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

Abstract Background Breast cancers display phenotypic and functional heterogeneity. 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. Here, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers and enriched in brain metastases compared to patient matched primary tissues. We go on to investigate the molecular mechanism underpinning the role of Id in CSC biology in the TNBC subtype. Methods To address the function of Id1 expressing cells within tumors, we developed three independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. We also developed an inducible Id knock down system which allowed us to deplete Id proteins in the 4T1 metastatic murine cell line. Results Using the Id1C3-Tag,p53−/− and 4T1 models, we found that 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 Id 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. Conclusions Id proteins play a critical role in the proliferative, self renewal and metastatic CSC phenotypes in TNBC. Our work has shed light on how Id acts on Robo1 to possibly control a Myc network and could be used to target the CSC population in the TNBC subtype.