Navigating the Ovarian Cancer (Epi)Genome
Dr Brian Gloss –Kinghorn Cancer Centre, Garvan Institute, Sydney
AMATA 2013

CAN...
Ovarian cancer

• 5th most
common cause
of cancer death
in women
• Mostly presents
at advanced
stage
• Poor outcome
• Comp...
Aims
• Ovarian cancer presents at advanced
stage
– Identify and develop epigenetic
biomarkers of ovarian cancer for clinic...
Epigenetic Biomarker Discovery

• Target validation
(Sequenom)
– Cell lines
– Tumours

• Clinical application
(headloop PC...
Epigenetic Biomarker Screening

• Target validation
(Sequenom)
– Cell lines
– Tumours

• Clinical application
(headloop PC...
Epigenetic Biomarker Validation

• Target validation
(Sequenom)

ImNormal mortal

Cancer

– Cell lines
– Tumours

• Clinic...
Epigenetic Biomarker Application

• Target validation
(Sequenom)
– Cell lines
– Tumours

• Clinical application
(headloop ...
Epigenetic Biomarker: ZNF300P1

• Target validation
(Sequenom)
– Cell lines
– Tumours

• Clinical application
(headloop PC...
A novel noncoding gene
chr5 (q33.1)

5p15.2

14.3

14.1 p13.3 13.2

p12

5q11.2

• ZNF300P1

13.2

14.1

5q14.3

5q15 q21....
A novel noncoding gene
100 kb

Scale
chr5:

150,150,000

DCTN4
DCTN4
DCTN4

SMIM3

CpG: 36

CpG: 36

150,200,000

hg18
150...
A Major Player in Cancer?
• Accumulating
methylation in
multiple cancer
types
• Candidate
lncRNA

Khalil et Al. PNAS 2009
...
siRNA expression profiling
-Single specific siRNA
(not affecting ZNF300)
-Biological triplicate knockdown
(immortalised no...
Cell Motility: Scratch/Wound Assay
Immortal
Ovarian
Epithelium

siRNA
Knockdown

Gloss et al under review 2013

CANCER RES...
Scratch

Cell Polarity

Direction of (normal) movement

Front
Golgi

Control

Knockdown

Middle

Ovarian cancers spread by...
Conclusions
• Insights into epigenetics
of ovarian cancer using
many whole-genome
and targeted tools
• Novel noncoding gen...
Acknowledgments
• Dr Goli Samimi & Prof. Sue Clark
• Ovarian and Epigenetic Cancer
groups
• Cancer department
• Tissue cul...
Questions

xkcd.com

Thanks!
Relationship with ZNF300

CANCER RESEARCH
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Navigating the Ovarian Cancer (Epi) Genome - Brian Gloss

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Navigating the Ovarian Cancer (Epi) Genome - Brian Gloss

  1. 1. Navigating the Ovarian Cancer (Epi)Genome Dr Brian Gloss –Kinghorn Cancer Centre, Garvan Institute, Sydney AMATA 2013 CANCER RESEARCH
  2. 2. Ovarian cancer • 5th most common cause of cancer death in women • Mostly presents at advanced stage • Poor outcome • Complex biology Estimated new cases Siegel CA Cancer J Clin 2011 Naora H. Expert Rev Mol Med 2007 Estimated deaths CANCER RESEARCH
  3. 3. 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
  4. 4. Epigenetic Biomarker Discovery • Target validation (Sequenom) – Cell lines – Tumours • Clinical application (headloop PCR) – Archival tumour samples – Plasma • Novel ovarian cancer genes Number of samples tested – Indirect (expression) – Direct (methylation profiling) Number of regions evaluated • Screening CANCER RESEARCH
  5. 5. Epigenetic Biomarker Screening • Target validation (Sequenom) – Cell lines – Tumours • Clinical application (headloop PCR) – Archival tumour samples – Plasma • Novel ovarian cancer genes Gloss et Al. Cancer Letters 2012 CANCER RESEARCH Number of samples tested – Indirect (expression) – Direct (methylation profiling) Number of regions evaluated • Screening
  6. 6. Epigenetic Biomarker Validation • Target validation (Sequenom) ImNormal mortal Cancer – Cell lines – Tumours • Clinical application (headloop PCR) – Archival tumour samples – Plasma • Novel ovarian cancer genes Gloss et Al. Cancer Letters 2012 Methylation Level CANCER RESEARCH Number of samples tested – Indirect (expression) – Direct (methylation profiling) Number of regions evaluated • Screening
  7. 7. Epigenetic Biomarker Application • Target validation (Sequenom) – Cell lines – Tumours • Clinical application (headloop PCR) – Archival tumour samples – Plasma • Novel ovarian cancer genes Gloss et Al. Cancer Letters 2012 Normal Samples Methylated CANCER RESEARCH Number of samples tested – Indirect (expression) – Direct (methylation profiling) Cancer Samples Methylated Number of regions evaluated • Screening
  8. 8. Epigenetic Biomarker: ZNF300P1 • Target validation (Sequenom) – Cell lines – Tumours • Clinical application (headloop PCR) – Archival tumour samples – Plasma • Novel ovarian cancer genes: ZNF300P1 Gloss et Al. Cancer Letters 2012 Cancer Normal CANCER RESEARCH Number of samples tested – Indirect (expression) – Direct (methylation profiling) Number of regions evaluated • Screening
  9. 9. A novel noncoding gene chr5 (q33.1) 5p15.2 14.3 14.1 p13.3 13.2 p12 5q11.2 • ZNF300P1 13.2 14.1 5q14.3 5q15 q21.1 q21.3 5q23.1 q23.2 31.1 5q32 33.1 33.3 5q34 100 kb 150,200,000 IRGM – Unprocessed Pseudogene of ZNF300 (84%) • 2.4kb transcript • Chr 5q33.1 35.1 35.2 35.3 hg18 150,250,000 RefSeq Genes ZNF300 ZNF300 ZNF300 150,300,000 ZNF300P1 CpG Islands (Islands < 300 Bases are Light Green) CpG: 23 Gloss, O’Brien & Clark WO Patent 2,012,142,656, 2012 CpG: 25 CANCER RESEARCH
  10. 10. A novel noncoding gene 100 kb Scale chr5: 150,150,000 DCTN4 DCTN4 DCTN4 SMIM3 CpG: 36 CpG: 36 150,200,000 hg18 150,250,000 IRGM 150,300,000 RefSeq Genes ZNF300 ZNF300 ZNF300 150,350,000 ZNF300P1 150,400,000 GPX3 TNIP1 TNIP1 TNIP1 TNIP1 TNIP1 TNIP1 TNIP1 TNIP1 TNIP1 TNIP1 CpG Islands (Islands < 300 Bases are Light Green) CpG: 23 CpG: 25 CpG: 43 Ovarian Cancer Cell Lines • Targeted for silencing in cancer? TNIP1 GPX3 ZNF300P1 ZNF300 MST150 DCN4 Gloss, O’Brien & Clark WO Patent 2,012,142,656, 2012 Methylation Level CANCER RESEARCH
  11. 11. 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 Vita Lin Wenjia Qu Nicola Currey CANCER RESEARCH
  12. 12. siRNA expression profiling -Single specific siRNA (not affecting ZNF300) -Biological triplicate knockdown (immortalised normal epithelium) GO terms Networks (IPA) Affy Gene ST v1 Arrays GSEA KEGG pathways Cancer/TSG/Cell Death terms Cell Cycle DOWN Cell/Cell interaction and motility apparatus UP Gloss et al under review 2013 CANCER RESEARCH
  13. 13. Cell Motility: Scratch/Wound Assay Immortal Ovarian Epithelium siRNA Knockdown Gloss et al under review 2013 CANCER RESEARCH Dr Kim Moran Jones
  14. 14. Scratch Cell Polarity Direction of (normal) movement Front Golgi Control Knockdown Middle Ovarian cancers spread by direct means An initiating molecular event? Back Gloss et al under review 2013 CANCER RESEARCH Dr Kim Moran Jones
  15. 15. 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
  16. 16. 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
  17. 17. Questions xkcd.com Thanks!
  18. 18. Relationship with ZNF300 CANCER RESEARCH

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