Sarah De Clercq

Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death

Adult stem cells (SCs) are at high risk of accumulating deleterious mutations because they reside and self-renew in adult tissues for extended periods. Little is known about how adult SCs sense and respond to DNA damage within their natural niche. Here, using mouse epidermis as a model, we define the functional consequences and the molecular mechanisms by which adult SCs respond to DNA damage. We show that multipotent hair-follicle-bulge SCs have two important mechanisms for increasing their resistance to DNA-damage-induced cell death: higher expression of the anti-apoptotic gene Bcl-2 and transient stabilization of p53 after DNA damage in bulge SCs. The attenuated p53 activation is the consequence of a faster DNA repair activity, mediated by a higher non-homologous end joining (NHEJ) activity, induced by the key protein DNA-PK. Because NHEJ is an error-prone mechanism, this novel characteristic of adult SCs may have important implications in cancer development and ageing.

Small-molecule MDM2 antagonists as a new therapy concept for neuroblastoma.

Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G(1) cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification.

Evolutionarily conserved role of nucleostemin: controlling proliferation of stem/progenitor cells during early vertebrate development.

ABSTRACT Nucleostemin (NS) is a putative GTPase expressed preferentially in the nucleoli of neuronal and embryonic stem cells and several cancer cell lines. Transfection and knockdown studies indicated that NS controls the proliferation of these cells by interacting with the p53 tumor suppressor protein and regulating its activity. To assess the physiological role of NS in vivo, we generated a mutant mouse line with a specific gene trap event that inactivates the NS allele. The corresponding NS−/− embryos died around embryonic day 4. Analyses of NS mutant blastocysts indicated that NS is not required to maintain pluripotency, nucleolar integrity, or survival of the embryonic stem cells. However, the homozygous mutant blastocysts failed to enter S phase even in the absence of functional p53. Haploid insufficiency of NS in mouse embryonic fibroblasts leads to decreased cell proliferation. NS also functions in early amphibian development to control cell proliferation of neural progenitor cells. Our results show that NS has a unique ability, derived from an ancestral function, to control the proliferation rate of stem/progenitor cells in vivo independently of p53.

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.

Identification of the tumour transition states occurring during EMT

In cancer, the epithelial-to-mesenchymal transition (EMT) is associated with tumour stemness, metastasis and resistance to therapy. It has recently been proposed that, rather than being a binary process, EMT occurs through distinct intermediate states. However, there is no direct in vivo evidence for this idea. Here we screen a large panel of cell surface markers in skin and mammary primary tumours, and identify the existence of multiple tumour subpopulations associated with different EMT stages: from epithelial to completely mesenchymal states, passing through intermediate hybrid states. Although all EMT subpopulations presented similar tumour-propagating cell capacity, they displayed differences in cellular plasticity, invasiveness and metastatic potential. Their transcriptional and epigenetic landscapes identify the underlying gene regulatory networks, transcription factors and signalling pathways that control these different EMT transition states. Finally, these tumour subpopulations are localized in different niches that differentially regulate EMT transition states.Epithelial-to-mesenchymal transition in tumour cells occurs through distinct intermediate states, associated with different metastatic potential, cellular properties, gene expression, and chromatin landscape

Expanding roles for the evolutionarily conserved Dmrt sex transcriptional regulators during embryogenesis

Dmrt genes encode a large family of transcription factors characterized by the presence of a DM domain, an unusual zinc finger DNA binding domain. While Dmrt genes are well known for their important role in sexual development in arthropodes, nematodes and vertebrates, several new findings indicate emerging functions of this gene family in other developmental processes. Here, we provide an overview of the evolution, structure and mechanisms of action of Dmrt genes. We summarize recent findings on their function in sexual regulation and discuss more extensively the role played by these proteins in somitogenesis and neural development.

Widespread Overexpression of Epitope-Tagged Mdm4 Does Not Accelerate Tumor Formation In Vivo

ABSTRACT Mdm2 and Mdm4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or Mdm4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and Mdm4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the Mdm4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of Mdm4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the Mdm4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or Eμ-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy.

The Doublesex Homolog Dmrt5 is Required for the Development of the Caudomedial Cerebral Cortex in Mammals

Regional patterning of the cerebral cortex is initiated by morphogens secreted by patterning centers that establish graded expression of transcription factors within cortical progenitors. Here, we show that Dmrt5 is expressed in cortical progenitors in a high-caudomedial to low-rostrolateral gradient. In its absence, the cortex is strongly reduced and exhibits severe abnormalities, including agenesis of the hippocampus and choroid plexus and defects in commissural and thalamocortical tracts. Loss of Dmrt5 results in decreased Wnt and Bmp in one of the major telencephalic patterning centers, the dorsomedial telencephalon, and in a reduction of Cajal-Retzius cells. Expression of the dorsal midline signaling center-dependent transcription factors is downregulated, including Emx2, which promotes caudomedial fates, while the rostral determinant Pax6, which is inhibited by midline signals, is upregulated. Consistently, Dmrt5(-/-) brains exhibit patterning defects with a dramatic reduction of the caudomedial cortex. Dmrt5 is increased upon the activation of Wnt signaling and downregulated in Gli3(xt/xt) mutants. We conclude that Dmrt5 is a novel Wnt-dependent transcription factor required for early cortical development and that it may regulate initial cortical patterning by promoting dorsal midline signaling center formation and thereby helping to establish the graded expression of the other transcription regulators of cortical identity.

DMRT5 together with DMRT3 directly controls hippocampus development and neocortical area map formation

Mice that are constitutively null for the zinc finger doublesex and mab-3 related (Dmrt) gene, Dmrt5/Dmrta2, show a variety of patterning abnormalities in the cerebral cortex, including the loss of the cortical hem, a powerful cortical signaling center. In conditional Dmrt5 gain of function and loss of function mouse models, we generated bidirectional changes in the neocortical area map without affecting the hem. Analysis indicated that DMRT5, independent of the hem, directs the rostral-to-caudal pattern of the neocortical area map. Thus, DMRT5 joins a small number of transcription factors shown to control directly area size and position in the neocortex. Dmrt5 deletion after hem formation also reduced hippocampal size and shifted the position of the neocortical/paleocortical boundary. Dmrt3, like Dmrt5, is expressed in a gradient across the cortical primordium. Mice lacking Dmrt3 show cortical patterning defects akin to but milder than those in Dmrt5 mutants, perhaps in part because Dmrt5 expression increases in the absence of Dmrt3. DMRT5 upregulates Dmrt3 expression and negatively regulates its own expression, which may stabilize the level of DMRT5. Together, our findings indicate that finely tuned levels of DMRT5, together with DMRT3, regulate patterning of the cerebral cortex.

Abstract C62: Targeting cancer cell migration and invasion by immunomodulation of actin binding proteins using single domain antibodies (nanobodies)

Enhanced motility of cancer cells by remodeling of the actin cytoskeleton through the concerted actions of different classes of actin binding proteins is crucial in the process of cancer cell invasion and metastasis. It is therefore not surprising that expression of quite a few actin associated proteins like gelsolin, myopodin and plastin, fluctuates during tumorigenesis. Furthermore, many studies using overexpression or RNA interference showed a direct involvement of these structural proteins in malignant cell behavior. Specific inhibitors of most cytoskeletal proteins are lacking. In an attempt to fill this gap, we developed Camelid single domain antibodies (nanobodies or VHHs) against several actin binding proteins including myopodin, gelsolin, and L‐plastin. Recombinant nanobodies were used for the biophysical and biochemical characterization of their properties. Furthermore, we used intracellular expression of these nanobodies as intrabodies to develop functional protein domain ‘knock‐outs’ to answer the question how ablation of a biochemical property of a protein translates into changes in cell morphology and cell behavior. Results on gelsolin and L‐plastin nanobodies show that they can bind their target antigen with nanomolar affinity in vitro. Cellular assays (for instance a mitochondrial outer membrane recruitment assay) were further used to confirm the interaction between the VHH intrabody and the endogenous antigen in vivo. Epitope mapping revealed for each nanobody to which domain of the protein they bind, demonstrating their selective interaction with different (conformational) epitopes. In this way we isolated a gelsolin nanobody that only recognizes the activated, calcium‐bound conformation of gelsolin. In contrast, other gelsolin nanobodies bind to gelsolin both in the presence and absence of calcium. Functional actin binding assays led to the identification of a gelsolin nanobody that blocks actin monomer binding. Furthermore, this nanobody is also able to reduce the motility of MDA‐MB231 breast cancer cells. A nanobody that specifically interacts with the tandem actin binding domains in L‐plastin inhibits actin bundling in vitro. This ‘knock‐out’ nanobody inhibits filopodia formation, motility and invasion when expressed in PC‐3 prostate cancer cells. Remarkably, L‐plastin RNA interference (RNAi) showed no significant effect on filopodial integrity. In conclusion, single domain VHHs represent a potent tool to ablate protein function and to explore the cellular role of distinct biochemical activities of polypeptides without manipulating gene expression. Furthermore, they can evolve into new therapeutics for cancer treatment targeting currently ‘undruggable’ proteins. Citation Information: Cancer Res 2009;69(23 Suppl):C62.