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.
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.
Michael
M
Buchert
Marina
M
Papin
Caroline
C
Bonnans
Charbel
C
Darido
Warren S
WS
Raye
Véronique
V
Garambois
André
A
Pélegrin
Jean-François
JF
Bourgaux
Julie
J
Pannequin
Dominique
D
Joubert
Frédéric
F
Hollande
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