Catherine D. Moser

Mutations in isocitrate dehydrogenase 1 and 2 occur frequently in intrahepatic cholangiocarcinomas and share hypermethylation targets with glioblastomas

Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in gliomas, myeloid leukemias, chondrosarcomas and thyroid cancer. We discovered IDH1 and IDH2 mutations in 34 of 326 (10%) intrahepatic cholangiocarcinomas. Tumor with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3 lysine 79 (H3K79). Mutations in IDH1 or IDH2 were associated with longer overall survival (P=0.028) and were independently associated with a longer time to tumor recurrence after intrahepatic cholangiocarcinoma resection in multivariate analysis (P=0.021). IDH1 and IDH2 mutations were significantly associated with increased levels of p53 in intrahepatic cholangiocarcinomas, but no mutations in the p53 gene were found, suggesting that mutations in IDH1 and IDH2 may cause a stress that leads to p53 activation. We identified 2309 genes that were significantly hypermethylated in 19 cholangiocarcinomas with mutations in IDH1 or IDH2, compared with cholangiocarcinomas without these mutations. Hypermethylated CpG sites were significantly enriched in CpG shores and upstream of transcription start sites, suggesting a global regulation of transcriptional potential. Half of the hypermethylated genes overlapped with DNA hypermethylation in IDH1-mutant gliobastomas, suggesting the existence of a common set of genes whose expression may be affected by mutations in IDH1 or IDH2 in different types of tumors.

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;)

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;)

Identification of Rtl1 , a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis

We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the Dlk1-Dio3 imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of Retrotransposon-like 1 (Rtl1) expression as the only consistent alteration detected in all SB–induced tumors with Dlk1-Dio3 integrations, suggesting that Rtl1 activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple Dlk1-Dio3 domain members and HCC, we show here that direct modulation of a single domain member, Rtl1, can promote hepatocarcinogenesis in vivo. Overexpression of Rtl1 in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of RTL1 in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The Rtl1 locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying Rtl1 as a novel driver of HCC, our study represents one of the first direct in vivo demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.

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 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.

Combinations of biomarkers and Milan criteria for predicting hepatocellular carcinoma recurrence after liver transplantation.

Growing evidence suggests that pretransplant alpha‐fetoprotein (AFP) predicts outcomes of hepatocellular carcinoma (HCC) patients treated with liver transplantation. We aimed to determine whether pretransplant AFP, Lens culinaris agglutinin‐reactive alpha‐fetoprotein (AFP‐L3), and des‐gamma‐carboxyprothrombin (DCP) predicted HCC recurrence after transplantation. A retrospective cohort study of 313 HCC patients undergoing transplantation between 2000 and 2008 was conducted, and 48 (15.3%) developed recurrence during a median follow‐up of 90.8 months. The 127 patients with available serum drawn before transplantation were included; they included 86 without recurrence and 41 with recurrence. Serum was tested for AFP, AFP‐L3%, and DCP in a blinded fashion with the μTASWako i30 immunoanalyzer. All biomarkers were significantly associated with HCC recurrence. The hazard ratios (HRs) were 3.5 [95% confidence interval (CI), 1.9‐6.7; P < 0.0001] for DCP ≥ 7.5 ng/mL and 2.8 (95% CI, 1.4‐5.4; P = 0.002) for AFP ≥ 250 ng/mL. The HR increased to 5.2 (95% CI, 2.3‐12.0; P < 0.0001) when AFP ≥ 250 ng/mL and DCP ≥7.5 ng/mL were considered together. When they were combined with the Milan criteria, the HR increased from 2.6 (95% CI, 1.4‐4.7; P = 0.003) for outside the Milan criteria to 8.6 (95% CI, 3.0‐24.6; P < 0.0001) for outside the Milan criteria and AFP ≥ 250 ng/mL and to 7.2 (95% CI, 2.8‐18.1; P < 0.0001) for outside the Milan criteria and DCP ≥7.5 ng/mL. Our findings suggest that biomarkers are useful for predicting the risk of HCC recurrence after transplantation. Using both biomarkers and the Milan criteria may be better than using the Milan criteria alone in optimizing the decision of liver transplantation eligibility. Liver Transpl 21:599–606, 2015.

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.