High Podocalyxin levels promote cell viability partially through up- regulation of Annexin A2 Isabelle V. Leefa Chong San a , Ga € elle Prost a , Jennifer A. Williams b , Stephen E. Moss b , Ulrike A. Nuber a, * a Lund Strategic Center for Stem Cell Biology, Lund University, Lund, Sweden b Department of Cell Biology, University College London Institute of Ophthalmology, London, United Kingdom article info Article history: Received 5 July 2016 Received in revised form 20 July 2016 Accepted 23 July 2016 Available online 25 July 2016 Keywords: Podocalyxin Annexin A2 Cell viability Neural stem/progenitor cells MAPK pathway abstract Podocalyxin (PODXL) is a highly glycosylated and sialylated transmembrane protein that is up-regulated in various types of tumors and whose expression levels positively correlate with tumor grade. We previously found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells (NSPs). Here we investigated the effects of elevated Podxl levels in these cells. NSPs overexpressing Podxl did not form brain tumors upon intracranial transplantations, indicating that high levels of this gene alone are not sufficient for tumor initiation. However, Podxl overexpression had a positive effect on cell number, sphere formation and cell viability, indicating that it might in this way contribute to the development and/or maintenance of tumors. Proteome analyses of Podxl-overexpressing and control NSPs revealed increased levels of Annexin A2 (ANXA2). We also found increased transcript levels, indicating that PODXL stimulates expression of the Anxa2 gene. Lack of Anxa2 in Podxl-overexpressing NSPs resulted in reduced viability of these cells, suggesting that PODXL-mediated pro-survival effects can at least in part be explained by increased ANXA2 levels. Finally, our data indicate that Podxl overexpression activates the MAP kinase (MAPK) pathway which in turn up-regulates Anxa2 expression. Our data indicate a novel molecular connection between PODXL and ANXA2: both exert pro-survival effects in NSPs, and PODXL positively regulates ANXA2 expression through the MAPK pathway. © 2016 Elsevier Inc. All rights reserved. 1. Introduction Podocalyxin is a member of the CD34 family of sialomucins and was initially identified as the major protein on the apical domain of podocytes in the kidney [1]. High expression of the PODXL gene has been linked to tumors of several organs including breast, prostate, lung, liver, blood, colon, testicles, thyroid gland, pancreas, ovary, and brain [2e10] and is frequently associated with aggressive tu- mor variants [2,3,11]. With respect to brain tumors, a correlation between high PODXL expression and increasing glioma grade and decreasing patient survival rate has been reported [2]. Consistent with this, we found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells [12] and thus sought to investigate the consequences of Podxl up-regulation in these cells. The exact role of PODXL in tumors is not yet established. Several in vitro studies using tumor cell lines support a facilitation of cell migration and invasion by PODXL [6,8,10,13,14], which might in part be related to the anti-adhesion property conferred by sialic acid residues located on its extracellular mucin domain [14]. To investigate the effects of PODXL, most studies have either knocked-down or overexpressed the gene in tumor cell lines. The drawback of such lines is their phenotypic and genotypic drift occurring under the selection pressure of long term cell culture conditions. We followed a gain-of-function approach using NSPs which represent cells of origin for different brain tumor types [12] and as primary cells are not associated with the disadvantages of tumor cell lines. We show that overexpression of Podxl in NSPs has a positive effect on cell numbers and sphere formation in vitro, the latter reflecting in vitro self-renewal capacity. Our experiments further- more reveal that high PODXL levels increase cell viability in vitro and in vivo. We identified ANXA2 as a protein that is up-regulated in PODXL-overexpressing cells and our results indicate that this occurs through the MAPK pathway. Finally, we show that lack of * Corresponding author. Present address: Technical University Darmstadt, Schnittspahnstrasse 13, 64287 Darmstadt, Germany. E-mail address: nuber@bio.tu-darmstadt.de (U.A. Nuber). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2016.07.102 0006-291X/© 2016 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 478 (2016) 573e579