NATIONAL CHIAYI UNIVERSITY

Department of Microbiology, Immunology & Biopharmaceuticals

CD24 expression is important in male urothelial tumorigenesis and metastasis in mice and is androgen

regulated

Proc Natl Acad Sci USA. 2012 Dec 18; 109 (51) : E3588-96

MULTIDISCIPLINARY SCIENCES : 4/56Impact factor: 9.737

Advisor: Yi-Wen LiuProfessor

Speaker:Mezbahul HaqueMaster’s Student

Date: 10.01.14

INTRODUCTION

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The majority of bladder cancers are transitional cell carcinomas - this is by far the most common type of bladder cancer. About 95% of bladder cancers are this type.

Low-grade papillary tumours rarely become muscle-invasive and they frequently harbour gene mutations that constitutively activate the receptor tyrosine kinase–Ras pathway.

By contrast, most high-grade invasive tumours progress to life-threatening metastases and have defects in the p53 and FGFR pathways.

UROTHELIAL

TUMORIGENESIS

Nat Rev Cancer. 2005 Sep;5(9):713-25.

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UROTHELIAL

TUMORIGENESIS

Most urothelial carcinomas (UCs) of the bladder (75%) are present as superficial tumors at the time of diagnosis.

Tumor staging and grading are considered the best prognostic markers, but the development of molecular markers may provide useful information for more effective diagnoses and treatments of bladder cancer.

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CD24 is a cell-surface glycosyl phosphatidylinositol membrane anchor protein on the surfaces of most human B cells. It can inhibit B cell differentiation into antibody-forming cells.

Because CD24 as a ligand to P-selectin that is important for metastatic tumor progression and lung colonization.

Using a doxycycline-inducible system for the expression of CD24 in breast cancer cells, CD24 has been proved to play a critical role in proliferation, invasion, and motility in cancer cells.

CD24

Cancer Res (2005) 65:: 10783–10793

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J Immunol ( 1991) 147:1412–1416

Cancer Res (2000) 60: 6714–6722

CD24 is known to be overexpressed in various human malignancies, both solid and hematological and is usually tied with a more aggressive course of the disease.

Several studies have reported that the protein is broadly over expressed in numerous types of cancer cell from the lung, breast, prostate, liver, kidney, pancreas and ovary, as well as in lymphomas.

Previous study also revealed that the frequency of CD24 over-expression was significantly increased in invasive carcinomas compared with noninvasive carcinomas.

CD24

J. Biol. Chem. 2011, 286:40548-40555.

6Arch Pathol Lab Med2007, Vol 131, Feb

Butyl-(4-hydroxybutyl) nitrosamine (BBN) has been shown to be a highly potent and specific bladder carcinogen.

Bladder carcinomas were found in all mice, other toxic effects were absent.

Males developed bladder tumors significantly earlier than did females.

Early changes in the bladder epithelium were also delayed in the female.

BBN and bladder cancer

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Eur J Cancer (1972)8:587–594.

A potential mediator of sex-specific differences is the androgen receptor (AR).

The AR, a member of the nuclear receptor superfamily, is a ligand-dependent transcriptional factor that mediates the biologic effects of androgens.

Expression of the AR has been detected in normal bladder epithelium and in bladder carcinomas from both male and female patients.

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Androgen and bladder cancer

J Natl Cancer Inst (2007)99:558–568.

OBJECTIVES

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The authors wanted to evaluate the contribution of CD24 in bladder cancer incidence and progression within a defined genetic model by exposing Cd24a-deficient mice to a carcinogen.

And authors also want to find out the answer of a question, is there any relation between CD24 and androgen regulation to induce bladder tumorigenesis and metastasis in male?

Experimental Animals and Cell Lines: C57BL/6 mice deficient for Cd24a and wild type C57BL/6

mice used as controls. Cell lines: UM-UC-3, TCCSUP and HTB9 human urothelial

carcinoma cell lines.

METHODS: RT-PCR CD24 Promoter Reporter Assays. Computational Binding-Site Predictions. Chromatin Immunoprecipitation (ChIP). Histological Analysis of Murine and Human Tissues.

MATERIALS & METHODS

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RESULTS

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FLOW CHART

Experiments in CD24-deficient mice model

Investigation in Androgen effect of CD24 Expression

Human Bladder Cancer Cell Study

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Bladder Tumor induced by OH-BBN

OH-BBN specifically induces bladder tumors.

mCT scans.Necropsy images.Histological preparations

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Ratio of the incidence of malignancy. No differences in histology between grade-matched WT and Cd24a deficient mice.

Bladder Tumor Incidence ratio between WT and Cd24a-Deficient Mice

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Cancerous urothelium exposed to OH-BBN had significantly more Cd24a than normal urothelium, regardless of the sex of the mice.

Cd24a mRNA Expression Are Increased in OH-BBN–Induced Bladder Cancer

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After exposure to OH-BBN, bladders from WT mice with cancer were found to express higher levels of Cd24a protein than dysplastic counterparts.

Cd24a Protein Expression Are Increased in OH-BBN–Induced Bladder Cancer

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Loss of CD24 is protective against OH-BBN–induced bladder cancer in male mice but not in female mice.

Cd24a Deficiency in Male Mice Delays Bladder Malignancy

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Schematic of the experimental design for assessing metastatic incidence.

Harboring Primary Tumors to metastasis

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Lung metastatic tissues showed similar histological architecture.

Comparisons of WT and Cd24a-deficient females showed no statistically significant difference in metastasis.

Cd24a Deficiency Reduces Metastasis in Male Mice

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SUMMARY

Loss of Cd24a is associated with reduced tumor incidence

OH-BBN–induced bladder tumors have high levels of Cd24a mRNA and protein expression than displasia.

Loss of Cd24a appears to protect bladder urothelium from OH-BBN–induced tumorigenesis

OH-BBN– induced bladder tumor rates are higher in male WT mice than in female WT mice.

Loss of Cd24a decreases metastasis in male mice.

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FLOW CHART

Mice model CD24 Deficiency

Effect of Androgen on CD24 Expression

Human Bladder Cancer Cell Study

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Disease-free survival data for male

Tumor CD24 Expression is correlated to disease-free surviving in male Patients of bladder cancer.

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Disease-free survival data for female

Treatment with R1881 increases growth compared with DMSO.

Androgen-Induced Growth Is Dependent on CD24 Expression in UM-UC3 cells in vitro and in vivo

Tumors isolated from normal male mice expressed higher levels of CD24 than tumors resected from castrated male mice.

R1881 is a synthetic androgen

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Lysates were collected and analyzed for CD24 and AR protein expression. Casodex: Casodex is an anti-androgen agent.

CD24 Protein Expression Are Androgen Dependent in Bladder Cancer Cells.

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Treatment with R1881 increases CD24 expression, an effect that is abrogated when cells are transfected first with AR siRNA.

CD24 mRNA Expression of UM-UC 3 and TCCSUP cells

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SUMMARY

CD24 is a negative and prognostic factor to the disease-free surviving in male patients.

Tumor growth promoted by androgen signaling is dependent on CD24 in vitro and in vivo.

CD24 mRNA and protein expression is regulated by androgen.

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FLOW CHART

Mice model CD24 Deficiency

Effect of Androgen on CD24 Expression

Analysis of CD24 gene promoter affected by androgen

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R1881 treatment induced a 2.8-fold increase in the activity of a 1,896-bp CD24 promoter

This mutation blocks R1881-induced activity of the CD24 promoter.

Synthetic Androgen treatment enhances the specific CD24 promoter

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Genomatix MatInspector software

The 1,896-bp reporter no longer responds to R1881 when cells are treated with AR siRNA.

However, AR was not associated with a region of the CD24 promoter that did not contain a computationally predicted ARE.

Androgen activates CD24 gene expression through its specific ARE binding site in the promoter

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SUMMARY

AR Activation Stimulates CD24 Promoter Activity in Human Bladder Cancer Cells.

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Accordingly, the results from this study suggest that CD24 plays a significant role in bladder tumorigenesis and metastasis, especially in males.

These data also implicate CD24 as an important androgen-regulated effector in male human bladder cancer and thus provide a rationale for novel therapeutic approaches targeting the androgen receptor (AR) in male bladder cancer.

Conclusion

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Thank you

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Androgen-Induced Growth Is Dependent on CD24 Expression in UM-UC3 cells in vitro and in vivo

(C) Normal and castratedmale nude mice (n = 10) were injected s.c.with 5 × 105UM-UC-3 cells that had been vector transfected or CD24 transfected as described previously (5). Graph represents tumor size over time. Data show that castrationcan reduce growth of UM-UC-3 tumors and that stable exogenous expression of CD24 inUM-UC-3 cells can rescue this growth reduction. Error bars indicate SEM.

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CARCINOGENESIS TREATMENT PROTOCOL

Male and female 7-wk-old Cd24a-deficient mice and WT C57BL/6 control mice were supplied ad libitum with tap water containing 0.1% OH-BBN; bottles were refreshed twice a week.

Mice were inspected weekly and observed for signs of distress associated with bladder lesions, including hematuria and firm bladders. Cohorts of treated Cd24a-deficient and WT mice were killed at 16-, 24-, and 28-wk time points.

We harvested bladder, periaortic lymph nodes, liver, lung, and kidney tissues. A portion of each tissue was preserved in phosphate-buffered 10% formalin and paraffin embedding for eventual sectioning and staining with H&E. The remaining tissues were snap frozen in liquid nitrogen and stored at −80 °C for RNA and protein analyses.

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CD24 PROMOTER, REPORTER ASSAYS, AND COMPUTATIONAL BINDING-SITE

PREDICTIONS

In this study we used the promoter from UM-UC-3 cells called “CD24-1896,” Use of Genomatix software on CD24-1896 suggested that an ARE is located at −1501 to −1475. Mutation of this site was executed according to instructions from the site-directed mutagenesis protocol.

DNA transfections ofUM-UC-3 cells were carried out using Lipofectamine (Invitrogen) and 1 μg of plasmid. For specific experiments, siRNA (20pmol) against luciferase was cotransfected withDNA. After 24 h cell medium was replaced with medium containing charcoal-stripped serum.

Cells then were treated with vehicle (ethanol). After 24 h cells were lysed and assayed for luciferase activity according to the manufacturer’s instructions (Promega). Luciferase values were normalized to cell number generated from Cyquant analysis (Life Technologies).

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CHIP: UM-UC-3 and TCCSUP cells were grown to 70% confluence in two 15-

cm dishes per treatment. Medium was removed, & fresh medium containing charcoal-stripped serum and EtOH was added for 2 h.

Then , according to instructions in the Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling), paraformaldehyde was added directly to cell medium (1% final concentration) and quenched with 1 M glycine at room temperature, and cells (4 × 107) were collected.

Overnight ChIP was set up using 10 μg of chromatin and antibody . After purification of the immunoprecipitated DNA, three-step qRT-PCR was used to quantify the DNA levels.

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FUNCTION OF CD24 IN NORMAL CELL

+ CD24 is a glycoprotein expressed at the surface of most B lymphocytes and differentiating neuroblasts.

+ This gene encodes a sialoglycoprotein that is expressed on mature granulocytes and in many B cells.

+ The encoded protein is anchored via a glycosyl phosphatidylinositol (GPI) link to the cell surface.

+ An alignment of this gene's sequence finds genomic locations with similarity on chromosomes 3p26, 15q21, 15q22, 20q11.2 and Yq11.1.

+ Whether transcription, and corresponding translation, occurs at each of these other genomic locations needs to be experimentally determined (source: NCBI).

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BBN AS A GENOTOXIC COMPOUND

+ The alcoholic group of administered BBN is rapidly oxidized to a carboxyl group by the liver enzymatic system alcohol/aldehyde dehydrogenase; the metabolite formed by N-butyl-N-(3-carboxybutyl) nitrosamine (BCPN) is also a bladder carcinogen and comes in contact with the urothelium via the urine.

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+ Cancer Res. 1977 Feb;37(2):399-407.+ Mutagenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine, a bladder

carcinogen, and related compounds.+ Nagao M, Suzuki E, Yasuo K, Yahagi T, Seino Y.+ Abstract+ N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN), which specifically induces bladder

tumors, was shown to be mutagenic to Salmonella typhimurium strains TA 1535 and TA100 in the presence of an S-9 mix prepared from the liver of rats treated with polychlorinated biphenyl. Reduced nicotinamide adenine dinucleotide was a more effective cofactor than reduced nicotinamide adenine dinucleotide phosphate in the activation of BBN by the rat liver S-9 fraction, N-Butyl-N-(3-carboxypropyl)nitrosamine, reported to be the main urinary metabolite of BBN as well as of N,N-dibutylnitrosamine and to induce urinary bladder tumors specifically, was found to be mutagenic without metabolic activation by the S-9 mix. The mutagenicities of 31 compounds related structurally or metabolically to BBN and N,N-dibutylnitrosamine were tested. Of these compounds, 13 have previously been demonstrated to be carcinogenic, and nine have been shown to be noncarcinogenic. All the carcinogenic compounds were found to be mutagenic to strain TA1535 with or without the S-9 mix. Four of the nine noncarcinogenic compounds were also mutagenic. These "false-positive" compounds were predicted, in fact, to be carcinogenic.

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+ American Heritage+ Medical Dictionary+ surrogate  sur·ro·gate (sûr'ə-gĭt, -gāt', sŭr'-)

n. +  One that takes the place of another; a substitute. +  A person or an animal that functions as a substitute for another,

as in a social or family role. +  A figure of authority who takes the place of the father or mother

in a person's unconscious or emotional life. +  A surrogate mother.

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+ Mucins are a family of high molecular weight, heavily glycosylated proteins (glycoconjugates) produced by epithelial tissues in most metazoans.[1] Mucins' key characteristic is their ability to form gels; therefore they are a key component in most gel-like secretions, serving functions from lubrication to cell signaling to forming chemical barriers.[1] They often take an inhibitory role.

+ Glycosylation mainly refers in particular to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules. This enzymatic process produces one of the fundamental biopolymers found in cells (along with DNA, RNA, and proteins). Glycosylation is a form of co-translational and post-translational modification.

Proceedings of the National Academy of Sciences

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Surrogates of death: "a biomarker intended to substitute for a clinical endpoint".

A surrogate endpoint (or marker) is a measure of effect of a certain treatment that may correlate with a real clinical endpoint but doesn't necessarily have a guaranteed relationship. The National Institutes of Health (USA) define surrogate endpoint as "a biomarker intended to substitute for a clinical endpoint"

Medical Dictionarysurrogate  sur·ro·gate (sûr'ə-gĭt, -gāt', sŭr'-) n.  One that takes the place of another; a substitute.  A person or an animal that functions as a substitute for another, as in a social or family role.  A figure of authority who takes the place of the father or mother in a person's unconscious or emotional life.

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CD24

Figure 1. (A) A model of CD24. The antigen is composed of a small protein core of 27 amino acids which is attached to the membrane via a GPI-anchor. The protein core has many potential glycosylation sites for O-linked carbohydrates, rendering the molecule structurally similar to mucins. To indicate the high degree of glycosylation, carbohydrateside chains are tentatively depicted in the model. Note, that the number of side chains and the extent of modification is variable.

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Androgen and bladder cancer

J Natl Cancer Inst 2007;99: 558 – 68

Assessment of human tumor samples, revealed a similar relationship between CD24 expression and metastatic recurrence after radical primary therapy: High CD24 expression in tumors from males was associated with a reduction in disease-free survival.

Finally, because therapeutic targeting of CD24 has proven beneficial in multiple xenograft models of human bladder , colon, and pancreatic cancer, this work provides a rational for the use of androgen deprivation as a therapeutic modality to reduce CD24 expression in males with CD24-dependent cancers.

Conclusion

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