PMC:3583298 / 5615-24617 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3583298","sourcedb":"PMC","sourceid":"3583298","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3583298","text":"Results\n\nYAP expression promotes medulloblastoma cell tumorigenicity\nWe 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).\nWe 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.\n\nYAP expression promotes increased proliferation, survival and cell cycle checkpoints override in irradiated cerebellar neural precursors\nCGNPs are proposed to be the cell-of-origin for medulloblastomas associated with increased Shh pathway activity (Wechsler-Reya and Scott 1999). Consistent with this hypothesis, treatment of CGNPs in culture with exogenous Shh causes expression of many of the same proliferation markers seen in this class of medulloblastomas, including Gli1, N-myc and YAP. We wished to determine whether we could use in vitro primary CGNP cultures to mechanistically dissect the effects of YAP on proliferation and survival after irradiation. Cells prepared from post-natal day 4-5 mouse cerebella are maintained in serum free medium; addition of Shh sustains their proliferation. We transduced Shh-treated, proliferating CGNPs with retroviruses carrying GFP or YAP, then irradiated them (10 Gy) twenty-four hours later. As shown in Figure 2A, ectopic YAP expression is associated with increased CGNP proliferation as determined by cyclin D2 levels, and as we have previously reported (Fernandez et al 2009). At 20 hours post-irradiation, YAP-infected CGNPs had reduced levels of cleaved caspase 3 in comparison with GFP-infected CGNPs, and reduced numbers of apoptotic cells as determined by quantification of pyknotic nuclei (GFP: 62.5+/−0.5% vs YAP: 35.5+/−5.5%). YAP-transduced CGNPs were significantly more proliferative, as indicated by levels of cyclin D2, and by Ki67 and P-Hist3 staining (Figures 2A, B). Taken together, these results indicate that CGNP cultures recapitulate the in vivo effect of YAP expression on promoting survival and proliferation after irradiation. Moreover, they suggest that YAP-mediated radiation resistance may contribute to medulloblastoma recurrence by promoting increased tumor cell survival and proliferation.\nIonizing radiation (IR) causes the formation of double-strand DNA breaks (DSBs) that are responsible for the activation of the DNA Damage Response (DDR), a complex network of proteins required for cell-cycle checkpoint maintenance and DNA repair. The G1/S checkpoint prevents cells from entering S phase, while the G2/M checkpoint arrests cells with unrepaired DNA before entry into mitosis. We wanted to assess whether high YAP expression causes defects in cell cycle checkpoints, allowing cells to go through the cell cycle regardless of damage to their DNA. To examine the G1/S checkpoint, we exposed CGNPs to IR (10 Gy), and determined the percentage of cells in S phase 18 hours post-treatment by quantifying bromo-deoxyuridine (BrdU) incorporation during a one-hour pulse. Upon IR treatment both GFP and YAP-expressing CGNPs showed a strong reduction of the number of cells incorporating BrdU, however YAP-expressing cells have a significantly higher S-phase ratio (%BrdU-positive of treated/%BrdU-positive untreated), suggesting a mild defect in the G1/S checkpoint (Figure 2C). The infection efficiency was similar in both GFP- and YAP-infected cultures (representative images, Supplementary Figure 2). To determine whether the G2/M checkpoint was disabled in YAP-infected irradiated CGNPs, we evaluated P-Hist3 levels at an early time point (1 hour) after radiation. GFP-transduced cells showed an 80% reduction of the number of P-Hist3 positive cells as compared to untreated cells. In contrast, YAP-expressing CGNPs showed only a 50% reduction of cells undergoing mitosis, indicating that the G2/M checkpoint was defective (Figure 2C).\n\nYAP over-expression affects DNA repair and causes genomic instability\nDefects in cell cycle checkpoints might result in genomic instability. We asked at which level YAP was affecting the DDR. Breaks in DNA are marked by the formation of protein complexes termed IR-induced foci (IRIF), which can be identified when analyzed by immunostaining for proteins comprising these complexes. To determine whether YAP-infected cells had reduced DNA damage, we carried out immunostaining for p53 binding protein 1 (53BP1), a key mediator of the DDR often used as a marker of the IRIF. At 3 hours post-irradiation, GFP- and YAP-infected CGNPs had similar numbers of foci per cell (data not shown). However, at 9 and 24 hours post-irradiation, YAP-infected CGNPs had a significantly reduced number of cells containing foci as well as fewer foci per cell (Figure 3A). These findings would suggest that YAP expression either enables cells to repair DNA more rapidly, causing foci to resolve earlier, or that YAP expression inhibits DNA repair pathways, resulting in dismantling of the DNA repair complexes.\nTo distinguish between these two possibilities, we carried out comet assays to detect broken DNA, in irradiated GFP- or YAP-infected CGNPs. In the comet assay, cells are immersed in an agarose matrix extended on a microscope slide, and treated with a lysis solution. Nuclear DNA is unwound under alkaline conditions and fragments resulting from strand breaks can migrate away from the nuclei under an electric field, forming a “comet tail” when stained. We quantified the amount of DNA damage by measuring tail lengths. As shown in Figure 3B, comet tails can be detected in GFP-infected CGNPs at 9 hours post-irradiation, but to a lesser extent at 24 hours post-irradiation, indicating that most of the damaged DNA is repaired at that point.\nIn contrast, at 9 hours after irradiation, YAP-infected cells had strikingly pronounced comet tails. Comet tails persisted in YAP-infected CGNPs 24 hours after irradiation, a time point at which we can detect proliferation in these cells (Figure 2). In order to analyze whether the DNA repair defect leads to chromosomal alterations, we performed metaphase spreads on GFP- or YAP-infected CGNPs 24 hours after irradiation. As shown in Figure 3C, YAP infection is associated with an increase in the number of cells with persistent DNA breaks. We also observed that YAP-transduced Pzp53med cells (Berman et al 2002) showed a similar loss of irradiation-induced foci and increased number of cells with DNA breaks after irradiation (Supplementary Figure 3A, B). These observations indicate that YAP expression enables cells to proliferate with unrepaired DNA, a condition which can lead to genomic instability, a hallmark of cancer cells. To address whether the effect of YAP was specific for radiation-induced DNA damage, we treated GFP- and YAP-infected cells with the antibiotic Bleomycin, which causes double strand breaks in DNA. Similar to what we observed in irradiated cells, 9 and 24 hours after treatment there were significantly fewer cells that contained foci among those over-expressing YAP, which indicates that the role of YAP promoting focus dis-assembly is not specific to radiation-induced damage in the DNA (Supplementary Figure 3C).\n\nYAP expression promotes inactivation of the checkpoint regulators ATM and Chk2\nEntry into mitosis is marked by activation of the cyclin dependent kinase (Cdk)1: cyclin B1 complex. During G2, Cdk1 is maintained in an inactive state by phosphorylation on Thr 14 and Tyr 15 (Castedo et al 2002); de-phosphorylation by cdc25 disinhibits Cdk1. To confirm that YAP-expressing cells continued to enter mitosis after irradiation, indicating inactivation of the G2/M checkpoint, we carried out western blotting for Tyr 15-phosphorylated Cdk1. As shown in Figure 4A, irradiation results in increased Tyr15-phosphorylated Cdk1 in both GFP- and YAP-infected CGNPs. However, after three hours, YAP-infected CGNPs show reduced levels of Tyr15-phosphorylated Cdk1, indicating re-activation of Cdk1 at this early timepoint, when DNA breaks still persist. The reduction of inactive Cdk1 was associated with an increase in levels of cyclin B1, which is synthesized during interphase. Following metaphase, cyclin B1 is degraded; this degradation is required for completion of mitosis. Taken together, these results confirm that YAP-expressing irradiated CGNPs enter mitosis despite the presence of double-stranded DNA breaks, and they suggest that the G2/M arrest checkpoint is compromised by YAP expression.\nThese observations prompted us to examine whether YAP expression blocked activation of the G2/M checkpoint by affecting the activity of proteins involved in its regulation. Upon irradiation, the kinase ATM is activated by autophosphorylation and subsequently phosphorylates and activates Chk2 kinase. Chk2, in turn, phosphorylates cdc25c, resulting in its inactivation and preventing entry into mitosis (Reinhardt and Yaffe 2009). Irradiation resulted in increased levels of phosphorylated ATM and its substrate Chk2 in both GFP- and YAP-infected cells. However, in the presence of ectopic YAP, ATM and Chk2 were more rapidly dephosphorylated (Figure 4B, western blot with quantification below). We observed a similar trend towards Chk2 inactivation in medulloblastoma cells (MBC) obtained from SmoA1 medulloblastomas, cultured in vitro and transduced with either GFP or YAP. Our observation of ATM and Chk2 inactivation in YAP-infected CGNPs and MBC is consistent with relief from the G2/M checkpoint and consequently, re-entry into mitosis despite the presence of unrepaired DNA breaks. We also observed reduced phosphorylation of Histone H2AX in YAP-transduced irradiated CGNPs, further indicative of impaired DNA damage response pathway signaling (Supplementary Figure 4A). Interestingly, we did not observe differential effects of YAP expression on regulation of Chk1 or p53 activity after exposure to radiation in CGNPs or MBC, indicating that YAP’s downstream effectors preferentially target the ATM/Chk2 DNA damage response pathway (Figure 4B).\n\nIGF2 is a candidate YAP target mediating proliferation and survival after irradiation\nWe next wished to gain insight as to the mechanism through which YAP protects tumor cells from radiation-induced apoptosis and allows them to continue cycling. YAP functions in a complex with TEAD to regulate gene expression and proliferation (Zhao et al 2008). To determine which genes may be induced or suppressed by YAP in irradiated CGNPs, we carried out microarray analysis of mRNA prepared from GFP- or YAP-infected control or irradiated CGNPs. Among the genes most highly expressed in YAP-infected CGNPs was IGF2, confirmed by quantitative RT-PCR (Figure 5A). IGF2 mRNA expression was increased in both cell populations after irradiation, but the levels of IGF2 in the YAP-infected CGNPs were significantly higher than in the GFP-infected cell under either condition (non-irradiated or irradiated). When we analyzed IGF2 protein levels in GFP- or YAP-infected CGNPs we found that YAP-infected CGNPs expressed and secreted significantly more IGF2 (Figures 5B, C).\nIGF signaling is required for survival of CGNPs in vivo and in vitro, and increased activity of the IGF pathway is found in human medulloblastomas (Chrysis et al 2001, Corcoran et al 2008, Hahn et al 2000, Hartmann et al 2005, Rao et al 2004, Tanori et al 2010). Interestingly, when we queried a genetically characterized database of over 100 human medulloblastomas (Northcott et al 2009b) we found that IGF2 was most highly expressed in the subclass of medulloblastomas associated with activation of the Shh pathway (T test, p=3.553E-14) (Figure 5D). This class of tumors also expresses high levels of N-myc, Gli1, and miR17/92 (Kool et al 2008, Northcott et al 2009b, Pomeroy et al 2002). The increased level of IGF2 protein in YAP-expressing tumors was conserved in YAP-SmoA1 medulloblastomas, as determined by western blot and immunohistochemical analysis (Figure 5E, F). As we observed in CGNPs, IGF2 levels are increased in both GFP-SmoA1 and YAP-SmoA1 medulloblastomas after irradiation, but are higher in YAP-SmoA1 medulloblastomas.\nIGF2 acts as a secreted ligand, binding to and activating the IGF1 receptor. The predominant downstream effector of the activated IGF1 receptor is the kinase Akt, which plays multiple roles in survival and proliferation. We have previously shown that Akt cooperates with Shh signaling to promote proliferation through stabilization of N-myc (Kenney et al 2004). Akt activity has been linked to abrogation of the G2/M checkpoint after irradiation, through inactivation of ATM/Chk2 (Hirose et al 2005, Kandel et al 2002). Consistent with increased IGF1 receptor activity in response to IGF2 secretion, YAP-Smo-driven medulloblastomas exhibit higher levels of activated Akt (S473-phosphorylated), most notably in cells surrounding the vasculature (Figure 5G); interestingly, these cells also express the highest levels of YAP and they have been proposed to function as tumor repopulating cells after irradiation (Fernandez et al 2009, Hambardzumyan et al 2008). Taken together these observations raise the possibility that YAP-mediated IGF2 induction not only promotes survival and proliferation through Akt activation but may also affect the phosphorylation of ATM/Chk2, resulting in disruption of the G2/M checkpoint.\n\nIGF2 is required for YAP-mediated G2/M arrest override and cell survival and proliferation after irradiation\nTo address whether IGF2/Akt activity regulates the YAP-associated DNA damage response defect after radiation, we used Shh-treated CGNP cultures. Confirming our observations (Figure 5), YAP infection is associated with increased Akt activity (Figure 6A) as determined by phosphorylation of S473. Treatment of YAP-infected CGNPs with the drug LY294002, which inhibits phosphoinositide-3 kinase, the upstream activator of Akt, reduced levels of S473-phosphorylated Akt. After irradiation, we saw induction of ATM phosphorylation in GFP- and YAP-infected CGNPs, although to a lesser extent in the presence of ectopic YAP, indicating reduced activity of this kinase. In keeping with reduced ATM activity in the presence of YAP, we also observed reduced Chk2 phosphorylation. In the presence of LY294002, full ATM and Chk2 phosphorylation were recovered, indicating that YAP requires Akt activity for its suppressive effect on ATM and Chk2.\nWe next wished to determine whether IGF2 is necessary for the effects of YAP on the DNA damage response. To this end, we used retroviruses targeting IGF2 for short hairpinRNA-mediated knock-down. As shown in Figure 6B, YAP-infected CGNPs transduced with these retroviruses showed strikingly reduced levels of IGF2, in comparison with YAP-infected CGNPs transduced with retroviruses carrying a scrambled, non-specific short hairpin RNA sequence. Consistent with IGF2 being an upstream activator of Akt, IGF2 knockdown was associated with reduced Akt Ser473 phosphorylation. In addition, knocking down IGF2 in YAP-expressing cells led to a recovery of phospho-ATM and phospho-Chk2 levels, as well as phospho-Cdk1, comparable to those observed in GFP-infected cells. The increase in inactive Cdk1 when IGF2 is knocked down indicates cells arresting after radiation, due to a restored G2/M checkpoint. IGF2 knock-down also blocked the effects of YAP on DNA damage-dependent focus formation after irradiation (Figure 6C), causing a significant rescue of focus formation as determined by immunofluorescent staining for 53BP1. Moreover, IGF2 knock-down impaired the effects of YAP expression on CGNP survival after irradiation, as well as proliferation (Figure 6D). These results are consistent with IGF2 and its downstream effector Akt being necessary for the ability of YAP to inactivate the G2/M checkpoint, permitting ongoing proliferation and enhancing survival of irradiated 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