Tumor-associated E-cadherin mutations do not induce Wnt target gene expression, but affect E-cadherin repressors.
E-cadherin is a cell-cell adhesion molecule and tumor invasion suppressor gene that is frequently altered in human cancers. It interacts through its cytoplasmic domain with beta-catenin which in turn interacts with the Wnt (wingless) signaling pathway. We have compared the effects of different tumor-derived E-cadherin variants with those of normal E-cadherin on Wnt signaling and on genes involved in epithelial mesenchymal transition. We established an in-house cDNA microarray composed of 1105 different, sequence verified cDNA probes corresponding to 899 unique genes that represent the majority of genes known to be involved in cadherin-dependent cell adhesion and signaling ('Adhesion/Signaling Array'). The expression signatures of E-cadherin-negative MDA-MB-435S cancer cells transfected with E-cadherin variants (in frame deletions of exon 8 or 9, D8 or D9, respectively, or a point mutation in exon 8 (D370A)) were compared to that of wild-type E-cadherin (WT) transfected cells. From the differentially expressed genes, we selected 38 that we subsequently analyzed by quantitative real-time RT-PCR and/or Northern Blot. A total of 92% of these were confirmed as differentially expressed. Most of these genes encode proteins of the cytoskeleton, cadherins/integrins, oncogenes and matrix metalloproteases. No significant expression differences of genes downstream of the Wnt-pathway were found, except in E-cadherin D8 transfected cells where upregulation of three Tcf/Lef-transcribed genes was seen. One possible reason for the lack of expression differences of the Tcf/Lef-regulated genes is upregulation of SFRP1 and SFRP3; both of which are competitive inhibitors of the Wnt proteins. Interestingly, known E-cadherin transcriptional repressors, such as SLUG (SNAI2), SIP1 (ZEB2), TWIST1, SNAIL (SNAI1) and ZEB1 (TCF8), but not E12/E47 (TCF3), had a lack of upregulation in cells expressing mutated E-cadherin compared to WT. In conclusion, E-cadherin mutations have no influence on expression of genes involved in Wnt-signaling, but they may promote their own expression by blocking upregulation of E-cadherin repressors.
Institute of Pathology, Technical University, Munich, Germany.
Holger
H
Laux
Raju
R
Tomer
Michael T
MT
Mader
Jan
J
Smida
Jan
J
Budczies
Roland
R
Kappler
Heidi
H
Hahn
Michaela
M
Blöchinger
Udo
U
Schnitzbauer
Friederike
F
Eckardt-Schupp
Heinz
H
Höfler
Karl-Friedrich
KF
Becker
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics*; D045744:Cell Line, Tumor; D002999:Clone Cells; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012334:RNA, Neoplasm / Q000032:analysis; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D015854:Up-Regulation; D051153:Wnt Proteins
D015820:Cadherins; D011518:Proto-Oncogene Proteins; D012334:RNA, Neoplasm; D012097:Repressor Proteins; D051153:Wnt Proteins