Id proteins promote a cancer stem cell phenotype in triple negative breast cancer via negative regulation of Robo1

Teo, Wee Siang; Holliday, Holly; Karthikeyan, N.; Cazet, Aurélie; Roden, Daniel; Harvey, Kate; Konrad, Christina Valbirk; Murali, Reshma; Varghese, Binitha Anu; Archana, P. T.; Chan, Chia-Ling; McFarland, Andrea; Junankar, Simon; Ye, Sunny; Yang, Jessica; Nikolić, Iva; Shah, Jaynish S.; Baker, Laura A.; Millar, Ewan K.A.; Naylor, Mathew J.; Ormandy, Christopher J.; Lakhani, Sunil R.; Kaplan, Warren; Mellick, Albert S.; O’Toole, Sandra A.; Nair, Radhika; Swarbrick, Alexander

doi:10.1101/497313

Cited by 4 publications

(14 citation statements)

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“…We have already demonstrated that Id KD significantly compromises other key CSC phenotypes like self renewal and migration (10). As Id marks a CSC in the TNBC subtype, we next looked at the expression of the CSC markers CD24 and CD29 in the Id KD system (10, 14-17). Contrary to our hypothesis, we found that the percentage of cells marked by CD29+/CD24+ in the Id KD population is significantly higher in comparison to the controls (Supplementary Figure1A).…”

Section: Resultsmentioning

confidence: 98%

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Targeting the Id1-Kif11-Aurka axis in triple negative breast cancer using combination therapy

Murali

Varghese

Karthikeyan

et al. 2019

Preprint

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31Evidence points to breast cancer following a hierarchical model, with Cancer Stem Cells (CSCs) 32 driving critical phenotypes of the bulk tumor. Chemoresistant CSCs are not an abstract concept 33 but have clinical consequences as they drive relapse and ultimately lead to mortality in patients, 34 making it imperative to understand how these subpopulations of cells survive. Our previous work 35 (1-2) has demonstrated that the bHLH transcription factor, Inhibitor of Differentiation 1 (Id1) 36 and it's closely related family member Id3, have an important role in maintaining the CSC 37 phenotype in the Triple Negative breast cancer (TNBC) subtype. A genetic screen conducted to 38 further elucidate the molecular mechanism underlying the Id (Id1/3) mediated CSC phenotypes 39 in TNBC revealed critical cell cycle genes such as Kif11 and Aurka as putative Id targets. We 40 take this work forward by investigating how alteration in Kif11 and Aurka via Id proteins 41 promotes the CSC phenotype in TNBC. Cells lacking Id are poised in a state of G0/G1 arrest 42 from which they can re-enter the cell cycle. Intriguingly, depletion of Kif11 and Aurka 43 independently did not phenocopy the G0/G1 arrest observed in Id knockdown (Id KD) cells. We 44 have further explored the hypothesis that we can deplete the chemo resistant Id expressing CSC 45 population by combining chemotherapy with targeted therapy using existing small molecule 46 inhibitors (against Id target Kif11) to more effectively debulk the entire tumor. This work opens 47 up exciting new possibilities of targeting Id targets like Kif11, in the TNBC subtype which is 48 currently refractory to chemotherapy. 49 50 51 52 53Breast cancer is a heterogeneous disease with different molecular subtypes displaying distinct 55 pathological-clinical outcomes that have been successfully exploited in the management of the 56 disease (3). The TNBC subtype does not express molecular markers such as ER and Her2 that 57 are the basis of targeted therapies in other molecular subtypes of breast cancer (4). Consequently 58 patients presenting with TNBC are left with few therapeutic choices, resulting in lower five- 59year survival rates when compared to the other subtypes (4-5). There is hence an urgent need to 60 understand the molecular basis of TNBC in order to identify new drug targets. 61The critical role of a subpopulation of cells termed Cancer Stem Cells (CSCs) in self-renewal, 62 chemoresistance and metastasis has assumed great clinical importance in breast cancer (6-7). The 63Inhibitor of differentiation (Id) proteins are negative regulators of the basic helix-loop-helix 64 (bHLH) transcription factors. The Id proteins are important for maintaining the CSC population 65 and therefore tumour progression in TNBC(8) We have previously shown that Id1/3 (collectively 66 known as Id) are critical for the CSC associated phenotypes in the TNBC molecular subtype(1) . 67A detailed genetic screen analysis of Id knock down (Id KD) and Id1 expression models led to 68 the ide...

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Section: Resultsmentioning

confidence: 98%

“…It has been previously demonstrated that Id KD significantly affects pathways associated with cell cycle progression ((10-11). We first sought to validate this observation in our model system using the pSLIK (single lentivector for inducible knockdown) construct(12-13).…”

Section: Resultsmentioning

confidence: 99%

Targeting the Id1-Kif11-Aurka axis in triple negative breast cancer using combination therapy

Murali

Varghese

Karthikeyan

et al. 2019

Preprint

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“…Breast cancer is a heterogeneous disease with different molecular subtypes displaying distinct outcomes [ 1 , 2 ]. The triple-negative breast cancer (TNBC) subtype does not express molecular markers such as estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) that are the basis of targeted therapies in other molecular subtypes of breast cancer [ 3 , 4 ].…”

Section: Introductionmentioning

confidence: 99%

“…The Id proteins are important for maintaining the CSC population and therefore tumor progression in TNBC. We have previously shown that Id1/3 (collectively known as Id ) are critical for the CSC-associated phenotypes in the TNBC molecular subtype [ 1 ]. Genetic screen analysis of Id knockdown ( Id KD) and Id1 expression models led to the identification of Kif11 and Aurka as putative Id targets in this study.…”

Section: Introductionmentioning

confidence: 99%

Targeting the Id1-Kif11 Axis in Triple-Negative Breast Cancer Using Combination Therapy

et al. 2020

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The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1–Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1–Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.

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“…Rob Salomon and Mr. David Snowden for their help with flow sorting. This manuscript has been released as a pre-print at bioRxiv (Teo et al, 2018 ).…”

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confidence: 99%

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1

Teo

Holliday

Karthikeyan

et al. 2020

Front. Cell Dev. Biol.

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Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1 + cells. Id1 + cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

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Id proteins promote a cancer stem cell phenotype in triple negative breast cancer via negative regulation of Robo1

Cited by 4 publications

References 79 publications

Targeting the Id1-Kif11-Aurka axis in triple negative breast cancer using combination therapy

Targeting the Id1-Kif11-Aurka axis in triple negative breast cancer using combination therapy

Targeting the Id1-Kif11 Axis in Triple-Negative Breast Cancer Using Combination Therapy

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1

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