Anna Bafico

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 (KD = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (Mr 240K) with [125I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through β-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.

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 Xenopusdevelopmental model. We demonstrated that FRP antagonizes the Wnt-induced increase in uncomplexed β-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.

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 β-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on β-catenin regulation.

Prostate Cancer Cells Promote Osteoblastic Bone Metastases through Wnts

Prostate cancer produces painful osteoblastic bone metastases. Although prostate cancer cells produce numerous osteogenic factors, to date, none have been shown to mediate osteoblastic bone metastases in an in vivo model of prostate cancer. Wnts are a large family of proteins that promote bone growth. Wnt activity is antagonized by endogenous proteins including dickkopf-1 (DKK-1). We explored if prostate cancer cells mediate osteoblastic activity through Wnts using DKK-1 as a tool to modify Wnt activity. A variety of Wnt mRNAs were found to be expressed in prostate cancer cell lines and Wnt mRNA expression was increased in primary prostate cancer compared with nonneoplastic prostate tissue. In addition to expressing Wnts, PC-3 prostate cancer cells expressed the Wnt inhibitor DKK-1. To determine if DKK-1 masked Wnt-mediated osteoblastic activity in osteolytic PC-3 cells, the cells were stably transfected with DKK-1 short hairpin RNA. Decreasing DKK-1 enabled PC-3 cells to induce osteoblastic activity, including alkaline phosphatase production and mineralization, in murine bone marrow stromal cells indicating that DKK-1 blocked Wnt-mediated osteoblastic activity in PC-3 cells. Another prostate cancer cell line, C4-2B, induces mixed osteoblastic/osteolytic lesions. To determine if Wnts contribute to C4-2Bs ability to induce mixed osteoblastic/osteolytic lesions, C4-2B cells were stably transfected with either empty vector or DKK-1 expression vector to block Wnt activity. The cells were then injected in the tibiae of mice and allowed to grow for 12 weeks. Blocking Wnt activity converted the C4-2B cells to a highly osteolytic tumor. Taken together, these data show that Wnts contribute to the mechanism through which prostate cancer induces osteoblastic activity.

A Novel Mechanism for Wnt Activation of Canonical Signaling through the LRP6 Receptor

ABSTRACT LDL receptor-related protein 6 (LRP6) is a Wnt coreceptor in the canonical signaling pathway, which plays essential roles in embryonic development. We demonstrate here that wild-type LRP6 forms an inactive dimer through interactions mediated by epidermal growth factor repeat regions within the extracellular domain. A truncated LRP6 comprising its transmembrane and cytoplasmic domains is expressed as a constitutively active monomer whose signaling ability is inhibited by forced dimerization. Conversely, Wnts are shown to activate canonical signaling through LRP6 by inducing an intracellular conformational switch which relieves allosteric inhibition imposed on the intracellular domains. Thus, Wnt canonical signaling through LRP6 establishes a novel mechanism for receptor activation which is opposite to the general paradigm of ligand-induced receptor oligomerization.

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.

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.

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 β-catenin, which was not observed with PDGFB, ras or erbB2 transfectants. In transient transfection, Wnt-1 and -2 induced a rapid increase in cytosolic β-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 β-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.

The Mechanism of Endogenous Receptor Activation Functionally Distinguishes Prototype Canonical and Noncanonical Wnts

ABSTRACT Wnt glycoproteins are developmentally essential signaling molecules, and lesions afflicting Wnt pathways play important roles in human diseases. Some Wnts signal to the canonical pathway by stabilizing β-catenin, while others lack this activity. Frizzled serpentine receptors mediate distinct signaling pathways by both classes of Wnts. Here, we tandemly linked noncanonical Wnt5a with the C-terminal half of Dickkopf-2 (Dkk2C), a distinct ligand of the Wnt coreceptor LRP5/6. Whereas Wnt5a, Dkk2C, or both together were incapable of stimulating endogenous canonical signaling, the Wnt5a/Dkk2C chimera efficiently activated this pathway in a manner inhibitable by specific antagonists of either frizzled or LRP receptors. Thus, activation of the canonical pathway requires ligand coupling of an endogenous frizzled/LRP coreceptor complex, rather than Wnt triggering each receptor independently. Moreover, fusion of Wnt5a with Dkk2C unmasked its ability to signal to Dishevelled through multiple frizzleds, indicating that the lack of functional interaction with LRP distinguishes noncanonical Wnt5a from canonical Wnts in mammalian cells. These findings provide a novel mechanism by which the same receptor can be switched between distinct signaling pathways depending on the differential recruitment of a coreceptor by members of the same ligand family.

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.