Wnt/β-catenin signaling inhibits the Shh pathway and impairs tumor growth in Shh-dependent medulloblastoma

studies showing inhibitory effects of Wnt3a through noncanonical Wnt-signaling on GNps [1]. Next, we introduced a Ctnnb1(ex3)Fl/+ allele into Math1-cre::SmoM2Fl/+ mice, a model for shh-medulloblastoma [10]. Activation of the shh pathway in Math1-positive GNps resulted in a thickened external granule cell layer (eGL) and medulloblastoma (Fig. 1b, c, suppl. Fig. 1a). Additional activation of the Wnt-pathway (Math1-cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ mice) notably reduced growth of medulloblastoma and resulted in decreased cerebellar size (Fig. 1b, d, suppl. Fig. 1a). GNps within the cochlear nucleus also form shh-dependent medulloblastoma in Math1-cre::SmoM2Fl/+ mice [3]. Brain stem tumors generated in Math1-cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ mice were substantially smaller and showed decreased proliferation in an analogous manner when compared to tumors in Math1-cre::SmoM2Fl/+ mice (suppl. Fig. 1b). Importantly, Math1-cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ mice (n = 16) survived significantly longer than Math1cre::SmoM2Fl/+ animals (n = 53, p < 0.0001, Fig. 1e). To see how the additionally activated Wnt-signaling affected shh-signaling in medulloblastoma, we performed in situ hybridizations and immunohistochemistry. Compared to Math1-cre::SmoM2Fl/+ medulloblastomas, Math1cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ tumors showed high expression of the Wnt target Axin2 together with nuclear β-catenin, while the expression of the shh target Gli1 was strongly decreased (Fig. 1f). We further used FACs to isolate GFp+ cells from SmoM2Fl/+ or SmoM2Fl/+Ctnnb1(ex3)Fl/+ GNp cultures after Cre-IRES-GFP virus treatment, and quantitative rT-pCr analysis confirmed that Axin2 was upregulated in SmoM2Fl/+Ctnnb1(ex3)Fl/+ GNps, whereas Gli1 was downregulated (Fig. 1g). Hence, active canonical Wnt signaling is a potent inhibitor of the shh pathway in medulloblastoma. Next, we tested on a cellular level, whether this Wnt activity was the reason for the reduced tumor 30 % of medulloblastomas show activated sonic-hedgehog (shh) signaling. shh is crucial for the proliferation of cerebellar granule neuron precursors (GNps) [12], but constitutive activation of the shh pathway in GNps leads to medulloblastoma [10]. In contrast, canonical Wnt/β-catenin signaling is required for the maturation of GNps, and constitutive activation of this pathway causes significant proliferation deficits [6]. To develop new therapeutic strategies for patients with shh-medulloblastoma, we asked whether the physiological roles of Wnt/β-catenin signaling can be utilized to treat shhmedulloblastomas that have developed from GNps. SmoM2Fl/+ [7], Ctnnb1(ex3)Fl/+ [4], or SmoM2Fl/+ Ctnnb1(ex3)Fl/+ cerebellar GNps were cultured as described [6] and transduced with Cre-IRES-GFP viruses. Cre treatment of SmoM2Fl/+ cerebellar GNps led to a 2.6-fold proliferation increase due to active shh signaling (Fig. 1a, p = 0.008). Wnt pathway activation in Ctnnb1(ex3)Fl/+ GNps reduced proliferation to 52 % (Fig. 1a, p = 0.029). Importantly, Wnt-activation significantly decreased oncogenic GNp proliferation driven by SmoM2 (Ctnnb1(ex3)Fl/+SmoM2Fl/+ condition, Fig. 1a, p = 0.028), and cells reached proliferation levels similar to the wild type. Thus, the proliferation of shh-medulloblastoma cells may be blocked through canonical Wnt signaling. These results extend previous