Limin Xia

Upregulated FoxM1 expression induced by hepatitis B virus X protein promotes tumor metastasis and indicates poor prognosis in hepatitis B virus-related hepatocellular carcinoma

BACKGROUND & AIMS Forkhead box M1 (FoxM1) is a master regulator of tumor metastasis that plays an important role in the development of hepatocellular carcinoma (HCC). However, whether or not FoxM1 contributes to the progression of HBV-associated HCC (HBV-HCC) remains unknown. Therefore, we aimed at investigating the clinicopathologic significance of FoxM1 in HBV-HCC and the potential role of FoxM1 in hepatitis B virus X (HBx)-mediated invasiveness and metastasis. METHODS The expression of FoxM1 and its functional targets matrix metalloproteinase-7 (MMP-7), RhoC, and Rho-kinase 1 (ROCK1) in human HBV-HCC tissues was detected by immunohistochemistry. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure the transcriptional regulation of FoxM1 promoter by HBx. The effect of FoxM1 on HBx-mediated invasiveness and metastasis was analyzed by transwell assays and an orthotopic metastatic model. RESULTS FoxM1 overexpression correlated with multiple malignant characteristics and indicated poor prognosis of HBV-HCC patients. FoxM1 expression was an independent factor affecting the recurrence and survival of patients with HBV-HCC after surgical resection. FoxM1 promoted hepatoma cell invasion and metastasis by promoting MMP-7, RhoC, and ROCK1 expression, while FoxM1 overexpression was associated with elevated expressions of these proteins in HBV-HCC tissues. HBx upregulated FoxM1 expression through the ERK/CREB pathway, and FoxM1 inhibition significantly decreased HBx-enhanced hepatoma cell invasion in vitro and lung metastasis in vivo. CONCLUSIONS We report a new molecular mechanism for HBV-associated hepatocarcinogenesis that involves the activation of FoxM1 expression by HBx through the ERK/CREB pathway, thereby leading to invasion and metastasis of hepatoma cells.

Forkhead box Q1 promotes hepatocellular carcinoma metastasis by transactivating ZEB2 and VersicanV1 expression.

Forkhead box Q1 (FoxQ1) is a master regulator of tumor metastasis. However, the molecular mechanism of FoxQ1 in regulating hepatocellular carcinoma (HCC) metastasis remains unknown. Here we report a novel function for FoxQ1 in modifying the tumor microenvironment to promote HCC metastasis. FoxQ1 expression was an independent and significant risk factor for the recurrence and survival in two independent cohorts totaling 1,002 HCC patients. FoxQ1 induced epithelial‐mesenchymal transition (EMT) through the transactivation of ZEB2 expression by directly binding to the ZEB2 promoter. Knockdown of ZEB2 decreased FoxQ1‐enhanced HCC metastasis, whereas up‐regulation of ZEB2 rescued the decreased metastasis induced by FoxQ1 knocking down. Additionally, serial deletion, site‐directed mutagenesis, and a chromatin immunoprecipitation assays showed that VersicanV1, which promoted HCC metastasis and macrophage attraction, was a direct transcriptional target of FoxQ1. FoxQ1‐induced VersicanV1 expression promoted the secretion of chemokine (C‐C motif) ligand 2 (CCL2) from HCC cells. Chemotaxis assay showed that the culture media from FoxQ1‐overexpressing HCC cells increased the migratory activity of the macrophages. Inhibition of VersicanV1 and CCL2 expression significantly inhibited FoxQ1‐mediated macrophage migration. In animal studies, the up‐regulation of FoxQ1 in HCC cells promoted HCC metastasis and intratumoral tumor associated macrophage (TAM) infiltration, whereas knockdown of VersicanV1 reduced FoxQ1‐mediated HCC metastasis and intratumoral TAM infiltration. Depletion of macrophages using clodronate liposomes dramatically decreased FoxQ1‐enhanced HCC metastasis. In human HCC tissues, FoxQ1 expression was positively correlated with ZEB2 and VersicanV1 expression and intratumoral TAM infiltration. Patients with positive coexpression of FoxQ1 and ZEB2, FoxQ1, and VersicanV1, or FoxQ1 and intratumoral TAMs were associated with poorer prognosis. Conclusion: FoxQ1 promotes HCC metastasis by transactivating ZEB2 and VersicanV1 expression, resulting in the induction of EMT and the recruitment of macrophage infiltration. (Hepatology 2014;59:958–973)

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.

Transcriptional upregulation of HSP70-2 by HIF-1 in cancer cells in response to hypoxia.

Heat shock protein 70‐2 (HSP70‐2) can be expressed by cancer cells and act as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate HSP70‐2 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the expression of HSP70‐2 had a significant increase in cancer cells. Such increase was due to the direct binding of hypoxia‐inducible factor to hypoxia‐responsive elements (HREs) in the HSP70‐2 promoter. By luciferase assays, we demonstrated that the HRE1 at position −446 was essential for transcriptional activation of HSP70‐2 promoter under hypoxic conditions. We also demonstrated that HIF‐1α binds to the HSP70‐2 promoter and the binding is specific, as revealed by HIF binding/competition and chromatin immunoprecipitation assays. Consequently, the upregulation of HSP70‐2 enhanced the resistance of tumor cells to hypoxia‐induced apoptosis. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of HSP70‐2 expression in tumor cells.

Upregulation of IL-23 Expression in Patients with Chronic Hepatitis B Is Mediated by the HBx/ERK/NF-κB Pathway

IL-23 is a newly discovered proinflammatory cytokine that contributes to the maintenance and expansion of Th17 cells. IL-23 has recently been identified as playing a critical role in a number of chronic inflammatory diseases. However, the regulatory mechanism of IL-23 in chronic hepatitis B (CHB) remains largely unknown. The aims of this study were to detect the expression of IL-23 in CHB patients and to explore the molecular mechanism of hepatitis B virus (HBV)-induced IL-23 expression. Serum levels and hepatic expression of IL-23 were significantly upregulated in CHB patients. A positive correlation was found between IL-23 expression and the histological activity index score, HBV DNA load, and serum alanine aminotransferase and aspartate aminotransferase levels. HBx protein increased IL-23 expression in a dose-dependent manner. It also aided in the nuclear translocation of NF-κB, which directly bound to the promoters of IL-23 subunits p19 and p40 to facilitate their transcription. NF-κB inhibitors blocked the effect of HBx on IL-23 induction, and NF-κB subunits p65 and p50 increased the augmented IL-23 expression. Inhibition of ERK1/2 activation and transfection with ERK dominant-negative plasmid significantly blocked the HBx-induced IL-23 expression. Furthermore, PI3K and Ras–MEK–MAPK inhibitors significantly decreased the ERK1/2 activation and IL-23 expression. Thus, we report a new molecular mechanism for HBV-induced IL-23 expression, which involves the activation of the ERK/NF-κB pathway by HBx, leading to the transactivation of the IL-23 p19 and p40 promoters.

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.

Sox12, a direct target of FoxQ1, promotes hepatocellular carcinoma metastasis through up-regulating Twist1 and FGFBP1

Metastasis is the main reason for high recurrence and poor survival of hepatocellular carcinoma (HCC) after curative resection. However, the molecular mechanism underlying HCC metastasis remains unclear. Here, we report on a novel function of SRY (sex determining region Y)‐box 12 (Sox12), a member of the SYR‐related high mobility group box family proteins, in promoting HCC metastasis. Overexpression of Sox12 was significantly correlated with loss of tumor encapsulation, microvascular invasion, and a higher tumor‐nodule‐metastasis (TNM) stage and indicated poor prognosis in human HCC patients. Sox12 expression was an independent and significant risk factor for recurrence and reduced survival after curative resection. Overexpression of Sox12 induced epithelial‐mesenchymal transition by transactivating Twist1 expression. Down‐regulation of Twist1 decreased Sox12‐enhanced HCC migration, invasion, and metastasis, whereas up‐regulation of Twist1 rescued the decreased migration, invasion, and metastasis induced by Sox12 knockdown. Additionally, serial deletion, site‐directed mutagenesis, and chromatin immunoprecipitation assays showed that fibroblast growth factor binding protein 1 (FGFBP1) was a direct transcriptional target of Sox12. Knockdown of FGFBP1 decreased Sox12‐mediated HCC invasion and metastasis, whereas overexpression of FGFBP1 rescued the decreased invasion and metastasis induced by Sox12 knockdown. Furthermore, forkhead box Q1 (FoxQ1) directly bound to the Sox12 promoter and transactivated its expression, which contributed to Sox12 overexpression in human HCC. Knockdown of Sox12 dramatically decreased FoxQ1‐mediated HCC metastasis. In two independent cohorts of human HCC tissues, Sox12 expression was positively correlated with Twist1, FGFBP1, and FoxQ1 expression, and patients with positive coexpression of Sox12/Twist1, Sox12/FGFBP1, or FoxQ1/Sox12 were associated with poorer prognosis. Conclusion: Up‐regulated Sox12 induced by FoxQ1 promotes HCC invasion and metastasis by transactivating Twist1 and FGFBP1 expression. Thus, our study implicates Sox12 as a potential prognostic biomarker and a novel therapeutic target for HCC. (Hepatology 2015;61:1920–1933)

ACP5, a direct transcriptional target of FoxM1, promotes tumor metastasis and indicates poor prognosis in hepatocellular carcinoma.

Tartrate-resistant acid phosphatase 5 (ACP5), which is essential for bone resorption and osteoclast differentiation, promotes cell motility through the modulation of focal adhesion kinase phosphorylation. However, whether ACP5 contributes to the metastasis and progression of hepatocellular carcinoma (HCC) remains unknown. In this paper, a complementary DNA microarray, serial deletion, site-directed mutagenesis and a chromatin immunoprecipitation assays confirmed that ACP5 is a direct transcriptional target of Forkhead box M1 (FoxM1). ACP5 expression was markedly higher in HCC tissues compared with adjacent noncancerous tissues. ACP5 overexpression was correlated with microvascular invasion, poor differentiation and higher tumor-node-metastasis stage. HCC patients with positive ACP5 expression had poorer prognoses than those with negative ACP5 expression. A multivariate analysis revealed that ACP5 expression was an independent and significant risk factor for disease recurrence and reduced-patient survival following curative resection. Transwell assays and an orthotopic metastatic model showed that the upregulation of ACP5 promoted HCC invasion and lung metastasis, whereas ACP5 knockdown inhibited these processes. The knockdown of ACP5 significantly attenuated FoxM1-enhanced invasion and lung metastasis. Immunohistochemistry revealed that ACP5 expression was positively correlated with FoxM1 expression in human HCC tissues, and their coexpression was associated with poor prognoses. In summary, ACP5 is a direct transcriptional and functional target of FoxM1. This novel FoxM1/ACP5 signaling pathway promotes HCC metastasis and may be a candidate biomarker for prognosis and a target for new therapies.

Loss of Barx1 promotes hepatocellular carcinoma metastasis through up-regulating MGAT5 and MMP9 expression and indicates poor prognosis

Metastasis is the major dominant reason for poor prognosis of hepatocellular carcinoma (HCC) after surgical treatment. However, the molecular mechanism of metastasis has not been well characterzied. Here, we report a novel function of Barx homeobox1 (Barx1) in inhibiting HCC invasion and metastasis. Barx1 expression is significantly decreased in human HCC tissues than in adjacent non-tumorous tissues and normal liver tissues. Low Barx1 expression is correlated with higher tumor-nodule-metastasis stage and indicates poor prognosis. Down-regulation of Barx1 promotes HCC migration, invasion and metastasis, whereas up-regulation of Barx1 inhibits HCC migration, invasion and metastasis. Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase 5 (MGAT5) and matrix metallopeptidase 9 (MMP9) are direct target genes of Barx1. Knockdown of Barx1 up-regulates MGAT5 and MMP9 expression in HCC cells with low metastatic capability, whereas over-expression of Barx1 suppresses their expression in HCC cells with high metastatic capability. Knockdown of both MGAT5 and MMP9 significantly decreases the invasion and metastasis abilities induced by Barx1 knockdown. Barx1 expression is negatively correlated with MGAT5 and MMP9 expression in human HCC tissues. Patients with low expression of Barx1 and high expression of MGAT5 or MMP9 are associated with poorer prognosis. Thus, loss of Barx1 represents a prognostic biomarker in human HCC patients.Metastasis is the major dominant reason for poor prognosis of hepatocellular carcinoma (HCC) after surgical treatment. However, the molecular mechanism of metastasis has not been well characterzied. Here, we report a novel function of Barx homeobox1 (Barx1) in inhibiting HCC invasion and metastasis. Barx1 expression is significantly decreased in human HCC tissues than in adjacent non-tumorous tissues and normal liver tissues. Low Barx1 expression is correlated with higher tumor-nodule-metastasis stage and indicates poor prognosis. Down-regulation of Barx1 promotes HCC migration, invasion and metastasis, whereas up-regulation of Barx1 inhibits HCC migration, invasion and metastasis. Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase 5 (MGAT5) and matrix metallopeptidase 9 (MMP9) are direct target genes of Barx1. Knockdown of Barx1 up-regulates MGAT5 and MMP9 expression in HCC cells with low metastatic capability, whereas over-expression of Barx1 suppresses their expression in HCC cells with high metastatic capability. Knockdown of both MGAT5 and MMP9 significantly decreases the invasion and metastasis abilities induced by Barx1 knockdown. Barx1 expression is negatively correlated with MGAT5 and MMP9 expression in human HCC tissues. Patients with low expression of Barx1 and high expression of MGAT5 or MMP9 are associated with poorer prognosis. Thus, loss of Barx1 represents a prognostic biomarker in human HCC patients.

382 Upregulated FOXM1 Expression Induced by Hepatitis B Virus X Protein Promotes Tumor Metastasis and Indicates Poor Prognosis in Hepatitis B Virus-Related Hepatocellular Carcinoma

Background and Aims: Smad7 has been reported to inhibit inflammation. The role of Smad7 in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. We investigated the functional significance of Smad7 in HCC both In Vivo and In Vitro. Methods: Smad7 knockout (KO) and wildtype (WT) littermates were single injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days to induce HCC. The biological function of Smad7 was determined In Vitro in human HCC cell line (Hep3B) and primary cultures established from resected HCCs from KO and WT mice. Cell growth was evaluated by cell viability and colony formation, cell apoptosis by Annexin V/7-AAD double staining, cell cycle distribution by flow cytometry and cell invasion ability by Matrigel invasion assay. Signaling pathways and downstream effectors of Smad7 in HCC was evaluated by luciferase reporter activity assay and pathway-PCR-array. Results: Smad7 KO mice were more susceptible to DENinduced HCC than WT mice (78% vs 22%, P=0.018). HCCs from KO mice displayed a greater proliferative activity (P<0.01), and a reduced apoptotic index compared with the WT littermates (P<0.05). Smad7 was down-regulated in human HCC tissues compared to their adjacent non-tumor tissues (n=11, P<0.05). These In Vivo results indicated that Smad 7 may function as a potential tumor suppressor, which was further functionally tested In Vitro. Ectopic expression of Smad7 in Hep3B cells suppressed cell growth as evidenced by retarded cell growth curve (P<0.0001) and colony formation assay (P<0.01). Conversely, Smad7 increased cell apoptosis (P<0.05), consistent with enhanced cleaved caspase-9, caspase-3 and PARP, indicative of Smad7-induced apoptosis through the intrinsic caspase dependent pathway. Cell cycle analysis in Heb3B transfected with Smad7 revealed an increased proportion of cells arrested at G0/G1 phase (P<0.001), concomitant with a reduction in the proportion of cells in S-phase (P<0.001) as compared with control. Smad7 also repressed cell invasive ability (P<0.05). Moreover, primary culture cells form HCCs of KO mice showed a increased cell growth curve (P<0.01) and colony formation (P<0.01) compared with the cells generated from HCCs of WT mice. Moreover, pathway luciferase reporter assay indicated that Smad7 inhibited NF-κB and TGF-β signalings. Co-immunoprecipitation analysis showed that Smad7 directly interacts with TAB2, an upstream activator of NF-κB. The downstream targets of Smad7 was further revealed by TGF-β and NF-κB pathway PCR array as shown in Figure 1, which contribute to the tumor suppressive effect. Conclusion: Loss of Smad7 is sufficient to enhance susceptibility to HCC. Smad7 suppresses HCC growth through reducing cell proliferation and invasion, inducing G0/G1 phase arrest and apoptosis by inhibiting NF-κB and TGF-β signalings. Thus, Smad7 acts as a tumor-suppressor in the liver.