Role of Neural (N)-Cadherin in Breast Cancer Cell Stemness and Dormancy in the Bone Microenvironment

SIMPLE SUMMARY: Breast cancer (BrCa) patients experience tumour recurrence 7–25 years after mastectomy. Dormant BrCa cells often home to the bone and, upon reactivation, lead to secondary lesions. We previously demonstrated that, in the bone marrow, dormant BrCa cells are located near the endosteal niche enriched in spindle-shaped N-cadherin-high Osteoblasts (SNOs), show a stem signature like the LT-HSCs and express high levels of Notch2. Here, we observed that Notch2(High) human BrCa MDA cells are also N-Cadherin(High). When injected in vivo, N-Cadherin(High) MDA cells show lower aggressiveness, and higher ability to colonize the endosteal niche and to adhere to SNOs compared to N-Cadherin(Low) MDA cells. These cells have features typical of cancer stem cells, displaying a gene signature similar to the HSCs. In contrast, N-cadherin has a negative role in mouse 4T1 cell stemness and HSC mimicry. In brief, our results identified a role of N-Cadherin in BrCa dormancy and stemness, also highlighting the differences between human and mouse BrCa cell lines. ABSTRACT: Breast cancer cells that interact with spindle-shaped N-Cadherin(+) Osteoblasts (SNOs) are recognised to become dormant through a Notch2-dependent mechanism. We found that Notch2(High) human BrCa MDA-MB231 (MDA) cells also expressed high level of N-Cadherin. This prompted us to hypothesize that N-Cadherin could have a role in MDA-SNO interaction. Of note, the expression of N-Cadherin in MDA cells reduced tumour incidence and bone osteolysis in BrCa mouse model. Moreover, similarly to Notch2(High) MDA cells, the N-Cadherin(High) MDA cells revealed a high expression of the canonical Haematopoietic Stem cell (HSC) markers, suggesting an HSC mimicry, associated with higher ability to form mammospheres. Interestingly, N-Cadherin(High) MDA cells showed greater capacity to adhere to SNOs, while the inhibition of SNO-mediating MDA cell proliferation was unremarkable. To investigate whether these features were shared by mouse BrCa, we used the 4T1 cell line in which N-Cadherin expression was abolished and then rescued. At variance with MDA cells, 4T1 cells expressing N-Cadherin revealed that the latter was associated with a lower expression of the HSC marker, Cxcr4, along with a lower capacity to form mammospheres. Furthermore, the rescue of N-Cadherin expression increased cell-cell adhesion and reduced proliferation of 4T1 cells when they were co-plated with SNOs. In conclusion, we demonstrated that: (i) N-Cadherin(High) and Notch2(High) MDA cells showed similar HSC mimicry and dormancy features; (ii) N-Cadherin mediated BrCa-SNO adhesion; (iii) N-Cadherin had a positive Notch2-dependent role on SNO-induced dormancy and HSC mimicry in MDA cells, and a negative role in 4T1 cell stemness and HSC mimicry.