Abdirashid M. Shire

Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma.

It has been shown that the heparin‐degrading endosulfatase, sulfatase 1 (SULF1), functions as a liver tumor suppressor, but the role of the related sulfatase, sulfatase 2 (SULF2), in liver carcinogenesis remains to be elucidated. We investigated the effect of SULF2 on liver tumorigenesis. Expression of SULF2 was increased in 79 (57%) of 139 hepatocellular carcinomas (HCCs) and 8 (73%) of 11 HCC cell lines. Forced expression of SULF2 increased HCC cell growth and migration, whereas knockdown of SULF2 using short hairpin RNA targeting SULF2 abrogated HCC cell proliferation and migration in vitro. Because SULF1 and SULF2 desulfate heparan sulfate proteoglycans (HSPGs) and the HSPG glypican 3 (GPC3) is up‐regulated in HCC, we investigated the effects of SULF2 on GPC3 expression and the association of SULF2 with GPC3. SULF2‐mediated cell growth was associated with increased binding of fibroblast growth factor 2 (FGF2), phosphorylation of extracellular signal‐regulated kinase and AKT, and expression of GPC3. Knockdown of GPC3 attenuated FGF2 binding in SULF2‐expressing HCC cells. The effects of SULF2 on up‐regulation of GPC3 and tumor growth were confirmed in nude mouse xenografts. Moreover, HCC patients with increased SULF2 expression in resected HCC tissues had a worse prognosis and a higher rate of recurrence after surgery. Conclusion: In contrast to the tumor suppressor effect of SULF1, SULF2 has an oncogenic effect in HCC mediated in part through up‐regulation of FGF signaling and GPC3 expression. (HEPATOLOGY 2008.)

Utility of serum immunoglobulin G4 in distinguishing immunoglobulin G4-associated cholangitis from cholangiocarcinoma.

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;)

Highly Methylated Genes in Colorectal Neoplasia: Implications for Screening

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)

The oncogenic effect of sulfatase 2 in human hepatocellular carcinoma is mediated in part by glypican 3–dependent Wnt activation

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;)

The Transcription Factor GLI1 Mediates TGFβ1 Driven EMT in Hepatocellular Carcinoma via a SNAI1-Dependent Mechanism

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.

The Tumor Suppressor Function of Human Sulfatase 1 (SULF1) in Carcinogenesis

IntroductionHuman 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.DiscussionThe 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.ConclusionStrategies targeting SULF1 or the interaction between SULF1 and the related sulfatase 2 will potentially be important in developing novel cancer therapies.

Epigenetic DNA hypermethylation in cholangiocarcinoma: potential roles in pathogenesis, diagnosis and identification of treatment targets

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.

The JNK inhibitor SP600129 enhances apoptosis of HCC cells induced by the tumor suppressor WWOX.

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.

Additive effect of apicidin and doxorubicin in sulfatase 1 expressing hepatocellular carcinoma in vitro and in vivo

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.

The Human Sulfatase 2 Inhibitor 2,4-Disulfonylphenyl-tert-Butylnitrone (OKN-007) has an Antitumor Effect in Hepatocellular Carcinoma Mediated via Suppression of TGFB1/SMAD2 and Hedgehog/GLI1 Signaling

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.