Nuclear accumulation of cyclin D1 and cancer

Proc Amer Assoc Cancer Res, Volume 46, 2005 SY08-1 Progression through each phase of the cell cycle is driven by complexes composed of a regulatory, cyclin, and a catalytic, CDK, subunit. G1 phase progression is initiated by mitogenic stimulation, which in turn initiates the expression and assembly of the D-type cyclins (D1, D2, D3) with their catalytic partner CDK4/6. The D-type cyclins are unique in that they respond directly to mitogenic signaling pathways rather than to signals intrinsic to cell cycle progression. Cyclin D1 expression requires Ras-dependent activation of the Raf-Mek-Erk kinase module. While the MAPK module signals cyclin D1 expression, maximal accumulation of cyclin D1 also depends on PI-3K-dependent inactivation of glycogen synthase kinase 3 beta (GSK-3β). GSK-3β-dependent phosphorylation of cyclin D1 on threonine 286 (Thr-286) triggers the ubiquitination and proteolysis of cyclin D1 via the 26S-proteasome during S-phase. Although cyclin D1 accumulates in the nucleus during the G1 interval, it relocalizes to the cytoplasm during S-phase. It is now clear that phosphorylation of cyclin D1 at Thr-286 not only promotes cyclin D1 proteolysis, but also CRM1 binding, and thus, cyclin D1/CDK4 nuclear export. This temporal regulation of cyclin D1 wherein it accumulates in the nucleus during G1 and is relocalized to the cytoplasm during S-phase implies that while the essential functions of cyclin D1 may require its nuclear localization, the redistribution of cyclin D1/CDK4 complexes to the cytoplasm following G1 implies that regulation of cyclin D1 nucleo-cytoplasmic distribution is necessary for maintaining cellular homeostasis. Cyclin D1 overexpression has been documented in an increasing number of solid tumors including esophageal, colonic, breast, and head and neck carcinomas. In addition to involvement in solid tumors, cyclin D1 was identified as the BcL-1 oncogene associated with the chromosomal translocation t(11;14)(q13;q32) in mantle cell lymphoma (MCL). MCL is an aggressive B-cell lymphoma, with a variable initial response to standard chemotherapeutic treatments and high incidence of relapse resulting in the overall poor survival. Cyclin D1 is invariably expressed due to translocation in MCL. Noteworthy, initial attempts to establish cyclin D1 as a dominant B-cell oncogene via overexpression in transgenic mice in were unsuccessful while attempts to model genetic changes associated with other human B-cell malignancies, such as those associated with Burkitt (c-myc) and follicular lymphoma (BcL-2), have been successful. While overexpression of wild type cyclin D1 is not itself sufficient for oncogenic transformation even in vitro we have recently found that cyclin D1 mutants that remain constitutively nuclear due to impaired Thr-286 phosphorylation, can transform cultured murine fibroblasts. These results imply that the inability of overexpressed wild type cyclin D1 to promote B-cell lymphomas in transgenic mice reflects the capacity of lymphocytes to maintain proper temporal regulation of cyclin D1 activity through regulated nuclear export. In lieu of the transforming ability of the cyclin D1-T286A mutant compared with wild type D1, we have investigated the possibility that constitutively nuclear localization of cyclin D1 is sufficient to drive lymphoid malignancies in mice. We have generated mice expressing Flag-D1-T286A under the control of the immunoglobulin enhancer, Eμ. The product of the D1-T286A transgene binds with CDK4 and p27 but not with CDK6 in lymphocytes. Mice expressing the Eμ D1-T286A transgene have a decreased life span due to the onset of malignant B cell lymphomas with an average latency of 14 months. Tumors are uniformly B-cell lymphomas with some degree of disseminated disease. We have begun to investigate whether known tumor suppressor pathways are targeted through secondary mutations in the observed lymphomas. Thus far, we have observed a subset of tumors wherein the ARF/Mdm2/p53 pathway is disrupted. We have also found that a majority of tumors overexpress either the Bcl-2 or Bcl-xL anti-apoptotic proteins. Our results suggest the ability of cyclin D1 to promote neoplastic growth is dependent upon its constitutive nuclear localization throughout the cell cycle and collectively suggest that disruption of cyclin D1 nuclear export will contribute to human malignancy. Indeed, we have identified mutations that specifically disrupt cyclin D1 nuclear export in human cancer. Our data are consistent with a model wherein deregulation of cyclin D1 nuclear export contributes to human neoplastic growth.