CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G 2͞M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.apoptosis ͉ cancer therapy ͉ cell cycle ͉ cytokinesis ͉ replication origin T he cell cycle is a precisely controlled set of biochemical and morphological events driven by the sequential activation of cyclin-dependent kinases (CDKs) (1). Three CDKs and their activating cyclins (A, B, D, and E) play key roles in mammalian cell cycle regulation (2). It has been established that CDK4͞ cyclin D and CDK2͞cyclin E͞A promote the passage through G 1 and S phases, whereas CDK1͞cyclin B regulates the transition through late G 2 and mitosis (3). However, recent genetic and RNA interference studies in mammalian cells have revealed that CDK2 and CDK4 are not essential for cell cycle progression, thus leaving CDK1 as the only nonredundant cell cycle driver (4-6). Genetic studies in yeast and mammalian cells have established the critical role of CDK1 in mitosis, but the long list of its putative substrate proteins still is growing, and its precise functions in the context of the dividing cell are poorly understood (7,8). This deficiency is due, in part, to the lack of specific molecular tools for reversible modulation of CDK1 activity in vivo. Several potent small-molecule inhibitors have been reported, but their activity and cell cycle profiles are not consistent with specific CDK1 inhibition (9, 10). Here, we identify a selective and reversible inhibitor of the catalytic activity of human CDK1͞cyclin B1 and CDK1͞cyclin A complexes that allows synchronization of proliferating cells in the late G 2 phase and probing of CDK1 function in the cellular context. Results and DiscussionWe screened a diverse library of organic compounds for their ability to inhibit the catalytic activity of human CDK1͞cyclin B1. The hits then were tested for selectivity against CDK2͞cyclin E and CDK4͞cyclin D. A class of thiazolinone analogs emerged as a source of ATP-competitive CDK1 inhibitors that were then further optimized for potency, selectivity, and ability to modulate CDK1 in proliferating cells. One quinolinyl thiazolinone derivative, RO-3306, showed good potency, in vitro selectivity, and a cell cycle profile (G 2 ͞M arrest) consistent with CDK1 inhibition (Fig. 1A). RO-3306 inhibited CDK1͞cyclin B1 activity with K i of 35 nM, nearly 10-fold selectivity relative to CDK2͞ cyclin ...

FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p=4.89 × 10−57), high mitotic index (p=5.25 × 10−28), pleomorphism (p=6.31 × 10−19), ER negative (p=9.02 × 10−35), PR negative (p=9.24 × 10−24), triple negative phenotype (p=6.67 × 10−21), PAM50.Her2 (p=5.19 × 10−13), PAM50.Basal (p=2.7 × 10−41), PAM50.LumB (p=1.56 × 10−26), integrative molecular cluster 1 (intClust.1) (p=7.47 × 10−12), intClust.5 (p=4.05 × 10−12) and intClust. 10 (p=7.59 × 10−38) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p= 4.4 × 10−16) and multivariate analysis (p= 9.19 × 10−7). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps<0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps<0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps<0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps<0.05). We conclude that FEN1 is a promising biomarker in breast and ovarian epithelial cancer.

Discovery of cancer genes through interrogation of genomic dosage is one of the major approaches in cancer research. In this study, we report that phosphodiesterase subtype 4D (PDE4D) gene was homozygously deleted in 198 cases of 5,569 primary solid tumors (3.56%), with most being internal microdeletions. Unexpectedly, the microdeletions did not result in loss of their gene products. Screening PDE4D expression in 11 different types of primary tumor samples (n = 165) with immunohistochemistry staining revealed that its protein levels were up-regulated compared with corresponding nontransformed tissues. Importantly, depletion of endogenous PDE4D with three independent shRNAs caused apoptosis and growth inhibition in multiple types of cancer cells, including breast, lung, ovary, endometrium, gastric, and melanoma, which could be rescued by reexpression of PDE4D. We further showed that antitumor events triggered by PDE4D suppression were lineage-dependently associated with Bcl-2 interacting mediator of cell death (BIM) induction and microphthalmia-associated transcription factor (MITF) downregulation. Furthermore, ectopic expression of the PDE4D short isoform, PDE4D2, enhanced the proliferation of cancer cells both in vitro and in vivo. Moreover, treatment of cancer cells with a unique specific PDE4D inhibitor, 26B, triggered massive cell death and growth retardation. Notably, these antineoplastic effects induced by either shRNAs or small molecule occurred preferentially in cancer cells but not in nonmalignant epithelial cells. These results suggest that although targeted by genomic homozygous microdeletions, PDE4D functions as a tumor-promoting factor and represents a unique targetable enzyme of cancer cells.