Abstract 4077: Plexin B1 trafficking in prostate cancer.
Authors: Legendre Anne , Masters John R W , Williamson Magali 
Published: August 2014

Abstract
Prostate cancer is a leading form of cancer in men with 324,000 cases diagnosed/year in the European Union. Plexin B1 is a cell-membrane receptor for semaphorins; its ligand is semaphorin 4D. Plexin receptors are involved in axon guidance, angiogenesis, immunity as well as cancer biology. We have identified a high frequency of somatic missense mutations in the plexin B1 gene and overexpression of the protein in prostate cancer. The mutations found in prostate cancer are functionally significant, altering signalling pathways downstream of plexinB1 and affecting cell phenotype. These results suggest that plexin B1 has an important role in prostate cancer progression. Previous studies have documented the unexpected localization of cell membrane receptors such as EGFR and ErbB2, in the nucleus and their role as transcription factors. Preliminary immunohistochemistry results in the laboratory indicated the presence of plexin B1 protein in the nucleus in tumour cells, suggesting that plexins may have a role in the nucleus. The aim of this study was to determine if plexin B1 is translocated to the nucleus in prostate cancer cells and what is the nuclear localization signal recognized for the trafficking of the protein.
The different techniques were used; Immunofluorescence was performed on cell lines with antibodies specific to the intracellular domain of plexin B1. Colocalization studies were carried out using the Volocity Software®. A cell fractionation assay was performed and cell lysates analysed by immunoblotting. A construct of the cytoplasmic domain of plexin B1 fused with GFP was generated and its subcellular localization in transfected cells analysed by confocal microscopy. The plexin B1/GFP construct is currently mutated on the putative NLS signal using site-directed mutagenesis. The subcellular localization of the plexin B1 mutants will be observed by confocal microscopy.
Immunofluorescence of prostate cancer cell lines showed the presence of the intracellular domain of plexin B1 receptor in the nucleus, suggesting a translocation of the protein to the nucleus. Plexin B1 was found to co-localize with DAPI staining. Further immunofluorescence studies showed that the presence of plexin B1 in the nucleus is dependent on semaphorin 4D binding in a time-dependent manner. The translocation occurs 15min after ligand binding and decreases after 40min of ligand binding. A cell fractionation assay followed by immunoblotting confirmed the preliminary immunofluorescence data. To assess the nuclear trafficking with a functional assay, a GFP tag was attached to the intracellular domain of plexin B1 and its translocation to the nucleus was observed in transfected cells.
In conclusion, the translocation of plexin B1 to the nucleus suggests an interesting novel role for this receptor in the nucleus. Plexin B1 could act as a transcription co-factor and may have an impact on carcinogenesis.
Citation Format: Anne Legendre, John R W Masters, Magali Williamson. Plexin B1 trafficking in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4077. doi:10.1158/1538-7445.AM2013-4077