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CD24 controls Src/STAT3 activity in human tumors

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Abstract

CD24 is a glycosyl-phosphatidylinositol-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties in vitro and tumor growth in vivo. However, little is known about how CD24 mediates these effects. Here we have analyzed the functional consequences of CD24 knock-down or over-expression in human cancer cell lines. Depletion of CD24 reduced cell proliferation and adhesion, enhanced apoptosis, and regulated the expression of various genes some of which were identified as STAT3 target genes. Loss of CD24 reduced STAT3 and FAK phosphorylation. Diminished STAT3 activity was confirmed by specific reporter assays. We found that reduced STAT3 activity after CD24 knock-down was accompanied by altered Src phosphorylation. Silencing of Src, similar to CD24, targeted the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented Src-Y416 phosphorylation, the recruitment of Src into lipid rafts and the expression of STAT3-dependent target genes. An antibody to CD24 was effective in reducing tumor growth of A549 lung cancer and BxPC3 pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-phosphorylation in the tumor and altered the expression of STAT3 target genes. Our results provide evidence that CD24 regulates STAT3 and FAK activity and suggest an important role of Src in this process. Finally, the targeting of CD24 by antibodies could represent a novel route for tumor therapy.

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Abbreviations

ECM:

Extracellular matrix

FAK:

Focal adhesion kinase

GPI:

Glycosyl-phosphatidylinositol

mAb:

Monoclonal antibody

pAb:

Polyclonal antibody

siCD24:

siRNA specific for CD24

siGFP:

siRNA specific for green fluorescent protein (GFP)

STAT3:

Signal transducer and activator of transcription 3

qPCR:

Quantitative real-time PCR

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Acknowledgments

This work was supported by a grant from the DKFZ-BSP Alliance to G. M., T.S. and P. A. We thank Helena Kiefel for valuable comments on the manuscript and Volker Kloess for help and technical support.

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Authors and Affiliations

  1. Tumor Immunology Programme, D015, DKFZ, German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

    Niko P. Bretz, Alexei V. Salnikov, Claudia Perne, Sascha Keller, Xiaoli Wang, Natalie Erbe-Hofmann, Gerhard Moldenhauer & Peter Altevogt

  2. Institute of Experimental Physics I, University of Leipzig, Leipzig, Germany

    Claudia T. Mierke

  3. Department of Pathology, Kaplan Hospital, Rehovot, Israel

    Mina Fogel

  4. Bayer Healthcare AG, Wuppertal, Germany

    Thomas Schlange

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  1. Niko P. Bretz
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  2. Alexei V. Salnikov
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Correspondence to Peter Altevogt.

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Suppl. Fig. 1 Dot-blot analysis of apoptosis after siCD24 knock-down using PI- and Annexin-V staining.

Suppl. Fig. 2 List of genes identified after depletion of CD24 by siCD24-dependent knock-down. SiGFP served as a control.

Suppl. Fig. 3 Western-blot analysis of active (Y416) and inactive (Y527) Src in glioblastoma cell lines after CD24 depletion.

Suppl. Fig. 4 Effect of Src inhibition by the specific inhibitor PP2 on the regulation of STAT3-dependent genes.

Suppl. Fig. 5 (A) Intraperitoneal tumor growth of SKOV3ip ovarian cancer cells in CD1 mice treated with either PBS, isotype control, or SWA11 mAb. Antibodies (10 mg/kg) were given twice a week with eight injections in total. Animals were killed and the tumor mass was determined and used for further analysis. (B) MRNAs were extracted from the isolated tumors and analyzed by RT-PCR for the indicated STAT3-dependent genes. Note that p-Src Y527 staining was undetectable both in SWA11- and isotype-control treated groups.

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Bretz, N.P., Salnikov, A.V., Perne, C. et al. CD24 controls Src/STAT3 activity in human tumors. Cell. Mol. Life Sci. 69, 3863–3879 (2012). https://doi.org/10.1007/s00018-012-1055-9

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