YAP expression promotes medulloblastoma cell tumorigenicity
We have previously shown that YAP is highly expressed in both human and mouse medulloblastomas, the YAP locus is highly amplified in 3% of the human medulloblastomas analyzed, and YAP expression is sufficient to drive proliferation of CGNPs in the absence of their obligate mitogen Shh (Fernandez et al 2009). In NeuroD2-SmoA1 mouse medulloblastomas, YAP is most highly expressed in tumor cells surrounding blood vessels, a micro-environment known as the peri-vascular niche (PVN). These cells are proposed to have “tumor repopulating” properties, and indeed, after irradiation these YAP-expressing cells can be found proliferating, possibly contributing to the regrowth of the tumor. To determine whether YAP expression increases medulloblastoma cell tumorigenicity, we cultured cells from medulloblastomas arising in NeuroD2-SmoA1 mice, infected them with retroviruses carrying either YAP or GFP, then implanted these cells intra-cranially into post-natal (PN) day 2 NOD/SCID pups. We then monitored the animals for symptomatic evidence of medulloblastoma (head tilt, seizures), confirmed upon sacrifice and dissection of the tumors. Ectopic expression of YAP enhances medulloblastoma growth, as determined by the ability of YAP-infected NeuroD2-SmoA1 cells to form tumors in the recipient mice, more effectively than GFP-infected NeuroD2 SmoA1 cells (14/17 YAP-SmoA1 injected mice vs. 8/17 GFP-SmoA1 injected mice). A similar trend was observed when wild-type C57BL/6 pups were used as recipients (Supplementary Figure 1). Moreover, YAP expression results in a significant acceleration of lethality (Long-rank Test, p=0.0047; Figure 1A; p=0.0487; Supplementary Figure 1), suggesting that YAP-expressing medulloblastomas are more aggressive. YAP-SmoA1 medulloblastomas have increased proliferation as determined by increased levels of cyclin D2 and phosphorylated Histone H3 (P-Hist3), markers of G1/S transition and mitosis phase of the cell cycle respectively (Figure 1B, C). YAP-SmoA1 tumors also showed reduced levels of apoptosis as determined by decreased levels of cleaved caspase 3 compared with tumors arising from GFP-infected SmoA1 cells. In keeping with our previous results, YAP is found throughout the tumor but is most highly expressed in PVN cells. Interestingly, YAP-expressing SmoA1 medulloblastomas also bear evidence of increased angiogenesis, suggested by higher levels of vascular endothelial growth factor (VEGF) and the endothelial cell marker CD31 (Figure 1B, C).
We have previously shown that YAP-expressing cells in Shh-induced mouse medulloblastomas proliferate after the animals have been treated with radiation. We next wished to determine whether YAP expression promotes survival after irradiation. To this end, we treated GFP-SmoA1 and YAP-SmoA1 medulloblastoma-bearing mice with 2 Gray (Gy) ionizing radiation and analyzed cell death and proliferation 3 hours post-irradiation. We observed a marked induction of cleaved caspase 3 in both tumors, but the levels of cleaved caspase 3 were less in YAP-SmoA1 tumors as compared to GFP-SmoA1 tumors (Figure 1D). Moreover, irradiated YAP-SmoA1 medulloblastomas had significantly higher levels of P-Hist3 and Ki67 (Supplementary Figure 1B) in comparison with irradiated GFP-SmoA1 medulloblastomas. These lines of evidence indicate that YAP expression endows medulloblastoma cells with increased proliferation and survival capacities, and renders them radio-resistant.