Abstract
Our previous studies on melanoma antigens identified two new polypeptides, named MELOE-1 and MELOE-2, that are involved in immunosurveillance. Intriguingly, these antigens are coded by distinct open reading frames (ORF) of the meloe mRNA which is significantly expressed only in the melanocytic lineage. In addition, MELOE-1 and -2 specific T cell clones recognized melanoma cells but very poorly normal melanocytes suggesting differential translation of meloe in normal vs tumor cells. This prompted us to elucidate the mechanisms of translation of these antigens in melanoma cells. We first demonstrated that no splicing event or cryptic promoter could generate shorter meloe transcripts containing only one of the two ORFs. Triggering meloe RNA degradation with a siRNA close to the ORF coding for MELOE-2 abrogated expression of both MELOE-1 and MELOE-2, thus confirming that the two ORFs are always associated. Next we showed, in a bicistronic reporter system, that IRES activities could be detected upstream of MELOE-1 and MELOE-2 and finally confirmed their translation from full length meloe cDNA in melanoma cells with eGFP constructs. In conclusion, meloe is a polycistronic mRNA that generates both MELOE-1 and MELOE-2 antigens through IRES-dependent translation in melanoma cells and that may explain their tumor specificity.
Highlights
Several clinical trials have suggested that T cell immunotherapy may be worthwhile in cancer and in metastatic melanoma [1]
Considering that these two co-expressed antigens are coded by distinct open reading frame (ORF) within meloe mRNA, we looked for the presence of alternate meloe transcripts that may contain only one of the two ORFs
Meloe mRNA was described in our earlier publications as the source of two melanoma antigens, MELOE-1 and MELOE2 that could generate a T cell response [10,19], we did not explore the mechanisms governing its translation
Summary
Several clinical trials have suggested that T cell immunotherapy may be worthwhile in cancer and in metastatic melanoma [1] In this respect, tumor antigens that can be recognized by T lymphocytes have been classified into mutated, differentiation, tumor-specific and over-expressed antigens [2]. Translation of a short ORF from a processed pseudogene (i.e. in which the main ORF is invalidated) can lead to T cell epitope generation in melanoma as we previously reported [8] It was reported in prostate cancer and chronic myeloid leukemia, that a short ORF located downstream of the main ORF coding for myotrophin was translated by an IRES (Internal Ribosomal Entry Sequence)dependent mechanism to generate the novel MPD6 antigen [9]
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