Mutation search and association study of candidate genes in non melanoma skin cancer after organ transplantation

Prostaglandin E2 (PGE2) is a prostanoid with a variety of bioactivities, and has been implicated in various pathologies. PGE2 appears as a key molecule in tumour formation, involved in cell proliferation, angiogenesis, immune surveillance, and apoptosis. PGE2 is produced via three sequential enzymatic reactions: release of arachidonic acid (AA) from membrane glycerophospholipids by phospholipase A2 (PLA2), conversion of AA to the unstable intermediate prostanoid PGH2 by cyclooxygenase (COX), and isomerization of PGH2 to PGE2 by prostaglandin E synthase (PGES). PGE2 is released from cells and it interacts with four distinct receptors, EP1, EP2, EP3, and EP4. Over-expression of the inducible form of COX (Cox-2) and microsomal-prostaglandin E synthase-1 (mPGES-1), resulting in excessive prostaglandin E 2 (PGE2) production, has been observed in cancer of various tissues, including skin cancer. An over-expression of EP1 has been observed in skin cancer development induced by UVB. Alteration in gene expression, due to genetic variants located in the promoter region of the genes for prostaglandin synthase-2 (PTGS2/COX2), microsomal prostaglandin E synthase (mPGES-1), or prostaglandin E receptor 1 (PTGER1) may result in augmented PGE2 synthesis or in altered response to PGE2, and could represent risk factors for the development of non melanoma skin cancer (NMSC) in organ transplant recipients (OTRs). To determine if polymorphisms in these genes can be useful genetic markers of susceptibility that may contribute to early detection of individuals at greater risk of NMSC, a case-control study was performed. Two polymorphisms in the PTGS2 gene, three in the mPGES-1 gene, and three in the PTGER1 gene were genotyped in 286 OTRs, 144 NMSC cases and 142 controls. Allele –765G in the PTGS2 gene was more frequent in cases than in controls [p=0.015, OR=9.59 (1.36-192.66)], suggesting that this variant might represented a risk factor in the development of basal cell cancer (BCC) in individuals undergoing transplantation before 50 years of age. Analysis of polymorphisms –1760C>A (rs3810254), -1728G>A (rs3810255), and –1113C>T (rs2241359) in the 5’ proximal region of the PTGER1 gene showed that minor alleles of these variants were more represented in individuals with squamous cell cancer (SCC), when compared to matched controls who underwent transplantation before 50 years of age, but the difference did not reach significance. To verify if the 1452 bp fragment of the 5’ flanking region, which contains the three polymorphisms could exert promoter activity, we performed functional analysis in HeLa and HaCat cultured cells. Although a weak promoter activity was observed in HeLa cells, we were unable to demonstrate any promoter activity in HaCat cells, even after LPS stimulation. The 5’ flanking region of the mPGES-1 gene was screened by heteroduplex analysis to identify new variants. Three polymorphisms, located within conserved regions of the gene, and reported in NCBI databases as –664T>A (rs7873087), –663A>T (rs7859349) and –439T>C (rs7872802), were identified. The observed genotype distributions indicated complete linkage disequilibrium for the three polymorphisms, and no association with NMSC was observed. In conclusion, allele –765C in the PTGS2 gene seems to represent a protection factor against the development of BCC tumours in individuals undergoing transplantation before 50 years of age. Analysis of polymorphisms in the 5’ regions of the mPGES-1 and PTGER1 genes did not support the hypothesis that variants in these regions could play a major role in NMSC predisposition.