Gene Regulation Associated with IFN-Resistance Mechanisms in Melanoma

Abstract

Melanoma is the most aggressive form of skin cancer with rapidly increasing rates of incidence in fair-skinned populations worldwide. The most promising current treatment for advanced stage melanoma involves a combination of chemotherapy and interferon alpha (IFN-α), although complete response rates are unacceptably low. Loss of sensitivity to IFNs in melanoma cells has been shown to arise from a deficiency in the level of intracellular signalling molecules including STAT1, STAT2 & IRF9, which are important transcription factors in the IFN signalling pathway. Of these, deficiencies in STAT1 show the greatest correlation with IFN resistance. Greater understanding of the regulation of STAT1 gene transcription is important for predicting, and potentially improving, IFN mediated activity in malignant melanomas. In a previous study, distinct enhancer and repressor regions were shown to be present in the first and second introns of the human STAT1 gene. A gene segment comprising 123 base pairs located in the second intron was shown to confer specific repression of reporter gene constructs in melanoma cell lines. Within this regulatory region, we have identified a putative repressor element-1 (RE1) site, and shown binding of the RE1 silencer of transcription (REST) protein in melanoma cell lines by chromatin immunoprecipitation (ChIP) assays. In transient transfection assays, a luciferase gene reporter construct comprising the RE1 sequence from the STAT1 gene cloned immediately downstream of the STAT1 promoter and enhancer regions exhibited significant repression of reporter gene activity. Furthermore, progressively stronger repression was conferred by cloning tandem copies of the STAT1-RE1 sequence adjacent to the promoter/enhancer region, confirming the repressive activity of this element. The partial loss of repression of the STAT1 promoter by the RE1 element in a cell line expressing a low level of the REST protein further confirmed the functional significance of REST and the RE1 element within the STAT1 gene.