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 two 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.

Intratumoral heterogeneity is a major ongoing challenge in the effective therapeutic targeting of cancer. Accumulating evidence suggests that a fraction of cells within a tumor termed Cancer Stem Cells (CSCs) are primarily responsible for this diversity resulting in therapeutic resistance and metastasis. Adding to this complexity, recent studies have shown that there can be different subpopulations of CSCs with varying biochemical and biophysical traits resulting in varied dissemination and drug-resistance potential. Moreover, cancer cells can exhibit a high level of plasticity or the ability to dynamically switch between CSC and non-CSC states or among different subsets of CSCs. The molecular mechanisms underlying such plasticity has been under extensive investigation and the trans-differentiation process of Epithelial to Mesenchymal transition (EMT) has been identified as a major contributing factor. Besides genetic and epigenetic factors, CSC plasticity is also shaped by non-cell-autonomous effects such as the tumor microenvironment. In this review, we discuss the recent developments in understanding CSC plasticity in tumor progression at biochemical and biophysical levels, and the latest in silico approaches being taken for characterizing cancer cell plasticity with implications in improving existing therapeutic approaches.

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...