Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes.

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to ß-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.

MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells.

Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-ß-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer.

Upregulation of Key Molecules for Targeted Imaging and Therapy.

Targeted diagnosis and therapy enable precise tumor detection and treatment. Successful examples for precise tumor targeting are diagnostic and therapeutic radioligands. However, patients with tumors expressing low levels of the relevant molecular targets are deemed ineligible for such targeted approaches. METHODS: We performed a screen for drugs that upregulate the somatostatin receptor subtype 2 (sstr2). Then, we characterized the effects of these drugs on transcriptional, translational, and functional levels in vitro and in vivo. RESULTS: We identified 9 drugs that act as epigenetic modifiers, including the inhibitor of DNA methyltransferase decitabine as well as the inhibitors of histone deacetylase tacedinaline and romidepsin. In vitro, these drugs upregulated sstr2 on transcriptional, translational, and functional levels in a time- and dose-dependent manner. Thereby, their combinations revealed synergistic effects. In vivo, drug-based sstr2 upregulation improved the tumor-to-background and tumor-to-kidney ratios, which are the key determinants of successful sstr2-targeted imaging and radiopeptide therapy. CONCLUSION: We present an approach that uses epigenetic modifiers to improve sstr2 targeting in vitro and in vivo. Translation of this method into the clinic may potentially convert patients ineligible for targeted imaging and therapy to eligible candidates.

Indium-111 labeled gold nanoparticles for in-vivo molecular targeting.

The present report describes the synthesis and biological evaluation of a molecular imaging platform based on gold nanoparticles directly labeled with indium-111. The direct labeling approach facilitated radiolabeling with high activities while maintaining excellent stability within the biological environment. The resulting imaging platform exhibited low interference of the radiolabel with targeting molecules, which is highly desirable for in-vivo probe tracking and molecular targeted tumor imaging. The indium-111 labeled gold nanoparticles were synthesized using a simple procedure that allowed stable labeling of the nanoparticle core with various indium-111 activities. Subsequent surface modification of the particle cores with RGD-based ligands at various densities allowed for molecular targeting of the αvß3 integrin in-vitro and for molecular targeted imaging in human melanoma and glioblastoma models in-vivo. The results demonstrate the vast potential of direct labeling with radioisotopes for tracking gold nanoparticles within biological systems.

Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning.

Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. Through embryological and biochemical experiments we find that: (1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo; (2) CV2 complexes with Twisted gastrulation and BMP4; (3) CV2 is not a substrate for tolloid proteinases; (4) CV2 binds to purified Chordin protein with high affinity (K(D) in the 1 nM range); (5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes; (6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. We propose that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels.