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Molecular mechanisms of the regulation of mammary stem cell homeostasis and their subversion in cancer

Final Report Summary - MAMMASTEM (Molecular mechanisms of the regulation of mammary stem cell homeostasis and their subversion in cancer)

Cancer stem cells (CSCs) are thought to be integral to the development and progression of cancer, and their eradication might be vital to curing cancer. Nevertheless, we still know very little about molecular and biological nature of CSCs and how they differ from normal SCs. This is due, in part, to the lack of effective methods to identify and purify, in sufficient quantities, adult tissue SCs for experimental analysis. In the MAMMASTEM project, we set out to characterize the key molecular mechanisms governing normal human mammary stem cell (MaSC) biology and how these mechanisms are disrupted in breast cancer. To achieve this, we exploited a technique developed in our lab that allows us to propagate and isolate, to near-purity, MaSCs from tissue samples for biological, biochemical and genetic analysis.
The specific focus of our project was the cell fate determinant Numb, which is known to dictate cell fate decisions during neuronal development in Drosophila. Our previous investigations on Numb had highlighted its involvement in tumor suppression; we had shown that Numb protein levels are downmodulated in approximately 30% of breast cancers due to excessive degradation of the protein by the ubiquitin-proteasome system. Notably, loss of Numb expression was associated with an aggressive cancer phenotype and, consistently, patients with Numb-negative breast cancer displayed an overall poorer prognosis than those with Numb-positive cancers. Mechanistically, loss of Numb caused a downmodulation of the p53 tumor suppressor pathway and, concomitantly, upregulation of the oncogenic Notch pathway in breast cancer cells. We also observed that Numb divides asymmetrically during mitosis of mouse and human MaSCs. We, therefore, hypothesized that Numb, via the p53 and Notch pathways, could be a key regulator of asymmetric cell division in MaSCs and that loss of Numb in these cells could be linked to the generation of CSCs and breast tumorigenesis.
During the MAMMASTEM project, we performed a comprehensive investigation of these hypotheses using a mouse genetics approach complemented by experiments on human primary breast cancer cells. Our studies led to the elucidation of Numb as a central player in the regulation of MaSC asymmetric division. This function of Numb is mediated by the p53 and Notch pathways and has important consequences both for normal mammary gland morphogenesis and, when deregulated, for tumorigenesis. Importantly, our data indicate that targeting Numb dysfunction in breast cancer is a valid strategy for the development of anti-cancer therapies capable of eradicating CSCs.
Since we, and others, have shown that Numb is an endocytic protein, we also investigated whether an endocytic mechanism could be involved in tumor suppression by Numb. We performed a series of cellular and biochemical analyses measuring the subcellular localization of the Numb/Hdm2/p53 tricomplex, through which Numb regulates p53 levels. We further analyzed the precise role of Numb in endocytosis and the importance of the correct localization of Numb at the PM and in vesicles. Together, our data support the notion that endocytosis could be involved in the regulation of MaSC self-renewal by Numb and, consequently, could be linked to its tumor suppressor function.
The final aim of the MAMMASTEM project was to identify the E3 ligase(s) responsible for the excessive Numb degradation in Numb-negative breast tumors. Such ligases could represent novel druggable targets that, when inhibited, should result in restoration of Numb protein levels in Numb-negative tumors and, potentially, inhibition of tumor growth. To identify the E3 ligases, we developed a high-throughput siRNA-based assay to screen approximately 600 E3 ligases and components of the ubiquitination machinery for their involvement in Numb degradation. This screening yielded a list of potential candidates, which following validation, led to the identification of two E3 ligase components that mediate Numb degradation in human breast cancer cells and primary tumor cells.
In conclusion, our work has led to the elucidation of novel mechanisms governing regulation of key biological properties in normal and cancer SCs and has opened new avenues in the development of SC-specific strategies for cancer treatment.