Aneuploidy is a characteristic feature of established cancers and can promote tumor development. Aneuploidy may arise directly, through unequal distribution of chromosomes into daughter cells, or indirectly, through a tetraploid intermediate. The polo family kinase Plk4/Sak is required for late mitotic progression and is haploinsufficient for tumor suppression in mice. Here we show that loss of heterozygosity (LOH) occurs at the Plk4 locus in 50% of human hepatocellular carcinomas (HCC) and is present even in preneoplastic cirrhotic liver nodules. LOH at Plk4 is associated with reduced Plk4 expression in HCC tumors but not with mutations in the remaining allele. Plk4 +/− murine embryonic fibroblasts (MEFs) at early passage show a high incidence of multinucleation, supernumerary centrosomes, and a near-tetraploid karyotype. Underlying these phenotypes is a high rate of primary cytokinesis failure, associated with aberrant actomyosin ring formation, reduced RhoA activation, and failure to localize the RhoA guanine nucleotide exchange factor Ect2 to the spindle midbody. We further show that Plk4 normally localizes to the midbody and binds to and phosphorylates Ect2 in vitro. With serial passaging Plk4 +/− MEFs rapidly immortalize, acquiring an increasing burden of nonclonal and clonal gross chromosomal irregularities, and form tumors in vivo. Our results indicate that haploid levels of Plk4 disrupt RhoGTPase function during cytokinesis, resulting in aneuploidy and tumorigenesis, thus implicating early LOH at Plk4 as one of the drivers of human hepatocellular carcinogenesis. These findings represent an advance in our understanding of genetic predisposition to HCC, which continues to increase in incidence globally and particularly in North America.aneuploidy | hepatocellular carcinoma | Polo-like kinase | RhoA | Ect2

Some cancers have been stratified into subclasses based on their unique involvement of specific signaling pathways. The mapping of human cancer genomes is revealing a vast number of somatic alterations; however, the identification of clinically relevant molecular tumor subclasses and their respective driver genes presents challenges. This information is key to developing more targeted and personalized cancer therapies. Here, we generate a new mouse model of genomically unstable osteosarcoma (OSA) that phenocopies the human disease. Integrative oncogenomics pinpointed cAMP-dependent protein kinase type I, α regulatory subunit (Prkar1a) gene deletions at 11qE1 as a recurrent genetic trait for a molecularly distinct subclass of mouse OSA featuring RANKL overexpression. Using mouse genetics, we established that Prkar1a is a bone tumor suppressor gene capable of directing subclass development and driving RANKL overexpression during OSA tumorigenesis. Finally, we uncovered evidence for a PRKAR1A-low subset of human OSA with distinct clinical behavior. Thus, tumor subclasses develop in mice and can potentially provide information toward the molecular stratification of human cancers.

Pif1 is a 5 -to-3 DNA helicase critical to DNA replication and telomere length maintenance in the budding yeast Saccharomyces cerevisiae. ScPif1 is a negative regulator of telomeric repeat synthesis by telomerase, and recombinant ScPif1 promotes the dissociation of the telomerase RNA template from telomeric DNA in vitro. In order to dissect the role of mPif1 in mammals, we cloned and disrupted the mPif1 gene. In wild-type animals, mPif1 expression was detected only in embryonic and hematopoietic lineages. mPif1 ؊/؊ mice were viable at expected frequencies, displayed no visible abnormalities, and showed no reproducible alteration in telomere length in two different null backgrounds, even after several generations. Spectral karyotyping of mPif1 ؊/؊ fibroblasts and splenocytes revealed no significant change in chromosomal rearrangements. Furthermore, induction of apoptosis or DNA damage revealed no differences in cell viability compared to what was found for wild-type fibroblasts and splenocytes. Despite a novel association of mPif1 with telomerase, mPif1 did not affect the elongation activity of telomerase in vitro. Thus, in contrast to what occurs with ScPif1, murine telomere homeostasis or genetic stability does not depend on mPif1, perhaps due to fundamental differences in the regulation of telomerase and/or telomere length between mice and yeast or due to genetic redundancy with other DNA helicases.Homeostatic telomere length is achieved by a balance between the extension of the 3Ј telomeric repeats by telomerase or by homologous recombination between telomeric tracts and telomere erosion due to incomplete DNA replication or nucleolytic degradation (30). Telomerase acts to lengthen telomeres via the reverse transcription of an RNA template (encoded by TERC in mammals or TLC1 in Saccharomyces cerevisiae) by a telomerase reverse transcriptase (TERT or Est2, respectively) (30). In the absence of telomerase, telomeric repeats can also be maintained via homologous recombination (12). Excessive telomere lengthening is usually curtailed by cis-inhibitory telomere binding factors, such as Rif1 and Rap1 in S. cerevisiae and TRF1 in humans, or by the action of nucleases (30). In particular instances, telomeric tracts undergo saltatory attrition via the excision of telomeric DNA circles, thus preventing unregulated telomere lengthening (12,40,73).This equilibrium is not simply a push and pull between factors that strictly promote or oppose telomere extension. In S. cerevisiae, the trimeric protein complex composed of Cdc13, Stn1, and Ten1 plays a critical role in the protection of chromosome ends from nucleolytic degradation, and the complex serves both positive and negative roles in the access of telomerase to the telomere during late S phase (24,27,66). The Ku70/Ku80 heterodimer, while essential for nonhomologous end joining, also plays a key role both in the recruitment of telomerase to the telomere and in the protection of ends from degradation (9,25,65,74). Several DNA helicases and nucleases are also known to play ...

The distal end of mouse chromosome 7 (Chr 7) contains a large cluster of imprinted genes. In this region two cis-acting imprinting centers, IC1 (H19 DMR) and IC2 (KvDMR1), define proximal and distal subdomains, respectively. To assess the functional independence of IC1 in the context of Chr 7, we developed a recombinase-mediated chromosome truncation strategy in embryonic stem cells and generated a terminal deletion allele, DelTel7, with a breakpoint in between the two subdomains. We obtained germ line transmission of the truncated Chr 7 and viable paternal heterozygotes, confirming the absence of developmentally required paternally expressed genes distal of Ins2. Conversely, maternal transmission of DelTel7 causes a midgestational lethality, consistent with loss of maternally expressed genes in the IC2 subdomain. Expression and DNA methylation analyses on DelTel7 heterozygotes demonstrate the independent imprinting of IC1 in absence of the entire IC2 subdomain. The evolutionarily conserved linkage between the subdomains is therefore not required for IC1 imprinting on Chr 7. Importantly, the developmental phenotype of maternal heterozygotes is rescued fully by a paternally inherited deletion of IC2. Thus, all the imprinted genes located in the region and required for normal development are silenced by an IC2-dependent mechanism on the paternal allele.The 1-Mb imprinted domain on distal chromosome 7 (Chr 7) shares syntenic homology to the Beckwith-Wiedemann syndrome (BWS) region on human Chr 11p15.5 (69). Located less than 3 Mb from the telomere (Tel7q), this region contains two imprinting centers, IC1 and IC2. These conserved imprinting control elements are cis-acting sequences which carry opposite germ line DNA methylation marks and regulate the monoallelic expression of different flanking genes (9). In the proximal part of the Chr 7 domain, IC1 is located 2 kb upstream of the H19 promoter (Fig. 1A) (66). This sequence acquires a paternal DNA methylation imprint established during spermatogenesis and maintained throughout development (4,24,67). The methylated IC1 is required to initiate silencing of the paternal H19 allele (65), whereas the maternal IC1 controls the paternally expressed genes Igf2 and Ins2 via a methylation-sensitive insulator (6, 35). Distally, IC2 is located in intron 10 of Kcnq1 (Fig. 1A). This sequence is marked by a maternal DNA methylation imprint acquired during oogenesis (19). The unmethylated paternal IC2 is associated with the production of the Kcnq1ot1 noncoding RNA (ncRNA) and the silencing in cis of several genes in this subdomain (26). Consequently, these protein-coding genes are imprinted and expressed preferentially from the maternal Chr 7 homologue (62). These maternally expressed genes (MEGs) include transcripts expressed in the placenta and required for embryonic development, such as Ascl2 (31, 32), Cdkn1c (36), and Phlda2 (58). As in the case of IC1, IC2 appears to carry different allele-specific functions, such as promoter, enhancer, and CTCF binding insulator activities (2...