Combination Analysis of Activator Protein-1 Family Members, Sp1 and an Activator Protein-2α-Related Factor Binding to Different Regions of the Urokinase Receptor Gene in Resected Colorectal Cancers

Abstract

Purpose: Studies on the transactivation of genes via promoter elements have mostly been done on cell lines rather than resected tissues. This, however, is essential to address an in vivo or clinical relevance. We have previously shown tumor-specific binding of Sp1 and an activator protein (AP)-2^related factor to promoter region À152/À135 of the metastasis-related u-PAR gene in 60% of in vivo^resected cancer tissues. Cell lines have implicated an additional role, and potential synergism, of an AP-1 region (À190/À171) in u-PAR regulation. This study was done to (a) analyze AP-1 binding to this region in resected tumor and normal tissues, and define subgroups in which it is tumor-specific, and (b) to analyze transcription factor^binding patterns to both promoter motifs in resected tissues, supporting synergism, and draw first prognostic conclusions. Experimental Design: In 103 patients with colorectal cancer, electrophoretic mobility shift assay/supershift analysis for u-PAR promoter region À190/À171 was done in tumors and normal tissues. In 71 patients, region À152/À135 was also analyzed. U-PAR protein was measured by ELISA. Results: Tumor-specific AP-1 binding to region À190/À171 of the u-PAR promoter was found in 40% of patients. Subgroup analysis showed tumor-specific binding for c-Fos in 58%, for c-Jun in 50%, for JunD in 39%, and for Fra-1 in 4% of cases. AP-1 binding correlated significantly with u-PAR protein amounts in both normal and tumor tissues (P < 0.001), in contrast to a tumor-specific correlation with u-PAR of the AP-2/Sp1 region. In analyses for both promoter regions, 62% of cancers showed simultaneous binding for AP-1, AP-2, and Sp1, 11% for AP-1 and AP-2, 16% for AP-2 and Sp1, 4% for AP-2 only, 3% for AP-1 only, and 0% for Sp1 only. The binding of AP-1, AP-2, and Sp1 correlated significantly with each other (P < 0.001), the combination of AP-1 and AP-2 showing the highest correlation with u-PAR (P = 0.008). Preliminary survival analysis indicated a trend for poorer prognosis for binding of all three transcription factors. Conclusion: This is the first study differentiating transcription factor^binding to two important u-PAR promoter regions in a large series of resected tumors and normal tissues. The AP-1 site seems to be a less tumor-specific regulator than the Sp1/AP-2 motif. Nevertheless, data corroborate the hypothesis of synergism between both elements in resected tumors.Analyses of molecular pathways have largely been attempted in artificial cell line models which have acquired several molecular and phenotypic changes during culture. Therefore, their ability to reflect the situation in naturally occurring tumors, or tissues in general, has to be questioned. Specifically, research on transcriptional regulation has rarely been done in authentic resected

Src Induces Urokinase Receptor Gene Expression and Invasion/Intravasation via Activator Protein-1/p-c-Jun in Colorectal Cancer

“…Tissue specimens (tumor, normal mucosa) were collected after verification by a pathologist and frozen immediately in liquid nitrogen. Tissues were processed and analyzed for AP-1 binding to u-PAR promoter region À190/À171 and Src activity as described (15,20).…”

“…To support this notion by further ex vivo data from resected tissues of patients, the tumor and corresponding normal tissues of 20 patients undergoing surgery for colorectal cancer were analyzed for endogenous Src activity and for the binding of AP-1 complexes to the u-PAR promoter region À190/À171, with gelshift and Src assay methods (15,20,21). A significant association was found between specific Src activities and AP-1 binding in resected tumor (P = 0.01, linear regression, Fig.…”

“…Nuclear extracts, electrophoretic mobility shift assays (EMSA), and Supershift Analysis were carried out as described previously (20). The sequence of the upstream AP-1 consensus -containing region À190/À171 of the u-PAR promoter is 5 ¶-GTGATCACAACTCCATGAGTCAGGGCCGAG-3 ¶.…”

“…The sequence of the upstream AP-1 consensus -containing region À190/À171 of the u-PAR promoter is 5 ¶-GTGATCACAACTCCATGAGTCAGGGCCGAG-3 ¶. Gelshift analysis for AP-1 binding to region À190/À171 in resected tissues were done as described previously (20).…”

Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors

“…Nuclear extracts, EMSA and ChIP Preparation of nuclear extracts and EMSAs were done as described by Schewe et al (2005), using oligonucleotides spanning regions À152/À135 (5 0 -CCAGCCGGCCGCGCC CCGGGAAGGGA-3 0 ) and À380/À354 (5 0 -ATACCAG TATCCCTCCTGACAAAACTA-3 0 ) of the u-PAR promoter. ChIP assays were performed using anti-Sp1, anti-Sp3 and IgG according to the manufacturer's instructions (Upstate, Charlottesville, VA, USA).…”