Role of the PDZ-scaffold protein NHERF1/EBP50 in cancer biology: from signaling regulation to clinical relevance.

The transmission of cellular information requires fine and subtle regulation of proteins that need to interact in a coordinated and specific way to form efficient signaling networks. The spatial and temporal coordination relies on scaffold proteins. Thanks to protein interaction domains such as PDZ domains, scaffold proteins organize multiprotein complexes enabling the proper transmission of cellular information through intracellular networks. NHERF1/EBP50 is a PDZ-scaffold protein that was initially identified as an organizer and regulator of transporters and channels at the apical side of epithelia through actin-binding ezrin-moesin-radixin proteins. Since, NHERF1/EBP50 has emerged as a major regulator of cancer signaling network by assembling cancer-related proteins. The PDZ-scaffold EBP50 carries either anti-tumor or pro-tumor functions, two antinomic functions dictated by EBP50 expression or subcellular localization. The dual function of NHERF1/EBP50 encompasses the regulation of several major signaling pathways engaged in cancer, including the receptor tyrosine kinases PDGFR and EGFR, PI3K/PTEN/AKT and Wnt-ß-catenin pathways.

Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis.

Small ArfGAP1 (stromal membrane-associated protein 1, SMAP1), a GTPase-activating protein specific for ADP-ribosylation factor 6 (Arf6), which is a small GTPase acting on membrane trafficking and actin remodeling, is frequently mutated in various tumors displaying microsatellite instability (MSI), notably in MSI colorectal cancers (CRC). Genotyping of 93 MSI CRCs (40 stage II, 32 stage III and 21 stage IV) allowed us to underscore that SMAP1 mutation frequency was inversely correlated with disease stage (P=0.01). Analysis of 46 cancer cell lines showed that SMAP1 mutations occurred only in MSI tumors, and consisted exclusively in short insertion or deletion in the coding 10-adenine repeat, generating a premature termination codon located downstream the ArfGAP domain. SMAP1 transcript levels were significant decreased (P=0.006), and truncated SMAP1 protein could not be detected in cells displaying biallelic SMAP1 mutations, owing to its sensitivity to proteasome degradation. To investigate the role of SMAP1 mutations, we used the SMAP1-null HCT116 cell line and we established three isogenic SMAP1-complemented clones. Cell proliferation was first assessed in vivo using subcutaneous xenografts into immunodeficient mice. Tumors developed in all animals regardless of the cell line injected, but tumor volumes were significantly smaller for both SMAP1-complemented clones compared with HCT116 (P<0.0001, at the time of killing). In vitro, SMAP1 mutations also increased cell clonogenicity (P=0.02-0.04), cell proliferation (P=0.008) by shortening the G2/M phase and decreased cell invasiveness (P=0.03-0.003). In keeping, SMAP1-complemented HCT116 gained several mesenchymal markers (Snail, Slug and vimentin) considered as a hallmark of epithelial-to-mesenchymal transition. These observations are reminiscent of some clinical characteristics of MSI CRCs, notably their larger size and lower rate of metastasis. Our observations suggest that SMAP1 loss-of-function mutations in MSI CRC may contribute to the emerging oncogenic pathway involving abnormal Arf6 regulation.

Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells.

Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis through EGFR activation.

KSR and CNK: two scaffolds regulating RAS-mediated RAF activation.

The RAS-RAF-MEK-extracellular-regulated kinase (RAS/ERK) pathway is a major intracellular route used by metazoan cells to channel to downstream targets a diverse array of signals, including those controlling cell proliferation and survival. Recent findings suggest that the pathway is assembled by specific scaffolding proteins that in turn regulate the efficiency, the location and/or the duration of signal transmission. Here, through the angle of studies conducted in Drosophila and C. elegans, we present two such proteins, the kinase suppressor of RAS (KSR) and connector enhancer of KSR (CNK) scaffolds, and highlight their implication in a novel mechanism regulating RAS-mediated RAF activation. Based on recent findings, we discuss the possibility that KSR, a RAF-like protein, does not solely act as a scaffold, but directly induces RAF catalytic function by a kinase-independent mechanism apparently shared by RAF-like proteins.

Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer's disease.

Cerebral accumulation of beta-amyloid peptide (A beta) is a central event in the pathogenesis of Alzheimer's disease (AD). Endothelin-converting enzyme-1 (ECE-1) is a candidate A beta-degrading enzyme in brain, but its involvement in AD pathogenesis was never assessed. We first performed brain immunocytochemistry, using a monoclonal anti-ECE-1 antibody, and observed neuronal ECE-1 expression in various cortical regions of nondemented subjects. In the hippocampus, ECE-1 immunoreactivity showed a stereotypical pattern inversely correlated with susceptibility to A beta deposition, further suggesting a physiological role in A beta clearance. In order to undertake a genetic association study, we identified a functional genetic variant (ECE1B C-338A) located in a regulatory region of the ECE1 gene. We showed that the A allele is associated with increased transcriptional activity in promoter-reporter gene assays and with increased ECE-1 mRNA expression in human neocortex. In a case-control study involving 401 patients with late-onset AD and 461 aged controls, we found that homozygous carriers of the A allele had a reduced risk of AD (OR=0.47, 95% CI 0.25-0.88). This finding was strengthened by the analysis of two other genetic variants of the ECE1 gene, which showed that the genetic association is extended over at least 13 kilobases of the gene sequence. Our results suggest that ECE-1 expression in brain may be critical for cortical A beta clearance and offer new potential targets for therapeutic interventions in AD.