Face morphogenesis is promoted by Pbx-dependent EMT via regulation of Snail1 during frontonasal prominence fusion.

Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.

Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

Microvesicle-mediated Wnt/ß-Catenin Signaling Promotes Interspecies Mammary Stem/Progenitor Cell Growth.

Signaling mechanisms that regulate mammary stem/progenitor cell (MaSC) self-renewal are essential for developmental changes that occur in the mammary gland during pregnancy, lactation, and involution. We observed that equine MaSCs (eMaSCs) maintain their growth potential in culture for an indefinite period, whereas canine MaSCs (cMaSCs) lose their growth potential in long term cultures. We then used this system to investigate the role of microvesicles (MVs) in promoting self-renewal properties. We found that Wnt3a and Wnt1 were expressed at higher levels in MVs isolated from eMaSCs compared with those from cMaSCs. Furthermore, eMaSC-MVs were able to induce Wnt/ß-catenin signaling in different target cells, including cMaSCs. Interestingly, the induction of Wnt/ß-catenin signaling in cMaSCs was prolonged when using eMaSC-MVs compared with recombinant Wnt proteins, indicating that MVs are not only important for transport of Wnt proteins, but they also enhance their signaling activity. Finally, we demonstrate that the eMaSC-MVs-mediated activation of the Wnt/ß-catenin signaling pathway in cMaSCs significantly improves the ability of cMaSCs to grow as mammospheres and, importantly, that this effect is abolished when eMaSC-MVs are treated with Wnt ligand inhibitors. This suggests that this novel form of intercellular communication plays an important role in self-renewal.

Dysregulation of PAD4-mediated citrullination of nuclear GSK3ß activates TGF-ß signaling and induces epithelial-to-mesenchymal transition in breast cancer cells.

Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3ß (GSK3ß), both in vitro and in vivo. Among other functions, GSK3ß is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3ß protein levels, increased TGF-ß signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3ß is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3ß is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.

LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure.

Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulation were examined using gain (Crooked tail, Lrp6(Cd)) and loss (Lrp6(-)) of function mouse lines. Two features often associated with canonical Wnt signaling, dorsal-ventral patterning and proliferation, were no different from wild-type (WT) in the Lrp6(Cd/Cd) neural tube. Lrp6(-/-) embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6(Cd/Cd) and Lrp6(-/-) cranial folds were indicated by cell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6(Cd/Cd) or Lrp6(-/-) embryos exhibited elevated and decreased RhoA basal activity levels, respectively. While ligand-independent activation of canonical Wnt signaling, bypassing Lrp-Frizzled receptors, did not activate RhoA, non-canonical Wnt5a stimulation of RhoA activity was impaired in Lrp6(-/-) MEFs. RhoA inhibition exacerbated NTDs in cultured Lrp6 knockout embryos compared with WT littermates. In contrast, a ROCK inhibitor rescued Lrp6(Cd/Cd) embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity in Wnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6(Cd) protein where interaction with DAAM1 could account for observed elevated RhoA activity. Conversely, null mutation that eliminates Lrp6 interaction with DAAM1 led to lower basal RhoA activity in Lrp6(-/-) embryos. These results indicate that Lrp6 mediates not only canonical Wnt signaling, but can also modulate non-canonical pathways involving RhoA-dependent mechanisms to impact neurulation, possibly through intracellular complexes with DAAM1.

Atypical protein kinase Cι is required for Wnt3a-dependent neurite outgrowth and binds to phosphorylated dishevelled 2.

Previously we reported that Wnt3a-dependent neurite outgrowth in Ewing sarcoma family tumor cell lines was mediated by Frizzled3, Dishevelled (Dvl), and c-Jun N-terminal kinase (Endo, Y., Beauchamp, E., Woods, D., Taylor, W. G., Toretsky, J. A., Uren, A., and Rubin, J. S. (2008) Mol. Cell. Biol. 28, 2368-2379). Subsequently, we observed that Dvl2/3 phosphorylation correlated with neurite outgrowth and that casein kinase 1δ, one of the enzymes that mediate Wnt3a-dependent Dvl phosphorylation, was required for neurite extension (Greer, Y. E., and Rubin, J. S. (2011) J. Cell Biol. 192, 993-1004). However, the functional relevance of Dvl phosphorylation in neurite outgrowth was not established. Dvl1 has been shown by others to be important for axon specification in hippocampal neurons via an interaction with atypical PKCζ, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKCι but not PKCζ. Wnt3a stimulated PKCι activation and caused a punctate distribution of pPKCι in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCι expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCι/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the co-immunoprecipitation of endogenous PKCι and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCι, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (González-Sancho, J. M., Greer, Y. E., Abrahams, C. L., Takigawa, Y., Baljinnyam, B., Lee, K. H., Lee, K. S., Rubin, J. S., and Brown, A. M. (2013) J. Biol. Chem. 288, 9428-9437). Taken together, these results suggest that site-specific Dvl2 phosphorylation is required for Dvl2 association with PKCι. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.

Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling.

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/ß-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser(594), Thr(595), and Ser(597) of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates ß-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.

An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway.

Misregulated ß-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of ß-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear ß-catenin. We show that these inhibitors efficiently block Wnt/ß-catenin-induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling.

Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir.

Wnt proteins that signal via the canonical Wnt/ß-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of ß-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, ß-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.

Mammary stem cells and cancer: roles of Wnt signaling in plain view.

The likely roles of Wnt signaling in regulating mammary stem cell behavior have been much discussed, in part because they may underlie the oncogenic effects of Wnt signaling in mammary tissue. Two recent papers add important data to this field. One tests directly the effects of purified Wnt protein on mouse mammary stem cells in culture and finds a specific increase in the proportion of cells with self-renewing stem cell phenotypes. The second identifies a novel target gene of canonical Wnt signaling that may be expressed in stem cells and is induced in both mouse and human mammary tumors associated with Wnt pathway activation.

Wnt signaling promotes oncogenic transformation by inhibiting c-Myc-induced apoptosis.

Aberrant activation of the Wnt/beta-catenin signaling pathway is associated with numerous human cancers and often correlates with the overexpression or amplification of the c-myc oncogene. Paradoxical to the cellular transformation potential of c-Myc is its ability to also induce apoptosis. Using an inducible c-MycER expression system, we found that Wnt/beta-catenin signaling suppressed apoptosis by inhibiting c-Myc-induced release of cytochrome c and caspase activation. Both cyclooxygenase 2 and WISP-1 were identified as effectors of the Wnt-mediated antiapoptotic signal. Soft agar assays showed that neither c-Myc nor Wnt-1 alone was sufficient to induce cellular transformation, but that Wnt and c-Myc coordinated in inducing transformation. Furthermore, coexpression of Wnt-1 and c-Myc induced high-frequency and rapid tumor growth in nude mice. Extensive apoptotic bodies were characteristic of c-Myc-induced tumors, but not tumors induced by coactivation of c-Myc and Wnt-1, indicating that the antiapoptotic function of Wnt-1 plays a critical role in the synergetic action between c-Myc and Wnt-1. These results elucidate the molecular mechanisms by which Wnt/beta-catenin inhibits apoptosis and provide new insight into Wnt signaling-mediated oncogenesis.

Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2.

Wnts are lipid-modified secreted glycoproteins that regulate diverse biological processes. We report that Wnt5a, which functions in noncanonical Wnt signaling, has activity on endothelial cells. Wnt5a is endogenously expressed in human primary endothelial cells and is expressed in murine vasculature at several sites in mouse embryos and tissues. Expression of exogenous Wnt5a in human endothelial cells promoted angiogenesis. Wnt5a induced noncanonical Wnt signaling in endothelial cells, as measured by Dishevelled and ERK1/2 phosphorylation, and inhibition of canonical Wnt signaling, a known property of Wnt5a. Wnt5a induced endothelial cell proliferation and enhanced cell survival under serum-deprived conditions. The Wnt5a-mediated proliferation was blocked by Frizzled-4 extracellular domain. Wnt5a expression enhanced capillary-like network formation, whereas reduction of Wnt5a expression decreased network formation. Reduced Wnt5a expression inhibited endothelial cell migration. Screening for Wnt5a-regulated genes in cultured endothelial cells identified several encoding angiogenic regulators, including matrix metalloproteinase-1, an interstitial collagenase, and Tie-2, a receptor for angiopoietins. Thus, Wnt5a acts through noncanonical Wnt signaling to promote angiogenesis.

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin.

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.

HER2/neu-induced mammary tumorigenesis and angiogenesis are reduced in cyclooxygenase-2 knockout mice.

The inducible prostaglandin synthase cyclooxygenase-2 (Cox-2) is overexpressed in approximately 40% of human breast cancers and at higher frequencies in preinvasive ductal carcinoma in situ (DCIS). Cox-2 expression is particularly associated with overexpression of human epidermal growth factor receptor 2 (HER2/neu). To definitively interrogate the role of Cox-2 in mammary neoplasia, we have used a genetic approach, crossing Cox-2-deficient mice with a HER2/neu transgenic strain, MMTV/NDL. At 20 weeks of age, mammary glands from virgin MMTV/NDL females contained multiple focal tumors, or mammary intraepithelial neoplasias, which histologically resembled human DCIS. Mammary tumor multiplicity and prostaglandin E2 (PGE2) levels were significantly decreased in Cox-2 heterozygous and knockout animals relative to Cox-2 wild-type controls. Notably, the proportion of larger tumors was decreased in Cox-2-deficient mice. HER2/neu-induced mammary hyperplasia was also substantially reduced in Cox-2 null mice. Additionally, mammary glands from Cox-2 knockout mice exhibited a striking reduction in vascularization, and expression of proangiogenic genes was correspondingly reduced. Decreased vascularization was observed both in dysplastic and normal-appearing regions of Cox-2-null mammary glands. Our data provide the first genetic evidence that Cox-2 contributes to HER2/neu-induced mammary tumorigenesis. This finding may help to explain the reduced risk of breast cancer associated with regular use of nonsteroidal anti-inflammatory drugs.

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells.

Recent evidence suggests that mammary cells expressing R-spondin receptor and Wnt pathway regulator Lgr5, regarded as a stem cell marker in multiple tissues, might represent mammary stem cells (MaSCs). Whether L gr5 marks a multipotent subpopulation of Lin-CD24low/medCD49fhigh MaSCs remains controversial. To some extent the differing results reflect different assays used to assess properties of stemness, including lineage tracing in vivo, mammosphere culture, and mammary fat pad transplantation assays. To address this issue directly, we isolated Lgr5+ cells from mammary glands of Lgr5-lacZ mice and established organoids based on principles adapted from studies of Wnt-driven Lgr5+ cell populations in other organs. Mammary organoids were grown from single Lgr5+ mammary cells in Matrigel, the substratum of choice for intestinal organoids, and in a growth factor cocktail containing EGF, Wnt3a and R-spondin, designed to optimally activate the endogenous Wnt signaling program of stem cells. Colonies derived from single Lgr5+ cells manifest at least four distinct cell populations: Lgr5+ and Lgr5- basal cells and c-Kit+ and c-Kit- luminal cells that spontaneously organize into a ductal structure with basal cells around the periphery and luminal cells lining an interior cavity, reminiscent of normal mammary duct structure. Lgr5+ cell-derived organoids were sustainable during prolonged passaging. In contrast, although Lgr5- cells expand into primary colonies, colony-forming efficiency immediately dissipated upon passaging. Furthermore, reproductive hormones induce epithelial cell proliferation resulting in marked increases in lumen diameter accompanied by squamous transdifferentiation. We propose this estrogen-responsive, self-organizing duct-like structure derived from single murine Lgr5+ mammary cells represents a "mini-breast" organoid.

Twist is up-regulated in response to Wnt1 and inhibits mouse mammary cell differentiation.

Wnt1, initially identified as a mammary oncogene, can activate transcription via beta-catenin/TCF complexes. Twist, a transcription factor of the basic helix-loop-helix class, has also been suggested to have oncogenic properties. The aim of this study was to determine whether Twist is regulated by Wnt1 and might thus be a novel mediator of Wnt signaling. We found that Twist was up-regulated in C57MG and HC11 murine mammary epithelial cells in response to Wnt1 expression. Additionally, we detected Twist expression in normal mammary gland and found elevated Twist expression in approximately 70% of mammary tumors from Wnt1 transgenic mice. A murine Twist promoter fragment was shown to be responsive to beta-catenin, and its activity was enhanced by coexpression of c-jun and Ets factors of the PEA3 family. Both PEA3 factors and c-jun were highly expressed in tumors from Wnt1 transgenic mice and may therefore contribute to the increased Twist expression observed in these tumors. To evaluate functional consequences of Twist induction, we examined the effect of Twist on mammary cell differentiation. Strikingly, overexpression of either Wnt1 or Twist in HC11 mammary epithelial cells completely suppressed induction of the milk protein beta-casein in response to lactogenic hormones. Additionally, Wnt1, but not Twist, partially abrogated induction of WDNM1, another marker of lactogenic differentiation. Taken together, our data indicate that Twist expression is regulated by Wnt/beta-catenin signaling and that both Wnt1 and Twist can function as inhibitors of lactogenic differentiation, an effect that could contribute to mammary tumorigenesis.

Celecoxib, a selective cyclooxygenase 2 inhibitor, protects against human epidermal growth factor receptor 2 (HER-2)/neu-induced breast cancer.

Cyclooxygenase 2 (HER-2) (Cox-2), an inducible form of Cox, is overexpressed in HER-2/neu-positive human breast cancers. The aim of this study was to determine whether celecoxib, a selective Cox-2 inhibitor, protected against HER-2/neu-induced experimental breast cancer. Cox-2 protein was detected in breast carcinomas from mouse mammary tumor virus (MMTV)/neu mice. Treatment with celecoxib (500 ppm) significantly reduced the incidence of mammary tumors in MMTV/neu mice (P = 0.003) and caused about a 50% reduction in mammary prostaglandin E2 (PGE2) levels. Because mammary glands from MMTV/neu mice expressed all four PGE2 receptor subtypes, we speculate that signaling through PGE2 receptors is important for mammary tumorigenesis. These results strengthen the rationale for developing clinical trials to determine whether selective Cox-2 inhibitors possess anticancer properties in humans at risk for breast cancer.

Truncated mutants of the putative Wnt receptor LRP6/Arrow can stabilize beta-catenin independently of Frizzled proteins.

Secreted signaling proteins of the Wnt family are known to regulate a diverse range of developmental processes, and their signaling pathway through beta-catenin is frequently activated in cancer. The identification of both Frizzled and LRP5/6 (LRP: low-density lipoprotein receptor-related protein) proteins as components of cell-surface receptors for Wnt proteins has raised questions about their individual functions. We have investigated this issue through a structure-function analysis of Frizzled and LRP proteins that have been implicated in Wnt1 signaling. Consistent with other reports, we find that LRP6/Arrow proteins deleted for their extracellular domain are able to activate the Wnt/beta-catenin signaling pathway. Importantly, our results demonstrate that this signaling from LRP6/Arrow derivatives can occur in a Frizzled- and ligand-independent manner. Furthermore, we show that the PPSP motifs within the intracellular domain of LRP6 are required for signaling. In contrast to results with LRP6, overexpression of Frizzled proteins did not activate the pathway. Based on evidence of ligand binding to both Frizzled and LRP6, current models suggest that both proteins are components of a Wnt receptor complex that signals to beta-catenin. In light of these models, our data imply that LRP5/6/Arrow proteins constitute the distal signal-initiating component of these receptors. The results also support the notion that LRP5/6 are candidate oncogenes.

Is there a role for Notch signalling in human breast cancer?

Aberrant Notch signalling has been observed in several human cancers, including acute T-cell lymphoblastic leukaemia and cervical cancer, and is strongly implicated in tumourigenesis. Unregulated Notch signalling in the mouse mammary gland leads to tumour formation. These results raise the possibility that Notch signalling might play a role in human breast cancer. There are currently few reports that address this question directly and this appears to be an area worthy of further investigation.