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.
Autocrine Wnt signaling in the mouse mammary tumor virus model was the first identified mechanism of canonical pathway activation in cancer. In search of this transformation mechanism in human cancer cells, we identified breast and ovarian tumor lines with upregulation of the uncomplexed transcriptionally active form of beta-catenin without mutations afflicting downstream components. Extracellular Wnt antagonists FRP1 and DKK1 caused a dramatic downregulation of beta-catenin levels in these tumor cells associated with alteration of biological properties and increased expression of epithelial differentiation markers. Colorectal carcinoma cells with knockout of the mutant beta-catenin allele retained upregulated beta-catenin levels, which also could be inhibited by these Wnt antagonists. Together, these findings establish the involvement of autocrine Wnt signaling in human cancer cells.
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 C4-2B's 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. (Cancer Res 2005; 65(17): 7554-60)
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 GenBank TM 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 -catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on -catenin regulation.
BackgroundEctopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development.Methodology/Principal FindingsHere, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%). Stem cell activity can be enriched by >200 fold (over 80% of activity), based on high Lrp5 expression alone. Though Lrp5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22%) and Lrp5−/− mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16Ink4a and TA-p63.Conclusions/SignificanceThis is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component of normal mammary stem cell function.
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.The Wnt family of secreted signaling molecules is essential in embryonic induction, cell polarity generation, and cell fate specification (46). Deregulation of Wnt signaling results in defects in development and growth control. The canonical Wnt pathway involves activation of -catenin-dependent transcription and is evolutionarily conserved from Caenorhabditis elegans to humans. Mutations in components, which constitutively activate canonical signaling, have been identified in several tumor types, including colorectal cancer (34). Wnts bind to two coreceptors, the Frizzled-type seven-transmembrane-domain receptor (5,17,19,49) and the low-density receptor-related protein (LRP) 5/6 (35, 41) or the Drosophila melanogaster ortholog Arrow (44). These interactions cause -catenin stabilization through inhibition of its phosphorylation by glycogen synthase kinase 3 (GSK3), which is assembled in a large cytoplasmic complex that includes Dishevelled, casein kinase I, Axin, APC, and Frat (36). As a consequence, stabilized cytoplasmic -catenin is translocated to the nucleus and forms a complex with a family of high-mobility group-like transcription factors, including leukocyte enhancer factor-1 (LEF-1) and T-cell factors (TCF), activating transcription of target genes (4).Frizzled family members have been shown to possess various affinities for different Wnt ligands. For example, Drosophila frizzled, Dfz1 and Dfz2, bind to Wingless (Wg), the Drosophila orthologue of Wnt, to activate the canonical pathway. However, Dfz2 has a 10-fold higher affinity for Wg than Dfz1 and plays a predominant role in transducing the Wg canonical signal in vivo (37). Frizzled family members also signal through the planar polarity pathway, which similarly involves Dsh (25) but is mediated through JNK and RhoA rather than -catenin stabilization (7). In Drosophila, Dfz1 but not Dfz2 appears to regulate planar polarity signaling (8, 37). LRP5, LRP6, and arrow receptors specifically function in the canonical pathway. Inactivation of arrow in Drosophila results in a phenotype similar to that of the wingless mutant (41), and mice deficient in LRP6 exhibit developmental defects resembling ...
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.
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