Peroxidasin is associated with a mesenchymal-like transcriptional phenotype and promotes invasion in cutaneous metastatic melanoma

Cutaneous melanoma is a highly invasive, heterogeneous and treatment resistant cancer. It’s ability to dynamically shift between transcriptional states or phenotypes results in an adaptive cell plasticity that may drive cancer cell invasion or the development of therapy resistance. The expression of peroxidasin (PXDN), an extracellular matrix peroxidase, has been proposed to be associated with the invasive metastatic melanoma phenotype. We have confirmed this association by analysing the transcriptomes of 70 metastatic melanoma cell lines with variable levels of PXDN expression. This analysis highlighted a strong association between high PXDN expression and the undifferentiated invasive melanoma phenotype. To assess the functional role of PXDN in melanoma invasion, we performed a knockout of PXDN in a highly invasive cell line (NZM40). PXDN knockout decreased the invasive potential by ~50 % and decreased the expression of epithelial-mesenchymal transition and invasive marker genes as determined by RNAseq and substantiated by proteomics analysis. Bioinformatics analysis of differentially expressed genes following PXDN knockout highlighted decreases in genes linked to extracellular matrix formation, organization and degradation as well as signalling pathways such as the WNT pathway. This study provides compelling evidence that PXDN plays a functional role in melanoma invasion by promoting an invasive, mesenchymal-like transcriptional phenotype. NZM40 WT and PXDN KO cells (generated by CRISPR-Cas9 KO) were seeded at 700,000 cells in 10 mL of MEM-alpha medium with reduced FBS (0.75%) in a 10 cm cell culture dish for 48 h. After 48h, the cells were replenished with fresh 0.75% FBS medium for 5 hours before the medium was fully removed and replaced with 6 mL medium containing 0% FBS. After 22 h, the cells were washed twice with PBS, the buffer was removed thoroughly and the cells were harvested by scraping in residual PBS. The cells were snap frozen on dry ice and stored at -80 degrees until RNA extraction. Five biological replicates of each cell line were analysed, with cell pellets being harvested on different days.