Dean Tian

Overexpression of forkhead box C1 promotes tumor metastasis and indicates poor prognosis in hepatocellular carcinoma.

Recurrence and metastasis remain the most common causes of lethal outcomes in hepatocellular carcinoma (HCC) after curative resection. Thus, it is critical to discover the mechanisms underlying HCC metastasis. Forkhead box C1 (FoxC1), a member of the Fox family of transcription factors, induces epithelial‐mesenchymal transition (EMT) and promotes epithelial cell migration. However, the role of FoxC1 in the progression of HCC remains unknown. Here, we report that FoxC1 plays a critical role in HCC metastasis. FoxC1 expression was markedly higher in HCC tissues than in adjacent noncancerous tissues. HCC patients with positive FoxC1 expression had shorter overall survival times and higher recurrence rates than those with negative FoxC1 expression. FoxC1 expression was an independent, significant risk factor for recurrence and survival after curative resection. FoxC1 overexpression induced changes characteristic of EMT and an increase in HCC cell invasion and lung metastasis. However, FoxC1 knockdown inhibited these processes. FoxC1 transactivated Snai1 expression by directly binding to the Snai1 promoter, thereby leading to the inhibition of E‐cadherin transcription. Knockdown of Snai1 expression significantly attenuated FoxC1‐enhanced invasion and lung metastasis. FoxC1 expression was positively correlated with Snai1 expression, but inversely correlated with E‐cadherin expression in human HCC tissues. Additionally, a complementary DNA microarray, serial deletion, site‐directed mutagenesis, and a chromatin immunoprecipitation assay confirmed that neural precursor cell expressed, developmentally down‐regulated 9 (NEDD9), which promotes the metastasis of HCC cells, is a direct transcriptional target of FoxC1 and is involved in FoxC1‐mediated HCC invasion and metastasis. Conclusions: FoxC1 may promote HCC metastasis through the induction of EMT and the up‐regulation of NEDD9 expression. Thus, FoxC1 may be a candidate prognostic biomarker and a target for new therapies. (HEPATOLOGY 2013;)

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 & AIMSnForkhead 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.nnnMETHODSnThe 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.nnnRESULTSnFoxM1 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.nnnCONCLUSIONSnWe 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)

Upregulation of SATB1 promotes tumor growth and metastasis in liver cancer

Special AT‐rich binding protein‐1 (SATB1) reprograms chromatin organization and transcription profiles to promote tumour growth and metastasis.

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.

Interleukin-8 Induces Expression of FOXC1 to Promote Transactivation of CXCR1 and CCL2 in Hepatocellular Carcinoma Cell Lines and Formation of Metastases in Mice

BACKGROUND & AIMSnInflammation regulated by interleukin (IL) 8 promotes metastasis of hepatocellular carcinoma (HCC). The transcription factor forkhead box C1 (FOXC1) promotes metastasis by activating the epithelial to mesenchymal transition; its levels in liver tumors have been associated with shorter survival times of patients. We investigated whether FOXC1 activates inflammation signaling pathways in HCC cell lines.nnnMETHODSnWe performed studies in the human HCC cell lines Huh-7 and SMMC7721, as well as the metastatic cell lines MHCC97H and HCCLM3. Cell lines were incubated with IL8 and transcription of reporter genes was measured; cells were also incubated with kinase inhibitors. Levels of FOXC1 or IL8 were knocked down with small interfering messenger RNAs in Huh7 cells; cells were analyzed inxa0vitro in migration and invasion assays. To study metastasis, HCC cells were injected into flanks of BALB/C nude mice; 4 weeks later, the subcutaneous tumor fragments were collected and implanted into livers of the nude mice, and number and size tumors formed were measured. Chromatin immunoprecipitation assays were used to measure binding of transcription factors promoter regions of genes. We measured levels of FOXC1, IL8, CXCR1, and CCL2 in 2 groups of human HCC tissues collected from the Xijing or Tongji Hospitals in China (nxa0= 690 and nxa0= 312 samples, respectively) using immunohistochemistry.nnnRESULTSnIncubation of HCC cells with IL8 led to increased expression of FOXC1, via activation of phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. Knockdown of FOXC1 in HCC cells that overexpressed IL8 reduced the numbers of metastases formed in mice, compared with cells without FOXC1 knockdown. Transgenic overexpression of FOXC1 in HCC cells with IL8 knockdown increased the numbers of metastases formed in mice compared with cells without FOXC1 overexpression. CXCR1 and CCL2 were direct transcriptional targets of FOXC1. Knockdown of the combination of CXCR1 and CCL2 reduced the invasive activities of HCC cells that overexpress FOXC1 and formation of lung metastases in mice, and transgenic overexpression of CXCR1 increased cells invasive and metastatic abilities after knockdown of FOXC1. Liver metastases grown from cells that overexpressed FOXC1 were infiltrated by tumor-associated macrophages, and CCL2 knockdown decreased tumor-associated macrophage infiltration; depletion of macrophages from mice significantly reduced growth of metastases by cells that overexpressed FOXC1. In human HCC tissues, level of FOXC1 correlated with levels of IL8 and CXCR1 and CCL2 and infiltration of tumors by macrophage. In multivariate analysis, detection of FOXC1 and CCL2 were independent predictors forxa0postoperative recurrence of HCC and overall survival.nnnCONCLUSIONSnIn HCC cell lines, IL8 activates expression of FOXC1 via the phosphoinositide 3-kinase signaling to AKT and hypoxia-inducible factor 1α. FOXC1 expression leads to transactivation of CXCR1 and CCL2, promoting inflammation and the invasive and metastatic abilities of HCC 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)

Down-regulated KLF17 expression is associated with tumor invasion and poor prognosis in hepatocellular carcinoma

Although the role of Krüppel-like factor 17 (KLF17) in regulating epithelial–mesenchymal transition (EMT) has been explored in breast cancer, its influence on primary hepatocellular carcinoma (HCC) remains unclear. This study aims to investigate the expression status of KLF17 in hepatocellular carcinoma (HCC) and the correlation between KLF17 expression and metastatic potential of HCC. KLF17 expression in HCC and adjacent liver tissues was studied by real-time PCR and Western blot, and the relationship between KLF17 expression and the clinicopathological features of HCC was evaluated in 60 patients. By using RNA interference technique, the correlation of KLF17 expression and metastatic potential was investigated by down-regulating KLF17 expression in HepG2 cells, and the effects of KLF17 down-regulation on cell migration, and invasion were then analyzed. Furthermore, the correlation between KLF17 expression and the surgical outcomes of a cohort of HCC patients was analyzed. Reduced expression of KLF17 is associated with a short survival time in clinical patients (Pxa0=xa00.034). Low KLF17 expression is related to tumor T stage (Pxa0=xa00.045), tumor size (Pxa0=xa00.027), lymph node stage (Pxa0=xa00.030), M stage (Pxa0=xa00.048), and portal vein tumor thrombosis significantly in HCC. Reduced expression of KLF17 promoted motility and invasion ability of HepG2 cells and changed the expression of E-cadherin, ZO-1, Snai1, and vimentin (genes are associated with EMT). Overall, these findings suggest a repressing role of KLF17 in tumor invasion and a new prognostic indicator in directing therapy. It deserves further exploration.