Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1

Department of Cancer Biology, The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA.
Oncogene (Impact Factor: 8.56). 11/2006; 25(47):6291-303. DOI: 10.1038/sj.onc.1209644
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ABSTRACT Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.

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Available from: John Alan Diehl, Jul 26, 2015
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    • "For example, no consistent association of cyclin D1 amplification was found with response to therapeutic challenge in patients with hormone receptor-positive breast cancer (Lundgren et al, 2012; Rudas et al, 2008). Furthermore, mutations within the coding region of cyclin D1, found in cases of esophageal and endometrial cancer (Moreno-Bueno et al, 2003; Benzeno et al, 2006), have yet to be associated with clinical markers of disease progression, together suggesting that the observed oncogenic functions of cyclin D1 involve additional mechanisms of deregulation. Consistent with this concept, emerging clinical evidence suggests that alternative splicing of cyclin D1 transcript (cyclin D1a) to the shorter isoform cyclin D1b occurs frequently in human malignancy (Augello et al, 2014; Musgrove et al, 2011; Wang et al, 2008; Comstock et al, 2009), and appears to be a major mechanism though which cyclin D1 exerts its oncogenic activity. "
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    ABSTRACT: Clinical evidence suggests that cyclin D1b, a variant of cyclin D1, is associated with tumor progression and poor outcome. However, the underlying molecular basis was unknown. Here, novel models were created to generate a genetic switch from cyclin D1 to cyclin D1b. Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo. Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo. Further molecular interrogation uncovered unexpected links between cyclin D1b and the DNA damage/PARP1 regulatory networks, which could be exploited to suppress cyclin D1b-driven tumors. Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.
    EMBO Molecular Medicine 03/2015; 7(5). DOI:10.15252/emmm.201404242 · 8.25 Impact Factor
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    • "Furthermore, expression of exogenous wild type as well as phospho-mimetic S12E Fbx4 decreased steady-state cyclin D1 levels in TE8 cells with low endogenous Fbx4 activity (Figure 1e). As a negative control, expression of Fbx4 in TE7 cells (in which cyclin D1 is not efficiently exported to the cytoplasm (Benzeno et al., 2006) did not affect cyclin D1 levels. Taken together, these data suggest that expression of Fbx4 exerts a tumor suppressive effect in human cancer cells through regulation of cyclin D1. "
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    ABSTRACT: Fbx4 is an F-box constituent of Skp-Cullin-F-box (SCF) ubiquitin ligases that directs ubiquitylation of cyclin D1. Ubiquitylation of cyclin D1 requires phosphorylation of both cyclin D1 and Fbx4 by GSK3β. GSK3β-mediated phosphorylation of Fbx4 Ser12 during the G1/S transition regulates Fbx4 dimerization, which in turn governs Fbx4-driven E3 ligase activity. In esophageal carcinomas that overexpress cyclin D1, Fbx4 is subject to inactivating mutations that specifically disrupt dimerization, highlighting the biological significance of this regulatory mechanism. In an effort to elucidate the mechanisms that regulate dimerization, we sought to identify proteins that differentially bind to wild-type Fbx4 versus a cancer-derived dimerization-deficient mutant. We provide evidence that phosphorylation of Ser12 generates a docking site for 14-3-3ɛ. 14-3-3ɛ binds to endogenous Fbx4 and this association is impaired by mutations that target either Ser8 or Ser12 in Fbx4, suggesting that this N-terminal motif in Fbx4 directs its interaction with 14-3-3ɛ. Knockdown of 14-3-3ɛ inhibited Fbx4 dimerization, reduced SCF(Fbx4) E3 ligase activity and stabilized cyclin D1. Collectively, the current results suggest a model wherein 14-3-3ɛ binds to Ser12-phosphorylated Fbx4 to mediate dimerization and function.
    Oncogene 01/2011; 30(17):1995-2002. DOI:10.1038/onc.2010.584 · 8.56 Impact Factor
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    • "Cyclin D1 is subject to intricate posttranslational control to ensure its temporal regulation. Cyclin D1 nuclear export followed by Fbx4-dependent ubiquitylation and degradation during S-phase are key features that serve to restrain nuclear cyclin D1/CDK4 activity and ensure normal cell division (Alt et al., 2000; Benzeno et al., 2006; Lin et al., 2006). Previous work revealed that aberrant nuclear accumulation of cyclin D1 during S-phase promotes transformation in vitro (Alt et al., 2000) and drives both B cell lymphomas and mammary carcinomas in mice (Gladden et al., 2006; Lin et al., 2008). "
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    ABSTRACT: Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 coregulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth, including CUL4 repression, CDT1 overexpression, and DNA rereplication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.
    Cancer cell 10/2010; 18(4):329-40. DOI:10.1016/j.ccr.2010.08.012 · 23.89 Impact Factor
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