Abstract 1093: Legumain in colorectal cancer: Unorthodox localization and trafficking
Authors: Haugen Mads H. , Flatmark Kjersti , Damgaard Ingrid , Tamhane Tripti , Engesæter Birgit , Tveito Siri , Egeland Eivind V. , Solberg Rigmor , Johansen Harald T. , Pettersen Solveig J. , Boye Kjetil , Brix Klaudia , Mælandsmo Gunhild M. 
Published: May 2015

Abstract
The cysteine proteinase legumain is unique in cleaving substrates N-terminally of asparagine, and recent observations also revealed carboxypeptidase activity. This specificity combined with its up-regulation in a number of solid tumors prompts to take advantage of legumain for cancer therapy and as a prognostic biomarker. Traditionally legumain was thought to reside within the endo-lysosomes, but the proteinase has also been reported expressed elsewhere in the cell, and we recently described nuclear localization of proteolytically active legumain in colorectal cancer cells. Presently, trafficking of legumain in the cell remains enigmatic, particularly when not routed to the endo-lysosomes.
Our recent analysis of legumain expression in tissue from primary colorectal cancer (n=277) revealed that the subcellular localization pattern in tumor cells was diversified between the cases. Some had mostly dispersed and others more granulated cytoplasmic expression, while nuclear localization could be observed in about one third of the patients. We have thus established the association of legumain expression with clinicopathological parameters and outcome, and found that high levels of total or nuclear legumain in colon cancer cells were significantly negative for overall survival. Although an overall lower expression, nuclear localized legumain was also observed in normal tissue while in cytoplasm it was found polarized primarily to the apical side of the epithelial layer. We have previously observed legumain in the extracellular media of cancer cells and are in the process of analyzing wether it can also be found in exosomes. The unexpected observations in cellular localization are presumably due to altered trafficking. We have therefore analyzed legumain transport in colorectal cancer cells treated with tunicamycin to block N-glycosylation of de novo synthesized legumain, which had a striking effect on the trafficking and activation of the proteinase. Regarding functionality, we hypothesize that nuclear legumain may be acting as a transcription factor and have made fusion constructs of legumain and HaloTag® to identify potential genomic targets. The fusion protein has so far been verified to be highly expressed in the nuclei and will be used for chromatin capture.
While the unique features of legumain make it an attractive target to exploit for therapy and diagnosis of cancer, our results reveal that location and function of legumain in normal and cancerous tissue is not completely elucidated. Such factors may be of outmost importance in attempts to utilize the protease activity for diagnostic and/or therapeutic purposes and may also represent novel cancer treatment opportunities.
Citation Format: Mads H. Haugen, Kjersti Flatmark, Ingrid Damgaard, Tripti Tamhane, Birgit Engesæter, Siri Tveito, Eivind V. Egeland, Rigmor Solberg, Harald T. Johansen, Solveig J. Pettersen, Kjetil Boye, Klaudia Brix, Gunhild M. Mælandsmo. Legumain in colorectal cancer: Unorthodox localization and trafficking. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1093. doi:10.1158/1538-7445.AM2014-1093