Cripto-1 controls dynamic stemness states in human colon cancer cells

Cripto-1 (CR1) is a GPI-linked protein expressed in embryonic tissues. Although absent in normal adult tissues, CR1 expression is often reactivated in cancer cells, contributing to tumor propagation and dissemination. We found that CR1 is expressed in colon cancer stem cells (CSC) isolated from human tumor specimens and expanded in vitro as tumor spheroids. CR1 expression largely associated with other stemness markers such as CD133, Nanog, ALDH and Lgr5 and disappeared upon CSC differentiation. In CSC populations, CR1 expression was subjected to a marked fluctuation (20-80% positivity) that reflected alternating states of self-renewal capacity. CSC separation on the basis of CR1 expression yielded two populations with different colony-forming abilities and expression/localization of Nanog, Lgr5 and beta-catenin, suggesting that CR1 expression distinguishes hierarchically different CSC subsets. CR1 expression on sorted CSC populations changed rapidly, resulting in a conversion of CR1-negative cells in CR1-super-positive cells with elevated proliferative and tumor-forming ability. Despite the presence of both CR1 signaling partners Nodal and GRP78, only GRP78 inhibition was able to impact on colon CSC proliferation and self-renewal, indicating an involvement of CR1-GRP78 interactions in CSC regulation. Stable CR1 silencing strongly affected CSC proliferation, self-renewal and in vivo tumorigenicity, reducing stem cell content in tumor xenografts as demonstrated by clonogenic and serial transplantation assays. Notably, inducible CR1 silencing in established tumor xenografts significantly decreased tumor progression and reduced CSC content, indicating an impact of CR1 inhibition on full-formed tumors. Finally, the absence of CR1 compromised CSC metastatic abilities, as demonstrated by the lack of lung metastatization by CR1-silenced CSC. These results indicate that stemness is a dynamic condition in colon cancer cells and is regulated by CR1 expression.