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Cancer Research 68, 2803, April 15, 2008. doi: 10.1158/0008-5472.CAN-07-6463
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or Small Interfering RNA

Eyal Sagiv1,4, Alex Starr2,4, Uri Rozovski1,4, Rami Khosravi3, Peter Altevogt5, Timothy Wang6 and Nadir Arber1,4

1 The Integrated Cancer Prevention Center and 2 Lung and Allergy Institute, Tel Aviv Medical Center; 3 The David and Inez Myers Laboratory for Genetic Research; 4 Department of Molecular Genetics and Biochemistry, Tel Aviv University, Tel Aviv, Israel; 5 Tumor Immunology Programme, D010, German Cancer Research Center, Heidelberg, Germany; and 6 Columbia University, New York, New York

Requests for reprints: Nadir Arber, Integrated Cancer Prevention Center, Tel Aviv Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel. Phone: 972-3-697-4968; Fax: 972-3-695-0339; E-mail: narber{at}post.tau.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of colorectal tumors at a fairly early stage in the multistep process of carcinogenesis. Anti-CD24 monoclonal antibodies (mAb) induce a significant growth inhibition in colorectal and pancreatic cancer cell lines that express the protein. This study is designed to investigate further the effects of CD24 down-regulation using mAb or small interfering RNA in vitro and in vivo. Western blot analysis showed that anti-CD24 mAb induced CD24 protein down-regulation through lysosomal degradation. mAb augmented growth inhibition in combination with five classic chemotherapies. Xenograft models in vivo showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, stable growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA). The produced clones proliferated more slowly, reached lower saturation densities, and showed impaired motility. Most importantly, down-regulation of CD24 retarded tumorigenicity of human cancer cell lines in nude mice. Microarray analysis revealed a similar pattern of gene expression alterations when cells were subjected to anti-CD24 mAb or shRNA. Genes in the Ras pathway, mitogen-activated protein kinase, or BCL-2 family and others of oncogenic association were frequently down-regulated. As a putative new oncogene that is overexpressed in gastrointestinal malignancies early in the carcinogenesis process, CD24 is a potential target for early intervention in the prevention and treatment of cancer. [Cancer Res 2008;68(8):2803–12]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colorectal cancer (CRC) typically develops over decades. It is a multistep process, consisting of several genetic alterations, that create a change in the protein milieu of the cell and drive it to malignant transformation. The molecular pathways that lead to the malignant transformation of the colorectal mucosa have been defined better than for any other tumor (1, 2). Despite this ample knowledge, the precise mechanism by which these genetic alterations lead to the development of CRC remains to be resolved, and many key genes and proteins involved are still not known. Better understanding of the biology of this multistep process could be potentially translated into improved therapy.

CD24 is a fairly novel gene, described in a growing body of literature in relation to cancer, and overexpressed in various human malignancies (3). Its expression is often correlated with poor prognosis (4–7). We have recently reported, using gene expression array, increased expression of CD24 in transformed, but not nontransformed, enterocytes that was down-regulated to a normal level of expression after short (72 hours) and long (6 months) exposures to a selective cyclooxygenase 2 (COX2) inhibitor, celecoxib (Celebrex, Pfizer; ref. 8). The results were validated by immunohistochemical stainings in 389 human samples derived from a variety of gastrointestinal malignancies. A strong membrane expression of CD24 protein was seen already at an early step of carcinogenesis, the adenomatous polyp (8). CD24 was expressed in 90.7% of adenomas and 86.3% of CRC cases, whereas very low expression was seen in normal epithelium (16.6%). The up-regulation of CD24 during CRC progression and its down-regulation by a known chemopreventive agent (COX2 inhibitor) suggested the possibility that CD24 could be important in the oncogenic pathway.

The CD24 gene encodes a heavily glycosylated cell surface protein anchored to the membrane by phosphatidylinositol (3). Human CD24 consists of 31 amino acids with 16 potential O-glycosylation and N-glycosylation sites. Owing to this extensive glycosylation, CD24 has mucin-like characteristics (3). CD24 plays a crucial role in cell selection and maturation during hematopoiesis. It is expressed mainly on premature lymphocytes and certain epithelial and neural cells (9, 10). It also plays a role during the embryonal development of neural and pancreatic cells (11, 12). Analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM, as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that the CD24 receptor mediates intracellular signaling although it has no transmembrane domain (9, 13). CD24 is also known to be an alternative ligand for P-selectin and thus might function in metastases shedding (14–17). Anti-CD24 monoclonal antibodies (mAb) induced growth inhibition in lymphocytes precursors (18, 19). We have shown that the growth of human colon and pancreatic cancer cell lines is inhibited in response to CD24 mAb in a dose-dependent and time-dependent manner and in a close association with their CD24 expression level. This growth inhibition was a consistent finding and reproducible with three different mAbs (8, 20).

Bauman et al. (21) showed that ectopic overexpression of CD24 in a rat carcinoma cell line increases cell proliferation and adhesion through activation of integrins. Similarly, Smith et al. (22) have shown how transient down-regulation of CD24 expression in human carcinoma cell lines resulted in growth inhibition and reduced clonogenicity and cell migration through a change in the actin cytoskeleton. The current study shows that down-regulation of CD24 expression in vitro (using short-hairpin interfering RNA) and in vivo (using monoclonal antibodies) decrease cancer cell tumorigenicity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the short hairpin RNA expression plasmid. The pSUPER-RNAi System, with the specific vector pSUPER.retro.puro (OligoEngine) was used to generate a plasmid that expresses hairpin RNAs (23). We designed two small interfering RNA (siRNA) sequences according to the cDNA of CD24, corresponding to nucleotides 833 to 851 (TGTTTACATTGTTGAGCTA, pRS833) and 1099 to 1117 (TTGCATTGACCACGACTAA, pRS1099; Genbank accession no. M58664). BLAST research ensured that the sequences have no significant homology with other human genes. Two homologous ssDNA molecules were chemically synthesized. Annealing of the ssDNA molecules was performed by incubation of 1 µg/µL of each complementary ssDNA oligonucleotide in annealing buffer [0.1 mol/L NaCl, 10 mmol/L Tris (pH 7.4)] in a total volume of 20 µL. The annealing mixture was incubated at 95°C for 5 min followed by a gradual cooling to room temperature. The inverted motif that contains the 9-nt spacer and five T's cloned into the HindIII and BglII sites of plasmid (Fig. 1A ). Similarly, we subcloned the same oligo-sequences into a vector with a hygromycin resistance gene replacing the puromycin selectable marker region (received as a generous gift from Prof. Yosef Shiloh, Tel Aviv University). As control vectors, we used the same plasmids carrying siRNA sequences nonspecific to any human gene, designed to the sequences of the GFP (pRSGFP) and Luciferase (pRSluc) genes (received from Prof. Yosef Shiloh).


Figure 1
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  		Figure 1. A, CD24-targeted pSUPER vectors used for the formation of clones. pRS833 is directed to nucleotide 833 at the mRNA, carrying a puromycine selectable marker, and pRS1099 for nucleotide 1099, with a hygromycine selectable marker. B, CD24 mRNA levels shown via semiquantitive RT-PCR with CD24-specific primers. The housekeeping gene GAPDH served as the control for equal loading. C, Western blot analysis for CD24 expression, loading 20 µg of total lysate from each clone onto an SDS-PAGE. Membranes were incubated with a 1:500 dilution of SWA11 antibodies. β-Actin expression was used to normalize for equal loading. Flow cytometry after incubation of cells with anti-CD24 ML5 antibodies and FL-conjugated antimouse secondary antibodies was also used to assess CD24 expression levels on the membrane; the graph represents mean quantifications of fluorescence from each sample over three repetitions. D, Western blot analysis for the indicated Colo357 clones of pRS833 and controls performed similarly (loading 50 µg protein).

 
Cell culture and formation of clones. The human colorectal (HT29) and pancreatic (Colo357) cancer cell lines were obtained from the American Type Culture Collection, cultured in DMEM (Sigma) containing 5% to 10% fetal bovine serum (FBS; Biological Industries), 1% penicillin, and 1% streptomycin (complete medium) at 37°C in an atmosphere of 95% oxygen and 5% CO2.

Transfections were done using Lipofectamine and plus reagents (Invitrogen, Life Technologies) according to the manufacturer's instructions. A total of 7 x 105 cells were seeded in six-well plates. The next day, 50% confluent dishes were transfected with 1 µg vectors. At first, cells were transfected with pRS833, pRSluc, and PrsGFP; resistant cells were selected in complete medium with 1 µg/mL puromycin (Sigma) for 3 wk. After selection, drug-resistant (puro+) clones were isolated from two different plates [designated 833(1)-833(6), GFP(1)-GFP(3), and Luc.(1)-Luc.(3)]. Clone 833(4), which showed the most significant decrease in CD24 expression, was expanded and transfected similarly with pRS1099 and pRSGFP that have hygromycin as the selectable marker; resistant cells were selected in complete medium with 1 µg/mL puromycin and 450 µg/mL hygromycin (Sigma) for 3 wk. After selection, drug-resistant (puro+/hygro+) clones were isolated [designated 833+1099(A-G), 833+GFP(1-3)]. For confirmation of the results in another cell line, Colo357 were transfected similarly with the vectors pRS833, pRSluc, and pRSGFP, and puro+ clones were selected and expanded. Four resistant clones were randomly chosen, designated 833-1, 833-4, 833-10, and 833-12.

Protein extraction and Western blotting. Protein extraction from exponentially growing cells and Western blot analysis for 20 µg protein of each samples were performed as described before (8). As primary antibodies, SWA11 (anti-CD24) and polyclonal anti-actin (I-19; Santa Cruz Biotechnology) were used. Antimouse and antigoat (The Jackson Laboratory) were used as secondary antibodies, respectively. Protein analysis after therapy under chloroquine induced inhibition of lysosome, cells (7 x 105 per well) were plated onto six-well plates and treated on the morrow. Chloroquine was added 2 h before treatment. SWA-11 was added to the medium at 1:40 dilution. Cells were rubbed off the plate at the end of the treatment.

Reverse transcriptase–PCR. Total RNA was prepared from the cell lines using Tri Reagent (Molecular Research Center). Reverse-transcriptase–PCR (RT-PCR) reaction was preformed with 50 ng on PTC-100 programmable thermal controller (MJ Research, Inc.). Primers were designed as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH; housekeeping gene), forward, 5'-GGAGATTGTTGCCATCAACG-3' and reverse 5'-TTGGTGGTG CAGGATGCATT-3'; CD24, forward 5'-GGCACTGCTCCTACCCACGCAG-3' and reverse, 5'-GCCACATTGGAATTCCAGACGCC-3'. The PCR products were separated in 2% agarose/GelStar gel and visualized under UV light.

Cell proliferation rate. Cells were plated at a density of 5 x 104 per well in 12-well plates using 1 mL of complete medium (DMEM/5% FBS) or medium deficient with bovine serum (0.5% FBS), as indicated. Starting on the morrow, two wells for each cell lines were counted every other day using a Coulter particle counter (Coulter Electronics Luton). Media were replenished twice weekly during 21 d.

Cell migration assay. A three-dimensional cell migration assay was performed using the Transwell System (Corning), which allows cells to migrate through an 8-µm pore–sized polycarbonate membrane. Cells were trypsinized, washed, and resuspended in DMEM containing 5% calf serum (6 x 105 cells/mL). The cell suspension (100 µL) was plated onto the Transwell insert (the upper chamber). The lower chamber was filled with 600 µL of the same medium. After incubation for 48 h at 37°C, the cells were fixed for 10 min in 4% paraformaldehyde (Sigma), perforated with 0.01% Triton (Sigma) for 5 min, and stained for 5 min with crystal violet (Sigma). The filters were then rinsed thoroughly in distilled water, and the nonmigrating cells were carefully removed from the upper surface of the Transwell with a wet cotton swab. The wells were counted, and the number of transmigrated cells was assessed by color quantification using the TINA 2.0 software.

Xenografts model in mice for measuring in vivo tumor development. Athymic nude mice were housed in sterile cages and were handled with aseptic precautions, supplemented with ad libitum nutrition. Exponentially growing cells were harvested with brief treatment of 0.25% Tryspin-EDTA solution and resuspended at a final concentration of 5 to 7.5 x 106 cells per 0.15 mL PBS per injection as indicated. The cells were injected s.c. into two sites on the back of the mice. They were weighed, and the tumor growth was measured twice a week; tumor volumes were calculated as 4{pi}ab2/3. At the end of the experiment, mice were sacrificed by cervical dislocation after anesthesia and examined for the presence of further metastases. Tumors were excised and weighed, and the volume was measured with calipers. Each assay was revised at least twice. For measuring tumorigenicity of the formed clones, four mice (eight tumors) served for each cell line. For testing the therapeutic potential of the anti-CD24 antibodies, in each experiment, 10 mice were injected with HT29 cells (20 tumors) and randomized for a control (saline) or AB group. Therapy was injected twice weekly, with a volume of 0.15 mL, which is estimated to be around 0.3 mg AB (representing ~15 mg/kg bodyweight), yet the exact amount was not purified and defined.

Growth inhibition induced by monoclonal antibodies to CD24. The killing effect of the anti-CD24 antibodies and the chemotherapies was assessed in vitro using a methylene blue assay, as described before (8). SWA11 served as the anti-CD24 antibody. Paclitaxel, doxorubicin, and 5-florouracil were obtained from Sigma; oxaliplatin and irrinotecan were obtained from Aventis Pharma.

RNA preparation and hybridization. RNA was extracted from cells as described before, and quality was assessed on Agilent 2100 Bioanalyzer device (Agilent Technologies) from the indicated cell lines and clones. If mAb treatment is indicated, cells were exposed for 1:40 SWA11 mAb for 72 h. RNA was labeled and hybridized as described before (8), only this time using the human (HG-U133A) Genechip (Affymetrix, Inc.).

Analysis of the Genechip data. The algorithm, implanted in Affymetrix Suite version 5.0 (MAS5), generates signal value (which designates a relative measure of the abundance of the transcript), a detection P value (which indicates the reliability of the transcript's detection call), and detection call (present, absent, or marginal). For interarray comparisons, the data from each array were scaled using MAS5. The bioinformatics analysis was carried out using GeneSpring version7 software (Silicon Genetics). Normalization procedure is as follows: values below 0.01 were set to 0.01. Each measurement was divided by the 50.0th percentile of all measurements in that sample (per chip normalization). Each gene was divided by the median of its measurements in all samples (per gene normalization). Genes were filtered out if appearing "present" in only none or one of the eight samples. Functional classification in gene ontology of list of genes that discriminates subclasses of samples was preformed using "David." This is an on-line database-hosting tool for annotation analysis, among them, the EASE software application. Full results were uploaded to the publicly available GEO database by National Center for Biotechnology Information.7


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD24 down-regulation in human CRC cell line. For this study, we used selected clones, derivatives of the HT29 human CRC cell lines that were stably transfected with pRS833, designated clone 833(4), and two derivatives of this clone, designated E and G, after stable transfection with pRS1099 (Fig. 1A). HT29 cells transfected with pRSGFP and/or pRSluc served as the vector controls for the study.

All three clones showed a decrease in the mRNA transcripts level compared with the parental and vector control cells (Fig. 1B). Similar results were obtained for CD24 protein level as analyzed through both Western blot for complete cell lysates and flow cytometry for membrane CD24 levels. Overall, a significant reduction in the protein level was seen in clone 833(4) (P < 0.001), with a further reduction observed in clones E and G (Fig. 1C).

Similarly, clones of pRS833 vector were formed and selected from Colo357 pancreatic cancer cells, along with its matching controls. Three resistant clones were randomly chosen; all the clones showed a decrease in the CD24 protein level (Fig. 1D). In these cells, the reduction in CD24 protein level was dramatic, so we concluded that the selected sequence worked very efficiently for the knockdown of CD24.

Growth properties of CD24 underexpressing clones. HT29 cells display an aggressive malignant phenotype. The HT29 CD24 underexpressing clones posses a milder phenotype. The cells display a reduced exponential growth and a lower saturation density depending on the level of CD24 expression (Fig. 2A ). Furthermore, as HT29 cells are capable of growing in starvation conditions, the growth inhibition and reduction in saturation densities in the CD24-siRNA clones were further enhanced under such conditions, achieved by maintaining the cells in medium that contains 0.5% FBS (Fig. 2A). Notably, the same results were shown by all the 833 clones of Colo357 that were randomly chosen for analyses (Fig. 2B). Fluorescence-activated cell sorting analysis of HT29 and its derivatives showed no significant changes in cell cycle distribution and the level of apoptosis (Fig. 2C).


Figure 2
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  		Figure 2. Underexpression of CD24 slows cell growth. A, HT29 cells, the siRNA control cells (GFP), and clones 833(4), E, and G were plated in triplicates in 12-well plates in complete medium or starvation (0.5% FBS). Number of cells was determined every 2 to 3 d using a Coulter counter. B, Colo357, its clones 833 (4, 10, and 12), and their controls were similarly examined. Each experiment was repeated at least twice. C, cell cycle variables for exponentially growing HT29 and its discussed clones following propidium oidide staining for DNA content.

 
Down-regulation of CD24 impairs cell motility. It was previously shown that transient knockdown of CD24 in cancer cells results in changes in the actin cytoskeleton (22). Whereas the stable down-regulation of CD24 cells did not result in any morphologic changes, a significant decrease in cell motility was shown using a transwell assay. Clones E and G were almost incapable of transpassing through the pores to the lower chamber compared with the GFP-targeted siRNA-expressing clone (Fig. 3A and B ).


Figure 3
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  		Figure 3. A, CD24 underexpression impairs cell motility. 6 x 104 cells per well of HT29 cells and their CD24 underexpressing derivatives were seeded onto the upper chamber of a transwell plate (0.8-µm pore size, Corning). At 48 h later, cells were fixated to the plate using PFA and stained in crystal violet. Picture was taken after wiping off the cells from the upper chamber using a cotton-stick. B, number of cells was determined using TINA2.0 software by reading the mean color intensity per well. The experiment was performed twice in duplicates; results are presented in percentages compared with the control clone for each experiment. C and D, CD24 down-regulation results in lowered tumorigenicity. HT29 (C) and Colo357 (D) cells and their CD24 underexpressing clones were injected s.c. into each of two flanks of athymic nude mice (5–7 x 106 cells per injection, five mice per group). Tumor volumes were measured twice weekly. SE bars are presented at each measurement.

 
Down-regulation of CD24 reduces cell tumorigenicity in vivo. Animals injected s.c. with parental or control nonspecific siRNA harboring HT29 or Colo357 cells were successful in forming detectable tumors within 7 to 10 days. In clones expressing low CD24 level, tumor incidence and rate of tumor formation were reduced (Fig. 3C and D). The experiment was performed twice for each cell line producing similar results. These results provide strong evidence that underexpression of CD24 in colorectal and pancreatic cancers markedly inhibits tumor growth in vivo.

CD24 down-regulation using mAb also inhibits cell growth. We have previously shown that the growth of HT29, Colo357, and Panc-1 cancer cells expressing CD24 was inhibited after exposure to three different anti-CD24 mAb in time-dependent and dose-dependent manners in vitro (8, 20). The growth of SW480 and MIA-PaCa cells, which do not express CD24, was not inhibited by the mAbs. The mAbs in use were SWA11, ML-5, and the commercially available SN3 antibody (Santa Cruz Biotechnology). We observed that the growth inhibition is likely mediated through degradation of the CD24 protein, as reduced protein levels are observed within a few hours after treatment of HT29 and Colo357 cells (Fig. 4A and B , respectively). No change in the transcription level of CD24 was seen under the same conditions (data is not shown).


Figure 4
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  		Figure 4. A and B, Western blot analysis to HT29 (A) and Colo357 (B) cells treated with SWA-11 anti-CD24 mAb diluted 1:40 in complete medium for the indicated durations. Lysates were collected at the indicated times of treatment, and actin served as the reference for equal loading. C, HT29 under lysosomal inhibition by 0.5 mmol/L chloroquine was compared with normal cells in their CD24 levels under treatment with mAb or to cells under proteosomal inhibition induced by 0.1 mmol/L MG132. Inhibitors were added 2 h before treatment as indicated. D, evaluating the combined effect of mAb with chemotherapy. HT29 cells were treated with SWA11 anti-CD24 antibodies for 72 h at the indicated concentrations, in combination with the various concentrations of five accepted chemotherapeutic agents: oxaloplatin, 5-fluorouracil, doxorubicin, irrinotecan, and palitaxol. Experiments were performed in duplicates or repeated twice. The Y axes stand for cell viability quantified via methylene blue assay.

 
Anti-CD24 mAbs mediate lysosomal degradation of the protein. Preexposure of cells to 0.5 mmol/L chloroquine prevented the decrease in CD24 protein level that accompanied the growth inhibition induced by the mAbs. It suggests that CD24 is degraded in the lysosome. No such effect was observed while exposing the cells to 0.1 mmol/L MG132, an inhibitor of the proteosome (Fig. 4C).

Anti-CD24 antibodies act additively with standard chemotherapies in inhibiting cancer cell growth. HT29 cells were treated with varying concentrations of anti-CD24 mAb (as indicated in Fig. 4D) and exposed for 48 hours in the presence or absence of varying doses of oxaloplatin, 5-florouracil, doxorubicin, irrinotecan, and paclitaxel. Biological therapy enhanced the killing effect of all five chemotherapeutic agents. The increased efficiency varied according to the therapeutic agent in use, but in some combinations, the addition of the mAbs, at already comparatively low dilutions (1:100 of the mAb distributed in the medium), allowed to decrease the dose of the chemotherapeutic agent in >50% (e.g., doxorubicin and irrinotecan).

Anti-CD24 mAb efficiently reduces tumor growth rate in vivo. In a human xenograft model formed by s.c. injection of HT29 cells to nude mice, as was previously described, tumor growth was markedly inhibited by two different mAbs, ML-5 and SWA-11, injected i.v. through the tail vein (Fig. 5 ). Each injection consisted of ~0.3 mg mAb, doses which have been established in preliminary trials to be nontoxic to mice (data is not shown).


Figure 5
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  		Figure 5. A and B, anti-CD24 mAb inhibit tumor growth and metastases in vivo. Tumors were formed in nude mice by injection of HT29 cells as described; half was treated by anti-CD24 monoclonal antibodies (~300 µg), i.v., twice weekly, and half was not treated. At 5 x 106 cells per injection, treatment started 3 d after the injection of the cells using the ML5 mAb (A); at 7.5 x 106 cells per injection, treatment started along with the injection of the cells using SWA11 (B). SE bars are presented at each measurement. C, gene expression array analysis for cancer cells after CD24 down-regulation using siRNA or mAb. Nonsupervised cluster analysis using nonparametric filtering of genes differentiated between CD24 expression and down-regulated cells. Results were generated using the GeneSpring software. D, a schematic summary of the overall probes that displayed differential expression after mAb, siRNA, or both.

 
Gene expression array suggests genes and pathways involved in the growth inhibition achieved by down-regulation of CD24. Gene expression array using a human genome (HG-U133A) Genechip was used to evaluate changes in expression profile mediated by CD24 down-regulation through RNA interference (RNAi) or mAb. RNA was produced from samples, as indicated in Materials and Methods. Nonsupervised hierarchical clustering, applied to the samples derived from HT29 cells with CD24 siRNA and the controls (HT29 and HT-GFP) using 12,729 genes that were filtered on a nonparametric basis, organized the samples according to their biological difference (Fig. 5C). The analysis found HT-833 and HT-G to be closely identical, supporting the approach that HT-833 could be treated as equal to E and G in subsequent analyses. Furthermore, greater similarity was found between HT-GFP and the CD24 siRNA harboring clones than between parental HT29 cells and the clones, pointing to some level of a nonspecific siRNA induced change that should be considered during analyses.

Samples HT29 and HT-GFP were compared with HT-E, HT-G, and HT-833 in their median signals (outliers were manually excluded). A differential expression of 3-fold or greater after CD24-targeted siRNA expression was observed in 954 down-regulated genes and 618 up-regulated genes. Similarly, samples HT29 and HT29-AB and samples Colo357 and Colo-AB were compared, and mean differences were considered. Thus, a differential expression of 3-fold or greater under treatment with mAb was observed in 290 down-regulated genes and 291 up-regulated genes. A detailed list of these four groups of genes has been deposited in the NIH GEO databank.

Seven genes related to the Ras pathay showed a ≥3-fold decreased expression after CD24 down-regulation, in addition to several members of the mitogen-activated protein kinase (MAPK) family downstream of Ras (data not shown). Biocarta algorithm for signaling pathway analysis revealed a high prevalence of genes that participate in the phospholipase C signaling pathway (P = 0.005), which includes also genes of the MAPK family, and a down-regulation of the oncogenic pathway of vascular endothelial growth factor, hypoxia, and angiogenesis (P = 0.021). KEGG algorithm for functional systems found a high prevalence of down-regulation of genes involved in metabolism of carbohydrates (mainly sucrose, P = 3e–5, and fructose, P = 0.024) and folate metabolism under biosynthesis of nucleic acids (3.3e–4).

In this study, we aimed to focus on the genes that were affected by both siRNA or mAb. The intersections of these lists include 84 genes that were down-regulated along with the CD24 (Table 1A and Fig. 5D) and 55 genes that were up-regulated (Table 1B and Fig. 5D).


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  		Table 1. Genes that were >3-fold differentially expressed both after siRNA and mAb induction

 
Pathway analysis using the EASE software for the genes that were down-regulated along with CD24 revealed a significantly high prevalence of genes that participate in cell cycle (9.86%, P = 2.04e–4), genes that encode nuclear proteins (28.17%, P = 3.23e–4), and genes that are involved in protein phosphorylation (26.76%, P = 1.20e–6; Table 1A). Among the mutually up-regulated genes, evident were mostly genes that are of a nonspecific response to xenobiotic stimuli and stress, and pathway analysis could not find a correlation to any signaling pathway.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies show that CD24 is important in growth and survival of cancer cells. Down-regulation of CD24 by two separate approaches, i.e., siRNA and specific antibodies, resulted in growth inhibition of the cells and a less severe malignant phenotype. The potential for targeting CD24 in cancer therapy seems promising, as CD24 is overexpressed in many human cancers whereas it is barely detectable in normal tissues (3, 8). Most remarkably, cells with stable down-regulation of CD24 through RNAi showed decreased tumorigenicity in vivo; the same effect was achieved by interference with CD24 membrane expression using anti-CD24 monoclonal antibodies.

An evolving body of literature has linked CD24 with rapid cancer cell growth. Published studies in this field seem to be divided. On the one hand, CD24 was suggested in some studies to contribute to malignant transformation as a peptidoglycan carrying the appropriate glycans (the sialylLex residue) which allows it to bind P-selectin (24). These studies showed that CD24 expression could potentiate homotypic B-cell aggregation and heterotypic adhesion to activated endothelium (14). Under physiologic conditions, CD24 overexpression enhanced cancer cells rolling on and invading through vessel walls by increasing their adherence to platelets and endothelial cells (15). Thus, within the tumor microenvironment, CD24 binding with P-selectin enhances tumor development, because P-selectin was found to be crucial in CRC carcinogenesis. In P-selectin–deficient mice versus normal mice, human CRC cells injected s.c. into mice proliferated more slowly and produced fewer lung metastases than through i.v. injection (25), suggesting that the mucin-dependent interaction with P-selectin is an important feature of CRC cells.

Other studies, which the current study strengthens, suggest that CD24 also plays a role in intracellular changes, initiated probably by evoking an intracellular signal transduction through as yet defined pathways but which might include the Ras, MAPK, or BCL2 pathways, as suggested by our microarray results. It should also be noted that CD24 expression was correlated with changes in cell growth in a monolayer culture with the absence of selectins and not only in clinical correlations.

Thus, recruitment of Src kinases to lipid rafts domains (26), activation of MAPKs and thus caspases (19), changes in the balance of the BCl-2 superfamily (27), or elevation of cytoplasmic calcium levels (10) was tied with the excitation of the pathway by specific antibodies in hematopoietic cells. CD24 ectopic expression in breast cancer cells resulted in increased proliferation rates and the activation of the {alpha}3β1 and {alpha}4β1 integrins, which induce binding to selectins, collagen, and laminin and thus cell migration (21). Similarly, transient underexpression of CD24 using addition of CD24 targeted siRNA molecules to the growth medium of several epithelial cancer cell lines (breast, urothelial, and prostate carcinomas and osteosarcoma) led to slower cell growth and decreased clonogenicity in soft agar, as well as observed changes in the actin cytoskeleton that resulted in impaired motility (22). The current study shows the long-term effect of the CD24 underexpression in human colorectal and pancreatic carcinomas, where CD24 is typically overexpressed (8, 20), and confirms that stably formed cell lines that do not express CD24 maintain a less malignant phenotype as their growth and tumorigenicity in vivo are milder; changes in actin structures that were shown transiently are probably the cause of the loss of migratory capacities of the manipulated cancer cells. Alternatively, CD24 expression might affect the function of integrins as a recent study has shown that β1-integrin become selectively activated and recruited into lipid rafts in CD24-expressing cells (28). The glycoproteins of tumor cells are often abnormal, both in structure and in quantity. In particular, the mucin-type O-glycans, in which CD24 is enriched with, have several cancer-associated structures (29).

Gene expression analysis was performed to assist in the identification of genes and pathways that might be involved in the growth inhibitory effect associated with CD24 down-regulation. The method has limitations, because it typically does not detect changes that occur at the protein level, such as changes in phosphorylation or protein degradation, yet observing changes in transcription patterns can hint at potential proteomic changes downstream of these changes in gene expression. This experiment is powered to generate hypotheses that can serve as a starting point for a further investigation of the molecular interactions of CD24.

A comparatively large amount of genes are listed as significantly changed after CD24 manipulation via either siRNA or mAb. Genes that were down-regulated along with the CD24 suppression included many proproliferative genes, suggesting most significantly that the formed clones harbor a lower activity of the Ras oncogenic pathway. It should be noted that a prior study (22) reported an increase in CD24 transcripts level after overexpression of Ral-B, a transcription factor under the Ras pathway. Thus, a cross-talk between these two molecules (CD24 and Ral-B) is hypothesized. Also, BCl2-related genes, Akt1, integrin-{alpha}6, topoisomerase I, and a coactivator of the enzyme (TOPBP1) are down-regulated in the discussed clones and might explain their weaker phenotype.

Treatment with mAb affected a much smaller number of genes. Among these genes of biological interest were those that also overlapped with genes that had an altered expression after siRNA expression. These genes were the primary focus of this study, as we hypothesized that both methodologies should affect the same molecular pathways. Of interest were genes of the BCl-2 pathway: BCl-2 associated transcription factor (up-regulated) and BCl-2 interacting killer (down-regulated, which is a novel, highly potent death inducing gene; refs. 30, 31). Also, down-regulated genes were retinoic acid receptor-responsive gene, p21 (Ras activator), CDC27 (mitochondrial tumor suppressor), interleukin-1–associated kinase, kinetochore-associated protein (crucial for telophase), and retinoblastoma binding protein-1 (a suppressor of Rb protein).

Previous studies have shown that anti-CD24 monoclonal antibodies do not only inhibit aggregation but also induce growth inhibition in Burkitt's lymphoma cells (that express CD24) via the GEM-dependent mechanism, accelerated by cross-link with the B-cell receptor (9). This study shows that in CRC, anti-CD24 antibodies alone can reduce tumor burden in vivo. The results also indicate that the efficacy of the treatment is likely related to reducing the overabundance of membrane CD24, which would be consistent, as well, with the long-term effects seen with siRNA. We have previously shown the specificity of the antibodies to CD24-expressing cells (8, 20), and in this study, we thus show both its safety and efficacy in preliminary in vivo trials, which should undoubtedly be expanded to larger scaled trials, at the end of which, the protocol of the exact amount of purified antibody, together with a low dose of a specific chemotherapeutic agent, will be constituted.


   Acknowledgments
 
Grant support: Israel Science Foundation grant 1156/05. This study was supported by a nonrestrictive grant from Pfizer, Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was part of the requirements of Eyal Sagiv for his Ph.D. degree at the Sackler School of Medicine at Tel Aviv University.


   Footnotes
 
7 Results of gene expression analysis are available on: http://www.ncbi.nlm.nih.gov/geo/. Back

Received 12/ 1/07. Revised 1/21/08. Accepted 1/26/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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