SUN-018 Epigenetic Loss of the PIWI/piRNA Machinery in Gonadal Tumors in Androgen Insensitivity Syndrome

Abstract: There is a high frequency of Testicular Germ Cell Tumors (TGCT) in 46,XY individuals with disorders of sexual development (46,XY DSD), which include Androgen Insensitivity Syndrome (AIS). That TGCT risk leads to pragmatic gonadectomy in these patients with several further implications. A better knowledge of TGCT tumorigenesis would be helpful either for alternative treatments or tumoral markers. Retrotransposons are mobile DNA elements that duplicate by retrotransposition, a “copy and paste” mechanism using an RNA intermediate. They composed around 50% of whole human genome. Retrotransposition has been proven as a tumorigenic mechanism in several human cancer cases due to its ability to cause genomic instability. Despite of that high frequency, they are repressed by DNA methylation through the piRNA pathway. The loss of that methylation could cause retrotransposons derepression and further tumor development. In order to analyze a possible role for epigenetic loss of piRNA machinery in seminomas from AIS patients, we performed a methylation analyses using bisulfite modification coupled to pyrosequencing of the 5’ end promoter CpG islands of the main PIWI protein genes (PIWI1, PIWI2 and PIWI4) and of the transposable element LINE-1 (long intersped element - 1) in gonads samples with anatomopathological seminoma from patients with proved AIS (AR mutations: c.2521C>G and c.384_385delGA; both with complete AIS). As control, we used the contralateral gonad of those patients, without any evidence of gonadal tumor. All samples were analyzed in quintuplicate. The methylation status was analyzed using QUMA webtool. The methylation of each CpG island was compared between tumoral versusnon-tumoral gonads. There was a hypermethylation of the PIWI protein genes (1, 2 and 4; p=0.03, 0.001, and 0.01 respectively). RT-PCR of PIWI genes showed a reduced expression of PIWI genes in tumoral samples in comparison with non-tumoral samples (1, 2, and 4; p=0.01, 0.02, and 0.0005 respectively). The opposite (hypomethylation) occurred with LINE-1 sequencing in tumoral samples (p<0.001). PIWI-family genes, together with the piRNAs are able to provoke DNA methylation leading to transposons silencing and genome integrity. In semonima from AIS, the PIWI genes hypermethylation reduced the PIWI genes expression which was related with transposable LINE-1 hypomethylation. These epigenetic alterations suggest LINE-1 as a possible mechanism for testicular tumors in AIS through PIWI/piRNA pathway disruption. As it happened in a context without any androgen action (complete AIS), it’s possible that epigenetic loss is an androgen-independent mechanism. These results open possibilities for further research on treatment based on transposable elements (as reverse transcriptase inhibitors) and for serum markers derivate from LINE-1 proteins and/or RNA.