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. 1998 Aug 18;95(17):10164-9.
doi: 10.1073/pnas.95.17.10164.

A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals

S Tanaka  1 , T Akiyoshi, M Mori, J R Wands, K Sugimachi
Affiliations
Free PMC article

A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals

S Tanaka et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

A novel member of the human frizzled (Fz) gene family was cloned and found to be specifically expressed in 3 of 13 well differentiated (23%), 13 of 20 moderately differentiated (62%), and 12 of 14 poorly differentiated (86%) squamous cell esophageal carcinomas compared with the adjacent uninvolved normal mucosa. The FzE3 cDNA encodes a protein of 574 amino acids and shares high sequence homology with the human FzD2 gene particularly in the putative ligand binding region of the cysteine-rich extracellular domain. Functional analysis revealed that transfection and expression of the FzE3 cDNA in esophageal carcinoma cells stimulates complex formation between adenomatous polyposis coli (APC) and beta-catenin followed by nuclear translocation of beta-catenin. Furthermore, cotransfection of a mutant construct encoding a FzE3 protein with a C-terminal truncation completely inhibited the interaction of APC with beta-catenin in cells. Finally, coexpression of FzE3 with Lef-1 transcription factor enhanced beta-catenin translocation to the nucleus. These observations suggest that FzE3 gene expression may down-regulate APC function and enhance beta-catenin mediated signals in poorly differentiated human esophageal carcinomas.

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Figures

Figure 1
Figure 1
Predicted amino acid sequences of human frizzled proteins and the pattern of expression in esophageal tumor tissues and adjacent normal mucosa. (A) Partial amino acid sequence of 7 human frizzled proteins located between the YPERPII and WWVILSL consensus sequences (FzE1-E7). The FzE2, FzE5 and FzE6 sequences are identical to those previously reported for FzD2, Hfz5, and FzD3, respectively (see Materials and Methods). (B) Expression pattern of FzE1-E7 in tissue samples of esophageal carcinoma (T) compared with adjacent normal mucosa (N). The number indicates the clinical sample.
Figure 2
Figure 2
(A) Complete nucleotide sequence (above) and deduced amino-acid sequence of FzE3 (below). FzE3 cDNA encodes a protein of 574 amino acids. The conserved cysteine residues are present in the N terminus (bold) and the XTXV motif known to bind to PDZ domains is present in the C-terminal tail (underlined). (B) Hydropathy profile of FzE3 protein as predicted by the Kyte–Doolittle algorithm with a window size of 12 amino acids. The signal peptide and seven transmembrane domains are indicated by ■. (C) Alignment of the predicted amino acid sequences of human FzE3 and FzD2. The cysteine-rich domain is boxed and the conserved 10 cysteine residues are represented by ░⃞. The FzE3 shares 78% identity to FzD2 and has 93% identity to the cysteine-rich extracellular domain.
Figure 3
Figure 3
FzE3 expression in clinical samples of esophageal carcinoma (T), normal adjacent mucosa (N), metastatic lymph nodes (L+), tumor free lymph nodes (L−), and cultured cell lines as measured by RT-PCR. KSE1, KSE2, TE4, TE5, KYSE150, and KYSE170 cells are derived from esophageal squamous cell carcinomas. DLD-1 and CaR-1 are cell lines derived from colon adenocarcinomas. Equal expression of glyceraldehyde-3-phosphate dehydrogenase certified the quality of mRNA in each sample.
Figure 4
Figure 4
(A) Schematic illustration of the FzE3 and FzE3ΔC proteins. (B) Interaction of APC with β-catenin in KYSE150 esophageal carcinoma cells transfected with empty plasmid (mock) and plasmid expressing FzE3 or FzE3ΔC. Cell lysates were immunoprecipitated by the anti-APC antibody (IP) followed by immunoblotting by using the anti-β-catenin antibody (Bl). (C) Localization of endogenous β-catenin in transfected KYSE150 cells. Mock: transfection with empty plasmid, FzE3: transfection with FzE3 expressing plasmid, FzE3/Lef-1: cotransfection of FzE3 with Lef-1 transcription factor expressing plasmid, FzE3ΔC/Lef-1: cotransfectantion of FzE3ΔC mutant construct with Lef-1 expression plasmid.
Figure 5
Figure 5
Diagram illustrating the potential role of FzE3 in down-regulating APC function in esophageal carcinoma cells. Normal APC and GSK-3β proteins target cellular β-catenin for degradation (Left) whereas in colon carcinoma cells with mutated APC, the β-catenin is not degraded, accumulates and binds to Lef/Tcf transcription factors (Center). Expression of FzE3 in a esophageal carcinomas represents functional mimicry of mutant APC signals possibly due to inactivation of GSK-3β and results in the formation of APC-β-catenin complexes (Right).
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