CCN3/Nephroblastoma Overexpressed Is a Functional Mediator of Prostate Cancer Bone Metastasis That Is Associated with Poor Patient Prognosis.

Prostate cancer (PC) commonly metastasizes to the bone, resulting in pathologic fractures and poor prognosis. CCN3/nephroblastoma overexpressed is a secreted protein with a known role in promoting breast cancer metastasis to bone. However, in PC, CCN3 has been ascribed conflicting roles; some studies suggest that CCN3 promotes PC metastasis, whereas others argue a tumor suppressor role for CCN3 in this disease. Indeed, in the latter context, CCN3 has been shown to sequester the androgen receptor (AR) and suppress AR signaling. In the present study, we demonstrate that CCN3 functions as a bone-metastatic mediator, which is dependent on its C-terminal domain for this function. Analysis of tissue microarrays comprising >1500 primary PC patient radical prostatectomy specimens reveals that CCN3 expression correlates with aggressive disease and is negatively correlated with the expression of prostate-specific antigen, a marker of AR signaling. Together, these findings point to CCN3 as a biomarker to predict PC aggressiveness while providing clarity on its role as a functional mediator of PC bone metastasis.

Cell cycle-dependent localization of CHK2 at centrosomes during mitosis.

BACKGROUND: Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. RESULTS: Here, we show that CHK2 only localizes to centrosomes during mitosis. Using wild-type and CHK2-/- HCT116 human colon cancer cells and human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs we show that several CHK2 antibodies are non-specific and cross-react with an unknown centrosomal protein(s) by immunofluorescence. To characterize the localization of CHK2, we generated cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with the centrosomes in a Polo-like kinase 1-dependent manner during mitosis, from early mitotic stages until cytokinesis. CONCLUSION: Our findings demonstrate that a subpopulation of CHK2 localizes at the centrosomes in mitotic cells but not in interphase. These results are consistent with previous reports supporting a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis.

Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage.

BACKGROUND: CDC25B phosphatase is a cell cycle regulator that plays a critical role in checkpoint control. Up-regulation of CDC25B expression has been documented in a variety of human cancers, however, the relationships with the alteration of the molecular mechanisms that lead to oncogenesis still remain unclear. To address this issue we have investigated, in model cell lines, the consequences of unscheduled and elevated CDC25B levels. RESULTS: We report that increased CDC25B expression leads to DNA damage in the absence of genotoxic treatment. H2AX phosphorylation is detected in S-phase cells and requires active replication. We also report that CDC25B expression impairs DNA replication and results in an increased recruitment of the CDC45 replication factor onto chromatin. Finally, we observed chromosomal aberrations that are also enhanced upon CDC25B expression. CONCLUSION: Overall, our results demonstrate that a moderate and unscheduled increase in CDC25B level, as observed in a number of human tumours, is sufficient to overcome the S-phase checkpoint efficiency thus leading to replicative stress and genomic instability.

DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis.

Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senescence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent. Extensive research on tumor suppressor genes, oncogenes, the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are tied to cell proliferation, cell-cycle arrest, cellular senescence and apoptosis. DNA-damage responses are complex, involving "sensor" proteins that sense the damage, and transmit signals to "transducer" proteins, which, in turn, convey the signals to numerous "effector" proteins implicated in specific cellular pathways, including DNA repair mechanisms, cell-cycle checkpoints, cellular senescence and apoptosis. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/recombination and cellular senescence, effects that are generally distinct from their function in apoptosis. In this review, we report progress in understanding the molecular networks that regulate cell-cycle checkpoints, cellular senescence and apoptosis after DNA damage, and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

CHK1 phosphorylates CDC25B during the cell cycle in the absence of DNA damage.

CDC25B is one of the three human phosphatases that activate the CDK-cyclin complexes, thereby triggering cell-cycle progression and division. Commitment to early mitotic events depends on the activation of a centrosomal pool of CDK1-cyclin-B1, and CDC25B is thought to be involved in initiating this centrosomal CDK1-cyclin-B1 activity. Centrosome-associated checkpoint kinase 1 (CHK1) has been proposed to contribute to the proper timing of a normal cell division cycle by inhibiting the activation of the centrosomal pool of CDK1. Here, we show that CDC25B is phosphorylated by CHK1 in vitro on multiple residues, including S230 and S563. We demonstrate these phosphorylations occur in vivo and that they are dependent on CHK1 activity. S230 CHK1-mediated phosphorylation is detected in cell extracts during S phase and G2 phase in the absence of DNA damage. We show that the S230-phosphorylated form of CDC25B is located at the centrosome from early S phase until mitosis. Furthermore, mutation of S230 to alanine increases the mitotic-inducing activity of CDC25B. Our results support a model in which, under normal cell cycle conditions and in the absence of DNA damage, CHK1 constitutively phosphorylates CDC25B during interphase and thus prevents the premature initiation of mitosis by negatively regulating the activity of CDC25B at the centrosome.

CDC25B phosphorylation by Aurora-A occurs at the G2/M transition and is inhibited by DNA damage.

CDC25B is one of the three human dual-specificity phosphatases involved in the activation of cyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsible for the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-A kinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/M transition. Here we have investigated this phosphorylation event during the cell cycle, and in response to activation of the G2 DNA damage checkpoint. We show that accumulation of the S353-phosphorylated form of CDC25B at the centrosome correlates with the relocalization of cyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation of the G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated and consequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-A results in a bypass of the checkpoint that was partially overcome by a S353A mutant of CDC25B. Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01 results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These results strongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is an important step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclin B1 activation.

Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.

Bcl-xES, a BH4- and BH2-containing antiapoptotic protein, delays Bax oligomer formation and binds Apaf-1, blocking procaspase-9 activation.

Bcl-2 family members either negatively or positively regulate the apoptotic threshold of cells. Bcl-xES (extra short), a novel Bcl-x member, possesses a unique combination of BH4 and BH2 domains as well as a COOH-terminal hydrophobic transmembrane anchor domain. Bcl-xES contains sequences of hydrophobic alpha-6 helices but lacks sequences of alpha-5 helices, suggesting that it does not have pore channel-forming activity but functions uniquely as a trapping protein. mRNA expression analysis by reverse transcriptase-polymerase chain reaction and RNase protection assay reveal that Bcl-xES is expressed in a variety of human cancer cell lines and human tumors, including bone marrow from patients with acute lymphoblastic leukemia. Bcl-xES expression is much less pronounced in some specimens of normal human tissues, including the breast, ovary, testis and lung. Stable, transfected human B lymphoma Namalwa variant cells expressing Bcl-xES were derived to investigate its role in apoptosis. Bcl-xES had a preventive effect on cell death induced by tumor necrosis factor-alpha and various concentrations of anticancer drugs, including camptothecin, etoposide and cisplatin. Its protective action on cell death was correlated with the inhibition of mitochondrial cytochrome c release and caspase activation. In a yeast two-hybrid system, Bcl-xES interacted with most Bcl-2 family members, including those containing only a BH3 domain, and with the Ced-4 homolog Apaf-1. Co-immunoprecipitation and gel filtration chromatography experiments suggest that Bcl-xES delays drug-induced apoptosis by disturbing the formation of Bax oligomers and preventing cytochrome c release, but also by interacting with Apaf-1 and inhibiting procaspase-9 activation, thus averting the apoptogenic proteolytic caspase cascade and cell death.

Activation and role of caspases in chemotherapy-induced apoptosis.

The importance of caspase activation during apoptosis has become eminently apparent in the last few years. The caspases participate in a proteolytic cascade activated in response to various stimuli, including anticancer drugs, that results in the systematic and orderly eradication of the cell. The core machinery of caspase activation is now emerging and involves multiple molecular complexes. We describe the two best-studied models of caspase activation, the mitochondrial pathway and the cell death receptor pathway, and discuss their involvement in caspase activation induced by various anticancer drugs used in chemotherapy. Defective apoptosis contributes to tumor growth and drug resistance. Understanding the activation and role of caspases in apoptosis may help develop new therapeutic strategies to circumvent drug resistance. Copyright 1999 Harcourt Publishers Ltd.