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Defining the mechanism regulating mammary gland stem cell multipotency

par Centonze, Alessia
Président du jury Remmelink, Myriam
Promoteur Blanpain, Cédric
Co-Promoteur Van Keymeulen, Alexandra
Publication Non publié, 2022-08-23

Thèse de doctorat

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Résumé :

Glandular epithelia, including the mammary and prostate glands, are generally composed by basal (BCs) and luminal cells (LCs) organised in a bilayered structure. Lineage tracing studies demonstrate that many glandular epithelia develop from multipotent basal stem cells (BSCs) that are replaced in adult life by distinct pools of unipotent basal and luminal stem cells. However, adult unipotent BSCs can reactivate multipotency under regenerative conditions and upon oncogene expression. Particularly, transplantation of mammary gland (MG) BCs alone that are unipotent in lineage tracing give rise to MG outgrowth composed of BCs and LCs, promoting the multipotent potential of BCs when transplanted without LCs. In contrast, when BCs are transplanted together with LCs, BCs maintain their unipotent basal fate, suggesting that LCs may actively restrict BCs multilineage differentiation potential in physiological conditions. The nature of this mechanism is unknown.During my thesis I studied the mechanisms involved in the activation of BSC multipotency in different conditions to identify whether a common multipotent program is activated in the different multipotent conditions, as well as, in different glandular epithelia. I first studied the activation of BSC multipotency upon LC ablation in multiple epithelia both in vivo in mice and in vitro in organoids. Performing bulk and single-cell RNA sequencing after LC ablation we identified that BSCs activate a hybrid basal and luminal cell differentiation program before giving rise to LCs—reminiscent of the genetic program that regulates multipotency during embryonic development. Also, using mammary gland derived organoids, we could demonstrate that the communication between LCs and BCs is essential to maintain BC unipotency. By predicting ligand–receptor pairs from single-cell data, we found that TNF—which is secreted by LCs—restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and EGFR pathways are activated in BSCs and their progeny after LC ablation; blocking these pathways, or stimulating the TNF pathway, inhibits regeneration-induced BC multipotency. To identify whether a common BC multipotency program was activated upon the different multipotent conditions identified in the laboratory (embryogenesis, LC ablation, oncogenic hit, transplantation) we compared the genes upregulated by the multipotent BCs. The comparison of the upregulated genes in these conditions shows the common overexpression of Col1a1 and Col1a2 genes, which encodes for the two chains of the collagen 1 (col1). Col1 is one of the main structural components of the mammary gland stroma, giving stiffness to the tissue and playing an important role in the branching morphogenesis process during puberty. The common upregulation of collagen genes in the multipotency signatures suggests that the extracellular matrix (ECM) and its stiffness could be involved in the regulation of BC multipotency, since cell behaviour can be modulated by different signals from the stroma which induces mechanotransduction signalling. To address whether different collagen concentrations or increased stiffness can promote BC multipotency we used BC lineage tracing in MG organoids embedded in either col1 matrix or polyethylene glycol-(PEG)-hydrogel at different concentrations, which correspond to different stiffness. Increased col1 concentration and high PEG content (stiff matrix) promote BC multipotency, suggesting that col1 matrix and its stiffness are involved in the activation of multipotency. Using single cell RNA sequencing analysis, we identified the activation of different hybrid states in the collagen 1 and stiff matrix conditions and the activation of AP-1 family of transcription factors (TFs). Moreover, by blocking integrin signalling and FAK, we identified the important role of B1-integrin/FAK-signalling in promoting BC multipotency. These results suggest that the MG epithelial stem cells can sense their surrounding environment and activate multipotency through B1-integrin/FAK/AP1-signalling pathway. Altogether, this study will reveal the importance of ECM composition and stiffness in regulating cell fate maintenance in the mammary gland which can be lost in pathological conditions, such as cancer.