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Navigating the Ovarian Cancer (Epi)Genome
Dr Brian Gloss –Kinghorn Cancer Centre, Garvan Institute, Sydney
AMATA 2013
CANCER RESEARCH
• 5th most
common cause
of cancer death
in women
• Mostly presents
at advanced
stage
• Poor outcome
• Complex
biology
Ovarian cancer
CANCER RESEARCH Siegel CA Cancer J Clin 2011
Naora H. Expert Rev Mol Med 2007
Estimated deaths Estimated new cases
Aims
• Ovarian cancer presents at advanced
stage
– Identify and develop epigenetic
biomarkers of ovarian cancer for clinical
application
• Ovarian cancer is complicated
– Evaluate the role of novel genes identified
above in the development of ovarian
cancer
CANCER RESEARCH
Epigenetic Biomarker Discovery
• Screening
– Indirect (expression)
– Direct (methylation profiling)
• Target validation
(Sequenom)
– Cell lines
– Tumours
• Clinical application
(headloop PCR)
– Archival tumour samples
– Plasma
• Novel ovarian cancer
genes
CANCER RESEARCH
Num
ber
of re
gio
ns e
valu
ate
d
Num
ber o
f sam
ple
s te
ste
d
Epigenetic Biomarker Screening
• Screening
– Indirect (expression)
– Direct (methylation profiling)
• Target validation
(Sequenom)
– Cell lines
– Tumours
• Clinical application
(headloop PCR)
– Archival tumour samples
– Plasma
• Novel ovarian cancer
genes
CANCER RESEARCH Gloss et Al. Cancer Letters 2012
Num
be
r o
f re
gio
ns e
valu
ate
d
Num
ber o
f sam
ple
s te
ste
d
Epigenetic Biomarker Validation
• Screening
– Indirect (expression)
– Direct (methylation profiling)
• Target validation
(Sequenom)
– Cell lines
– Tumours
• Clinical application
(headloop PCR)
– Archival tumour samples
– Plasma
• Novel ovarian cancer
genes
CANCER RESEARCH Gloss et Al. Cancer Letters 2012
Normal Im-
mortal Cancer
Num
be
r o
f re
gio
ns e
valu
ate
d
Num
ber o
f sam
ple
s te
ste
d
Methylation Level
Epigenetic Biomarker Application
• Screening
– Indirect (expression)
– Direct (methylation profiling)
• Target validation
(Sequenom)
– Cell lines
– Tumours
• Clinical application
(headloop PCR)
– Archival tumour samples
– Plasma
• Novel ovarian cancer
genes
CANCER RESEARCH Gloss et Al. Cancer Letters 2012
Num
be
r o
f re
gio
ns e
valu
ate
d
Num
ber o
f sam
ple
s te
ste
d
Cancer Samples Methylated
Normal Samples Methylated
Epigenetic Biomarker: ZNF300P1
• Screening
– Indirect (expression)
– Direct (methylation profiling)
• Target validation
(Sequenom)
– Cell lines
– Tumours
• Clinical application
(headloop PCR)
– Archival tumour samples
– Plasma
• Novel ovarian cancer
genes: ZNF300P1
CANCER RESEARCH Gloss et Al. Cancer Letters 2012
Num
be
r o
f re
gio
ns e
valu
ate
d
Num
ber o
f sam
ple
s te
ste
d
Cancer Normal
A novel noncoding gene
CANCER RESEARCH Gloss, O’Brien & Clark WO Patent 2,012,142,656, 2012
• ZNF300P1
– Unprocessed
Pseudogene of
ZNF300 (84%)
• 2.4kb transcript
• Chr 5q33.1
Scalechr5:
100 kb hg18150,150,000 150,200,000 150,250,000 150,300,000 150,350,000 150,400,000
RefSeq Genes
CpG Islands (Islands < 300 Bases are Light Green)
DCTN4DCTN4DCTN4
SMIM3 IRGM ZNF300ZNF300ZNF300
ZNF300P1 GPX3TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1
CpG: 36 CpG: 36 CpG: 23 CpG: 25 CpG: 43
chr5 (q33.1) 5p15.2 14.3 14.1 p13.3 13.2 p12 5q11.2 13.2 14.1 5q14.3 5q15 q21.1 q21.3 5q23.1 q23.2 31.1 5q32 33.1 33.3 5q34 35.1 35.2 35.3
A novel noncoding gene
CANCER RESEARCH Gloss, O’Brien & Clark WO Patent 2,012,142,656, 2012
• Targeted for silencing in
cancer?
Scalechr5:
100 kb hg18150,150,000 150,200,000 150,250,000 150,300,000 150,350,000 150,400,000
RefSeq Genes
CpG Islands (Islands < 300 Bases are Light Green)
DCTN4DCTN4DCTN4
SMIM3 IRGM ZNF300ZNF300ZNF300
ZNF300P1 GPX3TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1TNIP1
CpG: 36 CpG: 36 CpG: 23 CpG: 25 CpG: 43
Methylation Level
TN
IP1
GP
X3
ZN
F300P
1
ZN
F300
MS
T150
DC
N4
Ovaria
n C
ancer C
ell L
ines
A Major Player in Cancer?
• Accumulating
methylation in
multiple cancer
types
• Candidate
lncRNA
Khalil et Al. PNAS 2009
Gloss, O’Brien & Clark WO Patent 2,012,142,656, 2012 CANCER RESEARCH
Vita Lin
Wenjia Qu
Nicola Currey
siRNA expression profiling
CANCER RESEARCH Gloss et al under review 2013
GO terms
KEGG pathways
Networks (IPA)
GSEA
Affy Gene ST v1
Arrays
-Single specific siRNA
(not affecting ZNF300)
-Biological triplicate knockdown
(immortalised normal epithelium)
Cancer/TSG/Cell Death terms
Cell Cycle DOWN
Cell/Cell interaction and motility apparatus UP
Cell Motility: Scratch/Wound Assay
Immortal
Ovarian
Epithelium
siRNA
Knockdown
CANCER RESEARCH Dr Kim Moran Jones
Gloss et al under review 2013
Control Knockdown
Cell Polarity Front
Back
Middle
CANCER RESEARCH Dr Kim Moran Jones
Gloss et al under review 2013
Golgi
Dire
ction
of (n
orm
al) m
ove
me
nt
Scratch
Ovarian cancers spread by direct means
An initiating molecular event?
Conclusions • Insights into epigenetics
of ovarian cancer using many whole-genome and targeted tools
• Novel noncoding gene that is silenced in multiple cancer types
• Expression profiling highlights loss of cell polarity as the likely culprit
Hanahan & Weinberg 2000 & 2011, Cell CANCER RESEARCH
Acknowledgments • Dr Goli Samimi & Prof. Sue Clark
• Ovarian and Epigenetic Cancer groups
• Cancer department
• Tissue culture
• ACRF
• Peter Wills Bioinformatics
• Support
• Marcel Dinger: Genome Informatics Research team – At AMATA: Mark Cowley, Mark
McCabe, Jesper Maag
CANCER RESEARCH
Questions
xkcd.com Thanks!
Relationship with ZNF300
CANCER RESEARCH