Silencing of Profilin-1 suppresses cell adhesion and tumor growth via predicted alterations in integrin and Ca2+ signaling in T24M-based bladder cancer models.
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.
Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.|Research and Development Department, Mosaiques Diagnostics GmbH, Hannover, Germany.
Maria
M
Frantzi
Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece.|Cell and Gene Therapy Laboratory, Biomedical Research Foundation of The Academy of Athens, Athens, Greece.
Zoi
Z
Klimou
Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Manousos
M
Makridakis
Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Jerome
J
Zoidakis
Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Agnieszka
A
Latosinska
Research and Development Department, GenomeScan B.V., Leiden, The Netherlands.
Daniel M
DM
Borràs
Research and Development Department, GenomeScan B.V., Leiden, The Netherlands.
Bart
B
Janssen
First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece.
Ioanna
I
Giannopoulou
Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Vasiliki
V
Lygirou
First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece.
Andreas C
AC
Lazaris
Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece.|Cell and Gene Therapy Laboratory, Biomedical Research Foundation of The Academy of Athens, Athens, Greece.
Nicholas P
NP
Anagnou
Research and Development Department, Mosaiques Diagnostics GmbH, Hannover, Germany.
Harald
H
Mischak
Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece.|Cell and Gene Therapy Laboratory, Biomedical Research Foundation of The Academy of Athens, Athens, Greece.
Maria G
MG
Roubelakis
Proteomics Laboratory, Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Antonia
A
Vlahou
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D018450:Disease Progression; D015536:Down-Regulation; D019332:Endothelin-1 / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D019042:Muscle Neoplasms / Q000473:pathology* / Q000556:secondary; D051304:Profilins / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D001743:Urinary Bladder / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000473:pathology*; D060449:Wnt Signaling Pathway; D023041:Xenograft Model Antitumor Assays
D000199:Actins; D019332:Endothelin-1; D039081:Integrin alpha5beta1; C495738:PFN1 protein, human; D051304:Profilins; D034741:RNA, Small Interfering; D002118:Calcium
Profilin-1; bladder cancer; gene silencing; metastasis; xenograft
Int J Cancer. 2015 Mar 1;136(5):E359-86
25220842
J Proteome Res. 2010 Jun 4;9(6):3243-59
20423150
Bioinformatics. 2015 Jan 15;31(2):166-9
25260700
Nucleic Acids Res. 2015 Jan;43(Database issue):D764-70
25593349
Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21547-52
21115820
Clin Cancer Res. 2014 Jun 15;20(12):3094-106
24737547
Nat Rev Cancer. 2015 Jan;15(1):25-41
25533674
J Biol Chem. 2001 Oct 26;276(43):40247-53
11509569
J Muscle Res Cell Motil. 1983 Oct;4(5):569-88
6643679
Eur Urol. 2014 Apr;65(4):778-92
24373477
Genome Biol. 2013 Apr 25;14(4):R36
23618408
Cell Signal. 2010 Mar;22(3):527-32
19922791
J Cell Biochem. 2008 Nov 1;105(4):1081-91
18773414
Clin Exp Metastasis. 2000;18(5):391-400
11467771
Development. 2006 Nov;133(21):4219-31
17021034
Trends Cell Biol. 2004 Aug;14(8):461-9
15308213
Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15780-5
20798032
Cancer. 2014 Jan 1;120(1):86-95
24122346
Cell Adh Migr. 2015;9(3):247-53
25495756
Am J Pathol. 2000 May;156(5):1515-25
10793063
Eur J Cell Biol. 2008 Nov;87(11):893-904
18707793
J Cell Sci. 2005 Jan 15;118(Pt 2):331-41
15615774
Biochem Biophys Res Commun. 2000 Sep 24;276(2):454-60
11027496
Nat Rev Urol. 2015 Jun;12(6):317-30
26032553
Stem Cells Dev. 2007 Dec;16(6):931-52
18047393
J Urol. 2012 Dec;188(6):2391-7
23088985
J Clin Invest. 2011 Jan;121(1):132-47
21183790
Stem Cell Rev. 2014 Jun;10(3):417-28
24500853
J Proteome Res. 2013 Sep 6;12(9):3969-79
23924207
FASEB J. 1999 Jan;13(1):181-9
9872942
Mol Cancer. 2014 Aug 07;13:187
25103363
Cell Adh Migr. 2012 Sep-Oct;6(5):442-9
23076048
World J Gastroenterol. 2015 Feb 28;21(8):2323-35
25741138
Cell. 2002 Sep 20;110(6):673-87
12297042
Breast Cancer Res Treat. 2014 Nov;148(1):61-72
25284724
Oncotarget. 2015 Apr 20;6(11):9220-39
25940704
PLoS One. 2014 Aug 01;9(8):e103868
25084196
Biophys J. 2008 Dec 15;95(12):5544-73
18835900
Bioinformatics. 2012 Oct 15;28(20):2678-9
22914218
Cancer Cell Int. 2015 Jul 05;15:70
26213494
Philos Trans R Soc Lond B Biol Sci. 2015 Feb 5;370(1661):20140042
25533099
Lancet Oncol. 2015 Jul;16(7):795-803
26095785
Nat Cell Biol. 2012 Oct;14(10):1046-56
23000962
Tumour Biol. 2014 Jun;35(6):5675-87
24584816
Cell Mol Bioeng. 2011 Mar 1;4(1):1-8
21566724
PLoS One. 2015 Sep 01;10(9):e0137217
26325675
Int J Cancer. 2010 Dec 15;127(12):2893-917
21351269
Nucleic Acids Res. 2012 Jan;40(Database issue):D565-70
22123736
Nucleic Acids Res. 2015 Jan;43(Database issue):D662-9
25352552
J Biol Chem. 2014 Jan 3;289(1):387-402
24273166
Biochemistry. 2013 Sep 17;52(37):6456-66
23947767
J Biol Chem. 2014 Apr 4;289(14):10029-44
24550382
EMBO J. 2003 May 15;22(10):2324-33
12743027
J Cell Physiol. 2009 May;219(2):354-64
19115233
Anal Quant Cytol Histol. 2001 Aug;23(4):291-9
11531144
Hepatology. 2014 Mar;59(3):1130-43
24123265
Mol Cell Proteomics. 2012 Apr;11(4):M111.009449
22159600
Oncogene. 2014 Apr 17;33(16):2065-74
23686314
Genome Biol. 2014;15(12):550
25516281