O6-methylguanine-DNA methyltransferase in equine sarcoids: molecular and epigenetic analysis

Abstract

BackgroundBovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to the loss of function of a number of oncosuppressor genes. In this study the putative role of O6-methylguanine-DNA methyltrasferase (MGMT) was investigated for sarcoids. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissues. In addition, the DNA methylation profile was analysed to assess the role of epigenetic mechanism in regulation of MGMT expression.ResultsA group of 15 equine sarcoids and two primary sarcoid cell lines (fibroblasts) were analyzed for the expression of MGMT protein by immunohistochemistry, immunofluorescence and Western blotting techniques. The sarcoid cell line EqSO4b and the tumour samples showed a reduction or absence of MGMT expression. To investigate the causes of deregulated MGMT expression, ten samples were analyzed for the DNA methylation profile of the CpG island associated to the MGMT promoter. The analysis of 73 CpGs encompassing the region of interest showed in 1 out of 10 (10%) sarcoids a pronouncedly altered methylation profile when compared to the control epidermal sample. Similarily the EqSO4b cell line showed an altered MGMT methylation pattern in comparison to normal fibroblasts.ConclusionAs previously demonstrated for the oncosuppressor gene FHIT, analysis of MGMT expression in sarcoid tissues and a sarcoid-derived fibroblast cell line further suggests that oncosuppressor silencing may be also involved in BPV-induced equine tumours. Abnormal DNA methylation seems to be one of the possible molecular mechanisms involved in the alteration of MGMT expression. Further studies are required to address other basic molecular mechanisms involved in reduced MGMT expression. This study underlines the possible role of DNA methylation in oncosuppressor inactivation in equine sarcoids.

Transcriptome analysis of equine sarcoids

“…A recent study by Mählmann et al revealed, in diseased tissues, elevated transcript levels of the FOXP3 ( Forkhead Box P3 ) gene, which encodes protein involved in immune response. In turn, Altamura et al and Strazzullo et al showed under‐expression of the DNA‐repair gene MGMT ( O‐6‐methylguanine‐DNA Methyltransferase ) and of the putative tumour suppressor gene FHIT ( Fragile Histidine Triad ). However, these observations were not confirmed in our study.…”

Host genetic influence on papillomavirus‐induced tumors in the horse

“…Equine sarcoid is the most common cancer of the horse . This nonmetastasizing skin tumor is strongly associated with infection by BPV1 and BPV2 2 and thus appears to be a rare example of cross‐species infection by a papillomavirus . However, there is also evidence for sequence variants of BPV1 in sarcoids, which may indicate that sarcoid is caused by horse‐specific BPV1 variants.…”

“…3 This nonmetastasizing skin tumor is strongly associated with infection by BPV1 and BPV2 2 and thus appears to be a rare example of cross-species infection by a papillomavirus. 26 However, there is also evidence for sequence variants of BPV1 in sarcoids, 27 which may indicate that sarcoid is caused by horse-specific BPV1 variants. The primary goal of this study was to determine if a SNP array-based GWAS would detect an association between susceptibility to sarcoid and allelic variants in the Major Histocompatibility Complex of the horse.…”

Equine Sarcoid

“…The E7 protein cooperates in evading innate immunity [19][20][21][22][23][24][25][26][27]. The oncogenesis of equine sarcoids also involves loss of expression of the Fragile Histidine Triad (FHIT) and of the O 6 -methylguanine-DNA methyltransferase (MGMT) tumor suppressor proteins [28,29]. Recent studies have evaluated the role of small non-coding RNAs that regulate gene expression (microRNAs) in the development of sarcoids, and the role of aberrant methylation (S100A14 gene) is under research [30][31][32][33][34].…”