Ablation of Siglec-E augments brain inflammation and ischemic injury.

Sialic acid immunoglobulin-like lectin E (Siglec-E) is a subtype of pattern recognition receptors found on the surface of myeloid cells and functions as a key immunosuppressive checkpoint molecule. The engagement between Siglec-E and the ligand α2,8-linked disialyl glycans activates the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular domain, mitigating the potential risk of autoimmunity amid innate immune attacks on parasites, bacteria, and carcinoma. Recent studies suggest that Siglec-E is also expressed in the CNS, particularly microglia, the brain-resident immune cells. However, the functions of Siglec-E in brain inflammation and injuries under many neurological conditions largely remain elusive. In this study, we first revealed an anti-inflammatory role for Siglec-E in lipopolysaccharide (LPS)-triggered microglial activation. We then found that Siglec-E was induced within the brain by systemic treatment with LPS in mice in a dose-dependent manner, while its ablation exacerbated hippocampal reactive microgliosis in LPS-treated animals. The genetic deficiency of Siglec-E also aggravated oxygen-glucose deprivation (OGD)-induced neuronal death in mouse primary cortical cultures containing both neurons and glial cells. Moreover, Siglec-E expression in ipsilateral brain tissues was substantially induced following middle cerebral artery occlusion (MCAO). Lastly, the neurological deficits and brain infarcts were augmented in Siglec-E knockout mice after moderate MCAO when compared to wild-type animals. Collectively, our findings suggest that the endogenous inducible Siglec-E plays crucial anti-inflammatory and neuroprotective roles following ischemic stroke, and thus might underlie an intrinsic mechanism of resolution of inflammation and self-repair in the brain.

The role of the Siglec-G ITIM domain during bacterial infection.

Siglecs, membrane-bound lectins of the sialic acid-binding immunoglobulin superfamily, inhibit immune responses by recruiting tyrosine phosphatases (e.g., SHP-1 and SHP-2) through their cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) domain. The role of Siglecs in infection has been extensively studied, but downstream signaling through the ITIM domain remains unclear. Here, we used a GST pull-down assay to identify additional proteins associated with the ITIM domain during bacterial infection. Gdi2 bound to ITIM under normal homeostasis, but Rab1a was recruited to ITIM during bacterial infection. Western blot analysis confirmed the presence of SHP-1 and SHP-2 in eluted ITIM-associated proteins under normal homeostasis. We confirmed the association of ITIM with Gdi2 or Rab1a by transfection of corresponding expression vectors in 293T cells followed by immunoprecipitation-western blot assay. Thus, ITIM's role in the inhibition of the immune response during bacterial infection may be regulated by interaction with Gdi2 and Rab1a in addition to SHP-1 and SHP-2.

A CD24-p53 axis contributes to African American prostate cancer disparities.

BACKGROUND: Using a functional analysis of prostate cancer cells, we found a CD24-dependent inactivation of mutant p53, but the clinical significance of this observation remained uncertain. Here, we validated these results with samples of human prostate cancer and explored the role of a CD24-p53 axis in racial disparities of prostate cancer. METHODS: Samples of formalin-fixed, paraffin-embedded prostate cancer from 141 European Americans (EAs) and 147 African Americans (AAs) in two independent sample cohorts were assessed for protein expression of CD24, mutant p53, mouse double minute 2 human homolog (MDM2), and cyclin dependent kinase inhibitor 2A (ARF) using immunohistochemical analyses. All samples were analyzed for TP53R175H and TP53R273H . RESULTS: CD24, mutant p53, MDM2, and ARF proteins were expressed in 55%, 24%, 39%, and 68% of prostate cancer samples, respectively. CD24 and mutant p53 were present more frequently in late-stage and metastatic prostate cancer. The presence of CD24 was associated with a greater than fourfold risk of metastasis, which included lymph node and distant metastases. H score analysis showed positive correlations of CD24 expression with mutant p53 (r = .308, P < .001) and MDM2 (r = .227, P = .004). There was a negative correlation for CD24 with ARF (r = -.280, P < .001). A racial disparity was evident for CD24 (AAs/EAs: 64% vs 47%; P = .004) but not for mutant p53 (AA/EA: 28% vs 21%; P = .152). In 32 CD24+ /mutant p53+ cases, a TP53R273H mutation was found in five cases, but no TP53R175H mutation was found. CONCLUSION: The CD24-p53 axis may contribute to aggressive and metastatic prostate cancers, especially those of AAs. This observation enhances understanding of the pathogenesis of prostate cancer and its associated racial disparities.

Siglec-E Negatively Regulates the Activation of TLR4 by Controlling Its Endocytosis.

TLR4 signaling is critical for providing effective immune protection, but it must be tightly controlled to avoid inflammation-induced pathology. Previously, we reported extensive and direct interactions between TLR and Siglec families of pattern recognition receptors. In this study, we examined the biological significance of this interaction during infection. We show that Siglec-E is required for Escherichia coli-induced endocytosis of TLR4. Siglec-E-deficient dendritic cells infected with E. coli fail to internalize TLR4. This leads to sustained TLR4 on the cell surface and activation of NF-κB and MAPK p38, resulting in high levels of TNF-α and IL-6 compared with wild-type dendritic cells. In contrast to the signaling events occurring at the plasma membrane, as a result of the inability to internalize TLR4, Siglec-E-deficient dendritic cells were also defective for TRIF-mediated IFN-ß production in response to E. coli infection. Furthermore, we found that accumulation of ubiquitinated TLR4 and binding of E3 ubiquitin ligase Triad3A to TLR4 was increased significantly in bone marrow-derived dendritic cells from wild-type mice, but not from Siglec-E-deficient mice, after E. coli infection. This represents a newly discovered mechanism that regulates the signaling of TLR4 during E. coli infection.

Induction of Siglec-1 by Endotoxin Tolerance Suppresses the Innate Immune Response by Promoting TGF-ß1 Production.

Sepsis is one of the leading causes of death worldwide. Although the prevailing theory for the sepsis syndrome is a condition of uncontrolled inflammation in response to infection, sepsis is increasingly being recognized as an immunosuppressive state known as endotoxin tolerance. We found sialylation of cell surface was significantly increased on LPS-induced tolerant cells; knockdown of Neu1 in macrophage cell line RAW 264.7 cells resulted in enhanced LPS-induced tolerance, whereas overexpression of Neu1 or treatment with sialidase abrogated LPS-induced tolerance, as defined by measuring TNF-α levels in the culture supernatants. We also found that the expression of Siglec-1 (a member of sialic acid-binding Ig (I)-like lectin family members, the predominant sialic acid-binding proteins on cell surface) was specifically up-regulated in endotoxin tolerant cells and the induction of Siglec-1 suppresses the innate immune response by promoting TGF-ß1 production. The enhanced TGF-ß1 production by Siglec-1 was significantly attenuated by spleen tyrosine kinase (Syk) inhibitor. Knockdown of siglec-1 in RAW 264.7 cells resulted in inhibiting the production of TGF-ß1 by ubiquitin-dependent degradation of Syk. Mechanistically, Siglec-1 associates with adaptor protein DNAX-activation protein of 12 kDa (DAP12) and transduces a signal to Syk to control the production of TGF-ß1 in endotoxin tolerance. Thus, Siglec-1 plays an important role in the development of endotoxin tolerance and targeted manipulation of this process could lead to a new therapeutic opportunity for patients with sepsis.

Siglec-G/10 in self-nonself discrimination of innate and adaptive immunity.

Siglec-G/10 is broadly expressed on B cells, dendritic cells and macrophage subsets. It binds strongly to CD24, a small glycosyl-phosphatidylinositol-anchored sialoprotein, in a sialylation-dependent manner. Targeted mutation of Siglecg dramatically elevates the level of natural IgM antibodies and its producer, B1 B cells. Incorporation of Siglec-G ligands to both T-dependent and T-independent immunogens reduces antibody production and induces B-cell tolerance to subsequent antigen challenges. By interacting with CD24, Siglec-G suppresses inflammatory responses to danger (damage)-associated molecular patterns, such as heat-shock proteins and high mobility group protein 1, but not to Toll-like receptor ligands. By a CD24-independent mechanism, Siglec-G has been shown to associate with Cbl to cause degradation of retinoic acid-inducible gene 1 and reduce production of type I interferon in response to RNA virus infection. The negative regulation by Siglec-G/10 may provide a mechanism for the host to discriminate between infectious nonself and noninfectious self, as envisioned by the late Dr. Charles A. Janeway.

Induction of 6-sulfated glycans with cell adhesion activity via T-bet and GATA-3 in human helper T cells.

BACKGROUND: Cell surface 6-sulfated glycans play important roles in various immunological events through cell-to-cell interactions. The 6-sulfation process is mediated by 6-sulfotransferase family isoenzymes. We previously demonstrated that GlcNAc6ST-1, one of the isoenzyme genes, is induced by GATA-3 and NF-κB in human helper T (Th) cells. However, transcriptional regulation of HEC-GlcNAc6ST, another isoenzyme important in Th cells, remains unclear. METHODS: 5'-RACE analysis, chromatin immunoprecipitation, and reporter assays were performed to reveal transcriptional regulation of HEC-GlcNAc6ST. RNA-knockdown and forced expression experiments were performed to demonstrate the contribution of HEC-GlcNAc6ST to the 6-sulfated glycan expression. RESULTS: We identified potential binding sites of Sp1, T-bet, and GATA-3 in the HEC-GlcNAc6ST promoter. Reporter assays indicated that transfection of Sp1 enhanced the activity, whereas mithramycin A, an Sp1-specific inhibitor, repressed it. Transfection of T-bet increased the activity, which was inhibited by introducing a mutation into the potential T-bet binding site. GATA-3 alone could not elevate the activity, although co-transfection of protein kinase A, which is known to enhance IL-5 transcription in Th2 cells through phosphorylation of GATA-3, caused elevation. RNA-knockdown and forced expression of HEC-GlcNAc6ST in Jurkat cells down- and up-regulated α2,6-sialylated 6-sulfo N-acetyllactosamine, a preferential ligand for B-cell-specific CD22 antigen, respectively. From these results, we concluded that T-bet and GATA-3 as well as Sp1 control the expression of glycan with cell-adhesion activity by regulating HEC-GlcNAc6ST transcription in Th cells. GENERAL SIGNIFICANCE: These results may provide a clue to biological regulation of Th-cell interaction with selectins and other carbohydrate-recognition molecules by T-bet and GATA-3.

Targeting intracellular Neu1 for coronavirus infection treatment.

There are currently no effective therapies for COVID-19 or antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and vaccines appear less effective against new SARS-CoV-2 variants; thus, there is an urgent need to understand better the virulence mechanisms of SARS-CoV-2 and the host response to develop therapeutic agents. Herein, we show that host Neu1 regulates coronavirus replication by controlling sialylation on coronavirus nucleocapsid protein. Coronavirus nucleocapsid proteins in COVID-19 patients and in coronavirus HCoV-OC43-infected cells were heavily sialylated; this sialylation controlled the RNA-binding activity and replication of coronavirus. Neu1 overexpression increased HCoV-OC43 replication, whereas Neu1 knockdown reduced HCoV-OC43 replication. Moreover, a newly developed Neu1 inhibitor, Neu5Ac2en-OAcOMe, selectively targeted intracellular sialidase, which dramatically reduced HCoV-OC43 and SARS-CoV-2 replication in vitro and rescued mice from HCoV-OC43 infection-induced death. Our findings suggest Neu1 inhibitors could be used to limit SARS-CoV-2 replication in patients with COVID-19, making Neu1 a potential therapeutic target for COVID-19 and future coronavirus pandemics.

CD24-Siglec G/10 discriminates danger- from pathogen-associated molecular patterns.

It is now well accepted that the innate immune system recognizes both damage (or danger)- and pathogen-associated molecular patterns (DAMP and PAMP, respectively) through pattern recognition receptors, such as Toll-like receptors (TLR) and/or Nod-like receptors (NLR). Less clear are whether and how the response to PAMP and DAMP are regulated differentially. The answers may reveal whether the primary goal of the immune system is to defend against infections or to alert the host of tissue injuries. We demonstrated recently that the host response to DAMP is controlled by a DAMP-CD24-Siglec axis. Here we propose a key role for the CD24-Siglec pathway in discriminating between DAMPs and PAMPs.

Targeted disruption of Gdi2 causes early embryonic lethality.

INTRODUCTION: GDI2 regulates the GDP/GTP exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP, dysregulation of GDI2 has been reported in many different cancers. Recently, we found that GDI2 bound to the ITIM domain of Siglec-G under normal homeostasis, whereas Rab1a was recruited to the ITIM domain during bacterial infection. Therefore, GDI2 and Rab1a may regulate the immune response through interaction with the ITIM domain during bacterial infection. However, the regulation of the inflammatory response by GDI2 in vivo and its regulatory mechanism remain unknown. METHODS: We generated a Gdi2 null mutant mouse with a trapped Gdi2 gene and examined the expression by X-gal and immunohistochemistry staining. TUNEL staining was used to determine the apoptosis cells. RESULTS: Here we show that Gdi2 is essential for embryonic development. One functional Gdi2 allele is sufficient for murine embryo development, but complete loss of Gdi2 leads to embryonic lethality. Developmental retardation of Gdi2-/- mice is apparent at E10.5 to E14.5, with no viable Gdi2-/- embryos detected after E14.5. Histological analysis revealed extensive cell death and cell loss in Gdi2-/- embryos. Apoptosis was confirmed by staining with cleaved caspase-3, suggesting that Gdi2 maintain homeostasis by regulating the apoptosis of the cells. There was no significant difference in cytokine production and survival between wild-type and Gdi2+/- mice after LPS challenge. DISCUSSION: These findings suggest that one Gdi2 allele is sufficient to maintain function. However, the detailed molecular mechanism underlying Gdi2 in regulating the embryonic development needs further identification.

Targeted disruption of Rab1a causes early embryonic lethality.

Guanosine nucleotide diphosphate (GDP) dissociation inhibitor 2 (GDI2) regulates the GDP/guanosine triphosphate (GTP) exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP. The present study aimed to determine the function of Rab1a in vivo, and thus generated mice with a trapped Rab1a gene. It was demonstrated that Rab1a is essential for embryonic development. It was also found that one functional Rab1a allele was sufficient for development in a heterozygous murine embryo, whereas a double mutant led to embryonic lethality. The dissection of uteri on embryonic day (E)10.5­14.5 yielded no homozygous embryos, indicating that homozygotes die between E10.5 to E11.5. The gene trap construct contains a ß­galactosidase/neomycin reporter gene, allowing for heterozygotes to be stained for ß­galactosidase to determine the tissue­specific expression of Rab1a. Rab1a was found to be highly expressed in the small intestine of both adult mice and embryos, although its expression levels were low in the brains of embryos. Moreover, there was no significant change in cytokine production and survival in wild­type and heterozygous Rab1a+/­ mice following a challenge with lipopolysaccharide. On the whole, the present study demonstrates that the disruption of the Rab1a gene causes embryonic lethality and homozygotes die between E10.5 and E11.5, suggesting that Rab1a is essential for the early development of mouse embryos.

CD24-Siglec axis is an innate immune checkpoint against metaflammation and metabolic disorder.

The molecular interactions that regulate chronic inflammation underlying metabolic disease remain largely unknown. Since the CD24-Siglec interaction regulates inflammatory response to danger-associated molecular patterns (DAMPs), we have generated multiple mouse strains with single or combined mutations of Cd24 or Siglec genes to explore the role of the CD24-Siglec interaction in metaflammation and metabolic disorder. Here, we report that the CD24-Siglec-E axis, but not other Siglecs, is a key suppressor of obesity-related metabolic dysfunction. Inactivation of the CD24-Siglec-E pathway exacerbates, while CD24Fc treatment alleviates, diet-induced metabolic disorders, including obesity, dyslipidemia, insulin resistance, and nonalcoholic steatohepatitis (NASH). Mechanistically, sialylation-dependent recognition of CD24 by Siglec-E induces SHP-1 recruitment and represses metaflammation to protect against metabolic syndrome. A first-in-human study of CD24Fc (NCT02650895) supports the significance of this pathway in human lipid metabolism and inflammation. These findings identify the CD24-Siglec-E axis as an innate immune checkpoint against metaflammation and metabolic disorder and suggest a promising therapeutic target for metabolic disease.

Reprogramming of pancreatic adenocarcinoma immunosurveillance by a microbial probiotic siderophore.

There is increasing evidence suggesting the role of microbiome alterations in relation to pancreatic adenocarcinoma and tumor immune functionality. However, molecular mechanisms of the interplay between microbiome signatures and/or their metabolites in pancreatic tumor immunosurveillance are not well understood. We have identified that a probiotic strain (Lactobacillus casei) derived siderophore (ferrichrome) efficiently reprograms tumor-associated macrophages (TAMs) and increases CD8 + T cell infiltration into tumors that paralleled a marked reduction in tumor burden in a syngeneic mouse model of pancreatic cancer. Interestingly, this altered immune response improved anti-PD-L1 therapy that suggests promise of a novel combination (ferrichrome and immune checkpoint inhibitors) therapy for pancreatic cancer treatment. Mechanistically, ferrichrome induced TAMs polarization via activation of the TLR4 pathway that represses the expression of iron export protein ferroportin (FPN1) in macrophages. This study describes a novel probiotic based molecular mechanism that can effectively induce anti-tumor immunosurveillance and improve immune checkpoint inhibitors therapy response in pancreatic cancer.

CD24 polymorphisms affect risk and progression of chronic hepatitis B virus infection.

UNLABELLED: T-cell immunity to hepatitis B virus (HBV) is involved in both viral clearance and the pathogenesis of cirrhosis and hepatocellular carcinoma following chronic HBV infection. It is therefore of great interest to analyze whether genetic polymorphism of genes involved in the immune response may determine the outcomes of chronic HBV infection. Here we report that CD24 polymorphisms affect the risk and progression of chronic HBV infection. Thus the CD24 P170(T) allele, which is expressed at a higher level, is associated with an increased risk of chronic HBV infection. Among the chronic HBV patients this allele shows recessive association with more rapid progression to liver cirrhosis and hepatocellular carcinoma in comparison to the P170(C) allele. In contrast, a dinucleotide deletion at position 1527-1528 (P1527(del)), which reduces CD24 expression, is associated with a significantly reduced risk of chronic HBV infection. To confirm the role for CD24 in liver carcinogenesis, we compared the size of liver tumor developed in CD24(-/-) and CD24(+/-) HBV transgenic mice. Our data demonstrate that targeted mutation of CD24 drastically reduced the sizes of spontaneous liver cancer in the HBV transgenic mice. CONCLUSION: These data demonstrate that genetic variation of CD24 may be an important determinant for the outcome of chronic HBV infection.

Selective Response to Bacterial Infection by Regulating Siglec-E Expression.

Interactions between microbes and hosts can be a benign, deleterious, or even fatal, resulting in death of the host, the microbe, or both. Sialic acid-binding immunoglobulin-like lectins (Siglecs) suppress infection responses to sialylated pathogens. However, most pathogens are nonsialylated. Here we determined Siglecs respond to nonsialylated Gram-negative bacteria (Escherichia coli 25922 and DH5α) and Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes). We found that Siglece-/- mice had higher mortality than wild-type mice following Gram-negative but not Gram-positive bacterial infection. Better survival in wild-type mice depended on more efficient clearance of Gram-negative than Gram-positive bacteria. Gram-negative bacteria upregulated Siglec-E, thus increasing reactive oxygen species (ROS); Tyr432 in the ITIM domain of Siglec-E was required to increase ROS. Moreover, Gram-negative bacteria upregulated Siglec-E via TLR4/MyD88/JNK/NF-κB/AP-1, whereas Gram-positive bacteria downregulated Siglec-E via TLR2/RANKL/TRAF6/Syk. Thus, our study describes a fundamentally new role for Siglec-E during infection.

The E3 ligase TRIM56 is a host restriction factor of Zika virus and depends on its RNA-binding activity but not miRNA regulation, for antiviral function.

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.

Abnormalities caused by carbohydrate alterations in Ibeta6-N-acetylglucosaminyltransferase-deficient mice.

Ibeta6-N-acetylglucosaminyltransferase (IGnT) catalyzes the branching of poly-N-acetyllactosamine carbohydrate chains. In both humans and mice, three spliced forms of IGnT have been identified, and a common exon is present in all of them. We generated mice deficient in the common exon to understand the physiological function of poly-N-acetyllactosamine branching. IGnT activity was abolished in the stomach, kidney, bone marrow, and cerebellum of the deficient mice, while a low level of the activity persisted in the small intestine. Immunohistochemical analysis confirmed the loss of I antigen from the lung, stomach, and kidney. The deficient mice had reduced spontaneous locomotive activity. The number of peripheral blood lymphocytes was also reduced and renal function decreased in the deficient mice. Furthermore, in aged mice, vacuolization occurred in the kidney, and epidermoid cysts were frequently formed. However, cataracts did not develop earlier in the deficient mice. Decreased levels of lysosomal proteins, LAMP-2 and synaptotagmin VII, were found in the kidney of the deficient mice and correlated with renal abnormalities.

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells.

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Intracellular CD24 disrupts the ARF-NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation.

CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.