Xiaonan Kang

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)

NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal transition through NODAL/SMAD3 signaling pathway.

NANOG is a major transcription factor essential to the stem cell self-renewal and is associated with tumor malignancy, but the NANOG signaling in cancer metastasis is still elusive. In this study, we determined the expression of NANOG in hepatocellular carcinoma (HCC) and investigated its underlying mechanism in the metastasis of HCC. The expression levels of NANOG were examined in tumor tissues by immunohistochemistry. Functional effect of NANOG was investigated both in vivo and in vitro. Our data shows that high level of NANOG expression correlates with metastasis and low survival rate in HCC. HCC cells overexpressing NANOG are characterized by active epithelial-mesenchymal transition (EMT), and exhibit increased ability of invasion, soft agar colonization, sphere formation and drug resistance, whereas SB-431542, an antagonist of activin receptor-like kinase (ALK) receptors, attenuates EMT and invasion of HCC cells. NANOG activates NODAL and CRIPTO-1 to promote SMAD3 phosphorylation and SNAIL expression. The transcriptional activity of NODAL gene is dependent on two NANOG binding motifs in its promoter region. This study shows a significant correlation between the NANOG expression and the expression of NODAL, P-SMAD3 or SNAIL, and the combination of NANOG and P-SMAD3 is a potential predictor of poor prognosis of HCC. Additionally, cells in the tumor edge area displays higher NANOG expression than cells in the tumor center. These results present novel mechanistic insight into an important role of NANOG in HCC metastasis, and suggest a potential application of NANOG in HCC prognosis and treatment.

iTRAQ-2DLC-ESI-MS/MS based identification of a new set of immunohistochemical biomarkers for classification of dysplastic nodules and small hepatocellular carcinoma.

The study aims to develop novel clinical immunohistochemical biomarkers for distinguishing small hepatocellular carcinoma (sHCC) from dysplastic nodules (DN). iTRAQ-2DLC-ESI-MS/MS technique was used to screen immunohistochemical biomarkers between precancerous lesions (liver cirrhosis and DN) and sHCC. A total of 1951 proteins were quantified, including 52 proteins upregulated in sHCC and 95 proteins downregulated in sHCC by at least 1.25- or 0.8-fold at p < 0.05. The selected biomarker candidates were further verified using Western blotting and immunohistochemistry. Furthermore, receiver operation characteristics (ROC) curves and logistic regression model were carried out to evaluate the diagnostic values of the biomarkers. Finally, aminoacylase-1 (ACY1) and sequestosome-1 (SQSTM1) were chosen as novel candidate biomarkers for distinction of sHCC from DN. A constructed logistic regression model included ACY1, SQSTM1, and CD34. The sensitivity and specificity of this model for distinguishing sHCC from DN was 96.1% and 96.7%. In conclusion, ACY1 and SQSTM1 were identified as novel immunohistochemical biomarkers distinguishing sHCC from DN. In conclusion, expression levels of CD34, ACY1, and SQSTM1 can be used to establish an accurate diagnostic model for distinction of sHCC from DN.

Sox2 expression predicts poor survival of hepatocellular carcinoma patients and it promotes liver cancer cell invasion by activating Slug

Sox2 is a major transcription factor essential to the stemness characteristics and is associated with various types of cancers. In this study, we investigated the expressions and functional roles of Sox2 in hepatocellular carcinoma (HCC). Our data show that high level of Sox2 expression correlates with metastasis and low survival rate in HCC. HCC cells overexpressing Sox2 are characterized by active epithelial-mesenchymal transition and exhibit increased ability of transwell invasion, soft agar colonization, and sphere formation. We also found Sox2 expression was correlated with the transcription activity of SLUG promoter region. These results present novel mechanistic insight into an important role of Sox2 in HCC and suggest a potential application of Sox2 in HCC prognosis and treatment.

Tumor-derived secretory clusterin induces epithelial–mesenchymal transition and facilitates hepatocellular carcinoma metastasis

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Metastasis is the major concern that causes death in HCC. The goal of this study was to identify tumor-derived proteins in serum during HCC metastasis using an orthotopic xenograft tumor model and explore the role of key protein in HCC metastasis. Serum samples collected from HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1 wk, 3 wks and 6 wks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Twenty tumor-derived proteins were identified through human specific peptides. Secretory clusterin (sCLU), which was significantly upregulated during cancer progression and metastasis, was chosen for further study. The expression of sCLU was significantly higher in metastatic HCC cell lines and samples from metastatic HCC patients. ShRNA-mediated down-regulation of sCLU resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary metastasis in vivo. Overexpression of sCLU in HepG2 cell line showed increased cell migratory ability. Further study found that sCLU contributed to HCC migration and epithelial-mesenchymal transition (EMT) in vitro, and metastasis in vivo. In addition, sCLU also plays an important role in the regulation of TGF-β1-smad3 signaling. These findings suggest that sCLU may promote HCC metastasis via the induction of EMT process and may be a candidate target for HCC therapy.

Clusterin protects hepatocellular carcinoma cells from endoplasmic reticulum stress induced apoptosis through GRP78.

Clusterin (CLU) is a stress-activated chaperone, which plays an important role in cancer development and progression through promoting cell survival. However, the exact mechanism of how CLU exerts its cell protective role under ER stress condition is still unclear. Therefore, in order to explore the molecular mechanisms by which CLU inhibited ER stress-induced apoptosis, HCC cell lines were treated with tunicamycin (TN), an ER stress inducer. We found that the expressions of both CLU and GRP78 were increased after TN treatment. Knockdown of CLU expression in SMMC7721 and HCCLM3 cells inhibited GRP78 expression after TN treatment and enhanced ER stress-induced apoptosis, whereas over-expression of CLU in HepG2 cells increased GRP78 expression after TN induction and abolished the effect of TN on cell apoptosis. Furthermore, knockdown of GRP78 expression in CLU-HepG2 cells abrogated the protective role of CLU under ER stress condition. Co-immunoprecipitation (co-IP) and confocal microscopy experiments confirmed the direct interaction between CLU and GRP78 under ER stress condition. The effect of CLU knockdown on GRP78 expression and cell apoptosis in HCC tumors were further determined in orthotopic xenograft tumor model. Knockdown of CLU expression in HCCLM3 cells inhibited GRP78 expression in tumor tissues, accompanied with increased number of apoptotic cancer cells. Moreover, the correlation between CLU and GRP78 expression was further determined in clinical HCC specimens. Taken together, these findings reveal that CLU protects HCC cells from ER stress induced apoptosis at least partially through interacting with GRP78.

Cell Surface Glycan Alterations in Epithelial Mesenchymal Transition Process of Huh7 Hepatocellular Carcinoma Cell

Background and Objective Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC) is high. It is well known that the epithelial mesenchymal transition (EMT) and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. Methodology HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. Results After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α) GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. Conclusions The findings of this study systematically clarify the alterations of cell surface glycan in cancer EMT, and may provide novel insight for 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.

N-linked glycan changes of serum haptoglobin β chain in liver disease patients

Human haptoglobin is a serum glycoprotein secreted by the liver with four potential N-glycosylation sites on its β chain. Many studies have reported glycan changes of haptoglobin in diseases such as breast cancer and pancreatic cancer. The objective of our study is to analyze N-linked glycan alterations of serum haptoglobin β chain obtained from patients with the hepatitis B virus (HBV), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). MALDI-QIT-TOF mass spectrometry revealed the intensity of m/z 1809.6, identified as a fucosylated glycan, was much higher in samples from patients with LC and HCC relative to the patients with HBV and healthy controls. Compared with LC patients, triantennary glycan was elevated and the biantennary structure was decreased in the haptoglobin β chain of HCC patients. Thus, alterations in the glycan structure of the haptoglobin β chain may constitute significant spectral signatures of cirrhosis and HCC disease.

Novel synergistic antitumor effects of rapamycin with bortezomib on hepatocellular carcinoma cells and orthotopic tumor model

BackgroundDespite recent advances in the treatment of hepatocellular carcinoma (HCC), the chemotherapy efficacy against HCC is still unsatisfactory. The mammalian target of rapamycin (mTOR) has been emerged as an important cancer therapeutic target. However, HCC cells often resistant to rapamycin because of the paradoxical activation of Akt by rapamycin. In this study, we investigated whether bortezomib could enhance the antitumor effects of rapamycin.MethodsThe effects of rapamycin and bortezomib on HCC proliferation, apoptosis, migration, and invasiveness in vitro were assessed by CCK-8 analysis, flow cytometry, Hoechst 33342 staining and transwell assays, respectively. Total and phosphorylated protein levels of Akt were detected by Western blotting. The effects of rapamycin and/or bortezomib on the mRNA expression levels of p53, p27, p21 and Bcl-2 family in HCCLM3 cells were evaluated by RT-PCR. The roles of rapamycin and bortezomib on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. The effects of rapamycin and bortezomib on cell proliferation and apoptosis in vivo were test by PCNA and TUNEL staining.ResultsBortezomib synergized with rapamycin to reduce cell growth, induce apoptosis, and inhibit cell mobility in vitro. Further mechanistic studies showed that bortezomib inhibited rapamycin-induced phosphorylated Akt, which in turn enhanced apoptosis of HCC cell lines. The alteration of the mRNA expression of cell cycle inhibitors p53, p27, p21 and apoptosis associated genes Bcl-2, Bax were also involved in the synergistic antitumor effects of rapamycin and bortezomib. P53 inhibitor PFT-α significantly attenuate the effect of rapamycin and bortezomib on cell apoptosis, which indicated that the pro-apoptotic effect of rapamycin and bortezomib may be p53-dependent. Treatment of HCCLM3-R bearing nude mice with rapamycin and bortezomib significantly enhanced tumor growth inhibition (72.4%), comparing with either rapamycin- (54.7%) or bortezomib-treated mice (22.4%). In addition, the lung metastasis was significantly suppressed in mice received the combination treatment (16.6%). The combination treatment of rapamycin and bortezomib significantly inhibited tumor cell proliferation and tumor angiogenesis in vivo.ConclusionThe combination of rapamycin with bortezomib could be a novel and promising therapeutic approach to the treatment of HCC.