Janine H. van Ree

Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule–kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule–kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

Whole chromosome instability and cancer: a complex relationship

Although chromosome mis-segregation is a hallmark of cancer cells, its genetic basis and role in malignant transformation remain poorly understood. In recent years, several mouse models have been generated that harbor gene defects that perturb high-fidelity chromosome segregation. Analysis of these models has revealed that whole chromosome instability (W-CIN) can cause, inhibit or have no effect on tumorigenesis. Here we propose that the effect of W-CIN on tumor development depends on the particular W-CIN gene that is defective, including its other cellular functions, the extent or nature of the gene defect, the affected tissue or cell type and the context of other cancer gene mutations.

Ran-dependent docking of importin-β to RanBP2/Nup358 filaments is essential for protein import and cell viability

RanBP2 captures RanGTP–importin-β complexes at cytoplasmic fibrils to ensure adequate classical NLS–mediated protein import and cell viability.

Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

Low expression levels of Cdc20 result in chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment.

Pten regulates spindle pole movement through Dlg1-mediated recruitment of Eg5 to centrosomes

Phosphatase and tensin homologue (Pten) suppresses neoplastic growth by negatively regulating PI(3)K signalling through its phosphatase activity. To gain insight into the actions of non-catalytic Pten domains in normal physiological processes and tumorigenesis, we engineered mice lacking the PDZ-binding domain (PDZ-BD). Here, we show that the PDZ-BD regulates centrosome movement and that its heterozygous or homozygous deletion promotes aneuploidy and tumour formation. We found that Pten is recruited to pre-mitotic centrosomes in a Plk1-dependent fashion to create a docking site for protein complexes containing the PDZ-domain-containing protein Dlg1 (also known as Sap97) and Eg5 (also known as Kif11), a kinesin essential for centrosome movement and bipolar spindle formation. Docking of Dlg1–Eg5 complexes to Pten depended on Eg5 phosphorylation by the Nek9–Nek6 mitotic kinase cascade and Cdk1. PDZ-BD deletion or Dlg1 ablation impaired loading of Eg5 onto centrosomes and spindle pole motility, yielding asymmetrical spindles that are prone to chromosome missegregation. Collectively, these data demonstrate that Pten, through the Dlg1-binding ability of its PDZ-BD, accumulates phosphorylated Eg5 at duplicated centrosomes to establish symmetrical bipolar spindles that properly segregate chromosomes, and suggest that this function contributes to tumour suppression.

Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors.

Mitotic kinase cascades orchestrating timely disjunction and movement of centrosomes maintain chromosomal stability and prevent cancer.

Centrosomes are microtubule-organizing centers that duplicate in S phase to form bipolar spindles that separate duplicated chromosomes faithfully into two daughter cells during cell division. Recent studies show that proper timing of centrosome dynamics, the disjunction and movement of centrosomes, is tightly linked to spindle symmetry, correct microtubule-kinetochore attachment, and chromosome segregation. Here, we review mechanisms that regulate centrosome dynamics, with emphasis on the roles of key mitotic kinases in the proper timing of centrosome dynamics and how aberrancies in these processes may cause chromosomal instability and cancer.

Transgenesis in Mouse Embryonic Stem Cells

Traditionally, transgenic mice are generated by pronuclear injection of exogenous DNA. This technique has several limitations, including limited control over transgene expression, transgene cytotoxicity, -promiscuity and silencing, and founder mouse sterility. Here we describe two protocols to generate transgenic mice from ES cell clones carrying stably integrated exogenous DNA with inducible transgene expression.

Deciphering the tumor suppressive mechanisms of Pten

Phosphatase and tensin homolog (Pten) suppresses neoplastic growth by negatively regulating PI(3)K signaling through its phosphatase activity. Indeed, most missense mutations detected in human tumors are located within the amino-terminal phosphatase domain of the protein and negatively impact enzymatic activity. Furthermore, mutations confined to the carboxy-terminus frequently affect protein stability or membrane localization thereby indirectly decreasing catalytic capacity. Another frequently mutated cancer-critical gene, p53, exerts its prominence in human tumor suppression through a broad spectrum of biological functions by a series of modular domains. Like p53, Pten is also a highly modular protein with at least 4 functional domains besides the phosphatase domain, including a phosphatidylinositol-4,5-biphosphate binding domain, a C2 domain, a C-terminal tail and a PDZ-binding domain (PDZ-BD). Lessons learned from p53 mouse models mimicking point mutations found in human cancers or targeting individual functional domains for inactivation have provided a wealth of information about the relative importance of p53’s diverse biological functions in tumor suppression. Studies designed to understand the in vivo functions of Pten have proven much more difficult than for p53, largely because, unlike p53, Pten is a protein essential for embryonic development. The seemingly insurmountable barrier of embryonic lethality has now been successfully bypassed in a mutant targeting the PDZ binding domain of Pten defined by the last 3 amino acids (TKV) of the protein. Mice homozygously lacking these residues, referred to as Pten, were not only viable but also produced normal amounts of Pten protein and appeared to have normal Akt signaling. Despite these preservations, Pten mice were cancer prone, providing compelling evidence for a tumor suppressive role of Pten that is independent of its catalytic role in negatively regulating PtdIns(3)K signaling. To elucidate this new tumor suppressive function of Pten, PDZ-domain-containing proteins that could potentially interact with Pten were depleted in wild-type mouse embryonic fibroblasts (MEFs) and monitored for their impact on chromosome segregation. Dlg1 was uncovered as a critical partner of Pten in faithful chromosome segregation, and likewise, Dlg1¡/¡ MEFs were a phenocopy of Pten DTKV/DTKV MEFs regarding their mitotic phenotype. So how does Pten-Dlg1 complex formation contribute to accurate chromosome segregation? Two key mechanisms that ensure high-fidelity chromosome segregation, the spindle assembly checkpoint (SAC) and the error correction machinery, were unperturbed in Pten MEFs, as was the initial step of bipolar spindle assembly, the disjunction of the duplicated centrosomes. However, the subsequent step, the movement of centrosomes to opposite poles, occurred with reduced velocity, resulting in the formation of asymmetrical bipolar spindles in which the centrosomes are not perpendicular to the metaphase plate. Such spindles are prone to syntelic or merotelic attachments, that, when uncorrected, lead to misaligned and lagging chromosomes, respectively. Spindle geometry defects due to aberrancies in centrosome dynamics are emerging as a prominent source of cancer-causing aneuploidization, as evidenced by mouse models for human tumors overexpressing cyclin B2 or Nup88. The mechanism by which Pten-Dlg1 complexes control centrosome movement turned out to involve Eg5, a plus-end directed motor protein that is loaded onto centrosomes in early prophase to move duplicated centrosomes to opposite sides of the cell by driving outward microtubule sliding. In late G2, Dlg1 forms a complex with Eg5, which is delivered to the mitotic centrosomes through the docking of Dlg1 to centrosome-associated Pten molecules. However, there is another level of control involved. Pten will only localize to mitotic centrosomes if its C-terminal tail is phosphorylated. Plk1 was identified as a likely candidate to mediate this phosphorylation event because this kinase had been shown to phosphorylate several residues in the carboxy-terminus of Pten. Furthermore, Plk1 had been shown to be part of a kinase cascade regulating disjunction of sister centrosomes through the release of rootletin and cNap1. This cascade also included cyclin B2/Cdk1, Aurora A, Mst2 and Nek2. Indeed, Plk1 activity was confirmed to be required for proper accumulation of Pten to centrosomes. Earlier work had documented that Eg5 loading onto

Abstract 2992: Mitotic role for Pten in accurate chromosome segregation

During mitosis, sister chromatids are separated and equally divided over two daughter cells. If missegregation of whole chromosomes occurs, progeny cells with an abnormal number of chromosomes will arise. This is referred to as aneuploidy, a hallmark of most human cancers. We found that inactivation of a single Pten allele in mouse splenocytes and mouse embryonic fibroblasts (MEFs) is sufficient to cause aneuploidy, suggesting a role for Pten in the chromosome segregation process. Upon further study by live cell imaging of haploinsufficient and null Pten MEFs, we indeed found many missegregation errors (59% and 79% respectively, compared to 15% in wildtype MEFs). These abnormalities were independent from the AKT pathway since overexpression of a myristoylated, constitutively active form of AKT in wildtype MEFs resembled a wildtype instead of a Pten heterozygous phenotype. In addition, overexpression of a catalytic mutant Pten (C124S) in Pten+/- MEFs corrected the missegregation errors. We therefore propose a novel tumor-suppressive function of Pten to prevent whole chromosome instability, independent of the increased cell proliferation and survival caused by the activation of AKT. BubR1 is a regulator of kinetochore-microtubule attachment and a potent inhibitor of Cdc20, a key co-activator of the anaphase promoting complex/cyclosome (APC/C) that drives cells into anaphase by targeting the anaphase inhibitors cyclin B1 and securin for degradation by the 26S proteosome. We found that overexpression of BubR1 completely restores accurate chromosome segregation in Pten+/- MEFs, alluding to a specific role for Pten in mitosis. As a first step to test the hypothesis that Pten9s mitotic role in preventing aneuploidization represents a key tumor suppressive function of this phosphatase, we bred a Flag-tagged BubR1 transgene into Pten+/- mice. We are currently in the process of studying the impact of the transgene on the development of prostatic intraepithelial neoplasia (PIN). Consistent with our hypothesis, preliminary analysis of two mice shows that BubR1 overexpression considerably decreases PIN lesion multiplicity. Data from the complete analysis of our double transgenic mouse cohort will be presented at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2011-2992