Zhenzhen Zhou

Transcriptional up-regulation of FoxM1 in response to hypoxia is mediated by HIF-1.

The proliferation‐specific Forkhead box M1 (FoxM1) transcription factor is overexpressed in cancer cells and acts as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate FoxM1 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the mRNA and protein levels of FoxM1 had a significant increase in cancer cells (HepG2, MCF‐7, and HeLa). Such increase was due to the direct binding of hypoxia‐inducible factor 1 (HIF‐1) to the HIF‐1 binding sites in the FoxM1 promoter. By deletion and mutation assays, we demonstrated that the HIF1‐1 and HIF1‐3/4 binding sites on the FoxM1 promoter were essential for transcriptional activation of FoxM1 by hypoxia. We also demonstrated that HIF‐1α directly bound to the promoter of FoxM1 and the binding was specific, as revealed by HIF‐1 binding/competition assay and chromatin immunoprecipitation assay. Consequently, the up‐regulation of FoxM1 accelerated the growth of hypoxic cancer cells by decreasing nuclear levels of p21 and increasing expression of cyclin B1 and cyclin D1. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of FoxM1 expression in tumor cells. J. Cell. Biochem. 106: 247–256, 2009.

HBx protein induces expression of MIG and increases migration of leukocytes through activation of NF-κB

Elevated expression of monokine induced by the interferon-gamma (MIG) has been shown in HBV carriers, and it is involved in the infiltration of inflammatory cells and liver damage after HBV infection. However, the molecular mechanisms by which HBV-induced MIG expression have not been characterized. Our results indicated that HBx protein induced MIG expression in a dose-dependent manner. Such increase was due to the direct binding of NF-kappaB to the MIG promoter. By luciferase, chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that the NF-kappaB binding site at positions -147 was essential for transcriptional activation of MIG promoter by HBx protein. Chemotaxis assay showed that the up-regulation of MIG protein levels enhanced the migration of peripheral blood lymphocytes (PBLs), and inhibition of NF-kappaB significantly decreased the chemotaxis activity. Our findings provide a new insight into how leukocytes migrate to liver, and disclose a new regulatory mechanism of MIG expression after HBV infection.

Sirtuin 6 promotes transforming growth factor‐β1/H2O2/HOCl‐mediated enhancement of hepatocellular carcinoma cell tumorigenicity by suppressing cellular senescence

Sirtuin 6 (SIRT6) can function as a tumor suppressor by suppressing aerobic glycolysis and apoptosis resistance. However, the negative effect of SIRT6 on cellular senescence implies that it may also have the potential to promote tumor development. Here we report that the upregulation of SIRT6 expression was required for transforming growth factor (TGF)‐β1 and H2O2/HOCl reactive oxygen species (ROS) to promote the tumorigenicity of hepatocellular carcinoma (HCC) cells. Transforming growth factor‐β1/H2O2/HOCl could upregulate SIRT6 expression in HCC cells by inducing the sustained activation of ERK and Smad pathways. Sirtuin 6 in turn abrogated the inducing effect of TGF‐β1/H2O2/HOCl on cellular senescence of HCC cells, and was required for the ERK pathway to efficiently suppress the expression of p16 and p21. Sirtuin 6 altered the effect of Smad and p38 MAPK pathways on cellular senescence, and contributed to the inhibitory effect of the ERK pathway on cellular senescence. However, SIRT6 was inefficient in antagonizing the promoting effect of TGF‐β1/H2O2/HOCl on aerobic glycolysis and anoikis resistance. Intriguingly, if SIRT6 expression was inhibited, the promoting effect of TGF‐β1/H2O2/HOCl on aerobic glycolysis and anoikis resistance was not sufficient to enhance the tumorigenicity of HCC cells. Suppressing the upregulation of SIRT6 enabled TGF‐β1/H2O2/HOCl to induce cellular senescence, thereby abrogating the enhancement of HCC cell tumorigenicity by TGF‐β1/H2O2/HOCl. These results suggest that SIRT6 is required for TGF‐β1/H2O2/HOCl to enhance the tumorigenicity of HCC cells, and that targeting the ERK pathway to suppress the upregulation of SIRT6 might be a potential approach in comprehensive strategies for the therapy of HCC.

β3 Integrin Promotes TGF-β1/H2O2/HOCl-Mediated Induction of Metastatic Phenotype of Hepatocellular Carcinoma Cells by Enhancing TGF-β1 Signaling

In addition to being an important mediator of migration and invasion of tumor cells, β3 integrin can also enhance TGF-β1 signaling. However, it is not known whether β3 might influence the induction of metastatic phenotype of tumor cells, especially non-metastatic tumor cells which express low level of β3. Here we report that H2O2 and HOCl, the reactive oxygen species produced by neutrophils, could cooperate with TGF-β1 to induce metastatic phenotype of non-metastatic hepatocellular carcinoma (HCC) cells. TGF-β1/H2O2/HOCl, but not TGF-β1 or H2O2/HOCl, induced β3 expression by triggering the enhanced activation of p38 MAPK. Intriguingly, β3 in turn promoted TGF-β1/H2O2/HOCl-mediated induction of metastatic phenotype of HCC cells by enhancing TGF-β1 signaling. β3 promoted TGF-β1/H2O2/HOCl-induced expression of itself via positive feed-back effect on p38 MAPK activation, and also promoted TGF-β1/H2O2/HOCl-induced expression of α3 and SNAI2 by enhancing the activation of ERK pathway, thus resulting in higher invasive capacity of HCC cells. By enhancing MAPK activation, β3 enabled TGF-β1 to augment the promoting effect of H2O2/HOCl on anoikis-resistance of HCC cells. TGF-β1/H2O2/HOCl-induced metastatic phenotype was sufficient for HCC cells to extravasate from circulation and form metastatic foci in an experimental metastasis model in nude mice. Inhibiting the function of β3 could suppress or abrogate the promoting effects of TGF-β1/H2O2/HOCl on invasive capacity, anoikis-resistance, and extravasation of HCC cells. These results suggest that β3 could function as a modulator to promote TGF-β1/H2O2/HOCl-mediated induction of metastatic phenotype of non-metastatic tumor cells, and that targeting β3 might be a potential approach in preventing the induction of metastatic phenotype of non-metastatic tumor cells.

AEG-1 promotes anoikis resistance and orientation chemotaxis in hepatocellular carcinoma cells.

Metastasis contributes to the poor prognosis of hepatocellular carcinoma (HCC). Anoikis resistance and orientation chemotaxis are two important and sequential events in tumor cell metastasis. The process of tumor metastasis is known to be regulated by AEG-1, an important oncogene that plays a critical role in tumor metastasis, though the effects of this oncogene on anoikis resistance and orientation chemotaxis in HCC cells are currently unknown. To directly assess the role of AEG-1 in these processes, we up-regulated AEG-1 expression via exogenous transfection in SMMC-7721 cells, which express low endogenous levels of AEG-1; and down-regulated AEG-1 expression via siRNA-mediated knockdown in MHCC-97H and HCC-LM3 cells, which express high endogenous levels of AEG-1. Our data directly demonstrate that AEG-1 promotes cell growth as assessed by cell proliferation/viability and cell cycle analysis. Furthermore, the prevention of anoikis by AEG-1 correlates with decreased activation of caspase-3. AEG-1-dependent anoikis resistance is activated via the PI3K/Akt pathway and is characterized by the regulation of Bcl-2 and Bad. The PI3K inhibitor LY294002 reverses the AEG-1 dependent effects on Akt phosphorylation, Bcl-2 expression and anoikis resistance. AEG-1 also promotes orientation chemotaxis of suspension-cultured cells towards supernatant from Human Pulmonary Microvascular Endothelial Cells (HPMECs). Our results show that AEG-1 activates the expression of the metastasis-associated chemokine receptor CXCR4, and that its ligand, CXCL12, is secreted by HPMECs. Furthermore, the CXCR4 antoagonist AMD3100 decreases AEG-1-induced orientation chemotaxis. These results define a pathway by which AEG-1 regulates anoikis resistance and orientation chemotaxis during HCC cell metastasis.

Expression of metadherin/AEG-1 gene is positively related to orientation chemotaxis and adhesion of human hepatocellular carcinoma cell lines of different metastatic potentials

Metastasis contributes to the poor prognosis of hepatocellular carcinoma (HCC). However, the mechanism through which a primary HCC cell develops into a metastatic phenotype is not well understood. The purpose of this study was to examine the correlation between metadherin (MTDH)/astrocyte elevated gene-1 (AEG-1) expression in HCC cell lines of different metastatic potentials and such metastatic phenotypes as orientation chemotaxis and adhesion. MTDH/AEG-1 expression was detected by RT-PCR and western blotting in HCC cell lines (HepG2, Huh7, Sk-HEP-1, MHCC-97H). Distribution of MTDH/AEG-1 was observed by immunofluorescence staining and confocal laser scanning microscopy. The abilities of orientation chemotaxis and adhesion and the index of interaction between HCC cell lines and microvascular endothelial cell lines (MVECs, including HUVECs and HPMECs) were measured by chemotaxis assay and adhesion assay, respectively. The results showed that MTDH/AEG-1 protein expression was significantly higher in high metastatic potential cancer cell lines (Sk-HEP-1, MHCC-97H) than in low metastatic potential cell lines (HepG2, Huh7) (P<0.05). The MTDH/AEG-1 protein was localized in the perinuclear region of HCC cells. Furthermore, the abilities of orientation chemotaxis and adhesion of HCC cells to HPMECs were increased as compared with those of HCC cells to HUVECs (P<0.05). The abilities of orientation chemotaxis and adhesion were much stronger in Sk-HEP-1 and MHCC-97H cells with MTDH/AEG-1 highly expressed than in HepG2 and Huh7 cells with MTDH/AEG-1 lowly expressed (P<0.05). These results suggested that the expression of MTDH/AEG-1 gene in HCC cell lines of different metastatic potentials was closely positively related to the abilities of orientation chemotaxis and adhesion of HCC cells. It was deduced that MTDH/AEG-1 might play a pivotal role in the lung-specific metastasis of HCC, which may be mediated through orientation chemotaxis and adhesion abilities of HCC cells. MTDH/AEG-1 may serve as a potential therapeutic target for HCC.SummaryMetastasis contributes to the poor prognosis of hepatocellular carcinoma (HCC). However, the mechanism through which a primary HCC cell develops into a metastatic phenotype is not well understood. The purpose of this study was to examine the correlation between metadherin (MTDH)/astrocyte elevated gene-1 (AEG-1) expression in HCC cell lines of different metastatic potentials and such metastatic phenotypes as orientation chemotaxis and adhesion. MTDH/AEG-1 expression was detected by RT-PCR and western blotting in HCC cell lines (HepG2, Huh7, Sk-HEP-1, MHCC-97H). Distribution of MTDH/AEG-1 was observed by immunofluorescence staining and confocal laser scanning microscopy. The abilities of orientation chemotaxis and adhesion and the index of interaction between HCC cell lines and microvascular endothelial cell lines (MVECs, including HUVECs and HPMECs) were measured by chemotaxis assay and adhesion assay, respectively. The results showed that MTDH/AEG-1 protein expression was significantly higher in high metastatic potential cancer cell lines (Sk-HEP-1, MHCC-97H) than in low metastatic potential cell lines (HepG2, Huh7) (P<0.05). The MTDH/AEG-1 protein was localized in the perinuclear region of HCC cells. Furthermore, the abilities of orientation chemotaxis and adhesion of HCC cells to HPMECs were increased as compared with those of HCC cells to HUVECs (P<0.05). The abilities of orientation chemotaxis and adhesion were much stronger in Sk-HEP-1 and MHCC-97H cells with MTDH/AEG-1 highly expressed than in HepG2 and Huh7 cells with MTDH/AEG-1 lowly expressed (P<0.05). These results suggested that the expression of MTDH/AEG-1 gene in HCC cell lines of different metastatic potentials was closely positively related to the abilities of orientation chemotaxis and adhesion of HCC cells. It was deduced that MTDH/AEG-1 might play a pivotal role in the lung-specific metastasis of HCC, which may be mediated through orientation chemotaxis and adhesion abilities of HCC cells. MTDH/AEG-1 may serve as a potential therapeutic target for HCC.

Knockdown of astrocyte elevated gene‑1 inhibits growth through suppression of IL‑6 secretion in HepG2 human hepatoma cells

Astrocyte-elevated gene-1 (AEG-1) has been reported to be associated with cancer progression in various types of human cancers, including liver cancer. However, to date, the molecular mechanism of AEG-1 action on the growth of liver cancer cells has been poorly elucidated. The present study aimed to investigate the effect of AEG-1 on the proliferation and apoptosis of liver cancer cells and the role of IL-6 in this process using the HepG2 human hepatoma cell line. shRNAs targeting AEG-1 were used to silence the expression of AEG-1. The effects on cell growth were detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, colony formation and cell cycle assays. Apoptosis was analyzed by flow cytometry. The expression of IL-6 was examined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, and the phosphorylation of Stat3 was detected by western blotting. AEG-1 knockdown was observed to induce cell proliferation inhibition and apoptosis through the suppression of IL-6 secretion. Stat3, a downstream target of IL-6 signaling, was suppressed in the AEG-1-silenced cells and target genes, including Bcl-2 and crystalin, αB, which are associated with cell survival, were downregulated. Overall, the findings suggest that aberrant AEG-1 expression promotes the growth of HepG2 liver cancer cells, contributing to the progression of liver cancer, which may partly be mediated by IL-6 signaling.

Suppressive effect of SATB1 on hepatic stellate cell activation and liver fibrosis in rats

Liver fibrosis is a worldwide clinical issue. Activation of hepatic stellate cells (HSCs) is the central event during liver fibrosis. We investigated the role of SATB1 in HSC activation and liver fibrogenesis. The results show that SATB1 expression is reduced during HSC activation. Additionally, SATB1 inhibits HSC activation, proliferation, migration, and collagen synthesis. Furthermore, CTGF, a pro‐fibrotic agent, is also significantly inhibited by SATB1 through the Ras/Raf‐1/MEK/ERK/Ets‐1 pathway. In vivo, SATB1 deactivates HSCs and attenuates fibrosis in TAA‐induced fibrotic rat livers. These data indicate that SATB1 plays an important role in HSC activation and liver fibrosis.