Interazione tra la nicchia endosteale e le cellule tumorali mammarie dormienti: caratterizzazione molecolare e funzionale

Breast Cancer (BrCa) is the most commonly diagnosed malignancy in women worldwide. Early diagnosis, screening programs and therapies increased the overall chances of survival for patients, with a ten-year survival rate above 95%. The main problem for patients remains the long-term recurrence, which can occur due to a small percentage of the BrCa cells colonizing distant organs that can acquire a quiescent status and remain resistant to conventional therapies for years. After this period, specific stimuli, not yet fully known, prompt the cells to reactivate, generating a new tumor. In this thesis, it has been provided an overview of BrCa features, diagnosis and treatment, describing in detail the general metastatic process, and in particular the mechanism involved in the development of BrCa bone metastases. Given that the project was focused on the process of cellular dormancy, this latter has been described in detail, with a special emphasis on the “niche” concept that, in the bone microenvironment, is physiologically involved in the maintenance of the Hematopoietic Stem Cell (HSC) pool indispensable for the generation of blood cells. Previous results published by our laboratory had demonstrated the relevance of the endosteal niche enriched in spindle-shaped N-Cadherin+ Osteoblasts (SNOs), known to be involved in the quiescence of Long-Term HSCs, for the maintenance of the quiescent status of dormant BrCa cells. In this article, it was demonstrated that SNOs can reduce the proliferation of human BrCa MDA-MB231 (MDA) cells expressing high level of Notch2, and that Notch2High BrCa cells also express genes typical of HSCs, known to physiologically interact with SNOs remaining quiescent. In the project presented in this thesis, we investigated N-Cadherin as another potential player of BrCa cell-SNO interaction that could induce BrCa cellular dormancy. We also extended the knowledge on the involvement of the Notch family in BrCa dormancy, comparing various cell lines expressing high levels of Notch1, Notch2 or both, and profiling the molecular signatures obtained by RNAdSeq in Notch1High and Notch2High MDA cells. The results demonstrated that: • N-CadherinHigh and Notch2High MDA cells show similar HSC-mimicry and SNO-induced dormancy features. • N-Cadherin mediates MDA-SNO adhesion in the endosteal niche. • High expression of Notch2, but not of Notch1, confers MDA cells molecular signatures of pluripotency, HSC-mimicry and dormancy • HSC genes appear not to be implicated in SNO-induced MDA cellular dormancy. 6 • Notch2High MDA cells express high level of CD177 glycosylphosphatidylinositol (GPI)-linked surface glycoprotein that recognizes the plasminogen activator urokinase receptor (uPAR), the CD11b chain of the Mac-1 (alphaMbeta2; Cd11b/CD18; complement receptor-3) integrin and the CarcinoEmbrionic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) expressed by SNOs. Based on these results, we hypothesize that the CD177 associated pathways could potentially contribute to the SNO-mediated dormancy of Notch2High MDA cells. To demonstrate this hypothesis, further work will be necessary beyond the study presented in this thesis. This goal will be achieved through functional studies that will make use of CD177 antagonists in the in vitro and in vivo models of dormancy described in this thesis.