Restoration of epigenetically silenced SULF1 expression by 5-aza-2-deoxycytidine sensitizes hepatocellular carcinoma cells to chemotherapy-induced apoptosis.

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: 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 number 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-dC on sulfatase activity and drug-induced apoptosis. RESULTS: SULF1 mRNA was down-regulated in 9/11 HCC cell lines but only 6/10 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.

Activation of the transcription factor GLI1 by WNT signaling underlies the role of SULFATASE 2 as a regulator of tissue regeneration.

Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced ß-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and ß-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

Methylated Bone Morphogenetic Protein 3 (BMP3) Gene: Evaluation of Tumor Suppressor Function and Biomarker Potential in Biliary Cancer.

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.