Institut für Pathologie

Trogerstrasse 18

D-81675 München

The lab page of Karl-Friedrich Becker

- Gene expression analysis -

Gene expression

Supported in part by:

DFG - Sonderforschungsbereich 456 (until Dec 31st, 2004)

and

in collaboration with:

Raju Tomer (Indian Institute of Technology - New Delhi) and

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Molecular analysis of gene expression in tumor pathology

Human cancers are diverse in their pathology and responsiveness to clinical treatment. This diversity is at least in part due to variations in cellular gene expression programs. Although the analyis of proteins - the key players in cells and potential drug targets - is advancing rapidly, there are situations in which the analysis of RNA rather than proteins can provide valuable information for the diagnosis of cancer. These situations include absense of an antibody for the protein of interest, expression of functionally defective proteins, expressed small nucleotide polymorphisms (SNPs), analysis of alternatively or abnormally spliced molecules, and functional analysis of splice site mutations. In this regard we focus on the analysis of RNA from clinical samples and demonstrate how gene expression studies on the RNA level using a variety of new tools can be useful for discovering new classes of tumors, for predicting clinical outcome or therapy response, and for designing novel personalized clinical interventions that can not be achieved with histology alone.

 

 

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E-cadherin defects activate a genetic program for tumor metastasis determined by cDNA microarray analysis

 

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.

 

© 2003-2006 Dr. Karl-Friedrich Becker, Institut für Pathologie, TU München

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Generation of our cadherin/catenin cDNA microarray

(larger view)

Reference:
Hofler H, Specht K, Becker KF. Molecular analysis of gene expression in tumor pathology. Adv Exp Med Biol. 2003;532:19-26

Reference:

Laux H, Tomer R, Mader MT, Smida J, Budczies J, Kappler R, Hahn H, Blochinger M, Schnitzbauer U, Eckardt-Schupp F, Hofler H, Becker KF. Tumor-associated E-cadherin mutations do not induce Wnt target gene expression, but affect E-cadherin repressors. Lab Invest. 2004 Oct;84(10):1372-86