Methylation-associated silencing of heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2 (3-OST-2) in human breast, colon, lung and pancreatic cancers

Abstract

Aberrant CpG methylations play important roles in cancer development and progression. In this study, aberrant methylations in human breast cancer were searched for using methylation-sensitive representational difference analysis (MS-RDA). A CpG island (CGI) in the 5 0 region of the heparan sulfate d-glucosaminyl 3-Osulfotransferase-2 (3-OST-2) gene was found to be hypermethylated, while its exon 2 was hypomethylated. In seven breast cancer cell lines, hypermethylation of the 5 0 region and loss of 3-OST-2 expression were observed. Treatment with a demethylating agent, 5-aza-2 0 -deoxycytidine, removed the methylation of the CGI in the 5 0 region and restored its expression, demonstrating silencing of the 3-OST-2 gene. Methylation-specific PCR (MSP) analysis in 85 primary breast cancers showed that the hypermethylation of the CGI in the 5 0 region was present in 75 (88%) of them. Quantitative reverse transcriptase-PCR (RT-PCR) analysis in 37 primary breast cancers showed that the average expression level was decreased in them. Further, MSP analysis in primary colon, lung and pancreatic cancers showed that hypermethylation of the CGI in the 5 0 region was present in the colon (8/10, 80%), lung (7/10, 70%) and pancreatic (10/10, 100%) cancers. These results showed that silencing of 3-OST-2 was present in a wide range of human cancers. The 3-OST-2 gene encodes an enzyme involved in the final modification step of heparan sulfate proteoglycans (HSPGs), and its silencing is expected to result in abnormal modification of HSPGs and abnormal signal transduction. From the high incidence, silencing of the 3-OST-2 gene is expected to have high diagnostic, and potentially therapeutic, values.

Aberrant promoter methylation of multiple genes during multistep pathogenesis of colorectal cancers

“…Primer sequences and conditions for RT-PCR product were as described previously. 17,26,31 The housekeeping gene GAPDH was used as an internal control to confirm the success of the RT reaction. Total RNA from human NME was obtained from Clontech (Palo Alto, CA).…”

“…13 In the present study, we determined the methylation status profile of 19 TSGs including RUNX3, 3OST2 and SOCS1 genes in nonmalignant colonic epithelia (NME), CAs and CRCs. The 19 genes were chosen for study because of their presumed or known roles in various cellular functions related to cancer development, including cell cycle regulation, tissue invasion and metastasis, JAK-STAT and TGF-b signal pathways, key components of retinoid activity, signal transduction, apoptosis, angiogenesis, putative cytokine, mitotic stress checkpoint, methyltransferase superfamily and O-sulfotransferase [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] and were selected from the 6 ''hallmarks of cancer'' 32,33 (Table I). The aims of our study were as follows: (i) to clarify the methylation status of TSGs not previously studied in detail in CRC and correlate methylation with gene expression; and (ii) to investigate the role of methylation during the multistage pathogenesis of CRC, comparing the tumor profile with that of CAs and NME.…”

The heparan sulfate sulfotransferase 3-OST3A (HS3ST3A) is a novel tumor regulator and a prognostic marker in breast cancer

“…11,15,31 Our study shows that 3-OST3A was repressed and subjected to hypermethylation in all breast cancer cell lines tested except in HER2+-SKBR3 cells in which, consistently, it was expressed at relatively high level. Further analysis of the chromatin interactors at the 3-OST3A promoter showed that the epigenetic regulation not only depends upon DNA methylation, but also on repressive histone marks, and involves the recruitment of PcG proteins.…”

“…Prompted by studies from us and others pointing to an epigenetic control of 3-OST genes 11,15 , we determined whether 3-OST3A was regulated epigenetically in breast cancer cells. 3 DNA methylation analysis of the 3-OST3A promoter CpG island ( Figure S1A) showed strong hypermethylation in T-47D and MDA-derived cells, while in MCF-7 cells, hypermethylation only occurred downstream from the TSS ( Figure 1B).…”

“…10 Recent studies highlight an aberrant modulation of 3-OST gene expression through epigenetic mechanisms in various tumors including breast, lung, brain, pancreatic, skin and colorectal cancers, suggesting an important role in these pathological conditions. 11,12 However, these reports reveal either anti-oncogenic or tumor-promoting effects, and the mechanisms and consequences of 3-OST dysregulation in the cancer process are thus not well understood. 13,14 We recently showed that epigenetic repression of 3-OST genes in chondrosarcoma cells leads to altered HSPG sulfation, and demonstrated tumor-suppressive properties of 3-O-sulfation in chondrosarcoma cells.…”