Effects of altered ephrin-A5 and EphA4/EphA7 expression on tumor growth in a medulloblastoma mouse model

Abstract

Background: Members of the Eph/ephrin gene families act as key regulators of cerebellar development during embryogenesis. Aberrant signaling of Eph family of receptor tyrosine kinases and their ephrin ligands has also been implicated in human cancers. Medulloblastoma is an aggressive primitive neuroectodermal tumor that originates from granule neuron precursors in the cerebellum. Previous studies have suggested a role for the ephrin-A5 ligand and its receptors, EphA4 and EphA7, in granule cell-precursor formation and in guiding cell migration. In the present study, we investigated the effects of genetic loss of ephrin-A5, EphA4, and EphA7 on the spatiotemporal development of medulloblastoma tumors in the context of the smoothened transgenic mouse model system.

Findings: Radiographic magnetic resonance imaging (MRI) was performed to monitor tumor growth in a genetically engineered mouse model of medulloblastoma. Tumor tissue was harvested to determine changes in the expression of phosphorylated Akt by Western blotting. This helped to establish a correlation between genotype and/or tumor size and survival. Our in vivo data establish that in ND2-SmoA1 transgenic mice, the homozygous deletion of ephrin-A5 resulted in a consistent pattern of tumor growth inhibition compared to their ephrin-A5 wild-type littermate controls, while the loss of EphA4/EphA7 failed to produce consistent effects versus EphA4/EphA7 wild-type mice. A positive correlation was evident between tumor size, p-Akt, and proliferating cell nuclear antigen (PCNA) expression in our transgenic mouse model system, regardless of genotype.

Conclusions: Taken together, our findings underscore the importance of targeting specific members of the Eph/ephrin families in conjunction with the Akt pathway in order to inhibit medulloblastoma tumor growth and progression.

Fig. 1

MRI-based morphometric analyses identified a reduction in tumor size in the ephrin-A5 ^−/− Smo mouse model. a Representative images are shown for ephrin-A5 ^−/− Smo mice and their ephrin-A5 ^+/+ Smo littermate controls, monitored by sequential MRI. b Volumetric tumor growth analysis. Data represent mean ± standard deviation from different tumor tissues (*p ≤ 0.05)

Fig. 2

Western blot analysis shows altered expression of p-Akt and PCNA that related to tumor size and genotype in ephrin-A5 ^−/− Smo mouse model. a Western blot analysis in representative tumor sections suggests a decrease in p-Akt and PCNA expression in smaller tumors (<10 mm³) with loss of the ephrin-A5 gene. Total Akt levels seem to be unchanged. b Densitometric analysis of p-Akt expression (*p ≤ 0.05) and c PCNA expression (p = not significant) in ephrin-A5 ^+/+ Smo versus ephrin-A5 ^−/− Smo tumors. Data represent mean ± standard deviation from different tumor tissues

Fig. 3

Representative MRI scans show that genetic alteration of EphA4/EphA7 genotype yields no consistent effect on tumor size in the Smo medulloblastoma animal model. EphA4 ^−/− EphA7 ^−/− Smo mice and their EphA4 ^+/− EphA7 ^−/− Smo and EphA4 ^+/+ EphA7 ^−/− Smo littermate controls were followed by serial MRI. Representative images are shown for a EphA4 ^+/+ EphA7 ^−/− Smo, EphA4 ^+/− EphA7 ^−/− Smo, and EphA4 ^+/− EphA7 ^−/− Smo pairs. b Volumetric tumor growth analysis. Data represent mean ± standard deviation from different tumor tissues. (p = not significant)

Fig. 4

Western blot analysis suggests a correlation between p-Akt and PCNA levels and tumor size regardless of genotype in EphA4 ^+/+ EphA7 ^−/− Smo, EphA4 ^+/− EphA7 ^−/− Smo, and EphA4 ^−/− EphA7 ^−/− Smo mice. a Western blot analysis in tumor tissues indicated expression of p-Akt and PCNA correlative of tumor size. Lysates were collected from mice and Western blot analysis was done to determine the expression of p-Akt, total Akt, and PCNA. b Densitometric analysis of p-Akt expression (*p ≤ 0.05) and c PCNA levels (p = not significant) in EphA4 ^+/+ EphA7 ^−/− Smo, EphA4 ^+/− EphA7 ^−/− Smo, and EphA4 ^−/− EphA7 ^−/− Smo tumors. Data represent mean ± standard deviation from different tumor tissues