Silencing of Profilin-1 suppresses cell adhesion and tumor growth via predicted alterations in integrin and Ca2+ signaling in T24M-based bladder cancer models
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Maria Frantzi1,2, Zoi Klimou3,4, Manousos Makridakis1, Jerome Zoidakis1, Agnieszka Latosinska1, Daniel M. Borràs5, Bart Janssen5, Ioanna Giannopoulou6, Vasiliki Lygirou1, Andreas C. Lazaris6, Nicholas P. Anagnou3,4, Harald Mischak2, Maria G. Roubelakis3,4, Antonia Vlahou1
1Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
2Research and Development Department, Mosaiques Diagnostics GmbH, Hannover, Germany
3Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece
4Cell and Gene Therapy Laboratory, Biomedical Research Foundation of The Academy of Athens, Athens, Greece
5Research and Development Department, GenomeScan B.V., Leiden, The Netherlands
6First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece
Maria G. Roubelakis, email: email@example.com
Keywords: Profilin-1, bladder cancer, xenograft, gene silencing, metastasis
Received: March 22, 2016 Accepted: September 13, 2016 Published: September 23, 2016
Bladder cancer (BC) is the second most common malignancy of the genitourinary system, characterized by the highest recurrence rate of all cancers. Treatment options are limited; thus a thorough understanding of the underlying molecular mechanisms is needed to guide the discovery of novel therapeutic targets. Profilins are actin binding proteins with attributed pleiotropic functions to cytoskeletal remodeling, cell adhesion, motility, even transcriptional regulation, not fully characterized yet. Earlier studies from our laboratory revealed that decreased tissue levels of Profilin-1 (PFN1) are correlated with BC progression to muscle invasive disease. Herein, we describe a comprehensive analysis of PFN1 silencing via shRNA, in vitro (by employing T24M cells) and in vivo [(with T24M xenografts in non-obese diabetic severe combined immunodeficient mice (NOD/SCID) mice]. A combination of phenotypic and molecular assays, including migration, proliferation, adhesion assays, flow cytometry and total mRNA sequencing, as well as immunohistochemistry for investigation of selected findings in human specimens were applied. A decrease in BC cell adhesion and tumor growth in vivo following PFN downregulation are observed, likely associated with the concomitant downregulation of Fibronectin receptor, Endothelin-1, and Actin polymerization. A decrease in the levels of multiple key members of the non-canonical Wnt/Ca2+ signaling pathway is also detected following PFN1 suppression, providing the groundwork for future studies, addressing the specific role of PFN1 in Ca2+ signaling, particularly in the muscle invasive disease.
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