Project description:Merkel cell carcinoma (MCC) is a highly malignant skin cancer that expresses epithelial-, neuroendocrine-, and lymphoid-associated genes. Here, we focused on B-cell differentiation, which is characterised by the co-expression of PAX5 and TdT. PAX5 is the master regulator of B-cell commitment and is expressed in 65% of MCC cases. Yet, little is known about the underlying molecular biology of the frequently reported PAX5 expression in MCC. Multi-omics analyses, including RNA next-generation sequencing, RT-qPCR, immunohistochemistry, and western blotting, were performed to assess PAX5 expression in MCC. Differential DNA methylation analysis at 61,043 PAX5 binding sites in enhancer and promoter elements was performed to detect differences between n=14 MCC tissues and n=91 various normal B-cell populations. RNA analysis revealed full-length PAX5 expression in MCC at the transcriptional level using both PAX5 transcription start sites. PAX5 protein expression was found in 40 of 41 MCCs and six out of seven MCC cell lines. DNA methylation array analysis revealed 1,084 hypermethylated loci of enhancer and promoter elements located in PAX5 binding sites in MCC. Of these, 702 loci were associated with 257 genes which are not expressed. The PAX5-associated regulatory elements of these 257 genes were enriched for interferon regulatory factor 4 (IRF4) and SPi-proto-oncogene (SPI1) binding motifs. Neither IRF4 or SPI1 could be detected in MCC on a RNA or protein level. Thus, because of the absence of these transcription factors we conclude that full length PAX5 alone cannot induce B-cell differentiation.
