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

Abstract

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.

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

“…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.…”

“…We used the metastatic 4T1 cell line as it is representative of the TNBC subtype [14], and Id proteins have been shown to play an important role in tumorigenesis in the TNBC subtype. We used an inducible known down system, as reported earlier [1], and observed a significant decrease in the proliferative capacity of cells upon doxycycline (Dox)-induced Id KD in comparison to control conditions.…”

“…4T1 cells were sourced from American Type Cell Culture Collection (ATCC) and cultured as per specifications. The 4T1 Id1GFP cells were generated by lentiviral infection of the 4T1 cells with the Id1 GFP reporter, as reported previously [1].…”

“…4T1 cells were put into the tumorsphere assay, as described previously [1]. Paclitaxel and ispinesib were added at a concentration of 10nM and 5nM, respectively, to assay for the effect of these drugs on self-renewal.…”

“…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].…”