Changes in human Discs large (Dlg) and Scribble protein expression during malignant progression and analysis of Dlg gene regulation

GARDIOL, DANIELA; CAVATORTA, ANA LAURA; GIRI, ADRIANA A.; BANKS, LAWRENCE

Trieste, Italia

Congreso; ICGEB DNA Tumour Virus Meeting; 2007

ICGEB

There is a great deal of evidence suggesting that Dlg and Scribble expression is lost in human tumours as the disease progresses towards the latter stages of malignancy. We performed immunohistochemistry analysis for Dlg and Scrib expression using samples derived from both HPV-associated and non HPV-associated cancers. We observed that alterations in the expression pattern of hDlg and of Scrib increase during tumour progression; and demonstrated that there is an inverse relationship between the levels of both proteins and the loss of cell polarity and tissue architecture in the different biopsies, and that this is most likely linked to tumour suppression. However the molecular mechanisms regulating Dlg and Scrib expression, which may be responsible for the changes in their abundance during tumour formation, are not fully understood. At least in some HPV-negative tumour derived cells Dlg and Scrib transcription levels are extremely low. We have therefore initiated studies to investigate the mechanisms that control Dlg gene expression. We performed the cloning and analysis of a genomic 5´flanking region of Dlg ORF with promoter activity, and determined minimal and cis elements required for efficient transcription. Bioinformatic analysis of these sequences revealed the presence of two CpG–rich regions compatible with CpG islands. We observed a consistent increase in Dlg expression by immunoblotting and immunofluorescence in low-level Dlg-expressing cells when treated with the methylase inhibitor 5-aza-2’-deoxycytidine. These results might indicate that epigenetic mechanisms may also play a role in Dlg regulation. Using RACE techniques, we identified an alternative splicing event in the 5’ region of Dlg mRNA that generates transcripts with two different 5’-UTRs. We showed by reporter assays that the Dlg 5’-UTR containing the alternatively-spliced exon interferes with the translation of a downstream ORF, suggesting that this splicing event can contribute to down-regulation of Dlg. Moreover, we found within the Dlg promoter region consensus binding sites for cancer-related transcription factors, and their involvement on Dlg regulation is being evaluated.