Metastasis of Colorectal Cancers

Colorectal cancer is the leading cause of cancer death in developed countries. Although the prognosis for colorectal carcinomas has improved during the last twenty years, nearly one-half of the patients succumb as a result of the formation of metastases. The finding that metastasis and primary tumor were as similar in their overall pattern of gene expression as were repeated samplings of the same primary tumor, suggests that the molecular program of a primary tumor may generally be retained in its metastases. Traditional methods of analysis have imposed a practical limit on the number of candidate genes, the expression of which can be conveniently and simultaneously studied. Highly parallel methods for studying gene expression promise to be a means for quantitative and comparative expression analysis and to lead to the identification of molecules relevant e.g. for carcinogenesis and metastasis. We compared the gene expression profiles of colonic carcinomas, mostly staged T=2, from patients with a good survival with those from patients with a poor prognosis. Since we suspected that normal mucosa of tumor patients is not as normal as expected, colonic mucosa of healthy individuals was taken as a reference point. In each case normal or cancerous colonic epithelium was captured using laser-microdissection. Processed and labeled RNA was hybridized to Affymetrix GeneChips U95A, U95B, U95C, U95D, and U95E containing about 60 000 sequences, most of them ESTs (expressed sequence tags). No additional amplification of the RNA was performed, in order to not perturb the original representation of the RNA-sequences. Sequences showing a more than 4-fold differential expression difference were further examined. Our strategy was to dovetail the expression profiles observed by the GeneChip analysis with information stored in the Unigene Database using a newly developed in house software called “Integrated Genetic Map Service” (IGMS). The IGMS is a comprehensive information system that identifies UniGene clusters that are differentially expressed in different types of cancer with respect to certain reference tissues by calculating the ratio of the number of ESTs extracted from tumor tissue vs. the number of ESTs extracted from normal tissues. In addition, the system provides information about the tissue type which is important to assess the quality of the corresponding affymetrix spots. The relevance of molecules which show a differential expression in normal and cancer tissue both by GeneChip analysis and by database screenings is evaluated in larger patients groups by quantitative RT-PCR. To this end we have established a highly quantitative one step multiplex TaqMan assay system. Expression of four out of 35 candidate genes arising from bioinformatic searches were found to be overexpressed e.g. in the high-risk group with a poor prognosis. Kaplan-Meier/log rank statistical tests of up to 100 patients in three of the four genes demonstrated significant association of gene expression with poor survival. Expression of the genes was localized to epithelial cells by in-situ hybridization.
In another approach, tumor-associated alterations of cell surface glycosylation which play a crucial role for adhesion and metastasis of carcinoma cells were studied by investigating the effect of a2,6-sialylation for the adhesion properties of carcinoma cells. Colorectal and mammary carcinoma cells were sense-transfected with sialyltransferase ST6Gal-I cDNA or antisense-transfected with a part of the ST6Gal-I sequence. Sense transfectants showed an enhanced ST6Gal-I mRNA expression, ST6Gal-I enzyme activity and an increased binding of the lectin SNA, specific for a2,6-linked sialic acid. Transfection with ST6Gal-I in the antisense direction resulted in less enzyme activity and SNA-reactivity. A sense-transfected clone carrying increased amounts of a2,6-linked sialic acid adhered preferentially to collagen IV, showed reduced cell-cell adhesion and an enhanced invasion capacity. Effects of a2,6-linked sialic acid on growth and metastatic potential are now under investigation in an orthotopic in vivo model.

Recent publications:

Petretti T, Kemmner W, Schulze B, Schlag PM, Altered mRNA expression of glycosyltransferases in human colorectal carcinomas and liver metastases. GUT, 2000,46:359-366.

Schneider F, Kemmner W, Haensch W, Franke G, Gretschel S, Karsten U, Schlag PM, Overexpression of sialyltransferase ST6GalNAc-II is related to poor patient survival in human colorectal carcinomas. Cancer Research, 2001,61:4605-4611.

Brett D, Kemmner W, Koch G, Roefzaad C, Gross S, Schlag PM. A rapid bioinformatic method identifies novel genes with direct clinical relevance to colon cancer. Oncogene, 2001,20:4581-4585.

Kemmner W, Invasion und Metastasierung, in Chirurgische Onkologie (edited by Beck HG, Hohenberger W, Junginger T, Schlag PM), Georg-Thieme-Verlag,2001,9-13.

Lin S, Kemmner W, Grigull S, Schlag PM, Cell surface a2,6-sialylation affects adhesion of breast carcinoma cells. Exp Cell Res, 2002,276:101-110.

Schaefer G, Cramer T, Suske G, Kemmner W, Wiedenmann B, Hoecker M. Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements. J Biol Chem,2003,278:8190-8.

Kemmner W, Roefzaad C, Koch G, Haensch W, Schlag PM, Glycosyltransferase
expression in human colorectal tissue examined by oligonucleotide arrays. Biochim Biophys Acta, 2003, 1621:272-279.

Kemmner W, Schlag PM, Die Moleküle der Metastasierung. Chir Praxis,2003,61:203-212.

Gretschel S, Haensch W, Schlag PM, Kemmner W, Clinical relevance of sialyltransferases ST6GAL-I and ST3GAL-III in gastric cancer. Oncology,2003,139-145

Strowski MZ, Cramer T, Schäfer G, Stefan Jüttner, Walduck A, Stipani E, Kemmner W, Wessler S, Wunder C, Weber M, ,Meyer TF, Wiedenmann B, Jöns T, Naumann M and Höcker M. Helicobacter Pylori stimulates host Vascular Endothelial Growth Factor-A (vegf-A) Gene expression via MEK/ERK-Dependent Activation of Sp1 and Sp3. FASEB J, 2003, in press

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