Pancreatic neuroendocrine tumours (PNETs) may occur as part of the multiple endocrine neoplasia type 1 (MEN1) syndrome or as non-familial (sporadic) tumours. PNETs, which include gastrinomas, insulinomas and non-functioning tumours occur in more than 80% of MEN1 patients and account for 50% of disease-specific deaths. This is because 2540% of patients with PNETs will have metastasis at presentation, and current treatments, which include surgery, chemotherapy and radiotherapy for such advanced NETs rarely achieve a cure. Thus, additional therapies are required and we hypothesised that there maybe NET-specific receptors that could be targeted. We have therefore previously undertaken phage display screening using a human neuroendocrine cell line (BON-1) from a metastatic pancreatic carcinoid, to identify NET-binding peptides and reported a 12-mer peptide (P1). Confocal microscopy confirmed that P1 binds BON-1 cells, and trypan blue exclusion and Caspase-Glo 3/7 assays showed that P1 mediated a 70% decrease (P<0.005) in BON-1 cell proliferation and a 1.23-fold (P<0.02) increase in apoptosis respectively. To identify proteins that may interact with P1 on the surface of NET cells, BON-1 cell lysate was passed through streptavidin affinity chromatography columns loaded with biotinylated P1 or scrambled peptide control, fractions collected and digested with trypsin followed by mass spectrometry and protein identification software (MASCOT) analysis. A total of 247 proteins were identified that were unique to the P1 fractions. Using a MASCOT cut-off score of 50, these proteins included isoform 2 of heterogeneous nuclear ribonucleoprotein M (HNRNPM), heat shock cognate 71kD protein (HSPA8), PRKAR2A protein (PRKAR2A), prohibitin (PHB), cell division control protein 42 (CDC42) and Ras-related protein Rab-3D (RAB3D), which are involved in regulating exocytosis, proliferation, migration and protein folding. Further characterisation of these proteins may help in elucidating the role of P1 and its possible receptors in NET biology and in developing novel therapies for targeting pancreatic NETs.
07 Nov 2016 - 09 Nov 2016