DNA methylation is a frequent epigenetic event in many human cancers
[19, 20]. The transcriptional silencing by the hypermethylation of CpG islands in the promoter region, is a well-known common mechanism for the inactivation of tumour suppressor genes in human malignancies
[21, 22]. In an ongoing study aiming at investigating the possible epigenetic changes of oncosuppressor genes involved in sarcoid carcinogenesis, we have recently demonstrated a noticeable alteration of expression of the FHIT protein (Strazzullo et al., 2012), which encouraged the investigation of the role of other oncosuppressor genes and of their regulative mechanisms in sarcoid molecular pathology. Since many lines of evidence support the association between HPV infection and MGMT expression alteration
[23, 24], we analyzed the role of the MGMT gene.
In this study we investigated the expression of the MGMT protein within a normal fibroblast cell line (E-Derm) and the fully transformed sarcoid fibroblast lines explanted from an equine sarcoid tumour (EqSO1a and EqSO4b), as well as the pattern of expression in normal skin and sarcoid tissues. Our data indicate, for the first time, a noticeable reduction and or absence of MGMT protein in naturally occurring sarcoid tumours as well as in sarcoid derived cell lines.
Sarcoids may exist as six different clinical types
, we have not found a correlation between down-regulation of MGMT expression and clinical appearance, suggesting the existence of a common mechanism underlying the reduction of protein expression that acts early during the development of equine sarcoids. Our results are in agreement with several previous studies indicating that the loss and or reduced expression of MGMT is frequent in a variety of tumours
, particularly in HPV-induced cervical lesions
[26–28]. It is possible that decrease of MGMT protein expression is a common mechanism of cancer development among different species.
It has been suggested that the activity of MGMT increases as the severity of neoplasia and its clinical stage also increases
[29, 30]. Sarcoid is considered as a “benign” non metastatising tumour. In this regard, it is reasonable to speculate that the reduction and/or absence of MGMT in sarcoids may be correlated to their relative benign biological behavior
The presence of a functional MGMT gene is essential to avoid occurring mutations in other important genes involved in cancerogenic processeses. The silencing of MGMT gene induces a mutator pathway affecting some oncogenes such as Ras
. It is worth noting that sarcoid tumours and sarcoid derived cell lines show an up-regulation of Ras activity (Altamura et al., manuscript in preparation). It is therefore possible to hypothesize a link between MGMT down-regulation and Ras constitutive activation. However, further investigations are needed to confirm this, which is at moment only a matter of speculation.
As previously observed for the FHIT protein, EqSO4b cells revealed lower MGMT expression in comparison to EqSO1a. MGMT reduced expression in vitro has been linked to its binding to HPV E6 oncoprotein which is able to enhance the ubiquitin-dependent proteolysis of this oncosuppressor
. Additionally, HPV-16 E7 is able to modulate the DNA methylation activity
. Since the EqSO4b cell line contains higher copy numbers of viral genome and oncoprotein transcripts than the EqSO1a
, it is possible that the observed decreased level of MGMT protein found may directly correlate with BPV oncogene expression levels and be due to a similar mechanisms induced by BPV E5/E7 oncoproteins. Further studies are needed to elucidate the molecular mechanisms underlying this peculiar aspect.
Promoter methylation is the primary epigenetic alteration associated with transcriptional silencing of tumour suppressor genes during tumourigenesis
[34–36]. To determine if DNA methylation was involved in altered MGMT expression in sarcoid tumours, we performed the sodium bisulfite sequencing analysis of the CpG island associated to the 5′ untranslated region of MGMT
. Our data indicated that in MGMT gene promoter heavy hypermethylation occurred in 1 out of the 10 analyzed sarcoid samples and in EqSO4b cells. The sarcoid sample and the EqSO4b cell line showed an overall similar hypermethylation pattern. Differences may be explained by the presence of some polymorphisms, a fortiori considering that the analysis was carried out for a not translated region. EqSO4b is a primary culture cell line and this may introduce some difference too. Moreover it’s to be considered that the region is very complex in its sequence structure and this may promote polymerase slippage.
Similar frequency of MGMT hypermethylation is observed in HPV induced cervical cancer
, thus supporting the hypothesis that MGMT promoter methylation is not a common feature of PVs induced tumours.
Our findings indicate an association between BPV infection and MGMT protein expression alteration suggesting the possibility of a mechanistic role for this gene as a cofactor triggering the development of equine sarcoid tumour in concert with BPV.