Abstract
BackgroundAs 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.MethodsHuman 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. Wnthigh and Wntlow subpopulations were isolated and evaluated by MTS, sphere formation, transwell migration and xenograft formation assays.ResultsR-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, Wnthigh and Wntlow cells display distinctive biologic properties. Moreover, Wnthigh cell-inoculated xenografts exhibit enhanced tumorigenicity and increased expression levels of R-spondin 2 compared to Wntlow cell-inoculated xenografts.ConclusionOur study reveals a novel regulatory mechanisms underlying the over-activation of β-catenin-mediated signaling in the pathogenesis of GBM.
Highlights
As newly identified Wnt enhancer, R-spondin gene family members have been linked to various cancers; their role in isocitrate dehydrogenase-wildtype subtype of human glioblastoma (GBM) cells remains unknown
Rspo2 potentiates Wnt/β‐catenin signaling in GBM cell lines We first determined the expression of RSPO2, RSPO3 and their receptors LGR4–6 in GBM cell lines U251 and U87 by real-time PCR
The results showed that while the expression level of RSPO2 was comparable between U87 and U251, RSPO3 was more highly expressed in U251
Summary
As newly identified Wnt enhancer, R-spondin gene family members have been linked to various cancers; their role in isocitrate dehydrogenase-wildtype subtype of human glioblastoma (GBM) cells remains unknown. -called cancer stem cells (CSCs) have been isolated from human tumors [2]. It has been well established that Wnt/β-catenin pathway contributes to cancer development since the discovery that Wnt was capable of promoting breast cancer 30 years ago [12]. This recognition has been largely strengthened by the findings that intracellular components of the Wnt/β-catenin signaling cascade such as APC, Axin, and β-Catenin (CTNNB1), are mutated in a large variety of human cancers [13]. Besides the numerous mutations found in the pathway components, the contribution of various family members of Wnt ligand has been recognized in human cancers [14]. It is conceivable that other regulatory mechanisms that trigger the over-activation of β-catenin-mediated signaling are involved in the pathogenesis of GBM [23, 24]
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