Abstract
Engrailed-2 (EN2) is a transcription factor involved in development, where it has multiple functions and is expressed in a caudal-to-rostral gradient in the midbrain. Its mRNA and protein expression are absent from most adult cells, but are switched back on in cancer cells. Although transcription factors are usually located in the nucleus, a number of previous reports have indicated that EN2 protein could be in the cell membrane and on the cell surface on tumour samples, from where some at least might be released, as EN2 has been found in the urine of prostate cancer patients - a more readily obtainable clinical sample than tumour biopsies. In this study EN2 protein is definitively shown to be on the membrane of prostate cancer cells and in the tumour microenvironment. The commercial anti-EN2 antibody was found to be non-specific and we therefore used a tagged version of EN2 to study its cellular distribution and behaviour. This revealed different modes of EN2 protein transport and secretory mechanisms in different cancer cell lines. Live cell imaging further revealed the generation of secretory vesicles from PC3 cells, which are derived from metastatic prostate cancer, but not WPMY-1 cells that are derived from normal prostate fibroblasts. The findings further suggest that EN2 protein switches roles during tumour progression, from a transcriptional regulator to a regulator of protein translation in localised regions of the cytoplasm. The latter mechanism is especially significant as EN2 cellular localisation becomes dysregulated in cancer, becoming widely cytoplasmic and available for packaging into luminal vesicles. The findings also indicate that the translation factor EIF4E is a potential binding partner of EN2, in prostate cancer. The study findings also indicate that a monoclonal antibody-drug conjugate targeting EN2 may not be the most effective method of targeting EN2-expressing cells. A blocking peptide or antibody would be more appropriate to prevent its secretion and transfer, both of which have been shown to be possible mechanisms of tumour progression. Alternatively, because less EN2 is secreted (and hence more is retained) in prostate cancer cell lines with low metastatic potential, such as LnCaP, T cells could be employed to target early-stage prostate cancer. To conclude, cancer cells have seemingly retained the ability to tightly regulate the expression of EN2 protein in a spatial and temporal manner, unlike normal adult cells. EN2 is secreted in large vesicles by cells from more advanced prostate tumours and thus monoclonal antibodies may not be the most effective approach to therapy.