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

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 assaysin vitroand for tumor initiationin 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-renewalin vitro, as well as primary tumor growth and metastatic colonization of the lungin 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.