Non-canonical Wnt/Ca2+ signaling is essential to promote self-renewal and proliferation in colon cancer stem cells

Abstract

IntroductionCancer Stem Cells (CSC) are responsible for maintaining tumor growth, chemoresistance, and metastasis. Therefore, understanding their characteristics is critical to progress in cancer therapy. While the contribution of the canonical Wnt/b-catenin signaling in both normal and CSCs had been well established, the function of non-canonical Wnt signaling cascades in stem cells is unclear. Recently, we reported that Wnt ligands trigger complex signaling in which the canonical and non-canonical responses can be simultaneously activated by one ligand in colon cancer cells, suggesting, therefore, that noncanonical Wnt pathways may also be important in CSCs.MethodsThe present work aimed to know the role of the Wnt/Ca2+ pathway in colon CSCs. We used tumorspheres as a model of CSCs enrichment of CRC cell lines with different Wnt/b-catenin contexts.ResultsUsing Wnt3a and Wnt5a as prototype ligands to activate the canonical or the non-canonical pathways, respectively, we found that both Wnt3a and Wnt5a promote sphere-formation capacity and proliferation without stimulating b-catenin-dependent transcription. Upregulation of sphere formation by Wnt5a or Wnt3a requires the downstream activation of Phospholipase C and transcriptional factor NFAT. Moreover, the single specific inhibition of PLC or NFAT, using U73122 and 11R-VIVIT, respectively, leads to impaired sphere formation.DiscussionOur results indicate that both types of ligands activate the Wnt/Ca2+ signaling axis to induce/maintain the self-renewal efficiency of CSCs, demonstrating to be essential for the functions of CSC in colon cancer.

WNT Signaling in Stem Cells: A Look into the Non-Canonical Pathway

“…Because no detection of β-catenin in mature blood cells was described, the dispensable role of β-catenin in the hematopoietic process under physiological conditions was proposed [ 22 , 23 ]. Consistent with this, it has been reported recently [ 24 ] that using Wnt3a and Wnt5a as prototype ligands to activate the canonical or the non-canonical pathways, respectively, in colon cancer stem cells (CSCs), both ligands promote sphere-formation capacity and proliferation in a β-catenin-independent manner. Both Wnt3a and Wnt5a were also found to induce or maintain sphere formation by the downstream activation of Phospholipase C and transcriptional factor NFAT.…”

“…Both Wnt3a and Wnt5a were found to induce or maintain sphere formation by triggering Phospholipase C (PLC) and transcriptional factor NFAT activation. Consistent with this, the inhibition of PLC or NFAT impaired sphere formation, indicating that the non-canonical Wnt/calcium signaling activated by both Wnt ligands is essential for self-renewal of CSC, discussed in detail later in the text [ 24 ].…”

“…Both Wnt3a and Wnt5a were also found to induce or maintain sphere formation by the downstream activation of Phospholipase C and transcriptional factor NFAT. The single specific inhibition of PLC or NFAT leads to impaired sphere formation, indicating, therefore, that the non-canonical Wnt/Ca2 + signaling activated by both Wnt ligands is essential to induce/maintain the self-renewal efficiency of the CSCs [ 24 ].…”

“…Wnt3a upregulated the canonical Wnt signaling dependent on Lrp5/6, while Ror2 and JNK activities were necessary for Wnt5a stimulus [ 27 ]. Indeed, recently it has been reported that using Wnt3a and Wnt5a as prototype ligands to activate the canonical or the non-canonical pathways, respectively, promotes sphere-formation capacity and proliferation through the β-catenin-independent manner in colon cancer cells [ 24 ]. Both Wnt3a and Wnt5a were found to induce or maintain sphere formation by triggering Phospholipase C (PLC) and transcriptional factor NFAT activation.…”

“…As mentioned before, it was shown recently that Wnt3a and Wnt5a stimulate sphere formation in colorectal CSCs in a β-catenin-independent transcriptional activity [ 24 ]. Interestingly, the spheres-derived cells mobilized calcium in response to both Wnt ligands and pointed out Wnt/calcium signaling as an essential pathway to induce and maintain the self-renewal capacity of colon CSCs.…”