Symplekin promotes tumorigenicity by up-regulating claudin-2 expression.
Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.
Buchert
Michael
M
Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut National de la Santé et de la Recherche Médicale U661, and Université Montpellier 1 and 2, Montpellier F-34094, France.
Papin
Marina
M
Bonnans
Caroline
C
Darido
Charbel
C
Raye
Warren S
WS
Garambois
Véronique
V
Pélegrin
André
A
Bourgaux
Jean-François
JF
Pannequin
Julie
J
Joubert
Dominique
D
Hollande
Frédéric
F
D000818:Animals; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D057167:Claudins; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D047368:Tumor Burden; D015854:Up-Regulation
D022762:CCAAT-Enhancer-Binding Proteins; C491875:CLDN2 protein, human; D057167:Claudins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C101043:SYMPK protein, human; C094867:YBX3 protein, human; D019938:Cyclin D1