CDK-Mediated Regulation of Cell Functions via c-Jun Phosphorylation and AP-1 Activation

Cyclin-dependent kinases (CDKs) and their targets have been primarily associated with regulation of cell-cycle progression. Here we identify c-Jun, a transcription factor involved in the regulation of a broad spectrum of cellular functions, as a newly recognized CDK substrate. Using immune cells from mouse and human, and several complementary in vitro and in vivo approaches including dominant negative protein expression, pharmacologic inhibitors, kinase assays and CDK4 deficient cells, we demonstrate the ability of CDK4 to phosphorylate c-Jun. Additionally, the activity of AP-1, a ubiquitous transcription factor containing phosphorylated c-Jun as a subunit, was inhibited by abrogating CDK4. Surprisingly, the regulation of c-Jun phosphorylation by CDK4 occurred in non-dividing cells, indicating that this pathway is utilized for cell functions that are independent of proliferation. Our studies identify a new substrate for CDK4 and suggest a mechanism by which CDKs can regulate multiple cellular activation functions, not all of which are directly associated with cell cycle progression. These findings point to additional roles of CDKs in cell signaling and reveal potential implications for therapeutic manipulations of this kinase pathway

Transcriptional role of cyclin D1 in development revealed by a “genetic-proteomic” screen

Author manuscript: 2010 September 22.Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers[superscript 1, 2]. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas—an organ that critically requires cyclin D1 function[superscript 3, 4]—cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term ‘genetic–proteomic’, can be used to study the in vivo function of essentially any protein

Targeting cell cycle and hormone receptor pathways in cancer

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.C E S Comstock, M A Augello, J F Goodwin, R de Leeuw, M J Schiewer, W F Ostrander Jr, R A Burkhart, A K McClendon, P A McCue, E J Trabulsi, C D Lallas, L G Gomella, M M Centenera, J R Brody, L M Butler, W D Tilley and K E Knudse

Cyclin d1 depletion interferes with cancer oxidative balance and sensitizes cancer cells to senescence

Expression of cyclin D1 is required for cancer cell survival and proliferation. This is presumably due to the role of cyclin D1 in RB inactivation. Here we investigated the prosurvival function of cyclin D1 in a number of cancer cell lines. We found that cyclin D1 depletion facilitated cellular senescence in several cancer cell lines tested. Senescence triggered by cyclin D1 depletion was more extensive than that caused by the prolonged CDK4 inhibition. Intriguingly, the senescence caused by cyclin D1 depletion was independent of RB status of the cancer cell. We identified a buildup of intracellular reactive oxygen species, in the cancer cells that underwent senescence upon cyclin D1 depletion, but not in CDK4 inhibition, and that ROS buildup was responsible for the senescence. Lastly, the senescence was found to be instigated by the p38/JNK-FOXO3a-p27 pathway. Therefore, expression of cyclin D1 prevents cancer cells from undergoing senescence, at least partially, by keeping the level of intracellular oxidative stress at a tolerable sub-lethal level. Depletion of cyclin D1 promotes the RB-independent pro-senescence pathway, and cancer cell succumbing to the endogenous oxidative stress