Lorne poster 2015_Deciphering the biology of cancer stem cells in triple negative breast cancer_Final

AimThis project aims to test the requirement of target genes of Id1 for the viability, self-renewal, and proliferation of cancer stem cells (CSCs). The aim is thus to test whether these candidates are potential therapeutic targets in triple negative breast cancer (TNBC) with the future perspective of developing targeted treatments.

Id1 expression system Id1 depletion system

RNA-Seq Gene Array

4267 9234Array dataId1 KD 4T1

RNA-seq dataId1+ C3Ttg

Bioinformaticanalyses

siRNA-mediated knockdown of target gene

Knockdown of taget gene affects self-renewal

Knockdown of taget gene does not affect self-renewal

Garvan Institute of Medical Research 384 Victoria Street Darlinghurst NSW 2010 Australia

Cancer Tumor Progression

Acknowledgements: This research is funded by the Cancer Counsel

NSW, Knud Højgaards Fond, Oticon Fonden, and Kræftens

Bekæmpelse.

Christina Konrad, Radhika Nair, Wee Teo, Kate Harvey, Daniel Roden, Ben Elsworth, Alexander Swarbrick The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, Sydney

Deciphering the biology of Cancer Stem Cells in triple negative breast cancer

Candidate targets of Id1To characterise the network of genes regulated by Id1, bioinformatic analyses were performed on gene array or RNA-Seq data from two different TNBC models. 34 high confidence targets of Id1 were identified.

Validation of target genesThe requirement of these candidate genes for the viability, proliferation, and self-renewal of CSCs is currently being tested in vitro by knockdown studies using the tumorsphere and proliferation assays.

Id1 targets that are important for the CSC phenotype are potential therapeutic targets with the future prospect of developing targeted treatment for TNBC patients.

BackgroundWhile targeted treatments have resulted in a significant decrease in mortality rates for Hormone receptor and Her2 positive subtypes of breast cancer, no targeted treatment exist for patients with the aggressive triple negative breast cancer subtype. Malignant progression in TNBC is known to be driven by a subpopulation of tumor cells termed cancer stem cells (CSCs). Effective therapeutic targeting of CSCs is thus essential for the complete eradication of a tumor and prevention of relapse in patients due to outgrowth of chemo resistant CSCs. Research by our group has shown that the tran-scriptional repressor, Inhibitor of Differentation (Id1), is expressed within the CSCs of TNBC tumors and is important for the CSC phenotype like proliferation, self-renewal, and metastasis.

Knockdown of Id1/Id3 in a TNBC cell line model results in decreased cell proliferation.

Knockdown of Id1/Id3 reduces self-renewal in a TNBC cell line model.

Knockdown of Id1/Id3 suppresses spontaneous lung metastasis in mice.

Self-renewal

Metastasis

Proliferation

siRNA-mediated knockdown

Tumorsphere assayA key property of CSCs is their ability to self-renew. This property is tested in the tumorsphere assay in which cells are exposed to low adherent and non-differentiating conditions. Only cells with high self-renewal capacity are able to form tumorspheres under these conditions and thus have a phenotype associated with CSCs.

Possible outcomesTumorsphere assayLabeled cells

4T1 tumorsphere assay +/- methylcellulose

Num

bero

fprim

ary

tum

orsp

here

s/10

00ce

lls

0

20

40

60

80Labeled 4T1 cells+ methylcellulose

Labeled 4T1 cells- methylcellulose

Parental 4T1 cells+ methylcellulose

Parental 4T1 cells- methylcellulose

5000 10,000 5000 10,000 5000 10,000 5000 10,000


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sphe

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cells

0

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1000 5000 1000 5000 1000 5000 1000 5000


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mor

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cells

0

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1000 5000 1000 5000 1000 5000 1000 5000


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1000 5000 1000 5000 1000 5000 1000 5000

Defined shapeIrregular shape

- Methylcellulose + Methylcellulose- Methylcellulose + Methylcellulose

~100% aggregates ~50% aggregates

Seeding number

Methylcellulose

A lower seeding number reduced the number of aggregates. However, even at the lowest seeding number aggregates were formed.

Addition of 1% methylcellulose to the tumorsphere culture medium resulted in reduced aggregation and spheres with a more defined shape.

GFP

RFP

Tumorspheres formed at different seedingnumbers of MDA-MB-231 cells

MDA-MB-231 seeding number20

000

1500

010

000

5000

0

20

40

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802000015000100005000

Num

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oftu

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Tumorspheres formed at different seedingnumbers of MDA-MB-231 cells

2000

015

000

1000

050

000

2

4

6

82000015000100005000

MDA-MB-231 seeding number

Num

ber

oftu

mor

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1000

cells

Optimization of tumorsphere assay

Documents

Lorne poster 2015_Deciphering the biology of cancer stem cells in triple negative breast cancer_Final