UAB30, a novel RXR agonist, decreases tumorigenesis and leptomeningeal disease in group 3 medulloblastoma patient-derived xenografts

Abstract

Background: Group 3 tumors account for approximately 25-30% of medulloblastomas and have the worst prognosis. UAB30 is a novel synthetic rexinoid shown to have limited toxicities in humans and significant efficacy in the pediatric neuroectodermal tumor, neuroblastoma. We hypothesized that treatment with UAB30 would decrease tumorigenicity in medulloblastoma patient-derived xenografts (PDXs).

Methods: Three group 3 medulloblastoma PDXs (D341, D384 and D425) were utilized. Cell viability, proliferation, migration and invasion assays were performed after treatment with UAB30 or 13-cis-retinoic acid (RA). Cell cycle analysis was completed using flow cytometry. A flank model, a cerebellar model, and a model of leptomeningeal metastasis using human medulloblastoma PDX cells was used to assess the in vivo effects of UAB30 and RA.

Results: UAB30 treatment led to cell differentiation and decreased medulloblastoma PDX cell viability, proliferation, migration and invasion and G1 cell cycle arrest in all three PDXs similar to RA. UAB30 and RA treatment of mice bearing medulloblastoma PDX tumors resulted in a significant decrease in tumor growth and metastasis compared to vehicle treated animals.

Conclusions: UAB30 decreased viability, proliferation, and motility in group 3 medulloblastoma PDX cells and significantly decreased tumor growth in vivo in a fashion similar to RA, suggesting that further investigations into the potential therapeutic application of UAB30 for medulloblastoma are warranted.

Keywords: Medulloblastoma; PDX; Retinoic acid; UAB30.

Figure 1.. RA and UAB30 induced cellular differentiation in MB cell lines.

a Whole cell lysates from human MB PDXs D425, D384, and D341 were examined with immunoblotting to determine RXR expression. All three PDXs had protein for RXR detected. b Human MB PDX cells were treated with RA or UAB30 at 30 μM for 72 hours and photographs were obtained. Cells were examined for the presence of neurite outgrowths indicating differentiation. In all cell lines, neurite outgrowths (black arrows) were seen following RA and UAB30 treatment. c Neurite outgrowth from three independent experiments was quantitated and presented in graphic form as mean ± SEM.

Figure 2.. RA and UAB30 treatment led to decreased MB cell survival and proliferation and increased apoptosis.

a D341, D384, D425 MB PDX cells were treated with RA or UAB30 at increasing concentrations. After 72 hours of treatment, cell viability was measured with alamarBlue® assays and data reported as mean ± standard error of the mean. There was a statistically significant decrease in viability in all cell lines following RA and UAB30 treatment starting at 10 μM. b Since these compounds resulted in decreased cell survival, we examined the effects of RA and UAB30 on apoptosis in the MB cell. MB PDX cells were treated with RA or UAB30 30 μM for 72 hours and lysates were examined with immunoblotting for PARP cleavage products. There was an increase in cleaved PARP in all cell lines, indicating apoptosis was occurring. c Cell proliferation was detected utilizing CellTiter96® assays. MB PDX cells were treated with increasing concentrations of RA or UAB30 for 5 days. There was a significant decrease in proliferation in all three PDX’s with both RA and UAB30. Bars represent mean ± SEM.

Figure 3.. RA and UAB30 decrease sphere forming capacity in MB PDXs.

a b c PDX cells were plated in 96 well plates with decreasing number of cells per well (1000, 100, 50, 20, 10, 1) in conditioned neurobasal media with vehicle, RA, or UAB30. After 5 days, the number of wells containing spheres was counted for each concentration. Extreme limiting dilution assay analysis was performed and RA and UAB30 both significantly decreased sphere forming capacity in a D341, b D384, and c D425 MB PDX cells. There was no difference in neurosphere formation between RA and UAB30 treatment groups in any PDX. Representative photographs of plates are presented on the graphs.

Figure 4.. RA and UAB30 treatment of MB PDXs led to cell cycle arrest.

a Representative histograms for cell cycle analysis of D341, D384 and D425 human MB PDX cells following treatment with RA or UAB30 (0 μM, 5 μM) for 48 hours. Cells were analyzed by flow cytometry following staining with propidium iodine. There was an increase in the percentage of cells in the G1 phase and a decrease in the percentage in S phase following treatment. b Graphic representation of cell cycle analysis in D341, D384 and D425 MB PDX cells treated with RA or UAB30. There was a significant increase in the G1 phase in cells from all three PDXs (*p ≤ 0.05) and a significant decrease in S phase in all 3 PDXs (*p ≤ 0.05) after treatment, indicating failure to progress through the cell cycle. Experiments were repeated at least in triplicate and reported as mean ± SEM.

Figure 5.. RA and UAB30 decreased MB PDX cell invasion and migration.

a For cell migration, cells (1 × 10⁶) were plated in a 6 well culture plates and treated with RA or UAB30 (0 μM, 10 μM, 30 μM) for 24 hours. Treated cells (1.5 × 10⁵) were plated in Transwell® culture plates in the upper well with neurobasal media; 10% fetal bovine serum was added to the lower chamber as a chemoattractant. The cells were allowed to migrate for 24 hours, then were fixed, stained and counted and migration reported as fold change ± SEM. Migration was significantly inhibited in all 3 PDXs with 10 μM RA and UAB30. Representative photomicrographs of the 10 μM treated migration plates are provided below the migration graphs. b Invasion was assessed in a similar fashion. Treated cells were plated in Transwell® culture plates with Matrigel™ coating the top side of the insert. Cells were allowed to invade for 24 hours, and then fixed, stained and counted. Invasion was reported as fold change ± SEM. Invasion was significantly decreased in all 3 MB PDXs beginning at 10 μM RA or UAB30. Representative photomicrographs of the 10 μM treated invasion plates are provided below the graphs.

Figure 6.. AKT, ERK1/2 and c-myc expression in MB PDX cells with RA or UAB30 treatment.

D341, D384, and D425 cells were treated with RA or UAB30 (0, 10, 30, 50 μM) for 48 hours and whole cell lysates obtained. a Immunoblotting revealed a variable change in AKT phosphorylation following treatment with RA or UAB30; AKT phosphorylation was increased in D341 cells, unchanged in D384 cells, and increased in D425 cells. Total AKT expression was unchanged. b Immunoblotting for ERK1/2 and demonstrated a decrease in total ERK1/2 in D341, D384, and D425 MB PDX cells with both RA and UAB30. c Immunoblotting for c-myc in D384 and D425 cell lysates showed decreased protein expression in both MB PDX cell lysates after RA or UAB30 treatment.

Figure 7.. RA and UAB30 decreased tumor growth in MB tumor bearing mice.

a D425 cells (2.5 × 10⁶) were injected into the right flank of athymic nude mice and animals were randomized to receive vehicle-treated, RA-treated (53 mg/kg/day), or UAB30-treated chow (100 mg/kg/day) (n = 10 mice per group). Flank tumors were measured twice per week. Animals treated with UAB30 had significantly smaller tumors than vehicle treated controls beginning at day 13. Tumors in RA treated animals were smaller than controls, but only reached statistical significance at 17 days. There were no significant differences in changes in tumor volumes between RA and UAB30 treated animals. b D341 cells (5 × 10⁵ cells) were injected into the cerebellum of nude mice and the animals were randomized to receive treatment with vehicle-treated, RA-treated (53 mg/kg/day), or UAB30-treated chow (100 mg/kg/day) (n = 9 mice per group). The mice were monitored twice daily for the development of neurologic symptoms (e.g. hemiparesis, lethargy). Animals receiving RA and UAB30 had significantly prolonged survival compared to animals receiving control chow. Open arrow in upper insert demonstrates intracerebellar location of the tumor and H&E staining shown in the bottom insert shows infiltrating MB tumor cells in the cerebellum (bottom panel, closed arrow). c Following intraventricular injection of D341 cells (5×10⁵ cells), animals were randomized to receive treatment with vehicle-treated, RA-treated (53 mg/kg/day), or UAB30-treated chow (100 mg/kg/day) (n = 8 mice per group). The mice were monitored daily for the development of neurologic symptoms twice daily. Bioluminescence imaging was utilized to follow development of tumors and representative bioluminescence images are shown. There are marked differences in tumor burden in the brains and spinal canals (black arrows) of the animals treated with vehicle alone compared to RA or UAB30 treated animals. There was a statistically significant difference in the tumor burden found in the brain and spinal canal between the animals treated with RA or UAB30 compared to control vehicle-treated animals. There was no significant difference in brain or spinal canal tumor burden between the RA or UAB30 groups. d Bioluminescence data for tumor burden in the brain. There were significant decreases in total flux in animals treated with RA or UAB30. There were no differences between RA and UAB30 treated animals. e Bioluminescence data for tumor burden in the spinal canal. There were significant decreases in total flux in animals treated with RA or UAB30 compared to control animals. There were no differences between RA and UAB30 treated animals. f H&E staining confirmed tumor cells were present in the spinal canal (left panel, black arrows, right panel black arrows).