Niclosamide sensitizes triple-negative breast cancer cells to ionizing radiation in association with the inhibition of Wnt/?-catenin signaling.
ABSTRACT: Triple-negative breast cancer (TNBC) is one of the most difficult breast cancers to treat because there is no targeted treatment, and conventional cytotoxic chemotherapy followed by adjuvant radiation therapy is the standard of care for patients with TNBC. We herein reported that ionizing radiation (IR) induced Wnt3a, LRP6 and ?-catenin expression and consequently activated Wnt/?-catenin signaling in TNBC MDA-MB-231, MDA-MB-468 and Hs578T cells. Moreover, depletion of ?-catenin by shRNA sensitized TNBC cells to IR, whereas treatment of Wnt3a protein or overexpression of ?-catenin resulted in radioresistance of TNBC cells. Niclosamide, a potent inhibitor of Wnt/?-catenin signaling, not only inhibited constitutive Wnt/?-catenin signaling, but also blocked IR-induced Wnt/?-catenin signaling in TNBC cells. In addition, niclosamide sensitized TNBC cells to IR, prevented Wnt3a-induced radioresistance, and overcame ?-catenin-induced radioresistance in TNBC cells. Importantly, animals treated with the combination of niclosamide and ?-ray local tumor irradiation had significant inhibition of MDA-MB-231 tumor growth compared with treated with local tumor irradiation alone. These findings indicate that Wnt/?-catenin signaling pathway plays an important role in the development of radioresistance of TNBC cells, and that niclosamide had significant radiosensitizing effects by inhibiting Wnt/?-catenin signaling in TNBC cells. Our study also provides rationale for further preclinical and clinical evaluation of niclosamide in TNBC management.
Project description:The canonical Wnt/?-catenin pathway is activated in triple-negative breast cancer (TNBC). The activation of this pathway leads to the expression of specific target genes depending on the cell/tissue context. Here, we analyzed the transcriptome of two different TNBC cell lines to define a comprehensive list of Wnt target genes. The treatment of cells with Wnt3a for 6h up-regulated the expression (fold change > 1.3) of 59 genes in MDA-MB-468 cells and 241 genes in HCC38 cells. Thirty genes were common to both cell lines. Beta-catenin may also be a transcriptional repressor and we found that 18 and 166 genes were down-regulated in response to Wnt3a treatment for 6h in MDA-MB-468 and HCC38 cells, respectively, of which six were common to both cell lines. Only half of the activated and the repressed transcripts have been previously described as Wnt target genes. Therefore, our study reveals 137 novel genes that may be positively regulated by Wnt3a and 104 novel genes that may be negatively regulated by Wnt3a. These genes are involved in the Wnt pathway itself, and also in TGF?, p53 and Hedgehog pathways. Thorough characterization of these novel potential Wnt target genes may reveal new regulators of the canonical Wnt pathway. The comparison of our list of Wnt target genes with those published in other cellular contexts confirms the notion that Wnt target genes are tissue-, cell line- and treatment-specific. Genes up-regulated in Wnt3a-stimulated cell lines were more strongly expressed in TNBC than in luminal A breast cancer samples. These genes were also overexpressed, but to a much lesser extent, in HER2+ and luminal B tumors. We identified 72 Wnt target genes higher expressed in TNBCs (17 with a fold change >1.3) which may reflect the chronic activation of the canonical Wnt pathway that occurs in TNBC tumors.
Project description:The canonical Wnt/β-catenin signalling pathway and autophagy play critical roles in cancer progression. However, the role of Wnt-mediated autophagy in cancer radioresistance remains unclear. In this study, we found that irradiation activated the Wnt/β-catenin and autophagic signalling pathways in squamous cell carcinoma of the head and neck (SCCHN). Wnt3a is a classical ligand that activated the Wnt/β-catenin signalling pathway, induced autophagy and decreased the sensitivity of SCCHN to irradiation both in vitro and in vivo. Further mechanistic analysis revealed that Wnt3a promoted SCCHN radioresistance via protective autophagy. Finally, expression of the Wnt3a protein was elevated in both SCCHN tissues and patients' serum. Patients showing high expression of Wnt3a displayed a worse prognosis. Taken together, our study indicates that both the canonical Wnt and autophagic signalling pathways are valuable targets for sensitizing SCCHN to irradiation.
Project description: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 ?-catenin, contributing to suppression of colorectal cancer growth in vitro and in vivo 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 ?-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 (?-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.
Project description:The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report that WP upregulation causes ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in a RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Our study demonstrates for the first time that beta-catenin-RAC1 cascade signals integrin-directed metastasis-associated tumor cell phenotypes in TNBC.
Project description:Basal-like breast cancers (BLBC) are poorly differentiated and display aggressive clinical behavior. These tumors become resistant to cytotoxic agents, and tumor relapse has been attributed to the presence of cancer stem cells (CSC). One of the pathways involved in CSC regulation is the Wnt/?-catenin signaling pathway. LRP6, a Wnt ligand receptor, is one of the critical elements of this pathway and could potentially be an excellent therapeutic target. Niclosamide has been shown to inhibit the Wnt/?-catenin signaling pathway by causing degradation of LRP6. TRA-8, a monoclonal antibody specific to TRAIL death receptor 5, is cytotoxic to BLBC cell lines and their CSC-enriched populations. The goal of this study was to examine whether niclosamide is cytotoxic to BLBCs, specifically the CSC population, and if in combination with TRA-8 could produce increased cytotoxicity. Aldehyde dehydrogenase (ALDH) is a known marker of CSCs. By testing BLBC cells for ALDH expression by flow cytometry, we were able to isolate a nonadherent population of cells that have high ALDH expression. Niclosamide showed cytotoxicity against these nonadherent ALDH-expressing cells in addition to adherent cells from four BLBC cell lines: 2LMP, SUM159, HCC1187, and HCC1143. Niclosamide treatment produced reduced levels of LRP6 and ?-catenin, which is a downstream Wnt/?-catenin signaling protein. The combination of TRA-8 and niclosamide produced additive cytotoxicity and a reduction in Wnt/?-catenin activity. Niclosamide in combination with TRA-8 suppressed growth of 2LMP orthotopic tumor xenografts. These results suggest that niclosamide or congeners of this agent may be useful for the treatment of BLBC.
Project description:Retinoblastoma is an angiogenesis-dependent ocular tumor, the clinical management of which remains a challenge. Agents that can target tumor cells and angiogenesis, as well as augment current chemotherapy efficacy, present a promising therapeutic strategy for retinoblastoma. We demonstrated that niclosamide, an FDA-approved anthelmintic drug, is effective against multiple aspects of retinoblastoma. Niclosamide inhibited proliferation via causing cell cycle arrest at the G2/M phase and induced caspase-dependent apoptosis in a panel of retinoblastoma cell lines, including Y79, RB116, and WERI-Rb-1. In addition, niclosamide inhibited retinoblastoma angiogenesis by disrupting capillary network formation, decreasing migration and proliferation, and inducing apoptosis of human primary retinal microvascular endothelial cells. We also demonstrated that niclosamide specifically suppresses the levels of p-LRP6, Dvl2, and ?-catenin, but not p-STAT3, in Y79 cells. It decreased ?-catenin activity and the mRNA expression levels of Wnt/?-catenin target genes. Stabilization of ?-catenin with the Wnt activator lithium or overexpression of ?-catenin reversed the inhibitory effects of niclosamide in Y79 cells, confirming Wnt/?-catenin as the molecular target of niclosamide in retinoblastoma cells. Importantly, niclosamide significantly enhanced the <i>in vitro</i> and <i>in vivo</i> efficacy of carboplatin and inhibited Wnt/?-catenin signaling in a retinoblastoma xenograft mouse model. Our data suggest that niclosamide is a promising candidate for the treatment armamentarium for retinoblastoma. Our work also highlights that targeting Wnt/?-catenin is a potential therapeutic strategy in retinoblastoma.
Project description:Dysregulation of the Wnt signaling pathway is an underlying mechanism in multiple diseases, particularly in cancer. Until recently, identifying agents that target this pathway has been difficult and as a result, no approved drugs exist that specifically target this pathway. We reported previously that the anthelmintic drug Niclosamide inhibits the Wnt/?-catenin signaling pathway and suppresses colorectal cancer cell growth in vitro and in vivo. In an effort to build on this finding, we sought to discover new Wnt/?-catenin inhibitors that expanded the chemotype structural diversity. Here, we asked a specific SAR question unresolved in previous SAR studies of Niclosamide's inhibition of Wnt/?-catenin signaling to identify a new structural class of Wnt/?-catenin signaling inhibitors based on a triazole motif. Similar to Niclosamide, we found that the new triazole derivatives internalized Frizzled-1 GFP receptors, inhibited Wnt/?-catenin signaling in the TOPflash assay and reduced Wnt/?-catenin target gene levels in CRC cells harboring mutations in the Wnt pathway. Moreover, in pilot SAR studies, we found the Wnt/?-catenin SAR trends in the anilide region were generally similar between the two chemical classes of inhibitors. Overall, these studies demonstrate the ability to use the SAR of the Niclosamide salicylanilide chemical class to expand the structural diversity of Wnt/?-catenin inhibitors.
Project description:Wnt/?-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream ?-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/?-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/?-catenin pathway activation, downregulated Dvl2, decreased downstream ?-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-?B or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.
Project description:The Wnt signaling pathway is critical for normal tissue development and is an underlying mechanism of disease when dysregulated. Previously, we reported that the drug Niclosamide inhibits Wnt/?-catenin signaling by decreasing the cytosolic levels of Dishevelled and ?-catenin, and inhibits the growth of colon cancers both in vitro and in vivo. Since the discovery of Niclosamide's anthelmintic activity, a growing body of literature indicates that Niclosamide is a multifunctional drug. In an effort to identify derivatives of Niclosamide with improved pharmacokinetic properties that maintain the multifunctional drug activity of Niclosamide for clinical evaluation, we designed DK419, a derivative containing a 1H-benzo[d]imidazole-4-carboxamide substructure, using the structure-activity relationships (SAR) of the Niclosamide salicylanilide chemotype. Similar to Niclosamide, we found DK419 inhibited Wnt/?-catenin signaling, altered cellular oxygen consumption rate and induced production of pAMPK. Moreover, we found DK419 inhibited the growth of CRC tumor cells in vitro, had good plasma exposure when dosed orally, and inhibited the growth of patient derived CRC240 tumor explants in mice dosed orally. DK419, a derivative of Niclosamide with multifunctional activity and improved pharmacokinetic properties, is a promising agent to treat colorectal cancer, Wnt-related diseases and other diseases in which Niclosamide has demonstrated functional activity.
Project description:BACKGROUND/AIM:Radiation therapy (RT) represents a therapeutic option in breast cancer (BC). Even if a great number of BC patients receive RT, not all of them report benefits, due to radioresistance that gets activated through several factors, such as the hormone receptor status. Herein, we analyzed the gene expression profiles (GEP) induced by RT in triple-negative BC (TNBC) MDA-MB-231, to study signalling networks involved in radioresistance. MATERIALS AND METHODS:GEP of MDA-MB-231 BC cells treated with a high dose of radiation, went through cDNA microarray analysis. In addition, to examine the cellular effects induced by RT, analyses of morphology and clonogenic evaluation were also conducted. RESULTS:A descriptive report of GEP and pathways induced by IR is reported from our microarray data. Moreover, the MDA-MB-231 Radioresistent Cell Fraction (RCF) selected, included specific molecules able to drive radioresistance. CONCLUSION:In summary, our data highlight, the RT response of TNBC MDA-MB-231 cell line at a transcriptional level, in terms of activating radioresistance in these cells, as a model of late-stage BC.