RSPO3 is a glycoprotein secreted by mammary basal cells that induces canonical Wnt-Pathway activation, EMT and aggressive tumorigenic behavior in vivo

TOCCI, J. M.; FELCHER, C. M.; GODDIO, M. V.; CASTRO, O. A.; COSO, O. A.; KORDON, E. C.

Bariloche

Simposio; Third South American Spring Symposium in Signal Transduction and Molecular Medicine; 2015

R-spondins (RSPOs) is a family of four secreted proteins. Over the last years, they have been implicated in significantprocesses like embryonic development, tissue differentiation and human diseases, and they have been postulated as potentstem cells growth factors for therapeutic applications. RSPOs, which are usually co-expressed with WNT proteins, aredescribed as positive modulators of both the canonical and non-canonical WNT signalling pathways. It has beendemonstrated (by our group and others) that RSPOs, particularly RSPO3, are frequently mutated and over-expressed inMouse Mammary Tumor Virus (MMTV) induced tumors. We have analyzed RSPO3 expression in human and mousemammary cancer cells (not infected by MMTV) and we found high levels of Rspo3 in basal or triple-negative tumor lines. Todetermine whether this protein may play a role in normal mammary development and function, we investigated itsexpression profile in mammary epithelial cells sorted by flow-cytometry. Interestingly, Rspo3 mRNA was detected in thebasal-stem cell enriched compartment. In addition, our results show that RSPO3 addition to non-tumorigenic mammaryepithelial cells in culture induces morphological and molecular changes associated to epithelial-mesenchymal transition(EMT). Accordingly, stable silencing of Rspo3 in cultured basal mouse mammary cancer cells, by specific shRNA transfection,causes inhibition of migration (by wound healing assay), decrease of vimentin expression, and repression of the canonicalWNT signaling pathway, analyzed by activation of the TOP-FLASH reporter. Importantly, when transplanted into mice, theshRSPO3 cells generated less tumors, which grew significantly later than control cells. Finally, we believe that in the normalgland the RSPO3 glycoprotein is secreted by basal epithelial cells, acting only on this compartment, where it remains due toits high affinity to the extracellular matrix. This hypothesis is supported by the fact that RSPO3 levels in conditioned mediumof cultured basal mammary cells can be significantly increased upon treatment with soluble heparin. Taking together, ourresults indicate that RSPO3 is expressed by basal/stem cells of the normal mammary gland, being probably relevant for themaintenance of this compartment. In addition, our data show that over-expression and abnormal activity of this protein inundifferentiated or luminal mammary cells trigger oncogenic pathways, which lead to cancer development most likelythrough canonical WNT-pathway activation.