R-spodin2 enhances canonical Wnt signaling to maintain the stemness of glioblastoma cells

As newly identified Wnt enhancer, R-spondin gene family members have been linked to various cancers; however, their role in isocitrate dehydrogenase-wildtype subtype of human glioblastoma (GBM) cells remains unknown.

Human U87 and U251 cell lines were used to perform the experiments. GBM stem-like cells were enriched in stem cell growth media and induced to differentiate using retinoid acid or growth factor deprivation. Wnt^high and Wnt^low subpopulations were isolated and evaluated by MTS, sphere formation, transwell migration and xenograft formation assays.

R-spondin 2 but not R-spondin 3 potentiates Wnt/β-catenin signaling in GBM cell lines. While R-spondin 2 does not affect cell growth, it induces the expression of pluripotent stem cell markers in combination with Wnt3A. GBM stem-like cells are endowed with intrinsic high activity of β-catenin signaling, which can be further intensified by R-spondin 2. In addition, R-spondin2 promotes stem cell self-renewal and suppresses retinoid acid- or growth factor deprivation-induced differentiation, indicating R-spondin 2 maintains stem cell traits in GBM. On the other hand, we identify subpopulations of GBM cells that show distinctive responsiveness to Wnt/β-catenin signaling. Interestingly, Wnt^high and Wnt^low cells display distinctive biologic properties. Moreover, Wnt^high cell-inoculated xenografts exhibit enhanced tumorigenicity and increased expression levels of R-spondin 2 compared to Wnt^low cell-inoculated xenografts.

Our study reveals a novel regulatory mechanisms underlying the over-activation of β-catenin-mediated signaling in the pathogenesis of GBM.