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

Johns Hopkins University, Baltimore, Maryland, United States
Oncogene (Impact Factor: 8.46). 11/2006; 25(47):6291-303. DOI: 10.1038/sj.onc.1209644
Source: PubMed


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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    • "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.
    Full-text · Article · Mar 2015 · EMBO Molecular Medicine
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    • "We showed a TGFβ-induced nuclear localization of cyclin D1 in these metastatic breast cancer cell lines. It has been demonstrated that oncogenic actions of cyclin D1 are predominantly nuclear in many cancers, as carcinogenic mutations and deletions often occur at the T286 site, which controls cyclin D1 protein turnover and nuclear export [71,72]. Mutated cyclin D1 with constitutive nuclear localization and impaired degradation not only enhanced cyclin D1 transformation efficiency in vitro, but also promoted tumor formation in vivo [73]. "
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    ABSTRACT: Deregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis, and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFbeta) in breast cancer progression. Cyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion. We found TGFbeta to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFbeta-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFbeta-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFbeta promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21. Thus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFbeta-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.
    Full-text · Article · Jun 2013 · Breast cancer research: BCR
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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.
    Full-text · Article · Jan 2011 · Oncogene
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