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, Austral ia

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 a TNBC 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 in TNBC, and is enriched in metastatic lesions (Gupta et al., 2007). Id1 is expressed by a minority of cells within TNBC tumours (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 microarray and RNA-Seq datasets from two distinct TNBC models 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 cells showed upregulated expression of multiple surface proteins 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

The relationship between Id1 and Wnt-β-catenin signalling in the 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. Based on our results, a possible mechanism by which Id1 controls 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

Axinβ-catenin

APC

GSK3βDestruction

complex

ProteosomalDegradation

Ub

Ub

Ub

β-cateninUb

UbUb

Ub

Ub

Frizzled

β-catenin

β-catenin

LEFTCF

AxinAPC

GSK3β

WntLrp5/6 Frizzled

Id1

Robo1

Robo1

Akt

Ccnd1c-MycLef1Axin2

Id1GFP+ cellsId1GFP- 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 expressionFunctional analyses for CSC phenotype

Id1+

Id1-Id1+

Id1-

Id1GFP+ cellsId1GFP- cells

Id1GFP+ cellsId1GFP- cells

RNA-Seq

Tumor

Digestion of tumor

Id1GFP C3Ttg mouse model

InduciblePromoter Id1-shRNA Id3-shRNA Constitutive

Promoter ActivatorVenus

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

DownregulatedRobo1

UpregulatedOxtr SctrLgr6

DownregulatedCcnd1c-MycLef1Axin2

Cell cycleCytoskeleton remodellingCell adhesion and migrationChemotaxis