SummaryLoss of p53 function is invariably associated with cancer. Its role in tumor growth was recently linked to its effects on cancer stem cells (CSCs), although the underlying molecular mechanisms remain unknown. Here, we show that c-myc is a transcriptional target of p53 in mammary stem cells (MaSCs) and is activated in breast tumors as a consequence of p53 loss. Constitutive Myc expression in normal mammary cells leads to increased frequency of MaSC symmetric divisions, extended MaSC replicative-potential, and MaSC-reprogramming of progenitors, whereas Myc activation in breast cancer is necessary and sufficient to maintain the expanding pool of CSCs. Concomitant p53 loss and Myc activation trigger the expression of 189 mitotic genes, which identify patients at high risk of mortality and relapse, independently of other risk factors. Altogether, deregulation of the p53:Myc axis in mammary tumors increases CSC content and plasticity and is a critical determinant of tumor growth and clinical aggressiveness.
The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.
Tumour suppressor genes are frequently affected by somatic alterations in cancer, and the impairment of their normal function provides a strong contribution to tumourigenesis. Short-hairpin (sh) RNA library screens have been employed as powerful genetic tools to uncover important new players in human cancer 1-5 . To identify potential novel tumour suppressor genes acting in the p53 pathway, we performed an shRNA screen using a cell-based model in which only a single additional genetic event disrupting the p53 pathway is required to obtain in vitro transformation. By using this approach, we report here on the identification of the Frizzled-ligand Norrin (Norrie disease protein) as a candidate tumour suppressor. Inhibition of Norrin expression promotes anchorage-independent growth, confers a strong growth advantage to cells and causes a reduction in p53 protein levels. Conversely, recombinant human Norrin increases p53 levels in a β-catenin dependent fashion. Interestingly, Norrin expression is stimulated by oncogenic H-RAS and BRAF, suggesting that Norrin is part of an early fail-safe mechanism to suppress transformation, and that mutation or down regulation of Norrin could contribute to tumour progression. Indeed, we found that Norrin expression is significantly decreased in melanoma, breast, prostate and ovarian cancer. These findings support the existence of a novel autocrine/paracrine feedback loop that constrains tumourigenesis, in which the crosstalk between the RAS and -catenin pathways play an unanticipated role.
The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.
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