The E1 Copper Binding Domain of Full-Length Amyloid Precursor Protein Promotes Epithelial to Mesenchymal Transition in DU145 Cells in an Isoform-Specific Manner.

Epithelial to mesenchymal transition (EMT) confers migratory and dynamic properties on cells and, as such, plays a pivotal role in the development of metastatic, castration resistant prostate cancer. The amyloid precursor protein (APP), although most closely associated with the neurodegenerative condition Alzheimer's disease, has also been linked to the pathogenesis and prognosis of several cancers including prostate cancer. Aims: To investigate whether over-expression of APP could promote EMT in prostate cancer (PCa) DU145 cells and to determine the molecular prerequisites for this effect. Methodology: A range of APP molecular constructs were stably expressed in DU145 cells and their effects on EMT were monitored by morphological analysis and by immunoblotting for the EMT marker proteins, E-cadherin and vimentin. Results: Our results show that the full-length 695 amino acid isoform (APP695), but not APP751 or APP770, promoted EMT via a mechanism requiring an intact extracellular E1 copper binding domain and tyrosine687 within the cytosolic domain of the protein. Conclusion: Targeting the expression of APP695 or the E1 copper binding domain of the protein may, therefore, contribute to therapeutic strategies for the delay or prevention of prostate cancer metastasis.

Differential Regulation of E-Cadherin Expression by the Soluble Ectodomain and Intracellular Domain of Jagged1.

Aberrant Jagged1-mediated Notch activation is linked to cancer and induces epithelial-tomesenchymal transition through the repression of E-cadherin transcription. All three proteins are subject to sequential proteolytic events referred to as regulated intramembrane proteolysis. This process releases soluble protein ectodomains from the cell and, concomitantly, generates intracellular domains capable of nuclear translocation and transcriptional regulation. Aim: To determine the cognate roles of the Jagged1 ectodomain and intracellular domain fragments in the regulation of E-cadherin expression. Methodology: Human embryonic kidney cells were stably transfected with coding DNA constructs analogous to full-length Jagged1, the soluble Jagged1 ectodomain, or the intracellular domain fragment of the protein. Correct construct expression and processing were confirmed by immunoblot analysis of transfectant cell lysates and conditioned culture medium. The effects of the various Jagged1 constructs on endogenous E-cadherin expression and processing were subsequently monitored by immunoblot and RT-qPCR analyses. Results: Both full-length Jagged1 and the soluble Jagged1 ectodomain construct downregulated E-cadherin expression at the protein and RNA level. In contrast, the Jagged1 intracellular domain fragment construct enhanced E-cadherin expression but only at the RNA level. Conclusion: The soluble Jagged1 ectodomain is sufficient for the down-regulation of Ecadherin expression whereas the intracellular domain of the protein does not exhibit such an effect and actually increases E-cadherin RNA expression. These results raise the interesting possibility of E-cadherin regulation in cells distal to the site of soluble Jagged1 ligand generation.