The initial cloning of the cyclin D1 cDNA as a component of a chromosomal translocation from a parathyroid adenoma (Motokura et al., 1991
) immediately suggested the propensity of cyclin D1 to function as a potential initiating factor in neoplastic growth. Such implications were supported when a second group revealed that cyclin D1 was synonymous with the bcl-1
oncogene present in the 11;14 translocation present in nearly 100% of mantle cell lymphomas (Motokura et al., 1991
). Additional data revealing that cyclin D1-expression was acutely regulated by mitogen-dependent growth factors was also consistent with this notion (Sherr and Roberts, 1999
). Strikingly however, in vitro assays that evaluated the potential of cyclin D1 to function as a bona fide oncogene presented a paradox. Unlike ras
oncogenes, which readily transformed murine fibroblasts, neither cyclin D1, D2, nor D3 showed such potential, but rather could only potentiate Ras-dependent transformation in vitro (Quelle et al., 1993
). Results from transgenic animals were equally unrevealing. Transgenic expression of wild type cyclin D1 in murine lymphocytes failed to elicit any appreciable phenotype unless mice also expressed a c-myc
transgene (Bodrug et al., 1994
; Lovec et al., 1994
). Similar results were obtained in a transgenic mouse model of oral-esophageal cancer wherein cyclin D1 overexpression resulted in invasive oral-esophageal cancer only in combination with p53 deletion (Opitz et al., 2002
). The only hint of oncogenic activity came from mice programmed to overexpress cyclin D1 under the control of mouse mammary tumor virus (MMTV) promoter in mammary epithelial cells. These MMTV-D1 mice succumb to adenocarcinomas after a long latency and with incomplete penetrance (Wang et al., 1994
One interpretation of the above data is that cyclin D1 does not possess overt oncogenic activity. However, given the large amount of data demonstrating its overexpression in numerous cancers this seems unlikely. Thus a second and more likely possibility seems that cyclin D1 overexpression per se is not sufficient to drive neoplastic growth. If so, the latter would suggest that cells maintain additional regulatory mechanisms that maintain tight control over cyclin D1 activity thereby preventing manifestation of its neoplastic potential. Insight into this possibility stemmed from early observations that cyclin D1/CDK4 complexes shuttled between nuclear and cytoplasmic compartments during cell cycle progression (Alt et al., 2000
; Diehl et al., 1998
). This work revealed that the rate of nuclear import effectively maintains cyclin D1/CDK4 complexes in the nucleus during G1 phase; however, as cells enter S-phase, phosphorylation of cyclin D1 dramatically increased its rate of nuclear export (Diehl et al., 1997
). Nuclear export coupled with ubiquitin-dependent destruction of cyclin D1 in the cytoplasm during S-phase effectively limits access of the cyclin D1/CDK4 kinase to nuclear substrates during S-phase.
Experimental models support the idea that mutations that interfere with nuclear exclusion of cyclin D1/CDK4 during S-phase trigger neoplastic conversion. Indeed overexpression of a mutant cyclin D1 (D1T286A) that is defective in phosphorylation-mediated nuclear export and subsequent proteolysis, induces cell transformation in cell culture assays and triggers B-cell lymphoma in a mouse model of mantle cell lymphoma (Alt et al., 2000
; Gladden et al., 2006
). Furthermore, transgenic mice that over-express the identical mutant cyclin D1 driven by MMTV promoter (MMTV-D1T286A) developed mammary adenocarcinoma with a shorter latency relative to mice over-expressing the wild-type cyclin D1 (MMTV-D1) (Lin et al., 2008
). These observations suggest that subcellular localization and stabilization of cyclin D1 through its altered nuclear trafficking and proteolysis may exert more profound effects on tumorigenesis than its overexpression itself.
The experimental data described above stem from use of cyclin D1 mutants that are refractory to both CRM1-dependent nuclear export and Fbx4- and ubiquitin-dependent turnover. While such mutations have been observed in human cancers, they appear at low frequency (<3%). Mutations that inactivate Fbx4 and thereby stabilize cyclin D1 occur at a much higher frequency (~15 %), suggesting that loss of this E3 ligase is sufficient for the manifestation of the neoplastic potential of cyclin D1 (Barbash et al., 2008
). While no knockout mouse for Fbx4 is currently available to directly examine this possibility, in vitro studies are consistent with this notion. ShRNA-dependent reduction of Fbx4 not only resulted stabilized cyclin D1 but also constitutively nuclear cyclin D1/CDK4 and spontaneous transformation, a phenotype similar to expression of cyclin D1T286A (Barbash et al., 2008
). This would suggest that the Fbx4 E3 ligase functions as a critical off switch that prevents inappropriate nuclear accumulation of the D1/CDK4 complex and thereby prevents cell transformation.