Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.

Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Association of APC gene mutations and histological characteristics of colorectal adenomas.

Fifty-nine colonic adenomas and 6 hyperplastic colonic polyps were analyzed by single-strand conformation polymorphism analysis for mutations in the adenomatous polyposis coli gene (APC). Frameshifts and premature stop codons in at least one copy of APC were detected in 25 of these adenomas. Five adenomas carried 2 APC mutations. No mutations in APC were found in any of the 6 hyperplastic polyps. The detection of APC mutations increased with size and degree of dysplasia and in rectal as compared to colonic adenomas, although the association was not statistically significant. The frequency of detectable APC mutations was higher in tubulovillous and villous adenomas (10 of 13) than in tubular adenomas (15 of 45) (odds ratio, 6.67; 95% confidence limits, 1.39-41.83; P = 0.005). The significance of the association between the detection of APC mutations and a villous architecture was confirmed in multivariate analysis (relative risk, 6.67; 95% confidence limits, 1.54-28.8; P = 0.005). In conclusion, APC mutation plays a role in adenoma progression; its frequency is significantly higher in lesions with a more villous morphology.

Characterization of Wnt-1 and Wnt-2 induced growth alterations and signaling pathways in NIH3T3 fibroblasts.

Members of the Wnt family induce mouse mammary tumors and partially transform mammary epithelial cells in culture. However, their mechanism of transformation remains to be elucidated. In NIH3T3 mouse embryo fibroblasts, a standard transformation model, Wnt-1 and Wnt-2 were shown to induce altered properties including increased saturation density and growth in soft agar. Such cells also exhibited increased cell-cell adhesiveness. However, unlike oncogenes such as PDGFB or ras, Wnt-1 and -2 failed to induce detectable transformed foci following transfection, and stable NIH3T3 transfectants lacked tumor forming capacity. Wnt-1 and -2 transfectants exhibited increased uncomplexed, cytosolic beta-catenin, which was not observed with PDGFB, ras or erbB2 transfectants. In transient transfection, Wnt-1 and -2 induced a rapid increase in cytosolic beta-catenin but no detectable increase in the phosphorylated activated forms of MAP kinase. In contrast, ras was a potent activator of MAP kinase but had no effect on free beta-catenin levels. These findings establish that both Wnt signaling and pattern of growth alterations differ from those of oncogenes which activate proliferative signaling pathways in NIH3T3 cells.

Human frizzled 1 interacts with transforming Wnts to transduce a TCF dependent transcriptional response.

The human homologue of fz1 (Hfz1) was cloned from a cDNA library. Hfz1 was shown to couple to Wnt signal transduction pathways by its ability to enhance Wnt induced TCF dependent transcription in both autocrine and paracrine modes. Enhanced TCF dependent signaling was dose dependent with respect to both Wnt-3A and Hfz1. Moreover, Hfz1 deletion mutants with truncated carboxy termini showed markedly reduced capacity to enhance Wnt signal transduction. Specificity was demonstrated with respect to signal transduction by different Wnts. While Wnt-3a, -3, -1 and to a lesser extent Wnt-2 cooperated with Hfz1 in the paracrine assay for TCF dependent signaling, neither Wnt-4, -5a, -5b, -6, -7a nor -7b did so, despite similar levels of expression. However, coimmunoprecipitation of Hfz1 with both Wnt-3a and Wnt-5a indicated that TCF dependent signaling in response to Wnts is not determined solely by their ability to bind the receptor. All of these findings provide strong evidence that Hfz1 is a functional partner for certain Wnts in inducing TCF dependent transcription.

Genetic events in sporadic colorectal adenomas: K-ras and p53 heterozygous mutations are not sufficient for malignant progression.

Twenty-four sporadic colorectal adenomas were analysed for the presence of allelic loss on the short arm of chromosome 17 as well as mutations in the K-ras and p53 genes. Chromosome 17p13 allelic loss was not present in 14 out of 14 informative cases. K-ras mutations were observed in 15 out of 24 cases. A p53 gene mutation (GGC-->GAC at codon 245) was detected in two biopsies taken at a four year interval from a recurrent rectal villous adenoma. Both biopsies also contained the same K-ras gene mutation (GGT-->GTT at codon 12). The data from the recurrent rectal adenoma provide in vivo evidence that K-ras and p53 heterozygous mutations confer a proliferative advantage but together are not sufficient for malignant transformation.

Genomic PCR-SSCP analysis of the metastasis associated NM23-H1 (NME1) gene: a study on colorectal cancer.

To facilitate further mutational analysis of NM13-H1, a human metastasis suppressor gene, we have established its genomic organization. NM23-H1 is composed of five exons, spanning a genomic DNA fragment of 10 kb. Using oligonucleotide primers flanking each exon, PCR-SSCP analysis was performed on genomic DNAs of healthy individuals. A common polymorphism, a C to T transition, was detected 30 nucleotides upstream from the 5' splice site flanking exon 1. As NM23-H1 allele loss and altered expression have been reported in colorectal cancer, genomic DNAs of 20 colorectal tumors were analyzed for the presence of gene-specific mutations by PCR-SSCP: no abnormal sequences were detected within the coding and splice site regions of the NM23-H1 gene. This finding suggests that NM23-H1 mutations are rare events in human colorectal cancer.

Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma.

Lung cancer is the most common cause of cancer mortality worldwide. Non-small-cell lung carcinomas (NSCLCs), which represent around 80% of lung tumors, exhibit poor prognosis and are usually refractory to conventional chemotherapy. Elucidating the molecular and cellular mechanisms that are dysregulated in NSCLCs may lead to new possibilities for targeted therapy or enhanced efficacy of current therapies. Here we demonstrate Wnt pathway activation in around 50% of human NSCLC cell lines and primary tumors, through different mechanisms, including autocrine Wnt pathway activation involving upregulation of specific Wnt ligands. Downregulation of activated Wnt signaling inhibited NSCLC proliferation and induced a more differentiated phenotype. Together, our findings establish importance of activated Wnt signaling in human NSCLCs and offer the possibility of targeting upregulated Wnt signaling as a new therapeutic modality for this disease.

Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling.

Frizzled related proteins (FRPs) comprise a family of secreted molecules that contain an N-terminal cysteine-rich domain (CRD) highly similar to the CRDs of the frizzled family of membrane-anchored Wnt receptors. FRPs have been shown to interact with Wnt proteins and antagonize Wnt signaling in a Xenopus developmental model. We demonstrated that FRP antagonizes the Wnt-induced increase in uncomplexed beta-catenin in both transient cotransfection and stable transformation models, where Wnt-induced morphological alterations are inhibited as well. We showed further that FRP inhibits Wnt signaling in a paracrine mode using a T-cell factor luciferase reporter to measure Wnt function. Investigation of the mechanisms responsible for FRP inhibition revealed that FRP forms complexes with WNT-1 or WNT-2 through its CRD domain. Transfection analysis with FRPs containing different tags revealed that FRP itself forms complexes and that this ability is conferred by its CRD domain. Finally, we demonstrated by cotransfection that FRP forms complexes with a prototype frizzled. All of these findings are consistent with a model by which FRP inhibits Wnt signaling through interactions with Wnt and/or formation of nonfunctional complexes with the frizzled receptor.

Age of onset in familial adenomatous polyposis: heterogeneity within families and among APC mutations.

Heterogeneity among and within FAP pedigrees for the age of symptom onset and the age at death from colorectal cancer was studied in a sample of 583 patients of the Italian Polyposis Registry. The among pedigree variation was largely explained by clustering of families in two groups, 'early FAP' (most colorectal cancer deaths below 45 years of age) and 'late FAP' families (most deaths above age 45). The within-family variation was explained by a marked phenomenon of anticipation (15 years per generation, on the average), possibly not due to ascertainment bias. We then considered the pedigrees with identified mutation in the APC gene. Six families shared a common deletion at codon 1309 and showed the early FAP phenotype. Two families shared a mutation at codon 1061 and revealed the late FAP phenotype. Another two families (codons 453 and 302) clustered with the late FAP group, whereas a family with mutation at codon 835 clustered with the early FAP group. We suggest that there are at least two classes of mutations in the APC gene with different consequences at the phenotypic level. It seems that there are several critical points within the APC protein sequence at which truncation causes a more aggressive disease than truncation at other points.

Identification of APC gene mutations in Italian adenomatous polyposis coli patients by PCR-SSCP analysis.

The APC gene is a putative human tumor-suppressor gene responsible for adenomatous polyposis coli (APC), an inherited, autosomal dominant predisposition to colon cancer. It is also implicated in the development of sporadic colorectal tumors. The characterization of APC gene mutations in APC patients is clinically important because DNA-based tests can be applied for presymptomatic diagnosis once a specific mutation has been identified in a family. Moreover, the identification of the spectrum of APC gene mutations in patients is of great interest in the study of the biological properties of the APC gene product. We analyzed the entire coding region of the APC gene by the PCR-single-strand conformation polymorphism method in 42 unrelated Italian APC patients. Mutations were found in 12 cases. These consist of small (5-14 bp) base-pair deletions leading to frameshifts; all are localized within exon 15. Two of these deletions, a 5-bp deletion at position 3183-3187 and a 5-bp deletion at position 3926-3930, are present in 3/42 and 7/42 cases of our series, respectively, indicating the presence of mutational hot spots at these two sites.

Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling.

In an effort to isolate novel growth factors, we identified a human protein, designated Sk, that co-eluted with Neuregulin during chromatographic separation of conditioned medium from the SK-LMS-1 human leiomyosarcoma cell line. Degenerate oligonucleotides based on amino-terminal sequence analysis of the purified protein were used to isolate the corresponding cDNA from a library generated from this cell line. Sk is a novel 266-amino acid protein that contains a signal peptide sequence and two cysteine-rich domains with no similarity to other known growth factors. A single major 2-kilobase transcript was expressed in several embryonic tissues. Transfection of mammalian cells demonstrated that the protein was secreted and expressed as a doublet of approximately 35 kDa. In vitro translation and endoglycosylase analysis indicated that this doublet, which was also observed in cells expressing the endogenous protein, arises from posttranslational modification. A search of the GenBankTM data base revealed a match of Sk with Dkk-1, which is a novel secreted protein required for head induction in amphibian embryos and a potent Wnt inhibitor. When coexpressed with Wnt-2 in NIH3T3 cells, human Sk/Dkk-1 caused reversion of Wnt-2 induced morphological alterations and inhibited the Wnt-2 induced increase in uncomplexed beta-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on beta-catenin regulation.

Differential effects of hepatocyte growth factor isoforms on epithelial and endothelial tubulogenesis.

Hepatocyte growth factor (HGF)/scatter factor (SF) is a pleiotropic cytokine that acts as a mitogen, motogen, and morphogen for a variety of cell types. HGF/NK1 and HGF/NK2 are two naturally occurring truncated variants of HGF/SF, which extend from the NH2 terminus through the first and second kringle domain, respectively. Although these variants have been reported to have agonistic or antagonistic activity relative to HGF/SF in assays of cell proliferation and motility, their potential morphogenic activity has not been investigated. To address this issue, we assessed the ability of HGF/NK1 and HGF/NK2 to induce tube formation by (a) MCF-10A mammary epithelial cells grown within collagen gels and (b) human umbilical vein endothelial (HUVE) cells grown on Matrigel. We found that HGF/NK1 stimulated tubulogenesis by both MCF-10A and HUVE cells, whereas HGF/NK2 did not stimulate tubulogenesis, but efficiently antagonized the morphogenic effect of full-length HGF/SF. HGF/NK1 and HGF/NK2 also had agonistic and antagonistic effects, respectively, on MCF-10A cell proliferation and HUVE cell migration. These results demonstrate that HGF/NK1, which only consists of the NH2-terminal hairpin and first kringle domain, is sufficient to activate the intracellular signaling pathways required to induce morphogenic responses in epithelial and endothelial cells. In contrast, HGF/NK2, which differs from HGF/ NK1 by the presence of the second kringle domain, is devoid of intrinsic activity but opposes the effects of HGF/SF. The differential properties of the two HGF/SF isoforms provide a basis for the design of more potent HGF/SF agonists and antagonists.

Mutation in a splice-donor site of the APC gene in a family with polyposis and late age of colonic cancer death.

Adenomatous polyposis coli (APC) is an autosomal dominant disease characterized by the development of hundreds of colorectal adenomatous polyps during the first decades of life. The expression of the disease varies, as the age of onset of colonic cancer and the severity of extracolonic manifestations often differ between affected families. An attenuated form of APC has also been described in which a small number of polyps and a later age of onset of colonic cancer is observed. Cloning of the APC gene has allowed disease-causing mutations in APC families to be identified. Here, we report a novel splice site mutation (a G to T transversion at position +5 of the splice donor site in intron 9) in the APC gene of affected individuals in an Italian family. Characterization of the transcription products from this mutant APC allele revealed that normal splicing was disrupted: a shorter mRNA was expressed in which exon 8 was connected directly to exon 10. This created a shift in the reading frame and the introduction of a stop codon at position 1358. In addition, some normal APC transcript was produced from the mutant allele in lymphoblastoid cells. A comparison of the clinical features of affected members of this family with four unrelated Italian APC kindreds, in which the same AAAAG deletion at position 3926 has been found, showed a significant difference in the onset of disease symptoms and in the age of death attributable to colorectal cancer. Inefficient exon skipping may be, at least in part, responsible for the delay in the development of the disease in the reported family.