Marzia Ognibene

Gα13 regulation of proto-dbl signaling

Rho family GTPases play important roles in the regulation of intracellular signals induced by activated heterotrimeric G proteins of the α12/13 family. The α12/13 subunits activate Rho GTPases through direct binding to a group of Rho guanine nucleotide exchange factors (GEFs) characterized by the presence of a G protein signaling-like (RGL) domain. The Rho GEF proto-Dbl, that does not contain a RGL domain, was also found to link Gα12/13 signals to Rho. We have explored the effects of activated Gα13 and Gα13-associated G protein-coupled receptor (GPCR) agonists on proto-Dbl regulation. We show that activated Gα13, but not Gα12 or Gαq, induces translocation of proto-Dbl to the cell membrane with consequent enlargement of cell body and membrane ruffling. These effects were evident also when Gα13-associated GPCR agonists were used on cells expressing proto-Dbl and were accompanied by the activation of Cdc42 and RhoA GTPases and further downstream effector JNK and p38 kinases. Moreover, we show that both activated Gα13 and GPCR agonists stimulate proto-Dbl interaction with ezrin to promote ezrin translocation to the plasma membrane. These results suggest a mechanism by which proto-Dbl and its effector pathways are regulated by Gα13-mediated signals through association with ezrin.

The tumor suppressor hamartin enhances Dbl protein transforming activity through interaction with ezrin

The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between β1 and β2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.

Inhibition of PI3K induces Rac activation and membrane ruffling in proto-Dbl expressing cells

Proto-Dbl protein, a guanine nucleotide exchange factor (GEF) for Rho GTPases, is tightly regulated by a combination of mechanisms that involve intra- and intermolecular interaction and N- and C-terminal domain-dependent turnover of the protein. Moreover, the interaction of the PH domain of proto-Dbl with phosphoinositides regulates its subcellular localization and biological activity. Here we show that inhibition of the phosphatidylinositol 3-kinase (PI3K) by molecular and pharmacological inhibitors causes a strong inhibition of proto-Dbl-induced cell proliferation and transformation. Conversely, inhibition of PI3K results in the translocation of proto-Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. Furthermore, we investigated the signaling molecules involved in proto-Dbl-induced cell transformation and motility and observed that inhibition of PI3K in proto-Dbl expressing cells induces an increase in p38 activity and a decrease in ERK phosphorylation. Our results suggest that proto-Dbl activates distinct downstream effectors to induce morphological changes and cell transformation.

High frequency of development of B cell lymphoproliferation and diffuse large B cell lymphoma in Dbl knock-in mice.

Dbl is the prototype of a large family of GDP–GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.

Induction of Epithelial Mesenchimal Transition and Vasculogenesis in the Lenses of Dbl Oncogene Transgenic Mice

Background The Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. The numerous domains that are present in many Dbl family proteins suggest that they act to integrate multiple inputs in complicated signaling networks involving the Rho GTPases. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We generated transgenic mice introducing the cDNA of Dbl oncogene linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression in mouse lenses affected proliferation, migration and differentiation of lens epithelial cells. Results We used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of 2 days, 2 weeks, and 6 weeks old transgenic mice. We observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT), such as down-regulation of epithelial cell markers and up-regulation of fibroblast markers. Genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses where vascularization can be readily observed. Conclusion Onco-Dbl expression in mouse lens correlated with modulation of genes involved in the regulation of EMT, apoptosis and vasculogenesis leading to disruption of the lens architecture, epithelial cell proliferation, and aberrant angiogenesis. We conclude that onco-Dbl has a potentially important, previously unreported, capacity to dramatically alter epithelial cell migration, replication, polarization and differentiation and to induce vascularization of an epithelial tissue.

Immunohistochemical analysis of PDK1, PHD3 and HIF-1α expression defines the hypoxic status of neuroblastoma tumors

Neuroblastoma (NB) is the most common solid tumor during infancy and the first cause of death among the preschool age diseases. The availability of several NB genomic profiles improves the prognostic ability, but the outcome prediction for this pathology remains imperfect. We previously produced a novel prognostic gene signature based on the response of NB cells to hypoxia, a condition of tumor microenvironment strictly connected with cancer aggressiveness. Here we attempted to further define the expression of hypoxia-modulated specific genes, looking at their protein level in NB specimens, considering in particular the hypoxia inducible factor-1α (HIF-1α), the mitochondrial pyruvate dehydrogenase kinase 1 (PDK1), and the HIF-prolyl hydroxylase domain 3 (PHD3). The evaluation of expression was performed by Western blot and immunocytochemistry on NB cell lines and by immunohistochemistry on tumor specimens. Stimulation of both HIF-1α and PDK1 and inhibition of PHD3 expression were observed in NB cell lines cultured under prolonged hypoxic conditions as well as in most of the tumors with poor outcome. Our results indicate that the immunohistochemistry analysis of the protein expression of PDK1, PHD3, and HIF-1α defines the hypoxic status of NB tumors and can be used as a simple and relevant tool to stratify high-risk patients.

Constitutional 3p26.3 terminal microdeletion in an adolescent with neuroblastoma

ABSTRACT Background: Neuroblastoma (NB) is a common and often lethal cancer of early childhood that accounts for 10% of pediatric cancer mortality. Incidence peaks in infancy and then rapidly declines, with less than 5% of cases diagnosed in children and adolescents ≥ 10 y. There is increasing evidence that NB has unique biology and an chronic disease course in older children and adolescents, but ultimately dismal survival. Methods: We describe a rare constitutional 3p26.3 terminal microdeletion which occurred in an adolescent with NB, with apparently normal phenotype without neurocognitive defects. We evaluated the association of expression of genes involved in the microdeletion with NB patient outcomes using R2 platform. We screened NB patients tumor cells for CHL1 protein expression using immunofluorescence. Results: Constitutional and tumor DNA were tested by array-comparative genomic hybridization and single nucleotide-polymorphism-array analyses. Peripheral blood mononuclear cells from the patient showed a 2.54 Mb sub-microscopic constitutional terminal 3p deletion that extended to band p26.3. The microdeletion 3p disrupted the CNTN4 gene and the neighboring CNTN6 and CHL1 genes were hemizygously deleted, each of these genes encode neuronal cell adhesion molecules. Low expression of CNTN6 and CNTN4 genes did not stratify NB patients, whereas low CHL1 expression characterized 417 NB patients having worse overall survival. CHL1 protein expression on tumor cells from the patient was weaker than positive control. Conclusion: This is the first report of a constitutional 3p26.3 deletion in a NB patient. Since larger deletions of 3p, indicative of the presence of one or more tumor suppressor genes in this region, occur frequently in neuroblastoma, our results pave the way to the identification of one putative NB suppressor genes mapping in 3p26.3.

Growth arrest-inducing genes are activated in Dbl-transformed mouse fibroblasts

The Dbl oncogene is a guanine nucleotide exchange factor for Rho GTPases and its activity has been linked to the regulation of gene transcription. Dbl oncogene expression in NIH3T3 cells leads to changes in morphological and proliferative properties of these cells, inducing a highly transformed phenotype. To gain insights into Dbl oncogene-induced transformation we compared gene expression profiles between Dbl oncogene-transformed and parental NIH3T3 cells by cDNA microarray. We found that Dbl oncogene expression is associated with gene expression modulation involving upregulation of 51 genes and downregulation of 49 genes. Five of the overexpressed genes identified are known to exert antiproliferative functions. Our observations suggest that the expression of Dbl oncogene in NIH3T3 may lead to the induction of genes associated with cell cycle arrest, possibly through the activation of stress-induced kinases.

CHL1 gene acts as a tumor suppressor in human neuroblastoma

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

Neuroblastoma Cell Lines Are Refractory to Genotoxic Drug-Mediated Induction of Ligands for NK Cell-Activating Receptors

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, causes death in almost 15% of children affected by cancer. Treatment of neuroblastoma is based on the combination of chemotherapy with other therapeutic interventions such as surgery, radiotherapy, use of differentiating agents, and immunotherapy. In particular, adoptive NK cell transfer is a new immune-therapeutic approach whose efficacy may be boosted by several anticancer agents able to induce the expression of ligands for NK cell-activating receptors, thus rendering cancer cells more susceptible to NK cell-mediated lysis. Here, we show that chemotherapeutic drugs commonly used for the treatment of NB such as cisplatin, topotecan, irinotecan, and etoposide are unable to induce the expression of activating ligands in a panel of NB cell lines. Consistently, cisplatin-treated NB cell lines were not more susceptible to NK cells than untreated cells. The refractoriness of NB cell lines to these drugs has been partially associated with the abnormal status of genes for ATM, ATR, Chk1, and Chk2, the major transducers of the DNA damage response (DDR), triggered by several anticancer agents and promoting different antitumor mechanisms including the expression of ligands for NK cell-activating receptors. Moreover, both the impaired production of reactive oxygen species (ROS) in some NB cell lines and the transient p53 stabilization in response to our genotoxic drugs under our experimental conditions could contribute to inefficient induction of activating ligands. These data suggest that further investigations, exploiting molecular strategies aimed to potentiate the NK cell-mediated immunotherapy of NB, are warranted.