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A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

ABSTRACT: The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19?µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other "FOXO-resistant" cancers.

SUBMITTER: Salcher S

PROVIDER: S-EPMC6989399 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

Salcher Stefan S Spoden Gilles G Hagenbuchner Judith J Führer Sebastian S Kaserer Teresa T Tollinger Martin M Huber-Cantonati Petra P Gruber Thomas T Schuster Daniela D Gust Ronald R Zwierzina Heinz H Müller Thomas T Kiechl-Kohlendorfer Ursula U Ausserlechner Michael J MJ Obexer Petra P

Oncogene 20191007 5

The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date ...[more]

PMID: 31591479

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Project description:Forkhead box O class transcription factors are homeostasis regulators that control cell death, longevity and therapy-resistance. In neuroblastoma (NB), nuclear FOXO3 correlates with stage M disease and poor prognosis. To analyze whether FOXO3 contributes to drug-resistance in this childhood cancer, we investigated how different high-stage-derived NB cells respond to the activation of an ectopic FOXO3 allele. We found endogenous FOXO3 mostly localized to the nucleus-upon activation of an ectopic, 4OHT-activated FOXO3(A3)ER fusion protein two of the cell lines underwent apoptosis, whereas in the others FOXO3-activation even increased survival during drug-treatment. In the latter cell type, FOXO3 did not induce the BH3-only protein BCL2L11/BIM due to impaired binding of FOXO3 to the BIM-promoter, but still activated other FOXO3 targets. It was shown before that FOXO3 and TP53 physically interact with each other at two different regions-the TP53-N-terminus binds to the FOXO3-DNA binding domain (DBD) and the FOXO3-C-terminus interacts with the TP53-DBD. Interestingly, cell lines that undergo FOXO3-induced cell death carry homozygous point mutations in the TP53-DBD near the structural hotspot-mutation-site R175H, which abrogated FOXO3-TP53 interaction. In contrast, in FOXO3-death-resistant cells no point mutations in the TP53-DBD were found-in these cells FOXO3-TP53 complexes are formed and FOXO3-binding to the BIM-promoter, but not the induction of the detoxifying protein SESN3, were prevented, which in turn increased chemo-protection in this type of high-stage-derived NB cells. Our combined data suggest that FOXO3 steps in as a death inducer in case of TP53-mutation, whereas functional TP53 alters FOXO3-target-promoter-recognition, which prevents death induction by FOXO3 and instead increases chemo-protection and survival of NB cells. This novel mechanism may explain the low incidence of TP53 mutation in high-stage NB at diagnosis and suggests FOXO3 as a therapeutic target for this childhood malignancy.

Dataset Information

A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

Publications

A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

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