Muriel Quaranta

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.

Genotoxic-activated G2-M checkpoint exit is dependent on CDC25B phosphatase expression

Cell cycle arrest at the G2-M checkpoint is an essential feature of the mechanisms that preserve genomic integrity. CDC25 phosphatases control cell cycle progression by dephosphorylating and activating cyclin-dependent kinase/cyclin complexes. Their activities are, therefore, tightly regulated to modulate cell cycle arrest in response to DNA damage exposure. Here, we report that overexpression of CDC25B affects viability, reduces clonogenic efficiency, and increases sensitivity of cancer cells to a genotoxic agent. We show that ectopic expression of CDC25B results in bypass of a genotoxic-induced G2-M checkpoint. In addition, cancer cells constitutively expressing high level of CDC25B are shown to be prone to exit prematurely from the G2-M checkpoint arrest and to enter mitosis. Finally, we show that this exit is dependent on CDC25B expression. Together with previous results, our data strongly support a model in which CDC25B is the key phosphatase that controls entry into mitosis after DNA damage, thus emphasizing the relevance of its overexpression in many human tumors. [Mol Cancer Ther 2006;5(6):1446–51]

CDC25B Phosphorylated by pEg3 Localizes to the Centrosome and the Spindle Poles at Mitosis

The phosphatase CDC25B is one of the key regulators that control entry into mitosis throughthe dephosphorylation and subsequent activation of the cyclin-dependent kinases. Here westudy the phosphorylation of CDC25B at mitosis by the kinase pEg3, a member of theKIN1/PAR-1/MARK family. Using mass spectrometry analysis we demonstrate thatCDC25B is phosphorylated in vitro by pEg3 on serine 169, a residue that lies within the Bdomain. Moreover, using phosphoepitope-specific antibodies we show that serine 169 isphosphorylated in vivo, that this phosphorylated form of CDC25B accumulates duringmitosis, and is localized to the centrosomes. This labelling is abrogated when pEg3expression is repressed by RNA interference. Taken together, these results support a model inwhich pEg3 contributes to the control of progression through mitosis by phosphorylation ofthe CDC25 phosphatases.

IRC-083864, a novel bis quinone inhibitor of CDC25 phosphatases active against human cancer cells.

CDC25 phosphatases are key actors in cyclin‐dependent kinases activation whose role is essential at various stages of the cell cycle. CDC25 expression is upregulated in a number of human cancers. CDC25 phosphatases are therefore thought to represent promising novel targets in cancer therapy. Here, we report the identification and the characterization of IRC‐083864, an original bis‐quinone moiety that is a potent and selective inhibitor of CDC25 phosphatases in the low nanomolar range. IRC‐083864 inhibits cell proliferation of a number of cell lines, regardless of their resistance to other drugs. It irreversibly inhibits cell proliferation and cell cycle progression and prevents entry into mitosis. In addition, it inhibits the growth of HCT‐116 tumor spheroids with induction of p21 and apoptosis. Finally, IRC‐083864 reduced tumor growth in mice with established human prostatic and pancreatic tumor xenografts. This study describes a novel compound, which merits further study as a potential anticancer agent.

The membrane-tubulating potential of amphiphysin 2/BIN1 is dependent on the microtubule-binding cytoplasmic linker protein 170 (CLIP-170).

Amphiphysins are BIN-amphiphysin-RVS (BAR) domain-containing proteins that influence membrane curvature in sites such as T-tubules in muscular cells, endocytic pits in neuronal as well as non-neuronal cells, and possibly cytoplasmic endosomes. This effect on lipid membranes is fulfilled by diverse amphiphysin 2/BIN1 isoforms, generated by alternative splicing and showing distinct structural and functional properties. In this study, our goal was to characterize the functional role of a ubiquitously expressed amphiphysin 2/BIN1 by the characterization of new molecular partners. We performed a two-hybrid screen with an isoform of amphiphysin 2/BIN1 expressed in HeLa cells. We identified CLIP-170 as an amphiphysin 2/BIN1-interacting molecule. CLIP-170 is a plus-end tracking protein involved in microtubule (MT) stability and recruitment of dynactin. The binding between amphiphysin 2/BIN1 and CLIP-170 is dependent on the N-terminal part of amphiphysin 2 (mostly the BAR domain) and an internal coiled-coil region of CLIP-170. This partnership was confirmed by GST pull-down assay and by co-immunoprecipitation in HeLa cells that express endogenous amphiphysin 2 (mostly isoforms 6, 9 and 10). When overexpressed in HeLa cells, amphiphysin 2/BIN1 leads to the formation of intracellular tubules which can closely align with MTs. After MT depolymerization by nocodazole, amphiphysin 2-stained tubules disappear, and reappear after nocodazole washout. Furthermore, depletion of CLIP-170 by RNAi induced a decrease in the proportion of cells with amphiphysin 2-stained tubules and an increase in the proportion of cells with no tubules. This result suggests the existence of a mechanistic link between the two types of tubules, which is likely to involve the +TIP protein, CLIP-170. Amphiphysin 2/BIN1 may be an anchoring point on membranes for CLIP-170, and consequently for MT. Then, the pushing force of polymerizing MT could help amphiphysin 2/BIN1 in its tubulation potential. We propose that amphiphysin 2/BIN1 participates in the tubulation of traffic intermediates and intracellular organelles first via its intrinsic tubulating potential and second via its ability to bind CLIP-170 and MT.

Evaluation of Polo-like Kinase 1 inhibition on the G2/M checkpoint in Acute Myelocytic Leukaemia

Polo-Kinase 1 (PLK1) is a key cell cycle regulator that is necessary for checkpoint recovery after DNA damage-induced G2 arrest. We have examined the effects of PLK inhibition in Acute Myelocytic Leukaemia (AML) cells, whose resistance to genotoxic agents is thought to be associated with checkpoint reinforcement. We report that in U937 AML cells, PLK1 participates in checkpoint recovery, and that inhibition of PLK by the GW843682X compound results in mitotic accumulation and apoptosis. We also found that when challenged with VP-16, inhibition of PLK1 prevented U937 cells from checkpoint exit. Finally, we found that treatment with GW843682X slightly reduced genotoxic-induced inhibition of colony formation efficiency of primary leukaemia cells (CFU-L) from AML patients.

Interplay between chromatin modifying enzymes controls colon cancer progression through Wnt signaling

Cancer progression is associated with epigenetic alterations, such as changes in DNA methylation, histone modifications or variants incorporation. The p400 ATPase, which can incorporate the H2A.Z variant, and the Tip60 histone acetyltransferase are interacting chromatin-modifying proteins crucial for the control of cell proliferation. We demonstrate here that Tip60 acts as a tumor suppressor in colon, since mice heterozygous for Tip60 are more susceptible to chemically induced preneoplastic lesions and adenomas. Strikingly, heterozygosity for p400 reverses the Tip60-dependent formation of preneoplastic lesions, uncovering for the first time pro-oncogenic functions for p400. By genome-wide analysis and using a specific inhibitor in vivo, we demonstrated that these effects are dependent on Wnt signaling which is antagonistically impacted by p400 and Tip60: p400 directly favors the expression of a subset of Wnt-target genes and regulators, whereas Tip60 prevents β-catenin acetylation and activation. Taken together, our data underline the physiopathological importance of interplays between chromatin-modifying enzymes in the control of cancer-related signaling pathways.

H2A.Z depletion impairs proliferation and viability but not DNA double-strand breaks repair in human immortalized and tumoral cell lines.

In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z.

A new mitotic-cell specific monoclonal antibody

No abstract.