Maria-Magdalena Georgescu

Three-dimensional tissue culture based on magnetic cell levitation

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

PTEN tumor suppressor associates with NHERF proteins to attenuate PDGF receptor signaling

PTEN, a tumor suppressor frequently inactivated in many human cancers, directly antagonizes the activity of phosphatidylinositol‐3‐OH kinase (PI3K) by dephosphorylating phosphoinositides. We show here that PTEN interacts directly with the NHERF1 and NHERF2 (Na+/H+ exchanger regulatory factor) homologous adaptor proteins through the PDZ motif of PTEN and the PDZ1 domain of NHERF1 or both PDZ domains of NHERF2. NHERFs were shown to interact directly with platelet‐derived growth factor receptor (PDGFR), and we demonstrate the assembly of a ternary complex between PTEN, NHERFs and PDGFR. The activation of the PI3K pathway after PDGFR stimulation was prolonged in NHERF1(−/−) mouse embryonic fibroblasts as compared to wild‐type cells, consistent with defective PTEN recruitment to PDGFR in the absence of NHERF1. Depletion of NHERF2 by small interfering RNA similarly increased PI3K signaling. Phenotypically, the loss of NHERF1 enhanced the PDGF‐induced cytoskeletal rearrangements and chemotactic migration of the cells. These data indicate that, in normal cells, NHERF proteins recruit PTEN to PDGFR to restrict the activation of the PI3K.

PTEN Induces Cell Cycle Arrest by Decreasing the Level and Nuclear Localization of Cyclin D1

ABSTRACT PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancers that acts as a phosphatase on phosphatidylinositol-3,4,5-trisphosphate, antagonizing the activity of the phosphatidylinositol 3′-OH kinase. PTEN manifests its tumor suppressor function in most tumor cells by inducing G1-phase cell cycle arrest. To study the mechanism of cell cycle arrest, we established a tetracycline-inducible expression system for PTEN in cell lines lacking this gene. Expression of wild-type PTEN but not of mutant forms unable to dephosphorylate phosphoinositides reduced the expression of cyclin D1. Cyclin D1 reduction was accompanied by a marked decrease in endogenous retinoblastoma (Rb) protein phosphorylation on cyclin D/CDK4-specific sites, showing an early negative effect of PTEN on Rb inactivation. PTEN expression also prevented cyclin D1 from localizing to the nucleus during the G1- to S-phase cell cycle transition. The PTEN-induced localization defect and the cell growth arrest could be rescued by the expression of a nucleus-persistent mutant form of cyclin D1, indicating that an important effect of PTEN is at the level of nuclear availability of cyclin D1. Constitutively active Akt/PKB kinase counteracted the effect of PTEN on cyclin D1 translocation. The data are consistent with an oncogenesis model in which a lack of PTEN fuels the cell cycle by increasing the nuclear availability of cyclin D1 through the Akt/PKB pathway.

PTEN Tumor Suppressor Network in PI3K-Akt Pathway Control

The PI3K-Akt pathway is a major survival pathway activated in cancer. Efforts to develop targeted therapies have not been fully successful, mainly because of extensive internal intrapathway or external interpathway negative feedback loops or because of networking between pathway suppressors. The PTEN tumor suppressor is the major brake of the pathway and a common target for inactivation in somatic cancers. This review will highlight the networking of PTEN with other inhibitors of the pathway, relevant to cancer progression. PTEN constitutes the main node of the inhibitory network, and a series of convergences at different levels in the PI3K-Akt pathway, starting from those with growth factor receptors, will be described. As PTEN exerts enzymatic activity as a phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) phosphatase, thus opposing the activity of PI3K, the concerted actions to increase the availability of PIP(3) in cancer cells, relying either on other phosphoinositide enzymes or on the intrinsic regulation of PTEN activity by other molecules, will be discussed. In particular, the synergy between PTEN and the circle of its direct interacting proteins will be brought forth in an attempt to understand both the activation of the PI3K-Akt pathway and the connections with other parallel oncogenic pathways. The understanding of the interplay between the modulators of the PI3K-Akt pathway in cancer should eventually lead to the design of therapeutic approaches with increased efficacy in the clinic.

PTEN, NHERF1 and PHLPP form a tumor suppressor network that is disabled in glioblastoma

The phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway is activated in cancer by genetic or epigenetic events and efforts are under way to develop targeted therapies. phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is the major brake of the pathway and a common target for inactivation in glioblastoma, one of the most aggressive and therapy-resistant cancers. To achieve potent inhibition of the PI3K-Akt pathway in glioblastoma, we need to understand its mechanism of activation by investigating the interplay between its regulators. We show here that PTEN modulates the PI3K-Akt pathway in glioblastoma within a tumor suppressor network that includes Na+/H+ exchanger regulatory factor 1 (NHERF1) and pleckstrin-homology domain leucine-rich repeat protein phosphatases 1 (PHLPP1). The NHERF1 adaptor, previously characterized by our group as a PTEN ligand and regulator, shows also PTEN-independent Akt-modulating effects that led us to identify the PHLPP1/PHLPP2 Akt phosphatases as NHERF1 ligands. NHERF1 interacts via its PDZ domains with PHLPP1/PHLPP2 and scaffolds heterotrimeric complexes with PTEN. Functionally, PHLPP1 requires NHERF1 for membrane localization and growth-suppressive effects. PHLPP1 loss boosts Akt phosphorylation only in PTEN-negative cells and cooperates with PTEN loss for tumor growth. In a panel of low-grade and high-grade glioma patient samples, we show for the first time a significant disruption of all three members of the PTEN-NHERF1-PHLPP1 tumor suppressor network in high-grade tumors, correlating with Akt activation and patients abysmal survival. We thus propose a PTEN-NHERF1-PHLPP PI3K-Akt pathway inhibitory network that relies on molecular interactions and can undergo parallel synergistic hits in glioblastoma.

miR-29b and miR-125a regulate podoplanin and suppress invasion in glioblastoma.

Glioblastoma is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. Recently, it was demonstrated that podoplanin membrane sialo‐glycoprotein encoded by PDPN gene is over‐expressed and related to cellular invasion in astrocytic tumors; however the mechanisms of regulation are still unknown. MicroRNAs are noncoding RNAs that regulate gene expression and several biological processes and diseases, including cancer. Nevertheless, their roles in invasion, proliferation, and apoptosis of glioblastoma are not completely understood. In this study, we focused on miR‐29b and miR‐125a, which were predicted to regulate PDPN, and demonstrated that these microRNAs directly target the 3′ untranslated region of PDPN and inhibit invasion, apoptosis, and proliferation of glioblastomas. Furthermore, we report that miR‐29b and miR‐125a are downregulated in glioblastomas and also in CD133‐positive cells. Taken together, these results suggest that miR‐29b and miR‐125a represent potential therapeutic targets in glioblastoma.

Synergy in tumor suppression by direct interaction of Neutral Endopeptidase with PTEN

We show in this study that endogenous NEP and PTEN associate in cells directly through electrostatic interactions between a highly basic residue stretch in the intracellular domain of NEP and the major phosphorylation site in PTENs tail. NEP binds and engages in higher order complexes both phosphorylated and unphosphorylated PTEN. NEP recruits PTEN to the plasma membrane and enhances its stability and phosphatase activity. As a result, an enzymatically inactive NEP mutant preserves the ability to bind PTEN, inactivates the Akt/PKB kinase, and partially suppresses the growth of PC cells. This study demonstrates a molecular cooperation between NEP and PTEN tumor suppressors in which NEP constitutively recruits and activates PTEN to inhibit the PI3K/Akt oncogenic pathway.

Roles of NHERF1/EBP50 in Cancer

This review summarizes the emerging roles of NHERF1/EBP50 adaptor protein in tumorigenesis. NHERF1/EBP50 (Na(+)/H(+) exchanger regulating factor 1; ezrin-radixin-moesin (ERM) binding phosphoprotein of 50 kDa) is a PDZ domain-containing protein with physiological localization at the plasma membrane. We discuss in this review the functions of NHERF1/EBP50 as a linker between membrane proteins and the cytoskeleton network, as well as its involvement in different types of cancer, such as breast and liver cancers. Recent evidence obtained from our laboratory and from other groups shows that NHERF1/EBP50 is an important player in cancer progression. It appears that, depending on its subcellular distribution, NHERF1/EBP50 may behave either as a tumor suppressor, when it is localized at the plasma membrane, or as an oncogenic protein, when it is shifted to the cytoplasm. We provide here an overview of the mechanisms by which this adaptor protein controls cell transformation, and propose a model suggesting a dual role of NHERF1/EBP50 in cancer.

Cortical stabilization of β -catenin contributes to NHERF1/EBP50 tumor suppressor function

Anchorage-independent growth is a hallmark of tumor growth and results from enhanced proliferation and altered cell–cell and cell-matrix interactions. By using gene-deficient mouse embryonic fibroblasts (MEFs), we showed for the first time that NHERF1/EBP50 (Na/H exchanger regulator factor 1/ezrin-radixin-moesin binding phosphoprotein 50), an adapter protein with membrane localization under physiological conditions, inhibits cell motility and is required to suppress anchorage-independent growth. Both NHERF1 PDZ domains are necessary for the tumor suppressor effect. NHERF1 associates directly through the PDZ2 domain with β-catenin and is required for β-catenin localization at the cell–cell junctions in MEFs. Mechanistically, the absence of NHERF1 selectively decreased the interaction of β-catenin with E-cadherin, but not with N-cadherin. The ensuing disorganization of E-cadherin-mediated adherens junctions as well as the observed moderate increase in β-catenin transcriptional activity contributed most likely to the anchorage-independent growth of NHERF1-deficient MEFs. In vivo, NHERF1 is specifically localized at the apical brush-border membrane in intestinal epithelial cells and is required to maintain a fraction of the cortical β-catenin at this level. Thus, NHERF1 emerges as a cofactor essential for the integrity of epithelial tissues by maintaining the proper localization and complex assembly of β-catenin.

NHERF1/EBP50 Head-to-Tail Intramolecular Interaction Masks Association with PDZ Domain Ligands

ABSTRACT Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na+/H+ exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and β-catenin. An EB region composite structure comprising an α-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.