Discussion
Amplification of the YAP gene locus as well as YAP protein over-expression has been reported in a wide spectrum of human and murine tumors. We have previously shown that YAP is a downstream target of mitogenic Sonic hedgehog signaling that promotes proliferation and inhibits apoptosis of cerebellar granule cell precursors and we showed that YAP is over-expressed and amplified in the Sonic hedgehog-associated class of medulloblastomas (Fernandez et al 2009). Here, we report a new role for YAP in promoting IGF2/Akt-dependent radio-resistance and ongoing proliferation in the presence of unrepaired DNA, illustrating a novel link between Shh signaling, YAP, and the Akt pathway. We show that YAP activity markedly induces IGF2 transcription, in the absence and presence of radiation. IGF2, in turn, activates Akt downstream of PI-3 kinase, providing cells with a survival and proliferative advantage, and allowing them to inactivate cell cycle checkpoints (Figure 7). Many studies have implicated PI-3 kinase and Akt in cell survival, through its effects on FKHR proteins and Bcl2 family members (Brunet et al 2001, Burgering and Kops 2002, Duronio 2008), and Akt is essential for CGNP survival (Dudek et al 1997). Transcriptional profiling of human medulloblastomas has shown increased expression of IGF2 (Pomeroy et al 2002), and our report clearly links high levels of IGF2 expression with the Sonic hedgehog-associated subclass of medulloblastomas. IGF2 has been shown to be indispensable for the formation and progression of Shh-driven murine medulloblastomas (Corcoran et al 2008, Hahn et al 2000) and to synergize with Sonic Hedgehog in the induction of tumor formation (Rao et al 2004). IGF2 has previously been shown to promote proliferation of medulloblastoma cells and cerebellar neural cell precursors through the activation of Akt (Hartmann et al 2005).
Our results show that ectopic YAP expression accelerates medulloblastoma onset and aggressiveness, and also confers a survival advantage after tumor radiation. This has particularly important implications for medulloblastomas; due to their radiosensitivity, radiation therapy is a standard in children older than 3 years of age (Mueller and Chang 2009). We show here that 3 hours after radiation, YAP-driven SmoA1 medulloblastomas feature higher levels of proliferation markers and reduced apoptosis. In vitro, we found a similar effect when we irradiated GFP- or YAP-over-expressing CGNPs. CGNPs expressing YAP not only proliferated more than GFP-expressing cells after radiation, but also failed to arrest in response to radiation, skipping the G2/M checkpoint.
Defective G2/M checkpoint activation in the presence of DNA damage has been linked to Akt activity. Activation of Akt suppresses the checkpoint by inhibiting the DNA damage response pathways, either ATM/Chk2 or ATR/Chk1, depending on the cellular context (Hirose et al 2005, Kandel et al 2002, Shtivelman et al 2002, Xu et al 2010). In agreement with these studies, we observed a decrease in phosphorylated ATM and Chk2 in irradiated YAP-over-expressing CGNPs and medulloblastoma cells, compared to GFP-expressing cells, and YAP-transduced cells showed fewer DNA damage-induced foci. The effects of YAP on ATM and Chk2 was reversed in the presence of a PI-3 kinase inhibitor, and when we knocked down IGF2, which also abolished the accelerating effect of YAP on DNA damage-dependent focus disassembly. Interestingly, a previous study has shown that aberrant activation of the Shh pathway through Ptc loss-of-function impairs the function of ATR/Chk1, a separate signaling cascade (Leonard et al 2008). We did not detect effects of YAP on this cascade (data not shown); however the impact we report of YAP on ATM/Chk2 may synergize with an already defective DNA damage response due to Smoothened-mediated Chk1 impairment.
The ability of cells to maintain genomic integrity is fundamental for protection from cancer development. Genomic instability characterizes almost all sporadic human cancers, although mechanisms causing it are still not well understood. High-throughput sequencing studies suggest that mutations in DNA repair genes do not account for the presence of genomic instability in many sporadic cancers (Negrini et al 2010). Alternatively, activation of oncogenes, and more generally of growth signaling pathways, has been shown to induce genomic instability in mammalian cells cultured in vitro and in mouse models (Hernando et al 2004, Negrini et al 2010). Hyperactive Shh pathway activity has been shown to induce genomic instability and the development of spontaneous and ionizing radiation (IR)-induced tumors (Leonard et al 2008). Now we show that high levels of YAP expression further contribute to generation of genomic instability since irradiated YAP-expressing cells present a higher number of chromosomal alterations. The role of genomic instability has remained a controversial issue in cancer biology. Although it is still unclear if it is necessary for tumorigenesis to occur, genomic instability certainly provides the tumor with an advantage in terms of faster progression through the many stages of tumorigenesis (Sieber et al 2003). Indeed, we show that ectopic expression of YAP accelerates medulloblastoma onset and produces tumors that are highly aggressive and rapidly lethal.
YAP expression is particularly high in the peri-vascular niche (PVN) of medulloblastomas, where the tumor-repopulating cells reside. We have previously shown that 48 hours after radiation, the YAP positive population starts to expand (Fernandez et al 2009). Hambardzumyan et al. (Hambardzumyan et al 2008) elegantly demonstrated that the PI3K-Akt pathway regulates survival of these tumor-repopulating cells residing in the PVN. These results point to the importance of YAP regulating survival and proliferation post-radiation, allowing the cells to skip the cell cycle checkpoints and expand despite the un-repaired damage in their DNA, contributing ultimately to genomic instability. Further studies will address whether YAP-induced genomic instability is solely a consequence of the Akt-mediated cell cycle checkpoint suppression, or whether Akt lying downstream of YAP-induced IGF2 could also inhibit the DNA repair machinery, as previous studies have suggested (Plo et al 2008) (Figure 7). Our results suggest that targeting Akt downstream of YAP would be useful for medulloblastoma therapy in order to reduce the dose of radiation needed and to prevent tumor recurrence, by radiosensitizing PVN-residing tumor-repopulating cells.