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Background Ghrelin (GHRL), a gastric peptide encoded by the GHRL gene, is known to be involved in energy homeostasis via its G protein receptor, encoded by the growth hormone secretagogue receptor (GHSR) gene. Some studies have shown associations between plasma GHRL levels and GHRL single-nucleotide polymorphisms (SNPs), namely the Leu72Met polymorphism (rs696217 TG), with type 2 diabetes mellitus (T2DM) and insulin resistance (IR), while others have not. The controversies in these associations raise the issue of 'which SNPs in which populations.' The aim of this study was to investigate whether SNPs in GHRL and/or GHSR genes were associated with T2DM, IR, or plasma GHRL levels among Arab Saudis. Methods Blood was collected from 208 Saudi subjects with (n=107) and without (n=101) T2DM. DNA samples from these subjects were analyzed by real-time polymerase chain reaction to genotype five intronic SNPs in the GHRL (rs696217 TG, rs27647 CT, rs2075356 CT, and rs4684677 AT) and GHSR (rs509030 GC) genes. In addition, plasma GHRL levels were measured by a radioimmunoassay. Results None of the SNPs were associated with T2DM, IR, or plasma GHRL levels. The frequencies of the alleles, genotypes, and haplotypes of the five SNPs were comparable between the T2DM patients and the non-diabetic subjects. A large number of the GHRL haplotypes indicates the molecular heterogeneity of the preproghrelin gene in this region. Conclusion Neither the Leu72Met polymorphism nor the other intronic GHRL and GHSR SNPs were associated with T2DM, IR, or GHRL levels. Further investigations should be carried out to explain the molecular basis of the association of the GHRL peptide with T2DM and IR.

Background: Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer death worldwide. Sulfatase 1 (SULF1) functions as a tumor suppressor in HCC cell lines in vitro but also has an oncogenic effect in some HCCs in vivo. Aim: The purpose of this study was to examine the mechanisms regulating SULF1 and its function in HCC. Methods: First, SULF1 mRNA and protein expression were examined. Second, we examined SULF1 gene copy numbers in HCC cells. Third, we assessed whether DNA methylation or methylation and/or acetylation of histone marks on the promoter regulate SULF1 expression. Finally, we examined the effect of 5-aza-2′-deoxycytidine (5-Aza-dC) on sulfatase activity and drug-induced apoptosis. Results: SULF1 mRNA was downregulated in nine of eleven HCC cell lines, but only in six of ten primary tumors. SULF1 mRNA correlated with protein expression. Gene copy number assessment by fluorescence in situ hybridization showed intact SULF1 alleles in low-SULF1-expressing cell lines. CpG island methylation in the SULF1 promoter and two downstream CpG islands did not show an inverse correlation between DNA methylation and SULF1 expression. However, chromatin immunoprecipitation showed that the SULF1 promoter acquires a silenced chromatin state in low-SULF1-expressing cells through an increase in di/trimethyl-K9H3 and trimethyl-K27H3 and a concomitant loss of activating acetyl K9, K14H3 marks. 5-Aza-dC restored SULF1 mRNA expression in SULF1-negative cell lines, with an associated increase in sulfatase activity and sensitization of HCC cells to cisplatin-induced apoptosis. Conclusion: SULF1 gene silencing in HCC occurs through histone modifications on the SULF1 promoter. Restoration of SULF1 mRNA expression by 5-Aza-dC sensitized HCC cells to drug-induced apoptosis.

Elevated serum immunoglobulin G4 (sIgG4) is a feature of autoimmune pancreatitis (AIP) and IgG4-associated cholangitis (IAC); a >2-fold increase in sIgG4 is considered highly specific for these disorders. Many patients with IAC present with biliary strictures and obstructive jaundice, making cholangiocarcinoma (CCA) an important differential diagnosis. We determined the value of sIgG4 in distinguishing IAC from CCA. sIgG4 levels were measured in a test cohort of 126 CCA and 50 IAC patients. The results were confirmed in a validation cohort of 161 CCA and 47 IAC patients. Of the 126 CCA patients in the test cohort, 17 (13.5%) had elevated sIgG4 (>140 mg/dL) and four (3.2%) had a >2-fold (>280 mg/dL) increase. Primary sclerosing cholangitis (PSC) was present in 31/126 CCA patients, of whom seven (22.6%) had elevated sIgG4 and two (6.5%) had a >2-fold elevation. Of the 50 IAC patients, 39 (78.0%) had elevated sIgG4 and 25 (50.0%) had a >2-fold increase. The results in the validation cohort were consistent with those of the test cohort. Conclusion: Although elevated sIgG4 levels are characteristic of IAC, some patients with CCA, particularly with PSC, have elevated sIgG4 levels, including a small percentage with a more than a 2-fold increase in sIgG4. Therefore, sIgG4 elevation alone does not exclude the diagnosis of CCA. Depending on the prevalence of the two diagnoses, the use of a 2-fold cutoff for sIgG4 may not reliably distinguish IAC from CCA. At a cutoff of 4 times the upper limit of normal, sIgG4 is 100% specific for IAC. (HEPATOLOGY 2011;)

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide (1). Because of frequent de novo and acquired resistance of HCCs to chemotherapy, there are limited options for therapy of HCC (2, 3). There is therefore an urgent need for improved therapy of HCC. Consequently there is strong interest in identifying novel molecular targets for therapy of advanced HCC. The role of the extracellular heparan sulphate 6-O-endosulphatases, sulfatase 1 (SULF1) and sulfatase 2 (SULF2) in human carcinogenesis has not been completely elucidated (4, 5). SULF1 has been shown to function as a tumour suppressor in HCC, head and neck cancer, ovarian cancer and pancreatic cancer (5–10). SULF1 and SULF2 have also been reported to inhibit tumour growth in multiple myeloma (11). In contrast, SULF2 is upregulated in breast cancer and functions as an oncogene in HCC, pancreas cancer, lung cancer and chronic lymphocytic leukemia (12–16). Gene expression microarray analysis of 139 pairs of HCC tumour and adjacent benign tissue showed upregulation of SULF2 in 57% of HCCs (13). The 5-year survival rate for patients with HCCs with upregulated SULF2 was significantly worse than for those with down-regulated SULF2. Patients with upregulated SULF2 also had earlier recurrence of HCC after surgery. Immunohistochemical analysis of cell proliferation and apoptosis was performed in 30 of the HCCs (13). Tumours were classified into subclass A (poor prognosis) or subclass B (good prognosis) based on the prior gene expression profiling study by Lee et al. (17). Subclass A tumours were more frequent in the high SULF2 group (13 of 14 tumours) and less frequent in the low SULF2 group (two of 16 tumours; P=0.00001). HCCs with high SULF2 expression also had a significantly higher Ki-67 proliferation index (P= 0.007) and a significantly lower apoptosis index (P = 0.0001) than those with low SULF2 expression. SULF2 expression therefore correlated with increased proliferation and decreased apoptosis (13). In experiments to validate these results, we showed that SULF2 promoted proliferation and migration of HCC cells in vitro (13, 18). Mechanistically, SULF2 upregulated cell surface glypican 3 and promoted FGF signalling. Expression of SULF2 increased phosphorylation of Erk and Akt (13). SULF2 expression also increased phosphorylation of the anti-apoptotic Akt substrate GSK3β and stimulated Wnt/β-catenin signalling(19). Other investigators have also demonstrated that SULF2 promotes signalling by receptor tyrosine kinase ligands, Wnts and other growth factors (14, 20, 21). In terms of associations with other known pro-apoptotic molecules, SULF2 has been shown to be a transcriptional target of p53 in colon cancer, lung cancer, ovarian cancer and HCC cells, but the direct or indirect effects of SULF2 on apoptosis and apoptosis-related pathways in HCC have not been reported (22, 23). ERK, PI3K/Akt and JNK pathway inhibitors and histone deacetylase (HDAC) inhibitors induce apoptosis and are currently in clinical trials for cancer therapy (24–26). We studied the expression of SULF2 in HCCs and determined the role of SULF2 in modulating apoptosis induced by these kinase and HDAC inhibitors in HCC cells. The questions addressed in this study were: Is SULF2 mRNA expression correlated to protein expression in HCCs? Do changes in SULF2 expression affect cell viability, caspase activation and induction of apoptosis of HCC cells by ERK, PI3K, JNK or HDAC inhibitors? Does knockdown of SULF2 inactivate the Akt pathway? Does knockdown of SULF2 inhibit cell cycle progression as measured by cyclin D1 expression? Does SULF2 mediate its effects by regulating apoptosis-related Bcl-2, Bcl-XL and BAD protein expression?

Heparan sulfate proteoglycans (HSPGs) act as coreceptors or storage sites for growth factors and cytokines such as fibroblast growth factor and Wnts. Glypican 3 (GPC3) is the most highly expressed HSPG in hepatocellular carcinoma (HCC). Sulfatase 2 (SULF2), an enzyme with 6-O-desulfatase activity on HSPGs, is up-regulated in 60% of primary HCCs and is associated with a worse prognosis. We have previously shown that the oncogenic effect of SULF2 in HCC may be mediated in part through up-regulation of GPC3. Here we demonstrate that GPC3 stimulates the Wnt/β-catenin pathway and mediates the oncogenic function of SULF2 in HCC. Wnt signaling in vitro and in vivo was assessed in SULF2-negative Hep3B HCC cells transfected with SULF2 and in SULF2-expressing Huh7 cells transfected with short hairpin RNA targeting SULF2. The interaction between GPC3, SULF2, and Wnt3a was assessed by coimmunoprecipitation and flow cytometry. β-catenin–dependent transcriptional activity was assessed with the TOPFLASH (T cell factor reporter plasmid) luciferase assay. In HCC cells, SULF2 increased cell surface GPC3 and Wnt3a expression, stabilized β-catenin, and activated T cell factor transcription factor activity and expression of the Wnt/β-catenin target gene cyclin D1. Opposite effects were observed in SULF2-knockdown models. In vivo, nude mouse xenografts established from SULF2-transfected Hep3B cells showed enhanced GPC3, Wnt3a, and β-catenin levels. Conclusion: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3-mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis. (HEPATOLOGY 2010;)

Background/Aims The FRA16D fragile site gene WWOX is a tumor suppressor that participates in p53-mediated apoptosis. The c-jun N-terminal kinase JNK1 interacts with WWOX and inhibits apoptosis. We investigated the function of WWOX in human hepatocellular carcinoma (HCC) and the effect of JNK inhibition on WWOX-mediated apoptosis. Methods Allelic imbalance on chromosome 16 was analyzed in 73 HCCs using 53 microsatellite markers. WWOX mRNA in HCC cell lines and primary HCCs was measured by real-time RT-PCR. Effects of WWOX on proliferation and apoptosis and the interaction between WWOX and JNK inhibition were examined. Results Loss on chromosome 16 occurred in 34 of 73 HCCs. Of 11 HCC cell lines, 2 had low, 7 intermediate, and 2 had high WWOX mRNA. Of 51 primary tumors, 23 had low WWOX mRNA. Forced expression of WWOX in SNU387 cells decreased FGF2-mediated proliferation and enhanced apoptosis induced by staurosporine and the JNK inhibitor SP600129. Conversely, knockdown of WWOX in SNU449 cells using shRNA targeting WWOX increased proliferation and resistance to SP600129-induced apoptosis. Conclusions WWOX induces apoptosis and inhibits human HCC cell growth through a mechanism enhanced by JNK inhibition.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death world-wide. 1 Risk factors for HCC include chronic hepatitis and cirrhosis secondary to hepatitis B virus or hepatitis C virus infection, alcoholic cirrhosis, nonalcoholic steatohepatitis, hemochromatosis, alpha-1-antitrypsin deficiency, and dietary aflatoxin exposure.2 Survival of patients diagnosed with advanced HCC is dismal. Only a small proportion of HCCs are detected at an early stage at which potentially curative therapies are feasible. Because of frequent de novo and acquired resistance of HCCs to chemotherapy, standard chemotherapeutic agents are not effective against HCC.3 Recent trials have proven the efficacy of the multi-targeting Raf and receptor tyrosine kinase inhibitor sorafenib in advanced HCC.4 Therefore, there is considerable interest in identifying additional novel gene targets for therapy against HCC. The recently identified human sulfatase 2 (SULF2) gene at chromosome 20q13 encodes a protein with heparin-degrading endosulfatase activity.5 Cell-surface signaling by several heparin-binding growth factors, including fibroblast growth factor (FGF), heparin-binding epidermal growth factor, hepatocyte growth factor, and vascular endothelial growth factor, requires the binding of both the growth factor and its cognate cell-surface receptor tyrosine kinase to sulfated heparan sulfate glycosaminoglycans. 6,7 For these growth factors, sulfation of particular saccharide moieties of heparan sulfate proteoglycans (HSPGs) is required for growth factor signaling. It has been shown that desulfation of HSPGs by the related protein, sulfatase 1 (SULF1), inhibits binding of the growth factor to its receptor, abrogates growth factor signaling, and has a tumor suppressor effect.8–15 In contrast to SULF1, it has been reported that SULF2 messenger RNA (mRNA) is up-regulated in human breast cancers,16 and suppression of SULF2 expression decreases tumorigenesis of pancreatic cancer cell lines.17 Thus, although SULF2 and SULF1 share the same HSPG substrate, it appears that they have different functional effects.18 This model is supported by recent evidence that human SULF2 mobilizes growth factors and chemokines from heparin and consequently facilitates growth factor signaling. 19 Because our initial studies suggested that SULF2 has an oncogenic effect, we hypothesized that SULF2 increases HCC bcell growth by release of growth factors from cell surface or extracellular matrix HSPG storage sites, thereby enhancing binding of FGF2 and other receptor tyrosine kinase ligands to their corresponding receptors and leading to cell growth. Therefore, we studied the expression of SULF2 in HCCs and examined the role of SULF2 in modulating growth of HCCs. Glypicans are a family of HSPGs that are bound to the cell surface by a lipid glycosyl-phosphatidylinositol anchor. Glypican 3 (GPC3) is markedly overexpressed in a high proportion of HCCs and promotes the growth of HCCs.20–22 We therefore also investigated the potential interactions between SULF2, FGF2 signaling, and GPC3. The following questions were addressed: Is SULF2 expression increased in HCC? Does SULF2 expression affect growth and migration of HCC cells? Does SULF2 affect FGF2 binding to the cell surface? Does SULF2 modulate expression of GPC3? Does SULF2 predict prognosis of HCC patients?

Cholangiocarcinomas (CCs) are highly lethal malignant tumours arising from the biliary tract epithelium. The disease is notoriously difficult to diagnose and is usually fatal because of its typically late clinical presentation and the lack of effective non-surgical therapeutic modalities. The overall survival rate, including resected patients is poor, with less than 5% of patients surviving 5 years, a rate which has not changed significantly over the past 30 years. Although CC is a relatively uncommon tumor, interest in this disease is rising as incidence and mortality rates for intrahepatic cholangiocarcinoma are increasing markedly worldwide. A variety of risk factors, including primary sclerosing cholangitis, liver fluke infestation, and hepatolithiasis have been described. However, for most CCs the cause is unknown, and affected individuals have no history of exposure to, or association with, known risk factors. Recent advances in molecular pathogenesis have highlighted the importance of epigenetic alterations in the form of promoter region hypermethylation and histone deacetylation in addition to genetic changes in the process of cholangiocarcinogenesis. This review provides a comprehensive overview of the genes reported to be methylated in CC to date and their putative roles in cholangiocarcinogenesis. Future directions in the study of methylated genes and their potential roles as diagnostic and prognostic markers are also discussed.

Age-related macular degeneration (AMD) is the most common cause of irreversible visual loss in the developed world. Previous studies have demonstrated that the c.1204T>C, p.Tyr402His allelic variant in the complement factor H (CFH) gene is associated with an approximately three-fold increased risk for AMD in Caucasians of predominantly European descent. Both the prevalence as well as the phenotypic spectrum of AMD varies widely among persons of different ethnicities. We hypothesized that populations with a lower prevalence of AMD might also have a lower prevalence of the CFH risk allele. In this study we sought to determine the frequency of this sequence variant in control populations of Caucasians, African Americans, Hispanics, Somalis, and Japanese. Normal control populations were assembled for each ethnic group: Caucasian (n=148), Somali (n=128), African American (n=75), Hispanic (n=81), and Japanese (n=82). Individuals were genotyped using a restriction digest assay and the frequency of the C allele at nucleotide position 1204 of the CFH gene was determined. A bioinformatic approach was used to identify SNPs in linkage disequilibrium with rs1061170 (c.1204T>C, p.Tyr402His) from the human haplotype map project database (HapMap) in order to validate the findings. We found widely discordant frequencies of the risk allele between some of the different ethnic groups: Japanese 0.07±0.02, Hispanics 0.17±0.03, African-Americans 0.35±0.04, Caucasians 0.34±0.03, and Somalis 0.34±0.03. Allele frequencies generated by analysis of the HapMap database were consistent with these findings. This study suggests that there are other yet unidentified genetic factors important in the pathogenesis of AMD that may mitigate the effects of c.1204T>C, p.Tyr402His variant. Hum Mutat 27(9), 921–925, 2006. © 2006 Wiley-Liss, Inc.

Background: Although cholangiocarcinoma (CC) is an uncommon and highly lethal malignancy, early detection enables the application of potentially curative therapies and improves survival. Consequently, tools to improve the early diagnosis of CC are urgently needed. During a screen for genes epigenetically suppressed by methylation in CC that might serve as methylation markers for CC, we found that the BMP3 gene is methylated in CC cell lines, but the potential diagnostic value and the function of BMP3 in CC are unknown. Methods: We aimed to quantitatively assess BMP3 methylation in resected CC tumor specimens using methylation specific PCR and evaluate the tumor suppressor role of BMP3 in biliary cancer cell lines in comparison to an immortalized normal cholangiocyte cell line. Expression of BMP3 was quantified by mRNA levels before and after treatment with 5-Aza-2’-deoxycytidine and trichostatin A. After transfection with a BMP3-containing plasmid, cell viability was measured using the bromodeoxyuridine incorporation assay and apoptosis quantified by caspase assay. Results: In primary CC tumor tissue specimens significantly more methylated BMP3 copies were found when compared to matched benign bile duct epithelium from the same patient, with high specificity. BMP3 expression was absent in cell lines with BMP3 methylation; this suppression of BMP3 expression was reversed by treatment with a DNA demethylating agent and histone de-acetylase inhibitor. Transfection of a BMP3-expressing construct into a BMP3-negative biliary cancer cell line restored BMP3 mRNA expression and reduced cell proliferation and cell viability while increasing the rate of apoptosis. Conclusion: These findings strongly support a tumor suppressor role for BMP3 in CC and suggest that BMP3 methylation may be a new biomarker for early detection of CCs.of the peptidome are also involved.

The role of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established, however the regulatory mechanisms modulating this phenomenon remain unclear. Here, we demonstrate that transcription factor glioma-associated oncogene 1 (GLI1) modulates EMT through direct up-regulation of SNAI1 and serves as a downstream effector of the transforming growth factor-β1 (TGFβ1) pathway, a well-known regulator of EMT in cancer cells. Overexpression of GLI1 increased proliferation, viability, migration, invasion, and colony formation by HCC cells. Conversely, GLI1 knockdown led to a decrease in all the above-mentioned cancer-associated phenotypes in HCC cells. Further analysis of GLI1 regulated cellular functions showed that this transcription factor is able to induce EMT and identified SNAI1 as a transcriptional target of GLI1 mediating this cellular effect in HCC cells. Moreover, we demonstrated that an intact GLI1-SNAI1 axis is required by TGFβ1 to induce EMT in these cells. Together, these findings define a novel cellular mechanism regulated by GLI1, which controls the growth and EMT phenotype in HCC.

Human sulfatase 2 (SULF2) functions as an oncoprotein in hepatocellular carcinoma (HCC) development by promoting tumor growth and metastasis via enhancement of fibroblast growth factor-2/extracellular signal-regulated kinase and WNT/ β-catenin signaling. Recent results implicate that SULF2 activates the transforming growth factor beta (TGFB) and Hedgehog/GLI1 pathways in HCC. OKN-007 is a novel phenyl–sulfonyl compound that inhibits the enzymatic activity of SULF2. To investigate the antitumor effect of OKN-007 in HCC, we treated Huh7 cells, which express high levels of SULF2, with OKN-007 and found that it significantly promoted tumor cell apoptosis and inhibited cell proliferation, viability, and migration. To understand the action of OKN-007 on SULF2, we used Huh7 cells which normally express SULF2 and Hep3B cells that do not normally express SULF2. Utilizing Huh7 cells transfected with short hairpin RNA targeting SULF2 and transfection of Hep3B cells with a SULF2 plasmid to enhance SULF2 expression, we showed that the antitumor activity of OKN-007 was more pronounced in cells expressing SULF2. Furthermore, in vivo experiments verified that OKN-007 repressed tumor growth significantly. These results identify SULF2 as an important target of the antitumor effect of OKN-007. To determine the molecular mechanism of the antitumor effect of OKN-007, both TGFB1/SMAD and Hedgehog/GLI1 signaling pathway activity were measured by Western blot and SMAD- or GLI-reporter luciferase assays. We found that both signaling pathways were inhibited by OKN-007. Together, these results show that OKN-007 can suppress TGFB1/SMAD and Hedgehog/GLI1 signaling via its inhibition of SULF2 enzymatic activity. We conclude that OKN-007 or more potent derivatives may be promising agents for the treatment of HCC.

Background/Aims There are limited chemotherapy options for hepatocellular carcinoma (HCC). The heparin-degrading endosulfatase SULF1 functions as a liver tumor suppressor. We investigated the effects of the histone deacetylase inhibitor apicidin in combination with doxorubicin in SULF1-expressing HCC cells in vitro and in SULF1-expressing xenografts in nude mice. Methods We evaluated the effects of apicidin alone or combined with doxorubicin on apoptosis, caspase activity, and phosphorylation of Erk and Akt in SULF1-transfected Huh7 and Hep3B cells in vitro and in vivo . Results Apicidin induced HCC cell apoptosis and caspase activation in a dose- and time-dependent manner. Apicidin-induced caspase activation was significantly inhibited by the caspase inhibitor Z-Vad-fmk. Apicidin also decreased phosphorylation of both Erk and Akt. Expression of constitutively-active Mek1 and Akt significantly decreased apicidin-induced apoptosis. The combination of doxorubicin with apicidin significantly increased the anti-tumor effect in the SULF1-expressing Huh7 and Hep3B cells as compared to either apicidin or doxorubicin alone, both in vitro and in vivo . Conclusions The combination of a histone deacetylase inhibitor with doxorubicin may be a novel and promising therapeutic modality for HCCs, particularly for SULF1-expressing HCCs.

Human sulfatase 1 (SULF1) was recently identified and shown to desulfate cellular heparan sulfate proteoglycans (HSPGs). Since sulfated HSPGs serve as co-receptors for many growth factors and cytokines, SULF1 was predicted to modulate growth factor and cytokine signaling.The role of SULF1 in growth factor signaling and its effects on human tumorigenesis are under active investigation. Initial results show that SULF1 inhibits the co-receptor function of HSPGs in multiple receptor tyrosine kinase signaling pathways, particularly by the heparin binding growth factors--fibroblast growth factor 2, vascular endothelial growth factor, hepatocyte growth factor, PDGF, and heparin-binding epidermal growth factor (HB-EGF). SULF1 is downregulated in the majority of cancer cell lines examined, and forced expression of SULF1 decreases cell proliferation, migration, and invasion. SULF1 also promotes drug-induced apoptosis of cancer cells in vitro and inhibits tumorigenesis and angiogenesis in vivo.Strategies targeting SULF1 or the interaction between SULF1 and the related sulfatase 2 will potentially be important in developing novel cancer therapies.

Discriminant markers are required for accurate cancer screening. We evaluated genes frequently methylated in colorectal neoplasia to identify the most discriminant ones. Four genes specifically methylated in colorectal cancer [bone morphogenetic protein 3 (BMP3), EYA2, aristaless-like homeobox-4 (ALX4), and vimentin] were selected from 41 candidate genes and evaluated on 74 cancers, 62 adenomas, and 70 normal epithelia. Methylation status was analyzed qualitatively and quantitatively and confirmed by bisulfite genomic sequencing. Effect of methylation on gene expression was evaluated in five colon cancer cell lines. K-ras and BRAF mutations were detected by sequencing. Methylation of BMP3, EYA2, ALX4, or vimentin was detected respectively in 66%, 66%, 68%, and 72% of cancers; 74%, 48%, 89%, and 84% of adenomas; and 7%, 5%, 11%, and 11% of normal epithelia (P < 0.01, cancer or adenoma versus normal). Based on area under the curve analyses, discrimination was not significantly improved by combining markers. Comethylation was frequent (two genes or more in 72% of cancers and 84% of adenomas), associated with proximal neoplasm site (P < 0.001), and linked with both BRAF and K-ras mutations (P < 0.01). Cell line experiments supported silencing of expression by methylation in all four study genes. This study shows BMP3, EYA2, ALX4, and vimentin genes are methylated in most colorectal neoplasms but rarely in normal epithelia. Comethylation of these genes is common, and pursuit of complementary markers for methylation-negative neoplasms is a rational strategy to optimize screening sensitivity. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2686–96)

prognosis. (A) Immunohistochemistry analysis of FoxQ1 expression in 690 paired HCC tissues. (B) Representative FoxQ1 expression in primary and metastatic HCC tissues detected by immunohistochemical methods. (C)Western blot analysis of FoxQ1 expression in different human HCC cell lines. (D) Kaplan-Meier analysis of the correlation between FoxQ1 expression and the recurrence or overall survival of HCC patients. *P ,0.05.

Background: Nonalcoholic fatty liver disease (NAFLD), an important consequence of metabolic disorders such as obesity and type 2 diabetes (T2DM), has become highly prevalent in the US, and is now an important public health concern. Recent reports have identified the heparan sulfate degrading endosulfatase sulfatase 2 (Sulf2) as a suppressor of triglyceriderich lipoprotein (TRL) remnant clearance by hepatocytes in T2DM mice, thus enhancing postprandial hypertriglyceridemia. Further, inhibition of Sulf2 normalized the VLDL-binding capacity of hepatocytes from T2DM mice and abolished postprandial hypertriglyceridemia. We hypothesized that an increase in hepatic Sulf2 occurring during insulin resistance states such as obesity and T2DM decreases hepatic clearance of TRL remnants and contributes to the development of NAFLD, which can be characterized by non-alcoholic steatohepatitis (NASH) and liver fibrosis. Aims: To investigate (1) whether a fast food diet high in saturated fats, cholesterol, and fructose can produce insulin resistance and overexpression of Sulf2; and (2) whether depletion of Sulf2 can protect against NASH and liver fibrosis in obese mice on a fast food diet. Methods: Sulf2 knockout and wild-type mice (n=12 each) were fed a fast food diet rich in saturated fats and cholesterol along with fructose in their drinking water or normal rodent chow with tap water, beginning at 6-8 weeks of age. At 9 months, mice were sacrificed, blood was obtained for assessment of insulin resistance, alanine transaminase (ALT) and serum lipids, and the livers were harvested and examined for steatosis, steatohepatitis, and fibrosis. Results: The fast food diet produced obesity and insulin resistance. Sulf2 knockout mice fed the fast food diet had significantly less weight gain and lower serum ALT, total cholesterol and LDL-cholesterol than wild-type mice (p,0.05). The fast food diet also induced histopathological features of NASH, including hepatic microvesicular steatosis, inflammation and fibrosis. Hepatic fibrosis was significantly decreased in Sulf2 knockout mice compared to controls (p,0.05). Conclusions: Depletion of Sulf2 improves hypercholesterolemia, steatohepatitis and hepatic fibrosis in a mouse model of NASH induced by a fast food diet. Inhibition of SULF2 may be a potential preventive and therapeutic strategy for NASH with progressive fibrosis.

Background/Aim: Glioma-associated oncogene 1 (GLI1) is one of the most important transcription factors in the hedgehog (Hh) signaling pathway and has been shown to be involved in embryonic development and tumor progression. The epithelial-to-mesenchymal transition (EMT) is an important cellular program which contributes to invasion and metastasis of cancer. We have previously shown that GLI1 may promote HCC metastasis via EMT. Here, we demonstrate that GLI1 upregulates SNAI1 expression, induces EMT, and promotes HCC recurrence in vivo and in vitro. Methods: Gli1 mRNA was assessed in 139 HCCs by microarray and its correlation with HCC recurrence and SNAI1 mRNA expression was analyzed. In vitro, the mRNA and protein expression of GLI1 was measured in Gli1-negative Huh7 cells transfected with GU1 and GU1-expressing SNU398 cells transfected with antisense oligonucleotides targeting GU1 (GLI1 ASO). GLI1-dependent transcriptional activity was assessed with GU reporter luciferase assay and electrophoretic mobility shift assay (EMSA). The interaction between Gli1, SNAI1 and EMT was assessed by real-time PCR, Western immunoblotting and immunofluorescence. Effects of GLI1 on proliferation, viability, migration, and invasion of tumor cells were also assessed. Results: GLI1 mRNA was detected in 74 HCC tumors and found to be upregulated in 43 (58.1 %) HCCs. Increased Gli1 mRNA in HCCs was associated with tumor recurrence after resection. There was a positive correlation between GLI1 and SNAI1 mRNA expression. In HCC cells, GLI1 overexpression increased Gli1-dependent transcriptional activity, proliferation, viability, migration, and invasion of tumor cells. GU1 also increased SNAI1 expression and induced the EMT. Opposite effects were observed in GU1-knockdown models. Conclusion: Together, these findings identify a novel mechanism mediating HCC recurrence that includes GLI1-mediated activation of EMT via upregulation of SNAI1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B20.