1. The Kinghorn Cancer Centre, Garvan Institute of Medical Research 384 Victoria Street Darlinghurst NSW 2010 Australia
Cancer Tumour Progression
Christina Konrad, Radhika Nair, Wee Teo, Kate Harvey, Daniel Roden, Eoin Dodson, Holly Holliday, Ben Elsworth, Alexander Swarbrick
The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, Sydney, Australia
Deciphering the biology of Cancer Stem Cells in triple
negative breast cancer
Acknowledgements: This research is funded by the Cancer Counsel NSW
Aim
• Does Id1 mark CSCs in TNBC?
• Mechanism by which Id1 controls the CSC phenotype in TNBC
• Potential CSC surface markers and therapeutic targets for the
study of CSC biology and development of improved therapies
Cancer stem cells in TNBC
The development of targeted therapies have resulted in a
significant decrease in mortality rates for several breast cancer
types. However, the aggressive triple negative breast cancer
(TNBC) subtype lacks effective targeted treatments and thus
relies on chemo- or radiotherapy that are associated with relapse
and therapeutic resistance.
Cancer Stem Cells
A rare subpopulation of tumour cells, termed cancer stem
cells (CSCs), plays a critical role in driving tumour progression,
metastasis, drug resistance and relapse in TNBC.
An in depth understanding of the mechanisms driving the CSC
phenotype would provide better targeted therapies for TNBC
patients.
Id1 controls the CSC phenotype
Our research indicates a pivotal role of the Inhibitor of
differentiation 1 (Id1) protein in driving the CSC phenotype in
TNBC.
Depletion of Id1 in aTNBC metastatic model resulted in reduced
proliferation and self-renewal capacity in vitro, delayed primary
tumour growth and significantly impaired lung metastasis
formation in vivo.
Id1/Id3 depletion reduces self-renewal in a TNBC cell line model. (Courtesy by Dr. Wee Teo)
Self-renewal
Depletion of Id1/Id3 in a TNBC cell line model results in
decreased cell proliferation. (Courtesy by Dr. Wee Teo)
Proliferation
Depletion of Id1/Id3 suppresses spontaneous lung metastasis in mice. (Courtesy by Dr. Wee Teo)
Metastasis
Tumour growth
Id1/Id3 depletion results in delayed primary
tumour growth. (Courtesy by Dr. Wee Teo)
Id1 is deregulated in TNBC
Inhibitor of differentiation (Id) proteins are transcriptional
repressors that regulate cell differentiation and proliferation in
embryonic and tissue stem cells.
Id proteins are deregulated in many cancers.
Id proteins regulate the function of tissue specific transcription
factors by forming inactive complexes unable to bind DNA.
Id1 is expressed in oestrogen negative breast cancer, particularly
inTNBC, and is enriched in metastatic lesions (Gupta et al., 2007).
Id1isexpressedbyaminorityofcellswithinTNBCtumours(1-5%),
which may be CSCs.
Candidate targets of Id1
To elucidate the mechanism by which Id1 controls the CSC
phenotype, bioinformatic analyses were performed using
microarrayandRNA-SeqdatasetsfromtwodistinctTNBCmodels
characterized by Id1 depletion or expression.
Our results identified novel potential CSC surface markers and
showed evidence for Wnt-β-catenin pathway activation.
CSC surface markers
The inability of the current CSC markers to selectively enrich for
CSCs is a major limitation for the study of CSC biology.
RNA sequencing analysis of isolated Id1+ TNBC mouse tumour
cellsshowedupregulatedexpressionofmultiplesurfaceproteins
including Oxytocin receptor (Oxtr), Secretin receptor (Sctr), Lgr6
and Tmem252.
Oxtr, Sctr and Tmem252 are required for proliferation of TNBC
cells in vitro, thus possible therapeutic targets.
Our results identified novel potential CSC surface markers and
therapeutic targets in TNBC.
The ability of the surface proteins to selectively isolate the CSC population from
TNBC tumours is currently being tested by FACS and functional assays such as the
tumoursphere assay to test the CSC phenotype.
Mechanism of Id1 in CSC phenotype
TherelationshipbetweenId1andWnt-β-cateninsignallinginthe
CSC phenotype is currently being tested through knockdown
studies using reporter, tumoursphere and proliferation assays.
Knockdown of Wnt-β-catenin pathway inhibitors, such as
Robo1, rescued proliferation of TNBC cells depleted of Id1.
Basedonourresults,apossiblemechanismbywhichId1controls
the CSC phenotype in TNBC could be through Wnt-β-catenin
signalling by suppression of Robo1.
Id1 target genes driving the CSC phenotype are promishing
therapeutic targets for TNBC patients.
Akt
Lrp5/6
Axin
β-catenin
APC
GSK3β
Destruction
complex
Proteosomal
Degradation
Ub
Ub
Ub
β-catenin
Ub
Ub
Ub
Ub
Ub
Frizzled
β-catenin
β-catenin
LEFTCF
AxinAPC
GSK3β
Wnt
Lrp5/6 Frizzled
Id1
Robo1
Robo1
Akt
Ccnd1
c-Myc
Lef1
Axin2
Id1GFP+ cells
Id1GFP- cells
Tumor
Digestion of tumor
Id1GFP C3Ttg
mouse model
FACS
Id1+
CSC surface marker
Id1-
Id1+
Id1-
Isolation of cells positive and negative
for the putative CSC surface marker
Id1+
Id1-
Id1+
Id1- α-CSC marker
Id1-
Id1-
Id1+
Id1+
Validation of Id1 expression
Functional analyses for CSC phenotype
Id1+
Id1-
Id1+
Id1-
Id1GFP+ cells
Id1GFP- cells
Id1GFP+ cellsId1GFP- cells
RNA-Seq
Tumor
Digestion of tumor
Id1GFP C3Ttg
mouse model
Inducible
Promoter
Id1-shRNA Id3-shRNA
Constitutive
Promoter
Activator
Venus
Neo
+ Doxycycline
ConstitutiveInducible
4T1 cells
+ Dox
Microarray
+
Id1 expression system Id1 knockdown system
4T1 Id1/Id3 KD cells
- Dox
pSLIK inducible knockdown system
4T1 cells
Bioinformatic analyses
FACS
Potential CSC markers Downstream targets of Id1Mechanism of Id1
Downregulated
Robo1
Upregulated
Oxtr
Sctr
Lgr6
Downregulated
Ccnd1
c-Myc
Lef1
Axin2
Cell cycle
Cytoskeleton remodelling
Cell adhesion and migration
Chemotaxis