The dishevelled associated activator of morphogenesis protein 2 (Daam2) regulates neural tube closure.

BACKGROUND: The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood. RESULTS: Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo. CONCLUSIONS: Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

Hepatocystin is Essential for TRPM7 Function During Early Embryogenesis.

Mutations in protein kinase C substrate 80K-H (PRKCSH), which encodes for an 80 KDa protein named hepatocystin (80K-H, PRKCSH), gives rise to polycystic liver disease (PCLD). Hepatocystin functions as the noncatalytic beta subunit of Glucosidase II, an endoplasmic reticulum (ER)-resident enzyme involved in processing and quality control of newly synthesized glycoproteins. Patients harboring heterozygous germline mutations in PRKCSH are thought to develop renal cysts as a result of somatic loss of the second allele, which subsequently interferes with expression of the TRP channel polycystin-2 (PKD2). Deletion of both alleles of PRKCSH in mice results in embryonic lethality before embryonic day E11.5. Here, we investigated the function of hepatocystin during Xenopus laevis embryogenesis and identified hepatocystin as a binding partner of the TRPM7 ion channel, whose function is required for vertebrate gastrulation. We find that TRPM7 functions synergistically with hepatocystin. Although other N-glycosylated proteins are critical to early development, overexpression of TRPM7 in Xenopus laevis embryos was sufficient to fully rescue the gastrulation defect caused by loss of hepatocystin. We observed that depletion of hepatocystin in Xenopus laevis embryos decreased TRPM7 expression, indicating that the early embryonic lethality caused by loss of hepatocystin is mainly due to impairment of TRPM7 protein expression.

Design and development of a peptide-based adiponectin receptor agonist for cancer treatment.

BACKGROUND: Adiponectin, a fat tissue-derived adipokine, exhibits beneficial effects against insulin resistance, cardiovascular disease, inflammatory conditions, and cancer. Circulating adiponectin levels are decreased in obese individuals, and this feature correlates with increased risk of developing several metabolic, immunological and neoplastic diseases. Thus, pharmacological replacement of adiponectin might prove clinically beneficial, especially for the obese patient population. At present, adiponectin-based therapeutics are not available, partly due to yet unclear structure/function relationships of the cytokine and difficulties in converting the full size adiponectin protein into a viable drug. RESULTS: We aimed to generate adiponectin-based short peptide that can mimic adiponectin action and be suitable for preclinical and clinical development as a cancer therapeutic. Using a panel of 66 overlapping 10 amino acid-long peptides covering the entire adiponectin globular domain (residues 105-254), we identified the 149-166 region as the adiponectin active site. Three-dimensional modeling of the active site and functional screening of additional 330 peptide analogs covering this region resulted in the development of a lead peptidomimetic, ADP 355 (H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2). In several adiponectin receptor-positive cancer cell lines, ADP 355 restricted proliferation in a dose-dependent manner at 100 nM-10 µM concentrations (exceeding the effects of 50 ng/mL globular adiponectin). Furthermore, ADP 355 modulated several key signaling pathways (AMPK, Akt, STAT3, ERK1/2) in an adiponectin-like manner. siRNA knockdown experiments suggested that ADP 355 effects can be transmitted through both adiponectin receptors, with a greater contribution of AdipoR1. In vivo, intraperitoneal administration of 1 mg/kg/day ADP 355 for 28 days suppressed the growth of orthotopic human breast cancer xenografts by ~31%. The peptide displayed excellent stability (at least 30 min) in mouse blood or serum and did not induce gross toxic effects at 5-50 mg/kg bolus doses in normal CBA/J mice. CONCLUSIONS: ADP 355 is a first-in-class adiponectin receptor agonist. Its biological activity, superior stability in biological fluids as well as acceptable toxicity profile indicate that the peptidomimetic represents a true lead compound for pharmaceutical development to replace low adiponectin levels in cancer and other malignancies.

Efficacy of a leptin receptor antagonist peptide in a mouse model of triple-negative breast cancer.

Triple-negative breast cancers, which represent 10-20% of all mammary tumours, are characterised by the aggressive phenotype, are often found in younger women and have been associated with poor prognosis. Obesity increases the risk for triple-negative breast cancer development. Because triple-negative breast cancer patients are unresponsive to current targeted therapies and other treatment options are only partially effective, new pharmacological modalities are urgently needed. Here we examined if the leptin (obesity hormone) receptor is a viable target for the treatment of this cancer subtype. In human triple-negative breast cancer tissues, the leptin receptor was expressed in 92% (64/69) and leptin in 86% (59/69) of cases. In a model triple-negative breast cancer cell line MDA-MB-231, the leptin receptor antagonist peptide Allo-aca inhibited leptin-induced proliferation at 50 pM concentration. In an MDA-MB-231 orthotopic mouse xenograft model, Allo-aca administered subcutaneously significantly extended the average survival time from 15.4 days (untreated controls) to 24 and 28.1 days at 0.1 and 1mg/kg/day doses, respectively. In parallel, conventional treatment with 1mg/kg/day intraperitoneal cisplatin prolonged the average survival time to 18.6 days, while administration of 20mg/kg/day oral Tamoxifen (negative control) had no significant survival effects relative to controls. In normal CD-1 mice, Allo-aca produced no systemic toxicity up to the highest studied subcutaneous bolus dose of 50mg/kg, while, as expected, it induced a modest 6-10% body weight increase. Our results indicate that leptin receptor antagonists could become attractive options for triple-negative breast cancer treatment, especially in the obese patient population.