Potential Of Hepatocellular Carcinoma Cell Lines Research Articles

Transmembrane serine protease 2 cleaves nidogen 1 and inhibits extrahepatic liver cancer cell migration and invasion.

We aimed to confirm whether transmembrane serine protease 2 (TMPRSS2) regulates nidogen 1 (NID1) expression in extracellular vesicles (EVs) and metastatic hepatocellular carcinoma (HCC) cells. HCC cells, HUVEC cells, MRC-5 cells, HLE cells, MHCCLM3 cells, MHCC97L cells, H2P cells, H2M cells, as well as LO2 cells were cultured according to providers' instruction and EV models were established by using BALB/cAnN-nu mice to facilitate the verifications. We found that TMPRSS2 expression was inversely correlated with the metastatic potential of HCC cell lines. The expression of TMPRSS2 decreased in a time-dependent manner in tumor-bearing model mice implanted with MHCCLM3 cells compared with uninoculated mice. TMPRSS2 overexpression in MHCCLM3 and MHCC97L cells led to the significant downregulation of NID1 expression in total cell lysates and isolated EVs. In contrast, TMPRSS2 silencing resulted in the elevation of NID1 expression in cells and EVs. Administration of EVs from MHCCLM3 and MHCC97L cells with overexpressed or silenced TMPRSS2 inhibited or strengthened, respectively, the invasion, proliferation, and migration of LO2 tumor cells. EVs derived from MHCCLM3 and MHCC97L cells with overexpressed or depleted TMPRSS2 also deactivated or activated fibroblasts, respectively. These EVs secrete inflammatory cytokines and phosphorylated p65, facilitate the colonization of fibroblasts, and augment fibroblast growth and motility. These findings provide evidence for a new candidate drug targeting tumorigenic EV-NID1 to treat HCC.

Emerin knockdown induces the migration and invasion of hepatocellular carcinoma cells by up-regulating the cytoplasmic p21.

Emerin (EMD) plays diverse roles in cellular polarity organization, nuclear stability, and cell motility, however, the biological role of EMD relevant to the migration and invasion of hepatocellular carcinoma (HCC) cells has not yet been illustrated. In the present study, we initially found that the upregulation of EMD in HCC tissues, and EMD expression was negatively correlated with the spontaneous metastatic potential of HCC cell lines. Loss of EMD in HCC cells facilitated cell migration and invasion in vitro and metastasis in vivo. Meanwhile, we demonstrated that EMD knockdown induced EMT but enhanced p21 expression in HCC cells. Notably, silencing of EMD in HCC cells increased the cytoplasmic localization of p21 protein, whereas p21 knockdown partially abrogated the migratory and invasive ability, EMT, and the actin cytoskeleton rearrangement induced by EMD knockdown in HCC cells. Our results indicated a significant role of EMD knockdown in the HCC cell motility and metastasis through upregulating the cytoplasmic p21, unveiling a novel mechanism of cell motility regulation induced by EMD.

Ginsenoside CK Inhibits Hypoxia-Induced Epithelial-Mesenchymal Transformation through the HIF-1α/NF-κB Feedback Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a kind of malignant tumor with high morbidity and mortality rates worldwide. Epithelial–mesenchymal transformation (EMT) is crucial for HCC progression and prognosis. Characteristics of the tumor microenvironment, such as hypoxia, and excessive activation of the NF-κB signaling pathway have been identified as the key inducers of EMT in HCC. In our study, we verified the crosstalk between HIF-1α signaling and NF-κB pathway and their effects on EMT in HCC cells. The results show that CoCl2-induced hypoxia could promote IκB phosphorylation to activate NF-κB signaling and vice versa. Moreover, we found that ginsenoside CK, a metabolite of protopanaxadiol saponins, could inhibit the proliferation and colony formation of different HCC cell lines. Furthermore, ginsenoside CK could impair the metastatic potential of HCC cell lines under hypoxic conditions. Mechanistically, ginsenoside CK suppressed HIF-1α/NF-κB signaling and expression level of EMT-related proteins and cytokines in hypoxia-induced or TNFα-stimulated HCC cell lines. An in vivo study revealed that the oral delivery of ginsenoside CK could inhibit the growth of xenograft tumors and block HIF-1α and NF-κB signaling as well as EMT marker expression. Our study suggests that ginsenoside CK is a potential therapy for HCC patients that functions by targeting the HIF-1α/NF-κB crosstalk.

Deficiency in Embryonic Stem Cell Marker Reduced Expression 1 Activates Mitogen-Activated Protein Kinase Kinase 6-Dependent p38 Mitogen-Activated Protein Kinase Signaling to Drive Hepatocarcinogenesis.

Embryonic stem-cell-related transcription factors are central to the establishment and maintenance of stemness and pluripotency, and their altered expression plays key roles in tumors, including hepatocellular carcinoma (HCC), a malignancy with no effective treatment. Here, we report on the embryonic stem cell marker, reduced expression 1 (REX1; also known as zinc finger protein 42), to be selectively down-regulated in HCC tumors. Deficiency of REX1 in HCC was attributed to a combination of hypermethylation at its promoter as well as histone modification by methylation and acetylation. Clinically, hypermethylation of REX1 was closely associated with neoplastic transition and advanced tumor stage in humans. Functionally, silencing of REX1 potentiated the tumor-initiating and metastasis potential of HCC cell lines and xenografted tumors. Lentivirus-mediated Rex1 ablation in liver of male immunocompetent mice with HCC, induced by hydrodynamic tail vein injection of proto-oncogenes, enhanced HCC development. Transcriptome profiling studies revealed REX1 deficiency in HCC cells to be enriched with genes implicated in focal adhesion and mitogen-activated protein kinase (MAPK) signaling. From this lead, we subsequently found REX1 to bind to the promoter region of mitogen-activated protein kinase kinase 6 (MKK6), thereby obstructing its transcription, resulting in altered p38 MAPK signaling. Our work describes a critical repressive function of REX1 in maintenance of HCC cells by regulating MKK6 binding and p38 MAPK signaling. REX1 deficiency induced enhancement of p38 MAPK signaling, leading to F-actin reorganization and activation of nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, which collectively contributed to enhanced stemness and metastatic capabilities of HCC cells.

The application of whole exome sequencing in different metastatic potential of heptaocellular carcinoma

Objective Genome-wide exome sequencing was used to analyze DNA of hepatocellular carcinoma (HCC) tissues and cell lines with different metastatic potentials to explore the mutation genes and sites related to the HCC metastasis. Methods Genomic DNA extracted from twelve HCC tumors and its adjacent tissues and six HCC cells (HCC-LM3, MHCC-97L, MHCC-97H, HepG2, SMCC-7721 and PLC-RFP-5) with different metastatic potentials were analyzed by whole exome sequencing. Bioinformatics analysis and screening of mutations was used to investigated mutation genes and sites potentially associated with HCC metastasis. Results Comparing high and low metastatic potential HCC, high metastatic potential HCC group had more than three gene mutations including UIMC1 (rs3733876 C/T), SEMA3C (rs1527482 G/A), SAMD9 (rs10279499 G/T), RECK (rs16932912 G/A), PDLIM4 (rs4877 G/T, LIMK2 (rs3747154 A/G), CRIM1 (rs3821169 G/T) which was associated with HCC metastasis. Mutate genes and its sites such as CAPZB (rs79308175C/T), CSF1R (rs10079250A/G), HSPA5 (rs143920039A/T), SORBS3 (rs2449331T/C, rs3758036C/T) only occured in high metastatic potential HCC cells (HCC-LM3, MHCC-97L and MHCC-97H), while mutation sites RASAL3 (rs146624357G/A, rs142945276 C/T) only appeared in three low metastatic potential cells (HepG2, SMCC-7721 and PLC-RFP-5). The DNA comparative analysis of three homologous high metastatic potential HCC cell lines (HCC-LM3, MHCC-97Land MHCC-97H) showed that only EHBP1 (rs77403174T/C) was presented in HCC-LM3, while FAM189B (rs150296362C/T), SERPINB13 (rs144903442G/A) and ARHGEF1 (rs2303797C/T) only presented in MHCC-97L and MHCC-97H. Conclusion Mutations and its sites such as UIMC1 (rs3733876C/T), SEMA3C (rs1527482G/A), SAMD9 (rs10279499G/T) was detected to potentially related to the HCC metastasis by genome-wide exome sequencing. Key words: Whole exome sequencing; Hepatocellular carcinoma; Mutations; Metastasis

Mining featured biomarkers associated with vascular invasion in HCC by bioinformatics analysis with TCGA RNA sequencing data

This study aims to identify the feature genes associated with vascular invasion in hepatocellular carcinoma (HCC). Here, the RNA sequencing data related to vascular invasion in The Cancer Genome Atlas (TCGA) database, including 292 HCC patients with complete clinical data were included in our study as the training dataset for construction and E-TABM-36, including 41 HCC patients with complete clinical data was used as the validation dataset. Following data normalization, differentially expressed mRNA and copy number (CN) were selected between with and without vascular invasion samples. A support vector machine (SVM) classifier was constructed and validated in GSE9828 and GSE20017 datasets. Total 59 feature genes were found by the SVM classifier. Using Cox regression analysis, three clinical features, including Patholigic T, Stage and vascular invasion and 6 optimal prognostic genes, including ANO1, EPHX2, GFRA1, OLFM2, SERPINA10 and TKT were significantly correlated with prognosis. A risk score formula was developed to assess the prognostic value of 6 optimal prognostic genes, which were identified to possess the most remarkable correlation with overall survival in HCC patients. By performing in vitro experiments, we observed TKT was significantly increased, but OLFM2 was decreased in high metastatic potential HCC cell lines (SK-HEP-1 and MHCC-97 H) compared with low metastatic potential cell line Huh7 and normal human liver cell line LO2 using western blotting analysis. Knockdown of TKT in MHCC-97H or overexpression of OLFM2 in SK-HEP-1 significantly suppressed cell migration and invasion using transwell assays. Our results demonstrated that TKT and OLFM2 might be novel independent biomarkers for predicting survival based on the presence of vascular invasion in patients with HCC.

The Comprehensive Roles of ATRANORIN, A Secondary Metabolite from the Antarctic Lichen Stereocaulon caespitosum, in HCC Tumorigenesis.

Hepatocellular carcinoma (HCC) is one of the most deadly genetic diseases, but surprisingly chemotherapeutic approaches against HCC are only limited to a few targets. In particular, considering the difficulty of a chemotherapeutic drug development in terms of cost and time enforces searching for surrogates to minimize effort and maximize efficiency in anti-cancer therapy. In spite of the report that approximately one thousand lichen-derived metabolites have been isolated, the knowledge about their functions and consequences in cancer development is relatively limited. Moreover, one of the major second metabolites from lichens, Atranorin has never been studied in HCC. Regarding this, we comprehensively analyze the effect of Atranorin by employing representative HCC cell lines and experimental approaches. Cell proliferation and cell cycle analysis using the compound consistently show the inhibitory effects of Atranorin. Moreover, cell death determination using Annexin-V and (Propidium Iodide) PI staining suggests that it induces cell death through necrosis. Lastly, the metastatic potential of HCC cell lines is significantly inhibited by the drug. Taken these together, we claim a novel functional finding that Atranorin comprehensively suppresses HCC tumorigenesis and metastatic potential, which could provide an important basis for anti-cancer therapeutics.

PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation

Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently downregulated in hepatocellular carcinoma (HCC) and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis, and therapy resistance potential of HCC cell lines and patient-derived organoids. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a chemical-induced HCC PRMT6 knockout (PRMT6-/-) mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and showed that PRMT6 interacted with CRAF on arginine 100, which decreased its RAS binding potential and altered its downstream MEK/ERK signaling. Our work describes a critical repressive function for PRMT6 in maintenance of HCC cells by regulating RAS binding and MEK/ERK signaling via methylation of CRAF on arginine 100.

Autophagy activation contributes to glutathione transferase Mu 1-mediated chemoresistance in hepatocellular carcinoma.

Glutathione transferase Mu 1 (GSTM1) induces cancer drug resistance by hydrolyzing cancer chemotherapeutics or activating the anti-apoptosis pathway. However, the chemoresistance-inducing mechanism of GSTM1 in hepatocellular carcinoma (HCC) remains unknown. In the present study, the expression of GSTM1 was examined in three HCC cell lines. Oxaliplatin and sorafenib were selected as chemotherapeutic agents. Small interfering RNA was used to decrease GSTM1 expression. Cell death was measured using MTT and annexin V/propidium iodide assays. Activation of autophagy was evaluated by green fluorescent protein-light chain 3 redistribution and analysis of autophagy-related 5 expression in MHCC97-H and Huh-7 cells. A stepwise increase in GSTM1 expression with increasing metastatic potential of HCC cell lines was revealed. Cell death induced by oxaliplatin and sorafenib was significantly increased following GSTM1-knockdown in MHCC97-H and Huh-7 cells. Activation of autophagy was significantly inhibited by silencing GSTM1 expression. The results of the present study suggest that GSTM1 may protect HCC cells against the effect of oxaliplatin treatment through activating autophagy. The present study provides a novel perspective on HCC drug-resistance.

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

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. 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. 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. 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.

The effects of the CCR6/CCL20 biological axis on the invasion and metastasis of hepatocellular carcinoma.

Chemokines and their receptors have recently been shown to play major roles in cancer metastasis. Chemokine receptor 6 (CCR6) and its ligand, CCL20, were highly expressed in a variety of human cancers. In our present study, we aimed to clarify whether CCR6/CCL20 was correlated with the migration of hepatocellular carcinoma (HCC). RT-PCR and Western blot results showed that CCR6 was overexpressed in different invasive potential HCC cell lines (p < 0.05), while the expression of CCL20 had no obvious difference (p > 0.05). CCR6 was suppressed by siRNA in HCCLM6, and then the biological behaviors of HCCLM6 cells were observed. The results showed that the CCR6/CCL20 biological axis increased the capacity of proliferation and adhesion, as well as the chemotactic migration and the level of cytokines related to degraded extracellular matrix. In conclusion, these findings indicate that CCR6 indeed participates in regulating the migration and invasion of HCC, and it might become a prognostic factor of HCC.

Arsenic trioxide attenuates the invasion potential of human liver cancer cells through the demethylation-activated microRNA-491

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of metastasis and recurrence. In addition to treating acute promyelocytic leukemia (APL), arsenic trioxide (As2O3) also suppresses other solid tumors, such as HCC. However, the effects of As2O3 on the migration/invasion potential of liver cancer cells and the molecular mechanisms underlying in remain unclear. Here we found that As2O3 attenuated the migration/invasion potential of HCC cell lines by blocking matrix metalloproteinases (MMPs) activities and inducing a mesenchymal to epithelial transition (MET). Indeed, As2O3 elevated the expression of microRNA-491 (miR-491) via demethylation. On one hand, as a target miRNA of MMP9, miR-491 decreased the MMP9 expression. On the other hand, miR-491 blocked the activation of nuclear factor κB (NF-κB), which transcriptionally inactivated MMP2 and induced a MET (as determined by the increased expression of E-cadherin and decreased expressions of snail, slug, and vimentin). Knockdown of miR-491 abolished the As2O3-induced MMPs inactivation, MET, and the migration/invasion potential of HCC cell lines. By understanding a novel mechanism how As2O3 inhibits the migration/invasion potential of liver cancer cells, our study may help to identify potential therapeutic targets for liver cancer.

Suppression of Actopaxin Impairs Hepatocellular Carcinoma Metastasis Through Modulation of Cell Migration and Invasion

Early reports suggested that actopaxin, a member of the focal adhesion proteins, regulates cell migration. Here we investigated whether actopaxin is involved in hepatocellular carcinoma (HCC) progression and metastasis. We examined actopaxin expression in human HCC samples using immunohistochemistry and western blotting. The functional and molecular effect of actopaxin was studied in vitro by overexpression in a nonmetastatic HCC cell line, as well as repression in a metastatic cell line. The in vivo effect of actopaxin repression was studied in nonobese diabetic and severe combined immunodeficient mice. We found that actopaxin was frequently overexpressed in human HCC patients and its overexpression positively correlated with tumor size, stage, and metastasis. Actopaxin expression also correlated with the metastatic potential of HCC cell lines. Actopaxin overexpression induced the invasion and migration ability of nonmetastatic HCC cells, whereas down-regulation of actopaxin reverted the invasive phenotypes and metastatic potential of metastatic HCC cells through regulating the protein expression of certain focal adhesion proteins including ILK, PINCH, paxillin, and cdc42, as well as regulating the epithelial-mesenchymal transition pathway. Furthermore, there was a close association between actopaxin and CD29. HCC cells with stronger CD29 expression showed a higher actopaxin level, whereas actopaxin repression attenuated CD29 activity. Finally, actopaxin down-regulation enhanced the chemosensitivity of HCC cells towards oxaliplatin treatment by way of a collective result of suppression of survivin protein, β-catenin, and mammalian target of rapamycin pathways and up-regulation of p53. This study provides concrete evidence of a significant role of actopaxin in HCC progression and metastasis, by way of regulation of cell invasiveness and motility, an epithelial-mesenchymal transition process, and chemosensitivity to cytotoxic drugs.

MicroRNA‐491 is involved in metastasis of hepatocellular carcinoma by inhibitions of matrix metalloproteinase and epithelial to mesenchymal transition

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide; the prognosis of HCC patient remains poor owing to intrahepatic and extrahepatic metastasis and postsurgical recurrence. The aim of the present study is to determine the molecular mechanisms underlying the metastasis of HCC. HCC patients and treated HCC cells were molecularly characterized by miRNA microarray analysis, qRT-PCR, Western blots, transwell assay, and immunohistochemistry. Here, by employing a miRNAs microarray analysis, we found that miR-491 level was the most significant down-regulation in poorly differentiated HCC tissue compared with well-differentiated HCC tissue. We then selected HepG2 (very low migratory capacity), MHCC97L (low migratory capacity) and MHCC97H (high migratory capacity), as well as HCC tissues with different status to further investigate the effects of miR-491 on the metastasis of HCC. Our data showed that miR-491 levels were inversely correlated with different status of differentiation in HCC tissues and with migratory potential in HCC cell lines. In HepG2 cells, inhibition of miR-491 increased the expression of matrix metalloproteinase 2/9 (MMP-2/9) and the migratory potential; however, in MHCC97H cells, overexpression of miR-491 level decreased the expression of MMP-2/9 and the migratory capacity. Moreover, miR-491 had a positive relationship with E-cadherin level; however, it had a negative relationship with vimentin level both in cell lines and tissue samples of HCC. MiR-491 levels of non-metastasis HCC tissue are higher than that of metastasis HCC tissue. Our results suggest that miR-491 is involved in metastasis of HCC by blocking EMT and decreasing MMP-9 levels, which may provide a new clue for preventing tumour metastasis of HCC.

The expression of chitinase 3-like 1: a novel prognostic predictor for hepatocellular carcinoma

Chitinase 3-like 1 (CHI3L1) is associated with poor prognosis of various human cancers. However, the clinical and prognostic significance of CHI3L1 in hepatocellular carcinoma (HCC) is largely unknown. The aim of the present study is to investigate the expression of CHI3L1 in human HCC cell lines, clinical HCC specimens and its association with expressions of phosphorylated-Akt (p-Akt), E-cadherin and prognostic significance. The protein level of CHI3L1 in HCC cell lines was evaluated by western blot. The mRNA and protein levels of CHI3L1 in 19 self-paired HCC specimens were assessed by RT-PCR and western blot assays. The clinical and prognostic significance of CHI3L1 in 70 cases of HCC patients was determined by immunohistochemistry. In addition, expressions of p-Akt and E-cadherin were also assessed. The protein level of CHI3L1 paralleled with increased malignant potential of HCC cell lines (P < 0.05). The mRNA and protein levels of CHI3L1 in HCC tissues were up-regulated compared with those in adjacent peritumoral tissues and further increased in tumors with metastasis (P < 0.05). Clinicopathological analysis showed that positive CHI3L1 expression was significantly associated with larger tumor size, capsular invasion, advanced TNM stages and status of metastasis (P = 0.035, 0.003, 0.023 and 0.003, respectively). Furthermore, CHI3L1 expression was positively correlated with high level of p-Akt (r = 0.293, P = 0.014), but inversely correlated with expression of E-cadherin (r = -0.267, P = 0.026). Additionally, Kaplan-Meier survival analysis showed that HCC patients with positive CHI3L1 expression had a worse overall survival and disease-free survival compared with those with negative CHI3L1 expression (P < 0.001, respectively). Multivariate analysis identified CHI3L1 as an independent prognostic predictor for overall survival and disease-free survival of HCC patients (P = 0.044 and 0.031, respectively). CHI3L1 plays an essential role in HCC malignancies and may be served as a valuable prognostic biomarker for HCC patients.

B7-H3 is expressed in human hepatocellular carcinoma and is associated with tumor aggressiveness and postoperative recurrence.

B7-H3, a novel B7 family member, positively or negatively regulates T-cell responses. We investigated the clinical relevance and prognostic significance of B7-H3 in hepatocellular carcinoma (HCC). Western blotting showed B7-H3 upregulation in 17 of 24 (70.8 %) HCC tissues compared with nontumor liver tissues (p = 0.028). B7-H3 immunostaining on tissue microarrays containing 240 HCC patient samples indicated that 225 (93.8 %) tumors had aberrant B7-H3 expression, with strong intensity in 79 (32.9 %) cases, whereas B7-H3 expression in peritumor liver cells was weak in most cases (226; 94.2 %). Notably, patients with high/moderate tumor cell B7-H3 expression showed significantly poorer survival (p = 0.009) and increased recurrence (p = 0.002). After multivariable adjustment, high/moderate B7-H3 expression remained significant for an increased risk of recurrence (hazard ratio = 1.79; 95 % confidence interval = 1.19-2.70; p = 0.005). B7-H3 expression correlated with invasive phenotypes like vascular invasion and advanced tumor stage, and the metastatic potential of HCC cell lines. Flow cytometry showed that B7-H3 expression is inversely correlated with proliferation and interferon-γ production by infiltrating T cells. Interferon-γ stimulation significantly upregulated B7-H3 expression in HCC cells in vitro, implicating B7-H3 expression as a feedback mechanism to evade anti-tumor immunity. Importantly, the prognostic value of B7-H3 expression was validated in an independent cohort of 206 HCC patients. Collectively, our data suggest that B7-H3 was abundantly expressed in HCC and was associated with adverse clinicopathologic features and poor outcome. Thus, B7-H3 represents an attractive target for diagnostic and therapeutic manipulation in human HCC.

A possible connection between adhesion regulating molecule 1 overexpression and nuclear factor kappa B activity in hepatocarcinogenesis

Adhesion regulating molecule 1 (ADRM1), a 19S proteasome cap-associated protein, and nuclear factor kappa B (NF-κB), a protein transcription factor controlling DNA transcription, may play an important role in tumorigenesis. Overexpression of ADRM1 and activation of NF-κB are well-observed in hepatocellular carcinoma (HCC). However, little is known about whether both are functionally connected during hepatocarcinogenesis, and the mechanisms involved. In this study, using laboratory techniques including short hairpin RNA (shRNA)-mediated knockdown, immunohistochemistry (IHC), both semi-quantitative and real-time RT-PCR, western blotting, MTT assay, transwell assay, flow cytometry and electrophoretic mobility shift assay (EMSA), the expression of ADRM1, the effects of ADRM1 knockdown on NF-κB activity, as well as the biological behavior of HCC cells including proliferation, migration, invasion and apoptosis were investigated in the samples from HCC patients and HCC cell lines. We found that both mRNA and protein levels of ADRM1 were increased in HCC tissues and that this increase in ADRM1 expression was parallel to the metastatic potential of HCC cell lines. After ADRM1 knockdown in MHCC97-H cells, the expression of IκB-α was increased and the NF-κB activity was reduced. Furthermore, ADRM1 knockdown inhibited MHCC97-H cell proliferation and induced cell apoptosis, and the migration and invasion of MHCC97-H cells were significantly repressed. These results indicate that there is a clear functional connection between ADRM1 and NF-κB in hepatocarcinogenesis, despite the precise mechanisms through which the two work together still being unknown.

Liver Cancer Institute, Affiliated Zhongshan Hospital，Fudan University, Shanghai 200032, China

Objective By analyzing the expression of cluster of differentiation 74(CD74) in hepatocellular carcinoma (HCC) and HCC cell lines, the correlation between the level of CD74 expression and the patients′ prognosis was investigated. Methods The expression of CD74 in high metastatic potential HCC cell lines(MHCC-LM3，MHCC-97H), low metastatic potential HCC cell line(MHCC-97L), and no metastatic potential HCC cell line(Hep-G2) were estimated by Western blot. The paraffin embedded tissues which include intra-tumor and paratumor tissues were collected from 320 patients who had received HCC curative surgical resection and 5 normal liver tissues from the donors of liver tranplantation.The high density tissue micro-array was made of these specimens. Immunol-histochemistry was applied to discover the different levels of CD74 in tumor, paratumor and normal liver tissues. Survival curves were generated by the Kaplan-Meier method and verified by the Logrank test.Cox proportional hazards regression analysis was applied to estimate the prognostic factors in multivariate analysis. Results The expression level of CD74 was significantly higher in low metastatic potential and no metastatic potential HCC cell lines(MHCC-97L 1.224±0.014, Hep-G2 1.374±0.006) than that in high metastatic potential ones(MHCC-LM3 0.622±0.078, MHCC-97H 0.732±0.083).Significant differences can be found between the groups(t=-13.308，-16.849，-10.177，-13.436，-17.057； P 0.05), except gender (χ2=3.954, P<0.05). Kaplan-Meier method showed that patients with positively expression of CD74 had better prognosis than others (χ2=5.620, P<0.05).Cox proportional hazards regression analysis showed that CD74 was a significant and independent prognostic factor of survival ［ hazard ratio (HR)=0.721, 95% confidence interval (CI)=0.522-0.996, P<0.05］. Conclusion The expression of CD74 in hepatocellular carcinoma could be a biomarker of the prognosis and there′s some potential correlation with cancer cell apoptosis.（Chin J Lab Med, 2012,35:47-52） Key words: Carcinoma，hepatocellular; Liver neoplasms; Antigens，differentiation，B-lymphocyte; Histocompatibility antigens class Ⅱ; Prognosis

Thrombin is a therapeutic target for metastatic osteopontin-positive hepatocellular carcinoma

We previously identified osteopontin (OPN) as a promoter and thus a potential therapeutic target for hepatocellular carcinoma (HCC) metastasis. The serine protease thrombin interacts with OPN and can modify its biological activity. To explore the role of thrombin alone or in conjunction with OPN in HCC, we studied the correlation of thrombin levels to HCC prognosis in patients with various OPN levels, and evaluated the effects of OPN fragments generated by thrombin cleavage on proliferation and adhesion of HCC cells. We found that the thrombin level was strongly associated with the metastatic potential of HCC cell lines, and that thrombin was remarkably overexpressed in HCC tissue compared with adjacent nontumor tissue. In addition, HCC tissue from patients with recurrent disease displayed much higher thrombin levels, particularly in those with elevated OPN levels. Only HCCs with elevated OPN levels had a significant correlation between high thrombin levels and overall survival (OS; P < 0.01), or time to recurrence (TTR; P < 0.0001) of HCC. Multivariate analysis revealed that thrombin was an independent prognostic indicator. In vitro assays demonstrated that thrombin promotes the proliferation and adhesion of OPN+ HCC cells. Furthermore, thrombin activated the focal adhesion kinase (FAK) pathway of OPN+ HCC cells, which was blocked by the inhibition of integrin β1. Thrombin plays an important role in OPN-mediated aggressive phenotype of HCC through activation of integrin β1-FAK signaling, and is an independent poor prognostic factor for HCC. Thus, thrombin may be a potential therapeutic target to inhibit HCC metastasis in OPN+ patients.

Chemokine receptor CXCR7 regulates the invasion, angiogenesis and tumor growth of human hepatocellular carcinoma cells

BackgroundIn spite of recent advances in diagnostic and therapeutic measures, the prognosis of hepatocellular carcinoma (HCC) patients remains poor. Therefore, it is crucial to understand what factors are involved in promoting development of HCC. Evidence is accumulating that members of the chemokine receptor family are viewed as promising therapeutic targets in the fight against cancer. More recent studies have revealed that chemokine receptor CXCR7 plays an important role in cancer development. However, little is known about the effect of CXCR7 on the process of HCC cell invasion and angiogenesis. The aim of this study is to investigate the expression of CXCR7 in hepatocellular carcinoma tissues and cell lines and to evaluate the role of CXCR7 in tumor growth, angiogenesis and invasion of HCC cells.MethodsWe constructed CXCR7 expressing shRNA, and CXCR7shRNA was subsequently stably transfected into human HCC cells. We evaluated the effect of CXCR7 inhibition on cell invasion, adhesion, VEGF secretion, tube formation and tumor growth. Immunohistochemistry was done to assess the expression of CXCR7 in human hepatocellular carcinoma tissues and CD31 in tumor of mice. We also evaluated the effect of VEGF stimulation on expression of CXCR7.ResultsCXCR7 was overexpressed in hepatocellular carcinoma tissues. We showed that high invasive potential HCC cell lines express high levels of CXCR7. In vitro, CXCL12 was found to induce invasion, adhesion, tube formation, and VEGF secretion in SMMC-7721 cells. These biological effects were inhibited by silencing of CXCR7 in SMMC-7721 cells. In addition, we also found that VEGF stimulation can up-regulate CXCR7 expression in SMMC-7721 cells and HUVECs. More importantly, enhanced expression of CXCR7 by VEGF was founctional. In vivo, tumor growth and angiogenesis were suppressed by knockdown of CXCR7 in SMMC-7721 cells. However, silencing of CXCR7 did not affect metastasis of tumor in vivo.ConclusionsIncreased CXCR7 expression was found in hepatocellular carcinoma tissues. Knockdown of CXCR7 expression by transfected with CXCR7shRNA significantly inhibits SMMC-7721 cells invasion, adhesion and angiogenesis. Finally, down-regulation of CXCR7 expression lead to a reduction of tumor growth in a xenograft model of HCC. This study provides new insights into the significance of CXCR7 in invasion and angiogenesis of HCC.