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Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas.

ABSTRACT: Mutations in the adenomatous polyposis coli or beta-catenin gene lead to cytosolic accumulation of beta-catenin and, subsequently, to increased transcriptional activity of the beta-catenin-T cell-factor/lymphoid-enhancer-factor complex. This process seems to play an essential role in the development of most colorectal carcinomas. To identify genes activated by beta-catenin overexpression, we used colorectal cell lines for transfection with the beta-catenin gene and searched for genes differentially expressed in the transfectants. There are four genes affected by beta-catenin overexpression; three overexpressed genes code for two components of the AP-1 transcription complex, c-jun and fra-1, and for the urokinase-type plasminogen activator receptor (uPAR), whose transcription is activated by AP-1. The direct interaction of the beta-catenin-T cell-factor/lymphoid-enhancer-factor complex with the promoter region of c-jun and fra-1 was shown in a gel shift assay. The concomitant increase in beta-catenin expression and the amount of uPAR was confirmed in primary colon carcinomas and their liver metastases at both the mRNA and the protein levels. High expression of beta-catenin in transfectants, as well as in additionally analyzed colorectal cell lines, was associated with decreased expression of ZO-1, which is involved in epithelial polarization. Thus, accumulation of beta-catenin indirectly affects the expression of uPAR in vitro and in vivo. Together with the other alterations, beta-catenin accumulation may contribute to the development and progression of colon carcinoma both by dedifferentiation and through proteolytic activity.

SUBMITTER: Mann B

PROVIDER: S-EPMC15532 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas.

Mann B B Gelos M M Siedow A A Hanski M L ML Gratchev A A Ilyas M M Bodmer W F WF Moyer M P MP Riecken E O EO Buhr H J HJ Hanski C C

Proceedings of the National Academy of Sciences of the United States of America 19990201 4

Mutations in the adenomatous polyposis coli or beta-catenin gene lead to cytosolic accumulation of beta-catenin and, subsequently, to increased transcriptional activity of the beta-catenin-T cell-factor/lymphoid-enhancer-factor complex. This process seems to play an essential role in the development of most colorectal carcinomas. To identify genes activated by beta-catenin overexpression, we used colorectal cell lines for transfection with the beta-catenin gene and searched for genes differentia ...[more]

PMID: 9990071

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Project description:BackgroundDeregulation of Wnt/β-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein β-catenin. β-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes MYC and CCND1 as well as the genes encoding the basic helix-loop-helix (bHLH) proteins ASCL2 and ITF-2B. To identify genes commonly regulated by β-catenin in colorectal cancer cell lines, we analyzed β-catenin target gene expression in two non-isogenic cell lines, DLD1 and SW480, using DNA microarrays and compared these genes to β-catenin target genes published in the PubMed database and DNA microarray data presented in the Gene Expression Omnibus (GEO) database.ResultsTreatment of DLD1 and SW480 cells with β-catenin siRNA resulted in differential expression of 1501 and 2389 genes, respectively. 335 of these genes were regulated in the same direction in both cell lines. Comparison of these data with published β-catenin target genes for the colon carcinoma cell line LS174T revealed 193 genes that are regulated similarly in all three cell lines. The overlapping gene set includes confirmed β-catenin target genes like AXIN2, MYC, and ASCL2. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells and one pathway - the steroid biosynthesis pathway - was regulated in all three cell lines.ConclusionsBased on the large number of potential β-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T cells as well as the large overlap with confirmed β-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study Wnt/β-catenin signaling and associated colorectal carcinogenesis. Furthermore, the confirmed and the newly identified potential β-catenin target genes are useful starting points for further studies.

Project description:Glioblastoma multiforme (GBM) is the most prevalent type of adult brain tumor, and one of the deadliest tumors known to mankind. The genetic understanding of GBM is, however, limited, and the molecular mechanisms which facilitate GBM cell survival and growth within the tumor microenvironment are largely unknown. We applied digital karyotyping and single nucleotide polymorphism (SNP) arrays to screen for copy number changes in GBM samples and found that the most frequently amplified region is at chromosome 7p11.2. The high resolution of digital karyotyping and SNP arrays permits the precise delineation of amplicon boundaries and has enabled identification of the minimal region of amplification at 7p11.2, which contains two genes, EGFR and SEC61γ. SEC61γ encodes a subunit of a heterotrimeric protein channel located in the endoplasmic reticulum (ER). In addition to its high frequency of gene amplification in GBMs, SEC61γ is also remarkably overexpressed in 77% of GBMs, but not in lower-grade gliomas. The siRNA-mediated knockdown of SEC61γ expression in tumor cells led to growth suppression and apoptosis. Furthermore, we showed that pharmacological ER stress agents induce SEC61γ expression in GBM cells. Together, these results indicate that aberrant expression of SEC61γ serves significant roles in GBM cell survival, likely via a mechanism that is involved in the cytoprotective ER stress-adaptive response to the tumor microenvironment. hSETD1A was silenced in HCT116 cells using retrovirus harboring shRNA specifically against the hSETD1A gene. 10μg RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions (Applied Biosystems). cDNA products were treated with 100ng RNaseA and then purified using the Qiagen PCR purification kit according to the manufacturer’s instructions. NimbleGen Human Gene Expression array was purchased from Roche Applied Sciences. cDNA samples were labeled, Cy3 hybridized, and processed at the FSU NimbleGen Microarray Facility at Florida State University.

Project description:BackgroundLymphoid Enhancer Factor-1 (Lef-1) is a member of a transcription factor family that acts as downstream mediator of the Wnt/beta-catenin signalling pathway which plays a critical role in osteoblast proliferation and differentiation. In a search for Lef-1 responsive genes in human osteoblasts, we focused on the transcriptional regulation of the SLUG, a zinc finger transcription factor belonging to the Snail family of developmental proteins. Although the role of SLUG in epithelial-mesenchymal transition and cell motility during embryogenesis is well documented, the functions of this factor in most normal adult human tissues are largely unknown. In this study we investigated SLUG expression in normal human osteoblasts and their mesenchymal precursors, and its possible correlation with Lef-1 and Wnt/beta-catenin signalling.ResultsThe experiments were performed on normal human primary osteoblasts obtained from bone fragments, cultured in osteogenic conditions in presence of Lef-1 expression vector or GSK-3beta inhibitor, SB216763. We demonstrated that the transcription factor SLUG is present in osteoblasts as well as in their mesenchymal precursors obtained from Wharton's Jelly of human umbilical cord and induced to osteoblastic differentiation. We found that SLUG is positively correlated with RUNX2 expression and deposition of mineralized matrix, and is regulated by Lef-1 and beta-catenin. Consistently, Chromatin Immunoprecipitation (ChIP) assay, used to detect the direct Lef/Tcf factors that are responsible for the promoter activity of SLUG gene, demonstrated that Lef-1, TCF-1 and TCF4 are recruited to the SLUG gene promoter "in vivo".ConclusionThese studies provide, for the first time, the evidence that SLUG expression is correlated with osteogenic commitment, and is positively regulated by Lef-1 signal in normal human osteoblasts. These findings will help to further understand the regulation of the human SLUG gene and reveal the biological functions of SLUG in the context of bone tissue.

Dataset Information

Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas.

Publications

Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas.

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