Sarcoids account for an high percentage of all equine neoplasms and are the most common tumour diagnosed in horses [1
]. Few studies have been carried out so far concerning the molecular mechanisms underlying equine sarcoids carcinogenesis. It is well known that BPV is involved in the pathogenesis of the sarcoid, and the role of its oncoproteins in the carcinogenic process has been recently clarified [19
], however little is known about the molecular relationship between viral infection and neoplastic transformation. Recently, Yuan et al. [21
], have described the upregulation of MMP-1 and AP1 in sarcoid, underlying the invasivity potential of such tumour. To identify the key molecules in these phenomena and on the basis of the data deriving from other species, we analysed the role of the tumour suppressor FHIT gene. FHIT is a tumour suppressor gene and many lines of evidence support the association between HPV infection and FHIT expression alteration in different cancer types [22
]. However, FHIT downregulation has been recorded also in BPV induced bovine urinary bladder tumours which are composed of both epithelial and vascular (i.e. mesenchymal) tumours (personal observations).
In this study we have characterized the expression of FHIT protein by examining it in a normal fibroblast cell line (E-DERM) and fully transformed sarcoid fibroblast lines explanted from equine sarcoid tumours (EqSO1a and EqSO4b), as well as in normal skin and sarcoid tissues. The data we obtained document, for the first time, a reduction of FHIT protein expression in in vitro model of sarcoid-derived cell lines as well as in naturally occurring tumours. The consistency of data between in vivo and in vitro systems strenghten the validity of such cell culture system for gene expression analysis.
Our data indicate that FHIT protein is expressed in normal fibroblasts, whereas it is much reduced in both in vivo and in vitro tumours. It is worthwhile noting that EqSO4b has lower level of the protein compared to the EqSO1a. The EqSO4b cell line contains higher levels of viral genome and viral oncoprotein transcripts than EqSO1a [24
]. We may speculate that altered expression of FHIT protein may correlate with viral load and/or viral oncogenes expression. However, further studies are needed to clarify this point. Sarcoids may exist as six different clinical types [5
]; although we have examined a limited number of samples, we suggest a lack of correlation between down-regulation of FHIT expression and clinical appearance, indicating a common mechanism underlying the reduction of protein expression acting early during the development of equine sarcoids.
Our results are in agreement with several previous studies indicating that the loss of FHIT expression is frequent in a variety of tumors [25
] above all in PVs induced cervical lesions [11
]. Lowered levels of the FHIT protein were also documented in pagetoid variant of BPV induced urothelial tumour [18
Promoter methylation is the primary epigenetic alteration associated with transcriptional silencing of tumour suppressor genes during tumorigenesis [29
]. To determine the reasons for diminished FHIT expression in sarcoid tumour we performed the analysis of the methylation status of the FHIT 5'-CpG island. Our data indicated that FHIT gene promoter hypermethylation did not occur either in sarcoid tissues or in sarcoid derived cell lines, suggesting that this epigenetic mechanism may be not involved in FHIT aberrant expression in sarcoids. This is in accordance with a previous paper documenting no aberrant methylation causing FHIT promoter silencing in BPV induced vesical tumors of cattle [17
]. It is reasonable to assume that BPV does not alter the methylation status of FHIT promoter. It is worth noting that similar data have been obtained both in vitro and in vivo, thus again validating the sarcoid derived cell lines as a good model for the study of sarcoids [24
]. Our data show the presence of an alternative splicing of equine FHIT transcript in 5'UTR region; this is consistent with the data obtained in other species such as human and bovine [17
], in which several different 5'UTR regions were described. A well-known regulatory role has been described for the 5' and 3' UTR region in mammalian genome [33
]. The complex structure of the 5'UTR region of FHIT locus may suggest that this region might be target of post-transcriptional regulative mechanism.
In human species the mutational analysis of FHIT indicates the potential role of other mechanisms such as point mutation and deletions, with loss of heterozigosity (LOH). The human FHIT gene encompasses the FRA3B fragile site, a common target of genetic alterations [34
]. This locus is a hot spot for early inactivation in carcinogen-exposed tissue [13
]. FRA3B fragile site is also reported to be the site for HPV-16 integration and demonstrated to undergo frequent LOH in cervical carcinoma [36
]. Different PVs, such as HPV-16 and BPV-1, seems to share common processes of infection [10
]. We speculate that the involvement of BPV infection in equine tumorigenesis may be related with FHIT LOH, but this issue needs further investigation.
Our findings suggest an association between BPV infection and FHIT protein expression alteration raising the possibility of a mechanistic role for the FHIT gene as a cofactor with BPV in triggering the development of equine sarcoid tumour.