Pharmacological activation of mGlu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas

Abstract

Moving from the evidence that activation of type 4 metabotropic glutamate (mGlu4) receptors inhibits proliferation and promotes differentiation of cerebellar granule cell neuroprogenitors, we examined the expression and function of mGlu4 receptors in medulloblastoma cells. mGlu4 receptors were expressed in 46 of 60 human medulloblastoma samples. Expression varied in relation to the histotype (nodular desmoplastic>classic>>large-cell anaplastic) and was inversely related to tumor severity, spreading, and recurrence. mGlu4 receptors were also found in D283med, D341med, and DAOY medulloblastoma cell lines, where receptor activation with the selective enhancer PHCCC inhibited adenylyl cyclase and the phosphatidylinositol-3-kinase pathway without affecting the mitogen-activated protein kinase, Sonic Hedgehog, and Wnt pathways. Interestingly, mGlu4 receptor activation reduced DNA synthesis and cell proliferation in all three cell lines. This effect was abrogated by the phosphatidylinositol-3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. In in vivo experiments, repeated subcutaneous injections of N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) reduced the growth of D283med and DAOY cell xenografts in nude mice. More remarkably, subcutaneous or intracranial injections of PHCCC during the first week of life prevented the development of medulloblastomas in mice lacking one Patched-1 allele and x-irradiated 1 d after birth. These data suggest that mGlu4 receptor enhancers are promising drugs for the treatment of medulloblastomas.

Figure 1.

Expression of mGlu1a (A), mGlu2/3 (B), mGlu4 (C), and mGlu5 (D) receptors in medulloblastoma bioptic samples are shown. Sections are counterstained with hematoxylin–eosin. In A, immunolabeling of a cerebellar Purkinje cell occasionally present in the field provides a positive internal control for mGlu1a receptor expression (arrow). Note that in C, mGlu4 immunolabeling preferentially localizes to the cell membrane. Scale bar, 20 μm.

Figure 2.

Expression of mGlu4 receptors in human medulloblastomas in relation to the histological variant, tumor spreading, and clinical outcome. The extent of immunoreactivity (I.R.) was scored from 0 to 4 as outlined in Materials and Methods. A, The distribution of mGlu4 receptor expression in medulloblastoma samples from 60 patients. B, Receptor expression in relation to the histological variant. C–E, The extent of expression in samples from patients with or without spinal cord metastases (C), CSF spreading (D), and tumor recurrence (E). F, The extent of expression in relation to the clinical outcome (survival or death). The outcome was unknown in 23 patients. Values are means + SEM. *p < 0.05 (Student's t test) versus the corresponding control group.

Figure 3.

Medulloblastoma cell lines express functional mGlu4 receptors. A, B, Expression of mGlu4 receptor mRNA and protein in D283med, D341med, and DAOY cells. In A, the lack of contamination by genomic DNA is shown by the single β-actin amplimer (see Materials and Methods). M, Markers. In B, the monomeric 100 kDa band of mGlu4 receptors is shown. C, Inhibition of FSK (10 μm)-stimulated cAMP formation by l-AP-4 (100 μm), PHCCC (30 μm), or baclofen (10 μm) in D283med, D341med, and DAOY cells preincubated or not with PTX (10 nm, applied 16–18 h before). Values are means ± SEM of 6–12 determinations. p < 0.05 (one-way ANOVA and Fisher's PLSD) versus FSK alone (*) or versus FSK plus baclofen (#). D, Immunoblot analysis of the Gα_z protein in the three medulloblastoma cell lines. Note the absence of the protein in the rat cerebellum and in primary cultures of cerebellar granule cells. Cb, Rat cerebellum; Hp, rat hippocampus; CG, extracts from primary cultures of rat cerebellar granule cells grown in medium containing 25 mm K⁺ at 7 d in vitro.

Figure 4.

Inhibition of the PI-3-K pathway by PHCCC in medulloblastoma cells. D283med and DAOY cells were serum starved for 16–18 h and challenged with 10% FCS and/or PHCCC (30 μm) for 5 min. For densitometric analysis, phosphorylated-AKT (p-Akt) values were normalized by the amount of nonphosphorylated AKT for D283med cells and for the average amount of nonphosphorylated ERK1 and ERK2 for DAOY cells. Values are means ± SEM of 6–12 determinations. *p < 0.05 (Student's t test) versus the corresponding values obtained in the absence of PHCCC. Note that PHCCC has no effect on the stimulation of the MAPK pathway. The lanes are loaded with a different order for D283med and DAOY cells. Bas, Basal.

Figure 5.

PHCCC fails to affect the SHH pathway and the canonical Wnt pathway in medulloblastoma cells. A, Gli1 mRNA levels in D283med and DAOY cells treated or not with PHCCC (30 μm) for 24 h. B, Expression of a reporter gene (luciferase) controlled by Gli1-responsive elements (DGliRE) in D283med cells exposed to PHCCC (30 μm) for 20 or 24 h. C, The basal and Wnt7a-stimulated expression of a reporter gene (luciferase) controlled by TCF/LEF-responsive elements in D283med cells exposed to PHCCC (30 μm) for 20 or 24 h. Values are means ± SEM of 4–12 determinations. CNT, Controls.

Figure 6.

Activation of mGlu4 receptors inhibits DNA synthesis in medulloblastoma cells. [³H]Thymidine incorporation was assessed in D283med, D341med, and DAOY cells serum starved for 16–18 h and challenged with l-AP-4 (100 μm), PHCCC (0.3, 3, or 30 μm), DHPG (100 μm), LY379268 (0.1 μm), AMPA (100 μm), kainate (100 μm), NMDA (100 μm), and baclofen (10 μm). Values are means ± SEM of 6–60 determinations. *p < 0.05 (one-way ANOVA and Fisher's PLSD) versus controls (CNT).

Figure 7.

Continuous exposure to PHCCC reduces the growth of medulloblastoma cells. PHCCC (30 μm) was applied to D283med, D341med, and DAOY cells every other day for 9 d, starting 4 h after plating. Values are means ± SEM of 12 determinations. *p < 0.05 (Student's t test) versus the respective controls (vehicle).

Figure 8.

Inhibition of DNA synthesis by PHCCC (30 μm) in D283med cells incubated in the absence or presence of mGlu receptor antagonists or drugs that interfere with intracellular signaling pathways. FSK, 10 μm; 8Br-cAMP, 1 mm; UO126, 10 μm; LY294002, 10 μm; PTX, 10 nm (applied 16–18 h before PHCCC); MSOP, 200 μm; LY341495, 0.1 μm; MPEP, 1 μm; CPCCOEt, 10 μm. Values are means ± SEM of 6–24 determinations. *p < 0.05 (Student's t test) versus the corresponding values obtained in the absence of PHCCC.

Figure 9.

PHCCC inhibits the development of medulloblastomas in in vivo models. A, B, Systemic injections of PHCCC (5 mg/kg, s.c., once per day for 7 d) reduces the growth of DAOY (A) and D283med (B) cell xenografts implanted under the skin of nude mice. Tumor size was assessed on the day before the beginning of the treatment (time 0, corresponding to 7 d after implantation), after 4 and 7 d of treatment, and 7 d after drug withdrawal (corresponding to day 14). Values are means ± SEM of six to eight individual determinations. *p < 0.05 (Student's t test) versus the respective values obtained in mice treated with vehicle. C–G, Early postnatal treatment with PHCCC inhibits the growth of medulloblastoma in Ptc^neo67/+ mice irradiated after birth. C, The number of Ptc^neo67/+ mice x-ray irradiated at P1 and treated subcutaneously or intracerebrally with vehicle or PHCCC (5 mg/kg, s.c., once per day for 7 d; or 10/nmol/0.5 μl intracranially every other day for 7 d, during the first 7 d of life) expressing large-size tumors, small-size tumors, or pre-neoplastic lesions. D–G, Histological analysis of the cerebellum of Ptc^neo67/+ mice treated postnatally with vehicle or PHCCC and examined at 10 weeks of age. D, Large-sized tumor from a mouse treated systemically with vehicle (magnification, 100×). E, Small-sized tumor in one mouse treated systemically with PHCCC (magnification, 600×). F, Pre-neoplastic lesion in one mouse treated systemically with PHCCC (magnification, 400×). G, Presence of ectopic granule cells in the molecular layer of the cerebellar cortex (arrows) in a mouse treated intracranially with PHCCC (magnification, 200×).