Cellular Heterogeneity in Triple-Negative Breast Cancer is Regulated Through Symmetric/Asymmetric Divisions

Introduction: Differentiation events contribute to cellular heterogeneity within tumors and influence disease progression and response to therapy. Breast tumor cells expressing the cytokeratin K14 possess a differentiation state that is associated with that of normal luminal progenitors, while K14-negative cells contain a mature luminal cell gene signature. Triple-negative basal-like breast tumors contain both K14+ and K14- cell populations. In this study, we focused on ascertaining whether cells of this subtype transition between differentiation states thereby contributing to intratumoral heterogeneity and elucidated mechanisms which play a role in this process. Materials and Methods: Human triple-negative breast tumors, patient-derived xenografts, and basal-like breast cancer cell lines were analyzed for gene expression by RNA-seq and were scored for symmetric/asymmetric divisions. Regulators of K14 expression were determined by a functional screen in which 177 candidate transcription factors and signaling molecules were silenced by shRNAs and the percentage of K14+ cells was quantified by FACS. Results and Discussion: We show that cells can transition between the luminal progenitor and mature luminal states through asymmetric divisions, which produce one K14⁺ and one K14^– daughter cell, and that these asymmetric divisions contribute to the generation of cellular heterogeneity. We also demonstrate that the K14+ population is particularly tumorigenic. Several regulators that control the proportion of K14⁺ cells in the population were identified: EZH2 and Notch increase the numbers of K14⁺ cells and their rates of symmetric divisions, while FOXA1 has an opposing effect. Conclusion: Our findings demonstrate a role for asymmetric divisions in differentiation transitions, and identify pathways that control breast cancer cellular composition.