Odile Mondesert

Inhibition of human tumor cell growth in vivo by an orally bioavailable inhibitor of CDC25 phosphatases

Cell cycle regulators, such as the CDC25 phosphatases, are potential targets for the development of new anticancer drugs. Here we report the identification and the characterization of BN82685, a quinone-based CDC25 inhibitor that is active in vitro and in vivo. BN82685 inhibits recombinant CDC25A, B, and C phosphatases in vitro. It inhibits the growth of human tumor cell lines with an IC50 in the submicromolar range, independently of their resistance to chemotherapeutic agents. This inhibitory effect is irreversible on both the purified CDC25 enzyme in vitro and on tumor cell proliferation. The specificity of BN82685 towards the CDC25 phosphatases is shown by an increase in cyclin-dependent kinase 1 tyrosine 15 phosphorylation, by the reversion of the mitosis-inducing effect of CDC25B overexpression in HeLa cells, and by the lack of a growth inhibitory effect in an assay based on the use of a CDC25-independent fission yeast model. Finally, when administered p.o., BN82685 is shown to inhibit the growth of the human pancreatic tumor Mia PaCa-2 xenografted in athymic nude mice. BN82685 is therefore a promising new compound targeting CDC25, which confirms the interest of the inhibition of these enzymes as an anticancer therapeutic strategy.

A Novel Synthetic Inhibitor of CDC25 Phosphatases BN82002

CDC25 dual-specificity phosphatases are essential regulators that dephosphorylate and activate cyclin-dependent kinase/cyclin complexes at key transitions of the cell cycle. CDC25 activity is currently considered to be an interesting target for the development of new antiproliferative agents. Here we report the identification of a new CDC25 inhibitor and the characterization of its effects at the molecular and cellular levels, and in animal models. BN82002 inhibits the phosphatase activity of recombinant human CDC25A, B, and C in vitro. It impairs the proliferation of tumoral cell lines and increases cyclin-dependent kinase 1 inhibitory tyrosine phosphorylation. In synchronized HeLa cells, BN82002 delays cell cycle progression at G1-S, in S phase and at the G2-M transition. In contrast, BN82002 arrests U2OS cell cycle mostly in the G1 phase. Selectivity of this inhibitor is demonstrated: (a) by the reversion of the mitotic-inducing effect observed in HeLa cells upon CDC25B overexpression; and (b) by the partial reversion of cell cycle arrest in U2OS expressing CDC25. We also show that BN82002 reduces growth rate of human tumor xenografts in athymic nude mice. BN82002 is a original CDC25 inhibitor that is active both in cell and animal models. This greatly reinforces the interest in CDC25 as an anticancer target.

Development of Novel Thiazolopyrimidines as CDC25B Phosphatase Inhibitors

CDC25 inhibition by thiazolopyrimidines: CDC25 is an attractive target for cancer therapy, as it is overexpressed in numerous cancers and is often associated with tumor aggressiveness and poor prognosis. Based on an inu2005silico/inu2005vitro screen, we developed a series of thiazolopyrimidines to further improve inhibitory activity and cytotoxic properties.

Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D

BackgroundMultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D.MethodsCell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters.ResultsWe describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage.ConclusionOur data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models.

CDC25B Phosphorylation by p38 and MK-2

CDC25B is one of the three human phosphatases that are involved in the control of the activation of cyclin-dependent kinases. CDC25B participates in regulating entry into mitosis and appears to play a key role in the checkpoint response to DNA injury.CDC25B has been reported to be regulated by a number of kinases and controversial evidence suggests that it is phosphorylated by p38SAPK and/or MAPKAP Kinase-2. In this report, we clarify this issue using an approach combining mass spectrometry andthe use of specific antibodies against phosphorylated CDC25B residues. We report that MAPKAP Kinase-2 phosphorylates CDC25B on multiple sites including S169, S323, S353 and S375, while p38 phosphorylates CDC25B on S249. We show that theS323-phosphorylated form of CDC25B is detected at the centrosome during a normal cell cycle. Since most of these sites are also phosphorylated by several other kinases, our observations highlight the difficulty in characterising and understanding in vivo phosphorylation patterns.

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity

Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin‐dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers.

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.

5-Substituted [1]pyrindine derivatives with antiproliferative activity

We report herein the synthesis of 5-substituted [1]pyrindine derivatives and the evaluation of their antiproliferative properties on HeLa cells, a cervical carcinoma tumor cell line, and on the melanoma A2058 cell line. The most efficient compounds display cytotoxicity against tumor cells in the micromolar range but have interestingly no effect against the normal human fibroblasts CRL-2796. Generally, these pyrindines are active on both tumor cell lines. Compounds bearing large substituents with structural rigidity at position 5 such as phenyl-furyl show no inhibition of cell growth.

Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model.

Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.

Gap junctions contribute to anchorage-independent clustering of breast cancer cells

BackgroundCancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. We previously reported that cell-cell adhesion proteins, such as E-cadherin, and desmosomal proteins are involved in cell aggregation to form clusters independently of cell migration or matrix adhesion. Here, we investigated the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells.MethodsWe used live cell image acquisition and analysis to monitor the kinetics of MCF7 cell clustering in the presence/absence of GJIC pharmacological inhibitors and to screen a LOPAC® bioactive compound library. We also used a calcein transfer assay and flow cytometry to evaluate GJIC involvement in cancer cell clustering.ResultsWe first demonstrated that functional GJIC are established in the early phase of cancer cell aggregation. We then showed that pharmacological inhibition of GJIC using tonabersat and meclofenamate delayed MCF7 cell clustering and reduced calcein transfer. We also found that brefeldin A, an inhibitor of vesicular trafficking, which we identified by screening a small compound library, and latrunculin A, an actin cytoskeleton-disrupting agent, both impaired MCF7 cell clustering and calcein transfer.ConclusionsOur results demonstrate that GJIC are involved from the earliest stages of anchorage-independent cancer cell aggregation. They also give insights into the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.