Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes. PIK3CA and P53 (also known as TP53) are the two most frequently mutated genes and are associated with different types of human breast cancers. The cellular origin and the mechanisms leading to PIK3CA-induced tumour heterogeneity remain unknown. Here we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and the impact of mutations in this gene on tumour heterogeneity. Surprisingly, oncogenic Pik3ca(H1047R) mutant expression at physiological levels in basal cells using keratin (K)5-CreER(T2) mice induced the formation of luminal oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive tumours, while its expression in luminal cells using K8-CReER(T2) mice gave rise to luminal ER(+)PR(+) tumours or basal-like ER(-)PR(-) tumours. Concomitant deletion of p53 and expression of Pik3ca(H1047R) accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3ca(H1047R) in unipotent basal cells gave rise to luminal-like cells, while its expression in unipotent luminal cells gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that underwent cell fate transition upon Pik3ca(H1047R) expression in unipotent progenitors demonstrated a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures characteristic of the different cell fate switches that occur upon Pik3ca(H1047R) expression in basal and luminal cells, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3ca(H1047R) activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation.

Lineage tracing has become the method of choice to study the fate and dynamics of stem cells (SCs) during development, homeostasis, and regeneration. However, transgenic and knock-in Cre drivers used to perform lineage tracing experiments are often dynamically, temporally, and heterogeneously expressed, leading to the initial labeling of different cell types and thereby complicating their interpretation. Here, we developed two methods: the first one based on statistical analysis of multicolor lineage tracing, allowing the definition of multipotency potential to be achieved with high confidence, and the second one based on lineage tracing at saturation to assess the fate of all SCs within a given lineage and the "flux" of cells between different lineages. Our analysis clearly shows that, whereas the prostate develops from multipotent SCs, only unipotent SCs mediate mammary gland (MG) development and adult tissue remodeling. These methods offer a rigorous framework to assess the lineage relationship and SC fate in different organs and tissues.

SummaryThe mammary gland (MG) is composed of basal cells (BCs) and luminal cells (LCs). While it is generally believed that MG arises from embryonic multipotent progenitors (EMPs), it remains unclear when lineage restriction occurs and what are the mechanisms responsible for the switch from multipotency to unipotency during MG morphogenesis. Here, we performed multicolor lineage tracing and assessed the fate of single progenitors and demonstrated the existence of a developmental switch from multipotency to unipotency during embryonic MG development. Molecular profiling and single cell RNA-seq revealed that EMPs express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in EMPs promotes unipotent BC fate and was sufficient to reprogram adult LCs into BCs by promoting an intermediate hybrid multipotent like state. Altogether, this study identifies the timing and the mechanisms mediating the early lineage segregation of multipotent progenitors during MG development.

SummaryEpithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells.

Author contributions A.C., S.L. and C.B. designed the experiments and performed data analysis. A.C. and S.L. performed most of the biological experiments. E.T. performed the experiments and data analysis on prostate glands. A.Sifrim., M.M., Y.S., J.V.H. and T.V. performed the bioinformatic analysis. A.D. and G.B. provided technical help. C.D. performed FACS experiments. N.D. provided technical help with single-cell RNA sequencing. A.C., S.L., M.F., A.W. and A.V.K. performed immunostainings, blocking antibodies and small-molecule treatments and experiments with follow-up mice. A.Sahay contributed genetic tools. V.d.M. performed statistical analysis. C.W.S. provided the Notch antibodies. A.C., A.V.K. and C.B. wrote the manuscript. All authors read and approved the final manuscript.