Nuclear Legumain Activity in Colorectal Cancer

Abstract

The cysteine protease legumain is involved in several biological and pathological processes, and the protease has been found over-expressed and associated with an invasive and metastatic phenotype in a number of solid tumors. Consequently, legumain has been proposed as a prognostic marker for certain cancers, and a potential therapeutic target. Nevertheless, details on how legumain advances malignant progression along with regulation of its proteolytic activity are unclear. In the present work, legumain expression was examined in colorectal cancer cell lines. Substantial differences in amounts of pro- and active legumain forms, along with distinct intracellular distribution patterns, were observed in HCT116 and SW620 cells and corresponding subcutaneous xenografts. Legumain is thought to be located and processed towards its active form primarily in the endo-lysosomes; however, the subcellular distribution remains largely unexplored. By analyzing subcellular fractions, a proteolytically active form of legumain was found in the nucleus of both cell lines, in addition to the canonical endo-lysosomal residency. In situ analyses of legumain expression and activity confirmed the endo-lysosomal and nuclear localizations in cultured cells and, importantly, also in sections from xenografts and biopsies from colorectal cancer patients. In the HCT116 and SW620 cell lines nuclear legumain was found to make up approximately 13% and 17% of the total legumain, respectively. In similarity with previous studies on nuclear variants of related cysteine proteases, legumain was shown to process histone H3.1. The discovery of nuclear localized legumain launches an entirely novel arena of legumain biology and functions in cancer.

Proteolysis mediated by cysteine cathepsins and legumain—recent advances and cell biological challenges

“…However, the recent observations of activation under neutral conditions also point to an alternative activation process in which stabilization may facilitate the transition to its active endo-proteolytic form (Dall and Brandstetter 2013). In agreement with this, legumain has been observed in multiple compartments inside and outside the cell covering a wide range of conditions, suggesting that there may exist a diversity of compartment-specific biochemical pathways leading to legumain activation (Chen et al 1998;Haugen et al 2013). Just how legumain ends up in such a variety of intracellular and extracellular compartments, how regulation of its trafficking within endo-lysosomal compartments and its secretion into the extracellular space both remain poorly understood.…”

“…Enzymatic activities and proteins resembling, related, or being even identical with endo-lysosomal proteases have been detected in the cytoplasm, the mitochondria, and the nucleus (Dall and Brandstetter 2013;Duncan et al 2008;Goulet et al 2004;Haugen et al 2013;Maher et al 2014;Sullivan et al 2009;Tamhane et al 2014;Tedelind et al 2010). Below, we will outline possible pathways and cell biologically meaningful processes in an attempt to explain the occurrence of endolysosomal proteases and/or variants thereof in such highly unexpected locations.…”

“…One explanation is by their cytosolic translation and alternative folding to expose nuclear localization signals or through transport into the nucleus by hijacking proteins destined for nuclear entry (Bestvater et al 2005;Tedelind et al 2010). The nuclear appearance of legumain is easier to explain in this regard, because a nuclear localization sequence may be present (Haugen et al 2013).…”

Transcriptional regulation of the legumain gene by p53 in HCT116 cells

“…These results indicate that transcription of legumian and its activity were positively regulated by p53. Legumain plays an important role in tumor growth/metastasis [5][6][7][8][9][10][11], and cystatin E/M suppresses legumain activity and its processing [22,23]. …”

“…We further reported that legumain might have an important role in remodeling of the extracellular matrix through degradation of fibronectin in renal proximal tubular cells [4]. It has been suggested that legumain plays an important role in tumor growth/metastasis, carotid artery-atherosclerosis development [5][6][7][8][9][10][11][12], hemophagocytic syndrome [13] and formation of human unstable carotid plaque [14]. Recently, we reported that degradation of annexin A2 was reduced by knockdown of legumain in the mouse kidney [15].…”