RSPO3 IS A GLYCOPROTEIN SECRETED BY MAMMARY TUMOR CELLS THAT INDUCES WNTPATHWAY ACTIVATION AND EMT

TOCCI J; FELCHER C; GODDIO MV; CASTRO OA; COSO O; KORDON E

San Carlos de Bariloche

Encuentro; SISTAM 2015; 2015

R-spondins (RSPOs) is a family of four secreted proteins. Over the last years, they have been implicated in significant processes like embryonic development, tissue differentiation and human diseases, and they have been postulated as potent stem cells growth factors for therapeutic applications. RSPOs, which are usually co-expressed with WNT proteins, are described as positive modulators of both the canonical and non-canonical WNT signalling pathways. It has been demonstrated (by our group and others) that RSPOs, particularly RSPO3, are frequently mutated and over-expressed in Mouse Mammary Tumor Virus (MMTV) induced tumors. We have analyzed RSPO3 expression in human and mouse mammary cancer cells (not infected by MMTV) and we found high levels of Rspo3, by qRT-PCR and Western blot analysis, in basal or triple-negative tumor lines. To determine whether this protein may play a role in normal mammary development and function, we investigated its expression profile in mammary epithelial cells sorted by flow-cytometry. Interestingly, Rspo3 mRNA was detected only in the basal-stem cell enriched compartment. In addition, our results show that RSPO3 addition to non-tumorigenic mammary epithelial cells in culture induces morphological and molecular changes associated to epithelial-mesenchymal transition (EMT), which are associated to canonical WNT-pathway activation. Accordingly, stable silencing of Rspo3 in cultured basal mouse mammary cancer cells, by specific shRNA transfection, causes inhibition of migration (by wound healing analysis), vimentin expression, by qRT-PCR and repression of the canonical WNT signaling pathway, analyzed by activation of the TOP-FLASH reporter. Importantly, when transplanted into mice, the shRSPO3 transfected cells generated less tumors, which grew significantly later than control cells. We believe that in the normal gland the RSPO3 glycoprotein is secreted by basal epithelial cells, acting only on this compartment, where it remains due to its high affinity to the extracellular matrix. This hypothesis is supported by the fact that RSPO3 levels in conditioned medium of cultured basal mammary cells can be significantly increased upon treatment with soluble heparin. Taking together, our results indicate that RSPO3 is expressed by basal/stem cells of the normal mammary gland, being probably relevant for the maintenance of this compartment. In addition, our data show that over-expression and abnormal activity of this protein in undifferentiated or luminal mammary cells trigger oncogenic pathways, which lead to cancer development most likely through canonical WNT-pathway activation.