Dataset Information

Increased FLYWCH1 Expression is Negatively Correlated with Wnt/?-catenin Target Gene Expression in Acute Myeloid Leukemia Cells.

ABSTRACT: Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by ?-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear ?-catenin. Herein, we studied the FLYWCH1/?-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using methylcellulose-based and Propidium iodide/flow cytometry assays. We found that FLYWCH1 mRNA and protein were differentially expressed in the AML cell lines. C-Myc, cyclin D1, and c-Jun expression decreased in the presence of higher FLYWCH1 expression, and vice versa. There appeared to be the loss of FLYWCH1 expression in dividing cells. The sub-G0 phase was prolonged and shortened in the low and high FLYWCH1 expression cell lines, respectively. The G0/G1 arrest correlated with FLYWCH1-expression, and these cell lines also formed colonies, whereas the low FLYWCH1 expression cell lines could not. Thus, FLYWCH1 functions as a negative regulator of the Wnt/?-catenin pathway in AML.

SUBMITTER: Almars A

PROVIDER: S-EPMC6600431 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Increased FLYWCH1 Expression is Negatively Correlated with Wnt/β-catenin Target Gene Expression in Acute Myeloid Leukemia Cells.

Almars Amany A Chondrou Panagiota S PS Onyido Emenike K EK Almozyan Sheema S Seedhouse Claire C Babaei-Jadidi Roya R Nateri Abdolrahman S AS

International journal of molecular sciences 20190604 11

Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by β-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear β-catenin. Herein, we studied the FLYWCH1/β-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using meth ...[more]

PMID: 31167387

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Project description:Purpose: Wnt/?-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases ?-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3-mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/?-catenin and FLT3 inhibition in FLT3-mutant AML.Experimental Design: Wnt/?-catenin signaling was inhibited by the ?-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF.Results: We found significantly higher ?-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow-resident leukemic cells compared with circulating blasts. Disrupting Wnt/?-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3-mutated AML cells and stem/progenitor cells in vitro The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/?-catenin and FLT3 cooperatively decreased nuclear ?-catenin and the levels of c-Myc and other Wnt/?-catenin and FLT3 signaling proteins. Importantly, ?-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells.Conclusions: Disrupting Wnt/?-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3-mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3-mutated AML patients. Clin Cancer Res; 24(10); 2417-29. ©2018 AACR.

Project description:BackgroundAcute myeloid leukemia (AML) is initiated and maintained by a unique, small subset of leukemia stem cells (LSCs). LSCs are characterized by unrestricted self-renewal and contribute to the malignancy of leukemia. Aberrant protein fucosylation is associated with AML progression. However, it is still less understood that the miR-29b/Sp1/FUT4 crosstalk involved in the fucosylation-mediated LSCs malignancy in AML.MethodsAML cell lines were sorted by magnetic microbeads to obtain the CD34?+?CD38- sub-population. The key biomarkers for LSCs were identified by flow cytometry. Fucosyltransferase genes were screened by qRT-PCR, and FUT4 was focused. Effect of FUT4 on LSCs malignancy was determined by CCK8 assay, sphere formation assay, immunofluorescence staining, apoptosis and in vivo xenografts experiments. The linkage of FUT4 promoter and Sp1 was confirmed by dual-luciferase reporter gene assay. ChIP-PCR assay was used to show the directly binding of Sp1 and FUT4 promoter. Activity of Wnt//?-catenin pathway was determined by western blot. Overall survival curves were diagrammed by Kaplan-Meier analysis.ResultsHere, the expressional profiles of 11 fucosyltransferase genes were different comparing LSCs and non-LSCs of KG-1a and MOLM13 cells, whereas CD34?+?CD38- cells exhibited higher expression of FUT4. Functionally, alteration of FUT4 in CD34?+?CD38- cells modulated LSCs malignant behaviors both in vitro and in vivo. Transcriptional inhibitor actinomycin D (Act D) or translational inhibitor cycloheximide (CHX) prevented LSCs progression, and Sp1 was identified as the efficient regulator of FUT4 transcription. Moreover, miR-29b directly affected the binding of Sp1 and FUT4 promoter region, which further mediated LSCs proliferation, apoptosis and drug-resistance through fucosylated-CD44 via activation of Wnt/?-catenin pathway. Clinically, Sp1 and FUT4 were up-regulated and positively correlated with poor overall survival of AML patients.ConclusionThese data indicated that miR-29b/Sp1/FUT4 axis promoted the malignant behaviors of LSCs by regulating fucosylated CD44 via Wnt/?-catenin pathway. Identifying LSCs surface markers and targeting LSCs were important for the development of potential therapies in AML.

Dataset Information

Increased FLYWCH1 Expression is Negatively Correlated with Wnt/?-catenin Target Gene Expression in Acute Myeloid Leukemia Cells.

Publications

Increased FLYWCH1 Expression is Negatively Correlated with Wnt/β-catenin Target Gene Expression in Acute Myeloid Leukemia Cells.

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