Id proteins promote a cancer stem cell phenotype in triple negative breast cancer via Robo1-dependent c-Myc activation

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 tumour 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) are expressed by a diversity of stem and progenitor 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. To address the function of Id1 expressing cells within tumours, 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 tumoursphere assays in vitro and for tumour 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 tumour growth and metastatic colonization of the lung in vivo. We defined a novel mechanism of Id function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.