SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome
Houda Alachkar, … , Clara D. Bloomfield, Guido Marcucci
Houda Alachkar, … , Clara D. Bloomfield, Guido Marcucci
Published March 3, 2014
Citation Information: J Clin Invest. 2014;124(4):1512-1524. https://doi.org/10.1172/JCI70921.
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Research Article Oncology

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome

Abstract

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

Authors

Houda Alachkar, Ramasamy Santhanam, Kati Maharry, Klaus H. Metzeler, Xiaomeng Huang, Jessica Kohlschmidt, Jason H. Mendler, Juliana M. Benito, Christopher Hickey, Paolo Neviani, Adrienne M. Dorrance, Mirela Anghelina, Jihane Khalife, Somayeh S. Tarighat, Stefano Volinia, Susan P. Whitman, Peter Paschka, Pia Hoellerbauer, Yue-Zhong Wu, Lina Han, Brad N. Bolon, William Blum, Krzysztof Mrózek, Andrew J. Carroll, Danilo Perrotti, Michael Andreeff, Michael A. Caligiuri, Marina Konopleva, Ramiro Garzon, Clara D. Bloomfield, Guido Marcucci

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Figure 4

SPARC activates ILK/AKT/β-catenin signaling pathways in THP-1 cells and enhances β-catenin nuclear translocation and transcriptional activity.

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SPARC activates ILK/AKT/β-catenin signaling pathways in THP-1 cells and ...
THP-1 cells were transfected with pLenti-EV or pLenti-SPARC vectors. (A) Western blot measuring secreted SPARC protein levels in media collected from cell culture of THP-1 cells infected with pLenti-SPARC vector compared with cells infected with pLenti-EV. (B) SPARC and integrin coimmunoprecipitation assay. (C) ILK kinase activity assay. Lysates from THP-1/SPARC cells and THP-1/EV cells were immunoprecipitated with anti-ILK antibody and assayed utilizing GSK3 fusion protein (ILK substrate). Numbers represent ratio of band intensity to control band. (D) Western blot analysis of P-AKT(Ser473) and P-GSK3β(Ser9) levels in THP-1/SPARC cells compared with THP-1/EV in the presence of endogenous ILK or following ILK knockdown or (E) following blocking integrin receptors with integrin antibodies. Lanes separated by a line were run on the same gel but were noncontiguous. (F) Western blot analysis of P–β-catenin (Ser552) in THP-1/SPARC cells compared with THP-1/EV cells. (G) Immunofluorescence confocal microscopy images showing β-catenin (green) and cell nuclei (blue) in EV- or SPARC-transfected THP-1 cells stained with β-catenin antibody and Draq5 antibody (nuclear staining); arrows indicate cells exhibiting β-catenin nuclear translocation. Scale bars: 10 μm. (H) Luciferase activity measured in 293T cells following cotransfection with pBAR (TCF/LEF reporter vector) or pfuBAR (TCF/LEF reporter vector with mutated site) and pLenti-EV or pLenti-SPARC and (I) in the presence of control siRNAs or ILK siRNAs. (J) MYC mRNA expression (left panel) and protein expression (right panel) in THP-1/SPARC cells compared with THP-1/EV. Data represent mean ± SEM for 3 different experiments.