAT3 expression in different human HCC cell lines, including the highly metastatic HCCLM3 derived from orthotopic implantation and subsequent lung metastasis in athymic mice. The effects of STAT3 ASO treatment on HCC cells, metastasis, and animal survival following HCCLM3 orthotopic implantation were evaluated. Results: Specific suppression of phosphorylated STAT3 reduced its DNA-binding activity, inhibited the expression of vascular endothelial growth factor, survivin, matrix metalloproteinases 2 and 9, reduced cell proliferation and migratory potential, induced apoptosis in vitro, and inhibited intradermal angiogenesis and s.c. tumorigenesis upon injection in mice. In mice bearing orthotopically implanted HCCLM3, STAT3 inhibition following therapeutic treatment with STAT3 ASO reduced circulating vascular endothelial growth factor and basic fibroblast growth factor, decreased intratumor CD34-positive microvessel density, intrahepatic and intraperitoneal transmission, and lung metastasis. HCC tumor volume and weight were reduced and the survival time of mice bearing orthotopically xenografted HCC was approximately doubled in STAT3 ASO–treated mice (P < 0.05). Conclusions: Constitutively activated STAT3 is essential for the growth, survival, and metastasis of HCC, suggesting that STAT3-targeted therapy may have utility for HCC.

Involvement of protein kinase C β–extracellular signal‐regulating kinase1/2/p38 mitogen‐activated protein kinase–heat shock protein 27 activation in hepatocellular carcinoma cell motility and invasion

To understand the molecular mechanism that underlies the role of various prominent signal pathways in hepatocellular carcinoma (HCC) metastasis, a human signal transduction oligonucleotide microarray analysis was carried out in cultured HCC cell models with increasing spontaneous metastatic potential (MHCC97L, MHCC97H, and HCCLM6). The results revealed that the mitogen‐activated protein kinase (MAPK) pathway is the prominently upregulated pathway in HCC metastasis. Further study showed that basal phosphorylated levels of extracellular signal‐regulating kinase (ERK)1/2 and p38 MAPK consecutively increased from MHCC97L to MHCC97H to HCCLM6 cells, but not c‐Jun N‐terminal kinase. The phosphorylation of ERK1/2 and p38 MAPK was regulated by upregulated protein kinase Cβ (PKCβ) in HCC cells through the integrated use of PKCβ RNA interference, the PKCβ specific inhibitor enzastaurin and a PKC activator phorbol‐12‐myristate‐13‐acetate. Heat shock protein 27 (HSP27) was also verified as a downstream common activated protein of PKCβ–ERK1/2 and PKCβ–p38 MAPK. In vitro migration and invasion assay further showed that the depletion of PKCβ or inhibition of PKCβ activation effectively decreased HCC cell motility and invasion. Moreover, the motility and invasion of phorbol‐12‐myristate‐13‐acetate‐stimulated PKCβ‐mediated HCC cells was significantly negated by an ERK inhibitor, 1.4‐diamino‐2.3‐dicyano‐1.4‐bis[2‐aminophenylthio] butadiene, or a p38 MAPK inhibitor, 4‐(4‐Fluorophenyl)‐2‐(4‐methylsulfinylphenyl)‐5‐(4‐pyridyl)1H‐imidazole. It also showed that HSP27 is critical in PKCβ‐mediated HCC cell motility and invasion. Taken together, this study reveals the important role of this PKCβ‐ERK1/2/p38MAPK‐HSP27 pathway, which was verified for the first time, in modulating HCC cell motility and invasion. (Cancer Sci 2008; 99: 486–496)

The scent fingerprint of hepatocarcinoma: in-vitro metastasis prediction with volatile organic compounds (VOCs).

Background: Hepatocellular carcinoma (HCC) is a common and aggressive form of cancer. Due to a high rate of postoperative recurrence, the prognosis for HCC is poor. Subclinical metastasis is the major cause of tumor recurrence and patient mortality. Currently, there is no reliable prognostic method of invasion. Aim: To investigate the feasibility of fingerprints of volatile organic compounds (VOCs) for the in-vitro prediction of metastasis. Methods: Headspace gases were collected from 36 cell cultures (HCC with high and low metastatic potential and normal cells) and analyzed using nanomaterial-based sensors. Predictive models were built by employing discriminant factor analysis pattern recognition, and the classification success was determined using leave-one-out cross-validation. The chemical composition of each headspace sample was studied using gas chromatography coupled with mass spectrometry (GC-MS). Results: Excellent discrimination was achieved using the nanomaterial-based sensors between (i) all HCC and normal controls; (ii) low metastatic HCC and normal controls; (iii) high metastatic HCC and normal controls; and (iv) high and low HCC. Several HCC-related VOCs that could be associated with biochemical cellular processes were identified through GC-MS analysis. Conclusion: The presented results constitute a proof-of-concept for the in-vitro prediction of the metastatic potential of HCC from VOC fingerprints using nanotechnology. Further studies on a larger number of more diverse cell cultures are needed to evaluate the robustness of the VOC patterns. These findings could benefit the development of a fast and potentially inexpensive laboratory test for subclinical HCC metastasis.

Prediction of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma by SELDI-based serum decision tree classification

PurposeTo screen potential serological biomarkers and develop decision tree classifications of chronic hepatitis B, liver cirrhosis (LC) and hepatocellular carcinoma (HCC), respectively, with high prediction score for improving diagnosis of liver diseases.MethodsThe total serum samples were randomly divided into three training sets (41 HBV and 35 health; 36 LC and 35 health; 39 HCC and 35 health) and three testing groups (34 HBV and 38 health; 18 LC and 52 health; 42 HCC and 47 health). Selected WCX2 protein chip capture followed by SELDI-TOF-MS analysis was applied to generate the serum protein profiles. Subsequently serum protein spectra were normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard including baseline subtraction, mass accuracy calibration, automatic peak detection. Once the intensities of selected significant peaks from the training data set were transferred to further BPS analysis, an optimized classification tree with sequence-decision was established to divide training data set into disease group and control group successfully. A double blind test was employed to determine the clinical sensitivity and clinical specificity of three models.ResultsAfter comparative analysis of SELDI based serum protein profile between the cases of disease and healthy, a HCC decision tree classification with sensitivity of 94.872% and specificity of 94.286%; a LC decision tree classification with sensitivity of 91.667% and specificity of 94.286% and a HBV decision tree classification with sensitivity of 95.122% and specificity of 94.286% were produced by BPS respectively. When three decision tree models were challenged by the double-blind test samples, clinical sensitivity and clinical specificity of these models were predicted in diagnosis of three liver diseases (HCC: 90.48 and 89.36%; cirrhosis: 100 and 86.5%; HBV: 85.29 and 84.21%).ConclusionSELDI-based decision tree classifications showed great advantages over conventional serological biomarkers in the diagnosis of chronic hepatitis B, LC as well as HCC.

Gene expression profiles during activation of cultured rat hepatic stellate cells by tumoral hepatocytes and fetal bovine serum

PurposeHepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. Myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma (HCC). In this study, we determined gene expression changes in two different models of HSC activation, induction-activated HSCs (iHSCs) and culture-activated HSCs (cHSCs).MethodsHepatic stellate cells were isolated by density centrifugation and exposed to conditioned medium (CM) from the rat HCC cell line C5F, and fetal bovine serum (FBS). Expression of 27,100 genes in quiescent HSCs, cHSCs and iHSCs was analyzed by microarray and was confirmed on a subset of genes by real-time RT-PCR and Western blot.ResultsOne thousand nine hundred sixty-seven genes were differentially expressed in cHSCs and iHSCs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors such as Il1a, Vcam1, Ccl6, Ilr7, PRAP, osteopontin, Gp39, Raf1, Rac2, Adam17, Wnt6, MMP-9, and Cfd. C5F-CM-induced activation only partially reproduced the gene expression changes observed during FBS culture activation. iHSCs showed specific gene expression, suggesting that HCC cells can specifically induce HSC activation.ConclusionsInduction-activated HSCs’ gene expression patterns were partially similar to and different from that of cHSCs. iHSCs might play an important role in invasion and metastasis of HCC. This study provided theoretical foundations for investigating the biology of HSCs in HCC.

Long non-coding RNAs are differentially expressed in hepatocellular carcinoma cell lines with differing metastatic potential.

BACKGROUND Metastasis is a major reason for poor prognosis in patients with cancer, including hepatocellular carcinoma (HCC). A salient feature is the ability of cancer cells to colonize different organs. Long non-coding RNAs (lncRNAs) play important roles in numerous cellular processes, including metastasis. MATERIALS AND METHODS In this study, the lncRNA expression profiles of two HCC cell lines, one with high potential for metastasis to the lung (HCCLM3) and the other to lymph nodes (HCCLYM-H2) were assessed using the Arraystar Human LncRNA Array v2.0, which contains 33,045 lncRNAs and 30,215 mRNAs. Coding-non-coding gene co-expression (CNC) networks were constructed and gene set enrichment analysis (GSEA) was performed to identify lncRNAs with potential functions in organ-specific metastasis. Levels of two representative lncRNAs and one representative mRNA, RP5-1014O16.1, lincRNA-TSPAN8 and TSPAN8, were further detected in HCC cell lines with differing metastasis potential by qRT-PCR. RESULTS Using microarray data, we identified 1,482 lncRNAs and 1,629 mRNAs that were differentially expressed (≥1.5 fold-change) between the two HCC cell lines. The most upregulated lncRNAs in H2 were RP11-672F9.1, RP5-1014O16.1, and RP11-501G6.1, while the most downregulated ones were lincRNA-TSPAN8, lincRNA-CALCA, C14orf132, NCRNA00173, and CR613944. The most upregulated mRNAs in H2 were C15orf48, PSG2, and PSG8, while the most downregulated ones were CALCB, CD81, CD24, TSPAN8, and SOST. Among them, lincRNA-TSPAN8 and TSPAN8 were found highly expressed in high lung metastatic potential HCC cells, while lowly expressed in no or low lung metastatic potential HCC cells. RP5- 1014O16.1 was highly expressed in high lymphatic metastatic potential HCC cell lines, while lowly expressed in no lymphatic metastatic potential HCC cell lines. CONCLUSIONS We provide the first detailed description of lncRNA expression profiles related to organ-specific metastasis in HCC. We demonstrated that a large number of lncRNAs may play important roles in driving HCC cells to metastasize to different sites; these lncRNAs may provide novel molecular biomarkers and offer a new basis for combating metastasis in HCC cases.

Inhibition of T-cell responses by intratumoral hepatic stellate cells contribute to migration and invasion of hepatocellular carcinoma

The stroma of hepatocellular carcinoma (HCC) is markedly infiltrated with activated hepatic stellate cells (HSCs), and associated invasion and metastasis of HCC. However, little is known of the role of HSCs in immune responses in HCC. The Buffalo rat HCC model was established. Quiescent HSCs (qHSCs) and intratumoral HSCs (tHSCs) were isolated. Surface molecules of tHSC were detected by flow cytometry, and gene expression was analyzed by fluorescence quantitative RT-PCR. T cell proliferation was monitored by [3H]-thymidine (3H-TdR) incorporation into DNA, and cytotoxic activity was assessed by measuring the release of 51Cr. The level of cytokine expression by T cells was measured by enzyme-linked immunosorbent assay. T cell apoptosis was detected by double-stained terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and anti-CD3 antibodies. The migration and invasion of HCC was observed by transwell experiments. tHSCs express low levels of major histocompatibility complex (MHC) class I, MHC class II, and costimulatory molecules, and produce varying levels of cytokines. Addition of the tHSCs suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation. The tHSC-induced T-cell hyporesponsiveness was associated with enhanced T-cell apoptosis, and contributed to the migration and invasion of hepatoma cell. tHSCs was associated with markedly enhanced expression of B7-H1. Blockade of B7-H1/PD-1 ligation significantly reduced HSC immunomodulatory activity, and hepatoma cell migration and invasion. tHSCs can induce T cell apoptosis, suggesting an important role for B7-H1. The interactions between tHSCs and T cells may contribute to hepatic immune tolerance and invasion and migration of HCC.

T-cell apoptosis induced by intratumoral activated hepatic stellate cells is associated with lung metastasis in hepatocellular carcinoma

Profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro has recently been described in hepatocellular carcinoma (HCC). In the present study, we investigated the immune inhibitory activity of HSCs in vivo in an orthotopic rat HCC model with lung metastasis. Rats (n=24) were randomly sacrificed on days 7, 14, 21 and 28 (n=4 each). Lung tissues were stained with hematoxylin and eosin. Liver sections were stained for immunofluorescence analysis. T-cell apoptosis was detected using double staining for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Staining revealed marked and continuous accumulation of α-smooth muscle actin with tumor progression after orthotopic tumor implantation in rat liver. T lymphocyte numbers gradually increased following tumor progression, and subset analysis revealed an increase in the distribution of liver CD8+ and CD4+ T cells. Double staining for CD3 and TUNEL demonstrated T-cell apoptosis. Apoptotic T cells were more frequent in the HCC livers compared to the normal livers, and were spatially associated with intratumoral activated HSCs (tHSCs), suggesting a direct interaction. T-cell apoptosis was more frequently induced in the co-cultures of activated splenic T cells(aT)/tHSCs compared to aT/quiescent (q) HSCs or qT/tHSCs. tHSCs were positively correlated with T-cell apoptosis, and the percentage of T-cells undergoing apoptosis was positively correlated with the number of lung metastasis nodules. T-cell apoptosis may be promoted via an interaction with tHSCs, suggesting that tHSCs regulate T cells and contribute to lung metastasis in HCC.

Tetraspanin-8 promotes hepatocellular carcinoma metastasis by increasing ADAM12m expression

Recent evidence indicates that tetraspanin-8 (TSPAN8) promotes tumor progression and metastasis. In this study, we explored the effects of TSPAN8 and the molecular mechanisms underlying hepatocellular carcinoma (HCC) metastasis using various HCC cell lines, tissues from 149 HCC patients, and animal models of HCC progression. We showed that elevated expression of TSPAN8 promoted HCC invasion in vitro and metastasis in vivo, but did not influence HCC cell proliferation in vitro. Increased TSPAN8 expression in human HCC was predictive of poor survival, and multivariate analyses indicated TSPAN8 expression to be an independent predictor for both postoperative overall survival and relapse-free survival. Importantly, TSPAN8 enhanced HCC invasion and metastasis by increasing ADAM12m expression. We therefore conclude that TSPAN8 and ADAM12m may be useful therapeutic targets for the prevention of HCC progression and metastasis.

Prognostic Significance of Hes-1, a Downstream Target of Notch Signaling in Hepatocellular Carcinoma

BACKGROUND Hairy and enhancer of split 1 (Hes-1) protein is a downstream target of Notch signaling and is a basic helix-loop-helix transcriptional repressor. However, definitive evidence for a role in hepatocellular carcinoma (HCC) cells has not been reported. Here, Hes-1 was revealed to an important component of the Notch signaling cascade in HCC cell lines possessing different potential for lung metastasis. MATERIALS AND METHODS RNAi mediated by plasmid constructs was used to analyze the role of Hes-1 in MHCC-97L HCC cells by assessing proliferation, apoptosis, cell migration and matrigel invasion following transfection. Hes-1 protein expression analysis in HCC tissue was also conducted by immunohistochemistry. RESULTS Our studies revealed that Hes-1 was decreased in HCC cell lines with higher lung metastasis potential at both the mRNA and protein levels. Down-regulation of the Hes-1 gene in MHCC-97L cells resulted in increased cell proliferation, reduced apoptosis and increased migration and invasion. CONCLUSIONS Hes-1 has potential prognostic value in post-surgical HCC patients and may be an independent prognostic indicator for overall survival and tumor recurrence. These findings have important implications for understanding the mechanisms by which Hes-1 participates in tumor proliferation and invasion.