Genetic and Epigenetic Regulation of the Human Prostacyclin Synthase Promoter in Lung Cancer Cell Lines

Abstract

The importance of the arachidonic acid pathway has been established in colon and lung cancers, as well as in inflammatory diseases. In these diseases, prostacyclin I 2 (PGI2) and prostaglandin E 2 (PGE2) are thought to have antagonistic activities, with PGI2 exerting anti-inflammatory and antiproliferative activities, whereas PGE2 is proinflammatory and antiapoptotic. In human lung cancer, prostacyclin synthase (PGIS) and PGI2 are down-regulated, whereas PGE2 synthase (PGES) and PGE2 are up-regulated. Murine carcinogenesis models of human lung cancer reciprocate the relationship between PGIS and PGES expression. PGIS-overexpressing transgenic mice are protected from carcinogen-and tobacco smoke -induced lung tumor formation, suggesting that PGI2 may play a role in chemoprevention. We investigated several potential mechanisms for the down-regulation of PGIS in human lung cancer. Using transcription reporter assays, we show that single nucleotide polymorphisms in the PGIS promoter can affect transcriptional activity. In addition, PGIS expression in several human lung cancer cell lines is silenced by CpG methylation, and we have mapped these sites across the variable number of tandem repeats (VNTR) sequence in the promoter, as well as CpGs within exon 1 and the first intron. Finally, using fluorescence in situ hybridization, we show that human lung cancer cell lines and lung cancer tissues do not have a loss of the PGIS genomic region but multiple copies. These results show that an individual's PGIS promoter haplotype can play an important role in the predisposition for lung cancer and CpG methylation provides an epigenetic mechanism for the down-regulated PGIS expression. (Mol Cancer Res 2007;5(3):295 -308)

Functional Prostacyclin Synthase Promoter Polymorphisms. Impact in Pulmonary Arterial Hypertension

“…Transfection data (Figure 2) suggest that shorter VNTRs are associated with lower PGIS transcriptional activity. Although SNP3 alleles did not show significant variation in transcriptional activity in the cells studied here, we previously found the wt (C) alleles had lower activity in lung cancer cell lines (12). The unaffected carriers had a significant bias for SNP4 minor allele (A) compared with all PAH and control groups (Fisher exact test, P = 0.019).…”

“…SNPs 1, 2, and 3 along with the sequence numbering were previously described (9). and the 59 side of intron 1 (12). By unraveling this sequence complexity, in conjunction with functional transcriptional studies, we find more active PGIS promoter variants are significantly associated with unaffected carriers in HPAH families, suggesting that an enhanced level of endogenous PGIS expression is a potential mechanism for protection from HPAH disease.…”

“…The PGIS promoter region we focused on was determined to be a minimum promoter through directed deletion analysis (12). The PGIS promoter region is 231 bp proximal to the protein synthesis MET1 contained in exon 1 and is a CpG island.…”

“…All cloned PGIS promoter constructs, produced by PCR from genomic DNA, were made by fusing the 213 bp promoter variant in the native context of the MET1 of exon 1 of the PGIS as the MET1 of Luciferase (Luc) in pGL3-Basic (12). By constructing the PGIS promoter Luc plasmids in this way, each sequence polymorphism can be individually evaluated for promoter activity within a defined sequence context.…”

“…We expanded our investigation to include all of exon 1 and approximately 220 bp of the 59 end of intron 1, and discovered a new SNP (SNP4, C325A) since our earlier report (12). With the completion of the 1,000 Genomes Project (15), SNP4 was identified (rs116939356) as C/A with a minor allele frequency (MAF) of 6% in whites.…”