Reduced Expression of Sprouty1 Contributes to the Aberrant Proliferation and Impaired Apoptosis of Acute Myeloid Leukemia Cells

Abstract

In most of the acute myeloid leukemia patients there is an aberrant tyrosine kinase activity. The prototype of Sprouty proteins was originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. Usually, SPRY family members are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as a regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of adult acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in acute myeloid leukemia (AML) patients at diagnosis, suggesting a multistep regulation of RAS signaling in human cancers.

Keywords: FoxO3a; Sprouty1; acute myeloid leukemia (AML).

Figure 1

(A) Sprouty1 gene expression was assayed by qRT-PCR in BM and PB derived from both AML patients and normal subjects. The quantity is expressed as 2^−ΔΔCt after normalization with Abl housekeeping gene (** p ≤ 0.01 and *** p ≤ 0.001). (B) Western blot performed with anti Sprouty1 antibody on total protein derived from PB of four representative AML cells and one PB of a healthy donor. Vinculin is used as normalizer. (C) Immunofluorescence staining assay performed on cytospun BM cells of AML or control samples. The green signal corresponds to Sprouty1 while red propidium iodide is used to stain nuclei. BM, bone marrow; PB, peripheral blood; AML, acute myelocytic leukemia.

Figure 2

(A) Western blot analysis and quantification performed on Kasumi-1 cell lines transfected, respectively, with pCGN empty vector and pCGN-Sprouty1 vector. (B) Proliferation assay performed in Kasumi-1 cells transfected with empty or Sprouty1 vector. (C) Apoptosis evaluated by flow cytometry after FITC Annexin-V assay on Kasumi-1 cells transfected with pCGN-Sprouty1. (D) RPMI-Soft Agar colony assay on Kasumi-1 transfected cells. Representative colonies pictures were captured by Infinity Analyze 3 camera and processed by Lumenera software. All experiments were performed in triplicate.

Figure 3

(A) Immunofluorescence staining performed with anti-FoxO3a (green signal) on cells derived from control subjects and AML patients at diagnosis. (B) Western blot performed with an antibody against FoxO3a on lysates derived from the cytosolic and nuclear fraction of AML patients. (C) Western blot of FoxO3a and Sprouty1 in Kasumi-1 cells transfected with FoxO3a or empty plasmids. (D) Sprouty1 gene expression analysis on Kasumi-1 cells transfected with FoxO3a or empty plasmids. The quantity is expressed as 2^-ΔΔCt after normalization with Abl housekeeping gene. (E) Schematic representation of RAS/PI3K pathways and their negative regulation on FoxO3a. Representative western blot showing Sprouty1-dependent regulation of the RAS pathway in Kasumi-1 cells transfected with Sprouty1 plasmid. The relative intensity of each band is shown under the blot as fold change compared to non-transfected control, to which a value of 1 unit was assigned.