Progression through mitosis requires activation of cyclin B/Cdk1 and its downstream targets, including Polo-like kinase and the anaphase-promoting complex (APC), the ubiquitin ligase directing degradation of cyclins A and B. Recent evidence shows that APC activation requires destruction of the APC inhibitor Emi1. In prophase, phosphorylation of Emi1 generates a D-pS-G-X-X-pS degron to recruit the SCF^βTrCPubiquitin ligase, causing Emi1 destruction and allowing progression beyond prometaphase, but the kinases directing this phosphorylation remain undefined. We show here that the polo-like kinase Plk1 is strictly required for Emi1 destruction and that overexpression of Plk1 is sufficient to trigger Emi1 destruction. Plk1 stimulates Emi1 phosphorylation, βTrCP binding, and ubiquitination in vitro and cyclin B/Cdk1 enhances these effects. Plk1 binds to Emi1 in mitosis and the two proteins colocalize on the mitotic spindle poles, suggesting that Plk1 may spatially control Emi1 destruction. These data support the hypothesis that Plk1 activates the APC by directing the SCF-dependent destruction of Emi1 in prophase.

Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCF ^βTrCP ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.

The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1^lacZ/+), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1^lacZ/+ controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups. Significance Medulloblastoma is a common malignant pediatric brain tumor. Gene expression data have indicated that the tumors fall into four molecular subgroups, “Wnt,” “Hedgehog,” “group 3,” and “group 4.” With the exception of the Hedgehog subgroup, few functional data exist defining key molecular pathways driving tumor growth. Using a transposon mutagenesis approach, we identified genes that functionally cooperate with Hedgehog signalling to promote tumorigenesis in a Ptch1 mouse model of medulloblastoma. Surprisingly, the genes we identified were able to accurately define all four human molecular subtypes, not just Hedgehog, when used to interrogate published expression data. Thus, we have functionally defined key regulatory networks that illustrate both the differences and commonalities between tumor subgroups indicating a number of therapeutic strategies.

Various reports have implicated the virally encoded HBx protein as a cofactor in hepatocarcinogenesis. However, direct evidence of the role of HBx as a promoter of oncogenesis in response to an initiating factor such as DNA damage remains inadequate. Here, we report the effects of HBx in HepG2 cells exposed to UV light-induced DNA damage. HBx expression was found not to affect the morphology, viability, and cell cycle/apoptotic profiles or DNA repair machinery of untreated cells. Nonetheless, upon UV treatment, HBx protein levels increased concomitantly with p53 levels. Both HBx and p53 proteins were found to interact and colocalize primarily in the nucleus. The binding of HBx to p53 modulated (but did not inhibit) the transcriptional activation function of p53. Notably, HBx-expressing cells exhibited increased sensitivity to UV damage, resulting in greater G₂/M arrest and apoptosis of these cells. Additionally, these cells displayed a reduced DNA repair capacity in response to UV damage. In conclusion, this work suggests that DNA damage may be an initiating factor in hepatocarcinogenesis and that HBx may act as the promoting factor by inhibiting DNA repair. In hepatitis B virus-infected hepatocytes, a chronic infection may present the opportunity for such a DNA-damaging event to occur, and accumulated errors caused by the inhibition of DNA repair by HBx may result in oncogenesis.