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 PathologyThe Chinese University of Hong Kong

Bigner et al., Am J Pathol, 1999

1p/19q co-deletion is a genetic hallmark of oligodendroglial tumors (OTs)

•Frequency of 1p/19q codeletion 60-70%

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

Significance of chromosome 1p/19q co-deletion

Microsatellite Genetic markers distance Anaplastic oligodendroglioma oligoastrocytoma

(Mb) A9 A22 A24 A25 B16 B20 B22 B23 A19 B9 B27 B1 B2 B3 B7 B21 B24 A5 A11 A12 A13 A15 A23 A10 A17 B5 B10 B11 B15 B14 B26 A18 A2 A6 A14 A7 A26 B25 B4 B6 B12 B13 B17 B18 B19 A1 A16 A20 A21 A3 B8 A4

D1S171 1p36.32 2.41 ○ ○ ○ ○ – ○ ○ ○ ○ ○ – ● ● ● ● ● ● ○ ○ ○ – – ○ – ○ ● – ○ ○ – ● – ● ● ● ● ○ ○D1S468* 1p36.32 3.37 ○ ○ ○ ○ – ○ ○ ○ ○ ○ – – ● – ● – – ● ● ● ● ● ● ○ ○ ○ ○ – ○ – ○ ● – ● ● ○ ○ ○ ● ● – ● ● ● ● ● ● ● ● ○ ○ ●D1S2845 1p36.32 4.13 ○ ○ ○ ○ ○ ○ – – ● ○ – ● – ● ● ● – ○ ○ ○ ○ ○ ○ ● ● ○ ○ – – – ● ● ● ● ● ● ○ ○D1S2893 1p36.32 4.26 ○ ○ ○ ○ – – – ○ – – – ● ● – – – ● – ○ – – – ○ ○ – ○ ○ ○ ○ ● – – ● – – ● ○ ○D1S2660 1p36.32 4.48 – ○ ○ – – – ○ – – ● ● – – – – – ● – – – – – – – – – – – – – – – – – ● – – –D1S1608 1p36.32 4.67 ○ ○ ○ ○ ○ ○ – ○ – ○ ● ● ● ● – ● ● ○ ○ ○ ○ ○ ○ ○ ○ ● ○ – ○ – ● ● ● ● – ● ○ ○D1S2132 1p36.32 4.81 – ○ ○ – ○ ○ – ○ ○ ○ – ● ● ● – ● ● ○ – ○ ○ – ○ ○ ○ ● ○ ○ ○ – ● ● ● ● – ● ○ ○D1S2795* 1p36.31 5.27 – ○ ○ – – – ○ ○ ○ – ○ ● – ● ● ● – ● ● – ● ● ● ○ ○ – ○ ○ – ○ ○ ● ● ● ● – ○ ○ ● – ● ● – ● – ● ● ● ● – ○ –D1S2870 1p36.31 5.99 ○ – – ○ ○ ○ – – – – – – ● – ● – – ○ – – ○ ○ ○ ○ ○ ○ ○ – – – ● ● ● ● – – – ○D1S1646* 1p36.31 6.85 ○ – ○ ○ – ● – ● ● ● – ○ – ○ ● ● – – – ● ● – ● ○ –D1S1612* 1p36.23 7.83 – ○ ○ ○ – ● – ● ● ● ● ○ ○ ○ ● ● ● ○ ○ – ● ● – – ●D1S2667* 1p36.22 11.20 ○ ○ ○ ○ ○ ● ● – ● ● ● ○ ○ ○ – ● ● ○ ○ ● – ● ● ○ –D1S2647* 1p36.13 19.30 ○ ○ ○ ○ ○ ● ● – – ● ● ○ ○ ○ ● ● ● ○ ○ ● ● ● ● ○ ●D1S482* 1p36.12 23.32 – – – – – – ● – – – ● ○ ○ ○ ● ● ● – – ● – ● – ○ ●D1S470* 1p35.3 29.66 ○ ○ ○ ○ ○ ● ● ● ● ● ● ○ ○ ○ ● ● ● ○ ○ ● ● ● ● ○ ●D1S496* 1p34.3 34.83 ○ – ○ ○ – ● ● – ● ● ● ○ – – – ● ● ○ ○ ● – – ● ○ ●D1S2743* 1p34.2 40.13 – ○ ○ ○ – – ● – – – – – – ○ – – – ○ ○ – ● – ● ○ ●D1S2724* 1p33 48.61 – – – ○ – – ● – ● ● – ○ ○ ○ ● ● ● ○ – ● ● ● ○ –D1S2752* 1p32.1 58.48 ○ – ○ ○ – – ● – – ● – ○ – – – – – ○ ○ – ● – – ○ ●D1S473* 1p31.3 61.28 ○ ○ ○ ○ – ● ● ● ● ● ● – ○ – ● ● ● ○ ○ ● ● – ● ○ ●D1S1665* 1p31.1 73.60 ○ ○ – ○ – ● ● – – ● ● ○ ○ ○ – ● – ○ – ● ● ● – – ●D1S2807* 1p31.1 82.69 ○ ○ ○ ○ – ● ● – ● ● – – – – – ● ● ○ – ● ● ● ● – ●D1S188* 1p22.1 91.97 ○ ○ – ○ ○ ● – – – – ● – – ○ – ● – ○ ○ – ● – – – ●D1S2808* 1p21.3 98.72 – ○ ○ ○ ○ – – ● ● ● – ○ ○ ○ ● – ● ○ ○ – ● – – – –D1S2695* 1p13.3 109.99 – – – – – – ● – – – – – – – – – ● – – ● ● – – – –D1S514* 1p12 119.62 ○ ○ – ○ ○ ● – ● ● ● ● ○ ○ – ● – ● ○ ○ ● ● ● ● ○ ●

D1S215 1q25.2 176.87 – ○ ○ – ○ ○ ○ ○ ● ○ ○ ● ○ ○ ● ○ – – ○ ○ ○ ○ – ○ ○ ○ ○ ○ ○ ○ – ○ ○ ○ ○ ○ – ○ ○ ○ ○ ○ ○ ○ ○ – ● ○ ○ – ○ ○D1S2625 1q31.2 189.56 ○ ○ ○ ● – ○ ○ ○ ● – ○ ● ○ ○ ● ○ – ○ ○ ○ – ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ● ○ ○ ○ – ○ ○ ○ ● ○ ○ ○ ○ ○

D19S219 19q13.32 50.69 ● ○ ○ ○ ○ ● – ○ ○ – ● ● ● ● ● ● ● ● ● ● ● ● ● ○ ○ ○ ● ○ ○ ○ ● ● ● ● ● – ○ ○ ● ○ ● ● ○ ● ● ● ● ● ● ○ ○ ●D19S412 19q13.32 51.70 – ● – – ○ – ○ ○ ● – ○ ● – – – – ● ○ ○ – – – ○ ○ ○ ○ ● ○ – – ○ ○ ○ – – ○ ○ ○ ● – – ○ ○ – ● ● ● – ● – ○ –

AnaplasticCytoband OligoastrocytomaOligodendroglioma

Deletion mapping on chromosome 1p in 52 OTs

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

7/12 (58%)

CCDC27LOC100133612LOC284661

No detectable transcriptsIn all samples examined

•/ KIAA0495

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

p53 WT WT Mut Mut Mut

A172

U87MG

PDAMknockdown +cisplatin (DNA damage)

p53 ? cisplatin resistance

Why would p53-wild-type glioma cells become cisplatin-resistant when PDAM was knocked down?

Cisplatin is a DNA damaging agent p53- mediated DNA damage response cell death

p53 p21 p53 p21

A172 U87MG

Effects of PDAM knockdown and cisplatin on p53

Western blotting RT-PCR

PDAMknockdown +cisplatin (DNA damage)

p53stablization + upregulation

? cisplatin resistance

•DNA damage• response

PDAM knockdown antagonizes apoptosis

Anti-apoptosis Pro-apoptosis Cisplatin resistance

A172

U87MG

BCL-xL knockdown reverses PDAM-dependent cisplatin resistance

PDAMknockdown +cisplatin (DNA damage)

p53stablization +upregulation

BCL-xLupregulation +BCL2 partialderepression

cisplatin resistance

Anti-apoptosis

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

The Chinese University of Hong Kong Anatomical and Cellular PathologyNg Ho-KeungLi Ka Wai KayChung Yuk FeiLi Hiu MingYang Ling

SurgeryPoon Wai Sang

Clinical OncologyLui Wai Yan Vivian

Grant supportResearch Grants Council of Hong Kong

Sun Yat-sen University NeurosurgeryChen Zhong-ping

Fudan University, Huashan HospitalNeurosugeryZhou Liangfu

•Acknowledgements

•Thank you !