1. 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 : 4/56
Impact factor: 9.737
Advisor:
Yi-Wen Liu
Professor
Speaker:
Mezbahul Haque
Master’s Student
Date: 10.01.14

2. INTRODUCTION
2

3.  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.
3

4. 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. 4

5.  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
5
J Immunol ( 1991) 147:1412–1416
Cancer Res (2000) 60: 6714–6722

6.  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.
6
Arch Pathol Lab Med2007, Vol 131, Feb

7.  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
7
Eur J Cancer (1972)8:587–594.

8.  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.
8
Androgen and bladder cancer
J Natl Cancer Inst (2007)99:558–568.

9. OBJECTIVES
9
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?

10. 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
10

11. RESULTS
11

12. FLOW CHART
Experiments in CD24-deficient mice model
Investigation in Androgen effect of CD24
Expression
Human Bladder Cancer Cell Study
12

13. Bladder Tumor induced by OH-BBN
OH-BBN specifically induces bladder tumors.
mCT scans.
Necropsy images.
Histological preparations
13

14. 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
14

15. 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
15

16. 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
16

17. 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
17

18. Schematic of the experimental design for assessing metastatic incidence.
Harboring Primary Tumors to metastasis
18

19. 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
19

20. 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.
20

21. FLOW CHART
Mice model CD24 Deficiency
Effect of Androgen on CD24 Expression
Human Bladder Cancer Cell Study
21

22. Disease-free survival data for male
Tumor CD24 Expression is correlated to disease-free
surviving in male Patients of bladder cancer.
22
Disease-free survival data for female

23. 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
23

24. 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.
24

25. 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
25

26. 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.
26

27. FLOW CHART
Mice model CD24 Deficiency
Effect of Androgen on CD24 Expression
Analysis of CD24 gene promoter affected by
androgen
27

28. 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
28
Genomatix MatInspector software

29. 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
29

30. SUMMARY
AR Activation Stimulates CD24 Promoter Activity in Human
Bladder Cancer Cells.
30

31.  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
31

32. 32

33. 33
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 × 105
UM-UC-3 cells that had been vector transfected or CD24 transfected as described
previously (5). Graph represents tumor size over time. Data show that castration
can 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.

34. 34

35. 35

36.  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.
36

37.  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).
37

38.  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.
38

39. + 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).
39

40. + 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.
40

41. + 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.
41

42. + 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.
42

43. + 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

44. 44
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 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.

45. 45
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, carbohydrate
side chains are tentatively depicted in the model. Note, that the number of side chains
and the extent of modification is variable.

46. 46
Androgen and bladder cancer
J Natl Cancer Inst 2007;99: 558 – 68

47.  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
47