Wnt proteins bind to seven-transmembrane Frizzled receptors to mediate the important developmental, morphogenetic, and stem cell related tissue-regenerative effects of Wnt signaling. Dysregulated Wnt ...signaling is associated with many cancers. Currently, there are no drug candidates or even tool compounds that modulate Wnt-mediated receptor trafficking, and subsequent Wnt signaling. We examined libraries of FDA-approved drugs for their utility as Frizzled internalization modulators, employing a primary imaged-based GFP fluorescence assay that uses Frizzled1 endocytosis as the readout. We now report that the anti-helminthic niclosamide, a drug used for the treatment of tapeworm, promotes Frizzled1 endocytosis, downregulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated beta-catenin stabilization and LEF/TCF reporter activity. Additionally, following niclosamide-mediated internalization, the Frizzled1 receptor colocalizes in vesicles containing transferrin and agonist-activated beta(2)-adrenergic receptor. Therefore, niclosamide may serve as a negative modulator of Wnt/Frizzled1 signaling by depleting upstream signaling molecules (i.e., Frizzled and Dishevelled) and moreover may provide a valuable means of studying the physiological consequences of Wnt signaling.
The Wnt signaling pathway, known for regulating genes critical to normal embryonic development and tissue homeostasis, is dysregulated in many types of cancer. Previously, we identified that the ...anthelmintic drug niclosamide inhibited Wnt signaling by promoting internalization of Wnt receptor Frizzled 1 and degradation of Wnt signaling pathway proteins, Dishevelled 2 and β-catenin, contributing to suppression of colorectal cancer growth
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Here, we provide evidence that niclosamide-mediated inhibition of Wnt signaling is mediated through autophagosomes induced by niclosamide. Specifically, niclosamide promotes the co-localization of Frizzled 1 or β-catenin with LC3, an autophagosome marker. Niclosamide inhibition of Wnt signaling is attenuated in autophagosome-deficient ATG5
MEF cells or cells expressing shRNA targeting Beclin1, a critical constituent of autophagosome. Treatment with the autophagosome inhibitor 3MA blocks niclosamide-mediated Frizzled 1 degradation. The sensitivity of colorectal cancer cells to growth inhibition by niclosamide is correlated with autophagosome formation induced by niclosamide. Niclosamide inhibits mTORC1 and ULK1 activities and induces LC3B expression in niclosamide-sensitive cell lines, but not in the niclosamide-resistant cell lines tested. Interestingly, niclosamide is a less effective inhibitor of Wnt-responsive genes (β-catenin, c-Myc, and Survivin) in the niclosamide-resistant cells than in the niclosamide-sensitive cells, suggesting that deficient autophagy induction by niclosamide compromises the effect of niclosamide on Wnt signaling. Our findings provide a mechanistic understanding of the role of autophagosomes in the inhibition of Wnt signaling by niclosamide and may provide biomarkers to assist selection of patients whose tumors are likely to respond to niclosamide.
Signaling through β-arrestins is a recently appreciated mechanism used by seven-transmembrane receptors. Because G protein-coupled receptor kinase (GRK) phosphorylation of such receptors is generally ...a prerequisite for β-arrestin binding, we studied the roles of different GRKs in promoting β-arrestin-mediated extracellular signal-regulated kinase (ERK) activation by a typical seven-transmembrane receptor, the Gs-coupled V2 vasopressin receptor. Gs-and β-arrestin-mediated pathways to ERK activation could be distinguished with H89, an inhibitor of protein kinase A, and β-arrestin 2 small interfering RNA, respectively. The roles of GRK2, -3, -5, and -6 were assessed by suppressing their expression with specific small interfering RNA sequences. By using this approach, we demonstrated that GRK2 and -3 are responsible for most of the agonist-dependent receptor phosphorylation, desensitization, and recruitment of β-arrestins. In contrast, GRK5 and -6 mediated much less receptor phosphorylation and β-arrestin recruitment, but yet appeared exclusively to support β-arrestin 2-mediated ERK activation. GRK2 suppression actually increased β-arrestin-stimulated ERK activation. These results suggest that β-arrestin recruited in response to receptor phosphorylation by different GRKs has distinct functional potentials.
β-arrestins bind to G protein-coupled receptor kinase (GRK)-phosphorylated seven transmembrane receptors, desensitizing their activation of G proteins, while concurrently mediating receptor ...endocytosis, and some aspects of receptor signaling. We have used RNA interference to assess the roles of the four widely expressed isoforms of GRKs (GRK 2, 3, 5, and 6) in regulating β-arrestin-mediated signaling to the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2 by the angiotensin II type 1A receptor. Angiotensin II-stimulated receptor phosphorylation, β-arrestin recruitment, and receptor endocytosis are all mediated primarily by GRK2/3. In contrast, inhibiting GRK 5 or 6 expression abolishes β-arrestin-mediated ERK activation, whereas lowering GRK 2 or 3 leads to an increase in this signaling. Consistent with these findings, β-arrestin-mediated ERK activation is enhanced by overexpression of GRK 5 and 6, and reciprocally diminished by GRK 2 and 3. These findings indicate distinct functional capabilities of β-arrestins bound to receptors phosphorylated by different classes of GRKs.
β-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the ...consequences for cellular physiology have not been well understood. Here we demonstrate that β-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate β-arrestin but not G protein-dependent signaling, and are abrogated by β-arrestin-2 small interfering RNA. These findings establish a key role for β-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved.
Abnormal Wnt activity is a major mechanism responsible for many diseases, including cancer. Previously, we reported that the anthelmintic drug Niclosamide (NIC) inhibits Wnt/β-catenin signaling and ...suppresses colon cancer cell growth. Although the pharmacokinetic properties of NIC are appropriate for use as an anthelmintic agent, its low solubility, low bioavailability and low systemic exposure limit its usefulness in treating systemic diseases. To overcome these limitations, we conjugated NIC to recombinant chimeric polypeptides (CPs), and the CP-NIC conjugate spontaneously self-assembled into sub-100 nm near-monodisperse nanoparticles. CP-NIC nanoparticles delivered intravenously act as a pro-drug of NIC to dramatically increase exposure of NIC compared to dosing with free NIC. CP-NIC improved anti-tumor activity compared to NIC in a xenograft model of human colon cancer. Because NIC has multiple biological activities, CP-NIC could be used for treatment of multiple diseases, including cancer, bacterial and viral infection, type II diabetes, NASH and NAFLD.
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The Wnt signaling pathway plays a key role in regulation of organ development and tissue homeostasis. Dysregulated Wnt activity is one of the major underlying mechanisms responsible ...for many diseases including cancer. We previously reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. Niclosamide is a multi-functional drug that possesses important biological activity in addition to inhibition of Wnt/β-catenin signaling. Here, we studied the SAR of Wnt signaling inhibition in the anilide and salicylamide region of Niclosamide. We found that the 4′-nitro substituent can be effectively replaced by trifluoromethyl or chlorine and that the potency of inhibition was dependent on the substitution pattern in the anilide ring. Non-anilide, N-methyl amides and reverse amide derivatives lost significant potency, while acylated salicylamide derivatives inhibited signaling with potency similar to non-acyl derivatives. Niclosamide’s low systemic exposure when dosed orally may hinder its use to treat systemic disease. To overcome this limitation we identified an acyl derivative of Niclosamide, DK-520 (compound 32), that significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. The studies herein provide a medicinal chemical foundation to improve the pharmacokinetic exposure of Niclosamide and Wnt-signaling inhibitors based on the Niclosamide chemotype. The identification of novel derivatives of Niclosamide that metabolize to Niclosamide and increase its drug exposure may provide important research tools for in vivo studies and provide drug candidates for treating cancers with dysregulated Wnt signaling including drug-resistant cancers. Moreover, since Niclosamide is a multi-functional drug, new research tools such as DK520 could directly result in novel treatments against bacterial and viral infection, lupus, and metabolic diseases such as type II diabetes, NASH and NAFLD.
Regenerative medicine holds the promise of replacing damaged tissues largely by stem cell activation. Hedgehog signaling through the plasma membrane receptor Smoothened (Smo) is an important process ...for regulating stem cell proliferation. The development of Hedgehog-related therapies has been impeded by a lack of US Food and Drug Administration (FDA)-approved Smo agonists. Using a high-content screen with cells expressing Smo receptors and a β-arrestin2-GFP reporter, we identified four FDA-approved drugs, halcinonide, fluticasone, clobetasol, and fluocinonide, as Smo agonists that activate Hedgehog signaling. These drugs demonstrated an ability to bind Smo, promote Smo internalization, activate Gli, and stimulate the proliferation of primary neuronal precursor cells alone and synergistically in the presence of Sonic Hedgehog protein. Halcinonide, fluticasone, clobetasol, and fluocinonide provide an unprecedented opportunity to develop unique clinical strategies to treat Hedgehog-dependent illnesses.
Abstract Background Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor ...lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. Methods We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. Results This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). Conclusions dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. Trial registry ClinicalTrials.gov NCT00952692
The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with β-arrestins. This switches ...a receptor from G-protein activation to G-protein desensitization, receptor internalization, and β-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting β-arrestins to specific receptors has not been elucidated. Here we use the β2-adrenergic receptor (β2AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the β2AR and β-arrestins. We show that β2AR phosphorylation is required for high affinity β-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote β-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for β-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that β-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the β2AR and induce β-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures β-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of β-arrestin recruitment.
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