PDAM is frequently downregulated in oligodendroglial tumors
and its knockdown by siRNA induces cisplatin resistance
Ng Ho-Keung吳浩強
The State Key Laboratory of Oncology in South China
Department of Anatomical and Cellular Pathology
The Chinese University of Hong Kong
1p/19q co-deletion is a genetic hallmark of oligodendroglial tumors (OTs)
•Frequency of 1p/19q codeletion 60-70%
Bigner et al., Am J Pathol, 1999
Significance of chromosome 1p/19q co-deletion
 Frequent 1p/19q co-deletion suggests the presence of tumor suppressor genes
 1p/19q co-deletion is correlated with oligodendrogliomas with ‘classic’ histology
- an adjunct marker in OT diagnosis
 1p/19q co-deletion or 1p deletion alone is also associated with better survival and
chemosensitivity in OT patients
- first molecular marker in neuro-oncology for prognosis and chemosensitivity
 Investigation into the mechanism of 1p loss would provide insights into
- molecular pathogenesis of OTs
- mechanism underlying chemosensitivity
Deletion mapping on chromosome 1p in 52 OTs
Microsatellite
Genetic
Oligodendroglioma
markers Cytoband distance
(Mb) A9 A22 A24 A25 B16 B20 B22 B23 A19 B9 B27 B1 B2 B3 B7 B21 B24 A5 A11 A12 A13 A15 A23
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Oligoastrocytoma
A10 A17 B5 B10 B11 B15 B14 B26 A18 A2 A6 A14
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Anaplastic
oligoastrocytoma
Anaplastic oligodendroglioma
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A7 A26 B25 B4 B6 B12 B13 B17 B18 B19 A1 A16 A20 A21
A3 B8 A4
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D1S171
D1S468*
D1S2845
D1S2893
D1S2660
D1S1608
D1S2132
D1S2795*
D1S2870
D1S1646*
D1S1612*
D1S2667*
D1S2647*
D1S482*
D1S470*
D1S496*
D1S2743*
D1S2724*
D1S2752*
D1S473*
D1S1665*
D1S2807*
D1S188*
D1S2808*
D1S2695*
D1S514*
1p36.32
1p36.32
1p36.32
1p36.32
1p36.32
1p36.32
1p36.32
1p36.31
1p36.31
1p36.31
1p36.23
1p36.22
1p36.13
1p36.12
1p35.3
1p34.3
1p34.2
1p33
1p32.1
1p31.3
1p31.1
1p31.1
1p22.1
1p21.3
1p13.3
1p12
2.41
3.37
4.13
4.26
4.48
4.67
4.81
5.27
5.99
6.85
7.83
11.20
19.30
23.32
29.66
34.83
40.13
48.61
58.48
61.28
73.60
82.69
91.97
98.72
109.99
119.62
D1S215
D1S2625
1q25.2
1q31.2
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189.56 ○ ○ ○ ● – ○ ○ ○ ● – ○ ● ○ ○ ● ○ – ○ ○ ○ – ○ ○
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D19S219
D19S412
19q13.32 50.69
19q13.32 51.70
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1p loss frequency : 61%
Dong et al., Br J Cancer 2004
Candidate genes within the minimally deleted region on 1p36.3
~1.2 Mb
RT-PCR analysis of candidate genes in microdissected tumors
•/ KIAA0495
7/12 (58%)
CCDC27
LOC100133612
LOC284661
No detectable transcripts
In all samples examined
1.
Pang et al., Brain Pathol , in press
PDAM is downregulated in OTs
•p=0.01
64% (37 of 58) tumors showed
reduced PDAM expression by >2-fold
compared to normal brain, with 51%
showing >10-fold decreased level
Mechanisms responsible for PDAM downregulation
Allelic deletion
- 24 of 37 tumors with reduced PDAM expression showed chr 1p loss
(p=0.01)
Epigenetic modifications
PDAM promoter is hypermethylated
Reduced PDAM expression was associated with
promoter hypermethylation (p=0.004)
•PDAM expression is regulated by epigenetic modifications
*
* * *
* *
*
*
*
* P<0.05
Mechanisms responsible for PDAM downregulation
Of 37 cases with reduced PDAM expression:
- 20 (54%) exhibited both chr 1p loss and promoter hypermethylation
- 10 (27%) showed promoter hypermethylation but intact chr 1p
- 3 (8%) showed 1p deletion only
- 4 (11%) -- other mechanisms?
Hemizygous 1p deletion and epigenetic modifications are the major
mechanisms contributing to PDAM downregulation
Chr 1p loss is associated with chemosensitivity
Would knockdown of PDAM render glioma cells
sensitive to chemotherapeutic drugs?
Chemosensitivity assay
PDAM knockdown induces cisplatin resistance
A172
U87MG
p53
WT WT Mut Mut Mut
PDAM
knockdown
+
cisplatin
(DNA damage)
p53
?
cisplatin
resistance
Why would p53-wild-type glioma cells become cisplatinresistant when PDAM was knocked down?
Cisplatin is a DNA damaging agent  p53- mediated DNA damage response  cell death
Effects of PDAM knockdown and cisplatin on p53
Western blotting
RT-PCR
A172
p53
p21
U87MG
p53 p21
PDAM
knockdown
+
cisplatin
(DNA damage)
p53
stablization
+
upregulation
•DNA damage
• response
?
cisplatin
resistance
PDAM knockdown antagonizes apoptosis
A172
U87MG
Anti-apoptosis
Pro-apoptosis
Cisplatin
resistance
BCL-xL knockdown reverses PDAM-dependent cisplatin resistance
PDAM
knockdown
+
cisplatin
(DNA damage)
p53
stablization
+
upregulation
BCL-xL
upregulation
+
BCL2 partial
derepression
Antiapoptosis
cisplatin
resistance
PDAM may modulate apoptosis via regulation of p53-dependent anti-apoptotic genes
PDAM : P53-Dependent Apoptosis Modulator
Summary
 A novel gene PDAM is found to be frequently downregulated, mediated
through epigenetic modifications and genomic loss, in OTs. These findings
suggest that PDAM plays a role in the OT development
 A novel mechanism for cisplatin resistance is discoverd, in which PDAM may
possess the ability to modulate p53-dependent DNA damage response
•Acknowledgements
The Chinese University of Hong Kong
Sun Yat-sen University
Anatomical and Cellular Pathology
Ng Ho-Keung
Li Ka Wai Kay
Chung Yuk Fei
Li Hiu Ming
Yang Ling
Neurosurgery
Chen Zhong-ping
Surgery
Poon Wai Sang
Clinical Oncology
Lui Wai Yan Vivian
Grant support
Research Grants Council of Hong Kong
Fudan University, Huashan Hospital
Neurosugery
Zhou Liangfu
•Thank you !