ABSTRACT
Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. Although several Wnt signal transduction mechanisms have been described in detail, it is still largely unknown how cells are specified to adopt such different Wnt signaling responses. Here, we have used the stereotypic migration of the C. elegans Q neuroblasts as a model to study how two initially equivalent cells are instructed to activate either ß-catenin dependent or independent Wnt signaling pathways to control the migration of their descendants along the anteroposterior axis. We find that the specification of this difference in Wnt signaling response is dependent on the thrombospondin repeat containing protein MIG-21, which acts together with the netrin receptor UNC-40/DCC to control an initial left-right asymmetric polarization of the Q neuroblasts. Furthermore, we show that the direction of this polarization determines the threshold for Wnt/ß-catenin signaling, with posterior polarization sensitizing for activation of this pathway. We conclude that MIG-21 and UNC-40 control the asymmetry in Wnt signaling response by restricting posterior polarization to one of the two Q neuroblasts.
Subject(s)
Body Patterning , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/cytology , Cell Movement , Neurons/cytology , Repetitive Sequences, Amino Acid , Wnt Signaling Pathway , Animals , Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/chemistry , Cell Polarity , Extracellular Space/metabolism , Green Fluorescent Proteins/metabolism , Membrane Proteins/metabolism , Netrin Receptors , Neurons/metabolism , Receptors, Cell Surface/metabolism , Thrombospondins/chemistryABSTRACT
Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Carrier Proteins/physiology , Wnt Proteins/physiology , Animals , Caenorhabditis elegans Proteins/genetics , Carrier Proteins/genetics , Cell Line , Cell Membrane/physiology , Endosomes/physiology , Golgi Apparatus/physiology , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Kidney , Recombinant Proteins/metabolism , Transfection , Wnt Proteins/geneticsABSTRACT
About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.
Subject(s)
Melanoma/drug therapy , Melanoma/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Drug Resistance, Neoplasm/genetics , Frizzled Receptors/antagonists & inhibitors , Frizzled Receptors/genetics , Frizzled Receptors/metabolism , Humans , Indoles/pharmacology , Melanoma/metabolism , Mutation , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Sulfonamides/pharmacology , Up-Regulation/drug effects , Wnt Proteins/antagonists & inhibitors , Wnt Signaling Pathway/drug effects , Wnt-5a Protein , beta Catenin/metabolismABSTRACT
Elevated levels of nuclear ß-catenin are associated with higher rates of survival in patients with melanoma, raising questions as to how ß-catenin is regulated in this context. In the present study, we investigated the formal possibility that the secretion of WNT ligands that stabilize ß-catenin may be regulated in melanoma and thus contributes to differences in ß-catenin levels. We find that WLS, a conserved transmembrane protein necessary for WNT secretion, is decreased in both melanoma cell lines and in patient tumours relative to skin and to benign nevi. Unexpectedly, reducing endogenous WLS with shRNAs in human melanoma cell lines promotes spontaneous lung metastasis in xenografts in mice and promotes cell proliferation in vitro. Conversely, overexpression of WLS inhibits cell proliferation in vitro. Activating ß-catenin downstream of WNT secretion blocks the increased cell migration and proliferation observed in the presence of WLS shRNAs, while inhibiting WNT signalling rescues the growth defects induced by excess WLS. These data suggest that WLS functions as a negative regulator of melanoma proliferation and spontaneous metastasis by activating WNT/ß-catenin signalling.
Subject(s)
Catenins/metabolism , Cell Proliferation , Intracellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/metabolism , Melanoma/metabolism , Signal Transduction , Skin Neoplasms/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Cell Line, Tumor , Cell Movement , Humans , Lung Neoplasms/secondary , Melanoma/secondary , Mice , Skin Neoplasms/pathology , Transplantation, HeterologousABSTRACT
Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Repressor Proteins/metabolism , Wnt Proteins/metabolism , Animals , Animals, Genetically Modified , Axin Protein , Base Sequence , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA, Helminth/genetics , Female , Genes, Helminth , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Repressor Proteins/genetics , Signal Transduction , Vulva/growth & development , Vulva/metabolism , Wnt Proteins/geneticsABSTRACT
In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.