CD24 controls Src/STAT3 activity in human tumors

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.

Fig. 1

The silencing of CD24 decreases cell proliferation and enhances apoptosis. a The indicated tumor cell lines were treated with siRNA targeting CD24 or GFP as control for 96 h. Cells were stained with antibodies to CD24 (SWA11) followed by PE-conjugated goat anti-mouse IgG and subjected to FACS analysis. b Western-blot analysis of whole-cell lysates using SWA11 and peroxidase-coupled secondary IgG. c MTT proliferation assay of cells after siCD24 knock-down. Proliferation was tested at the indicated time points. d Induction of apoptosis was investigated 72 h after siCD24 transfection using PI- and Annexin-V staining. e Cell cycle analysis on SNB19 cells showing the strongest apoptotic effect was carried out in parallel. Pooled results of n = 3 experiments (d) are shown. Representative data of n = 4 experiments are shown for (a), (b), (c), and (e)

Fig. 2

CD24 knock-down alters cell adhesion and FAK phosphorylation. a The indicated tumor cell lines were subjected to siCD24-mediated knock-down of CD24. Cells were analyzed for cell binding to immobilized substrates FN-40, laminin and BSA for background control. The experiments were carried out in triplicates. b Analysis of FAK phosphorylation in CD24-depleted cells. Cell lysates of the indicated cells were tested for FAK, p-FAK Y925 (Src-dependent) and p-FAK Y397 (autophosphorylation) sites. Representative data of n = 3 experiments are shown

Fig. 3

CD24 knock-down mediates gene regulation and reduces STAT3 protein phosphorylation and activity. a Identification of a CD24-dependent target genes signature (OPG, STC-1, MMP-7, and STAT3) by quantitative RT-PCR analysis of mRNAs isolated after siCD24-mediated knock-down. Note that OPG, STC-1, and MMP-7 genes are up-regulated, whereas STAT3 is down-regulated in the indicated cell lines. Pooled results of n = 6 experiments are shown. b Analysis of STAT3 levels after siRNA-mediated knock-down of CD24. Note that all oligonucleotides specific for CD24 reduce STAT3 expression levels. c Western-blot analysis for STAT3 and p-STAT3 Y705 after siCD24-mediated depletion of CD24. d Decreased STAT3 activity after CD24 or STAT3 knock-down (as a positive control) was measured using a STAT3-specific luciferase reporter assay. Pooled results (b) and representative results (c, d) of n = 4 experiments are shown

Fig. 4

Loss of CD24 affects transcription of classical STAT3 target genes. a Known STAT3 target genes (cyclin D1, MCL-1, survivin) were analyzed by qPCR after CD24 or STAT3 knock-down in indicated tumor cell lines. Relative mRNA levels normalized to β-actin were compared to siGFP control. b Regulation of the CD24-dependent target gene signature expression after STAT3 silencing was determined by qPCR. Relative mRNA levels normalized to β-actin were compared to siGFP control. Pooled results of n = 4 experiments are shown (a, b)

Fig. 5

Silencing of Src affects phosphorylation of STAT3 and STAT3 target gene regulation. a Sucrose gradient analysis of lipid rafts after CD24 knock-down. Gradient fractions were probed with the indicated antibodies followed by peroxidase-conjugated secondary antibodies and ECL detection. b The indicated tumor cell lines were subjected to siRNA-mediated knock-down of Src and probed by qPCR for knock-down efficacy. c Phosphorylation of STAT3 after Src knock-down was analyzed by Western blotting. d Expression analysis of STAT3 target genes after specific knock-down of Src. Representative results (a, c) or pooled results (b, d) of at least n = 3 experiments are shown

Fig. 6

Effect of CD24 over-expression on phosphorylation and gene regulation. a Cytofluorographic analysis for CD24 expression in A125, A125-CD24low, and A125-CD24high-expressing cells. b Western-blot analysis of whole-cell lysates with the indicated phospho-specific antibodies. c Sucrose gradient analysis of lipid rafts as described in the legend to Fig. 5a. d Regulation of selected STAT-3-dependent target genes in the indicated cell lines. e A125 CD24high cells were treated with the inhibitors (20 μM) for overnight and cells were analyzed for the expression of STAT3-dependent genes. f A125 CD24high cells were depleted for Src by siRNA and the effect on the expression of STAT3-dependent genes was analyzed. Representative results of n = 3 analysis are shown in (a, b, c). Pooled results of n = 4 experiments are shown in (d, e, f)

Fig. 7

The antibody to CD24 affects phosphorylation of Src in vivo and has a therapeutic effect. a Subcutaneous tumor growth of A549 lung cancer cells in Scid/beige mice treated with either PBS, isotype control IgG2a, or SWA11 mAb. Antibodies (10 mg/kg) were given every 5 days with five injections in total. Tumor growth was measured using a caliper. Animals were killed and the isolated tumors were used for further analysis. b BxPC3 tumor cells were injected s.c. into Nod/Scid mice and treated with SWA11 mAb (10 mg/kg) every 3 days with five injections in total. Animals were killed and the isolated tumors were used for further analysis. c, d Immunofluorescence analysis of Src (Y527 and Y416) in SWA11 versus isotype control-treated animals. Note that p-Src Y416 is decreased and p-Src Y527 staining is increased in tumor areas where mAb SWA11 is bound. e, f) mRNAs were extracted from treated tumors and analyzed by qPCR for indicated STAT3-dependent genes. Pooled results of n = 4 animals per group are shown

Fig. 8

Schematic representation of the functional role of CD24 in tumors. a The presence of CD24 supports the localization and the activity of Src kinase in cholesterol enriched lipid rafts. Active Src is able to activate STAT3 molecules by phosphorylation at tyrosine 705. Activated STAT3 dimerizes and translocates into the nucleus. There, DNA binding of STAT3 dimers results in expression of specific target genes that might influence cellular properties, like survival and proliferation, contributing to oncogenicity of the tumor cell. Additionally, FAK activation might influence integrin-dependent cell adhesion to ECM substrates. b Silencing of CD24 and subsequent depletion from lipid rafts displaces Src kinase from rafts and impairs Src activity. Thereby, decreased STAT3 activation resulting in diminished expression of STAT3 target genes impairs tumor cell proliferation and enhances apoptosis. FAK-regulated adhesion to the ECM is also impaired