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

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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

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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

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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

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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

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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