Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion.

The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression. To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and three-dimensional cultures of MCF-10░A cells. We show that upon Dbl expression MCF-10░A cells undergo EMT. In addition, we found that Dbl overexpression sustain

Characterization of high- And low-risk hepatocellular adenomas by magnetic resonance imaging in an animal model of glycogen storage disease type 1A

Hepatocellular adenomas (HCAs) are benign tumors, of which the most serious complications are hemorrhage and malignant transformation to hepatocellular carcinoma (HCC). Among the various subtypes of HCA, the \u3b2-catenin-activated subtype (bHCA) is associated with greatest risk of malignant transformation. Magnetic resonance imaging (MRI) is an important tool to differentiate benign and malignant hepatic lesions, and preclinical experimental approaches may help to develop a method to identify MRI features associated with bHCA. HCAs are associated with various pathologies, including glycogen storage disease 1a (GSD1a). Here, we utilized a mouse model for GSD1a that develops HCA and HCC, and analyzed the mice in order to distinguish low-risk from high-risk tumors. Animals were scanned by MRI using a hepato-specific contrast agent. The mice were sacrificed after MRI and their lesions were classified using immunohistochemistry. We observed that 45% of the animals developed focal lesions, and MRI identified four different patterns after contrast administration: isointense, hyperintense and hypointense lesions, and lesions with peripheral contrast enhancement. After contrast administration, only bHCA and HCC were hypointense in T1-weighted imaging and mildly hyperintense in T2-weighted imaging. Thus, high-risk adenomas display MRI features clearly distinguishable from those exhibited by low-risk adenomas, indicating that MRI is a reliable method for early diagnosis and classification of HCA, necessary for correct patient management

Exosomal microRNAs from Longitudinal Liquid Biopsies for the Prediction of Response to Induction Chemotherapy in High-Risk Neuroblastoma Patients: A Proof of Concept SIOPEN Study

Despite intensive treatment, 50% of children with high-risk neuroblastoma (HR-NB) succumb to their disease. Progression through current trials evaluating the efficacy of new treatments for children with HR disease usually depends on an inadequate response to induction chemotherapy, assessed using imaging modalities. In this study, we sought to identify circulating biomarkers that might be detected in a simple blood sample to predict patient response to induction chemotherapy. Since exosomes released by tumor cells can drive tumor growth and chemoresistance, we tested the hypothesis that exosomal microRNA (exo-miRNAs) in blood might predict response to induction chemotherapy. The exo-miRNAs expression profile in plasma samples collected from children treated in HR-NBL-1/SIOPEN before and after induction chemotherapy was compared to identify a three exo-miRs signature that could discriminate between poor and good responders. Exo-miRNAs expression also provided a chemoresistance index predicting the good or poor prognosis of HR-NB patients

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 beta 1 and beta 2 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

Development of hepatocellular adenomas and carcinomas in mice with liver-specific G6Pase-\ua0 deficiency

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α), and is characterized by impaired glucose homeostasis and a high risk of developing hepatocellular adenomas (HCAs). A globally G6Pase-α-deficient (G6pc−/−) mouse model that shows pathological features similar to those of humans with GSD-1a has been developed. These mice show a very severe phenotype of disturbed glucose homeostasis and rarely live beyond weaning. We generated liver-specific G6Pase-α-deficient (LS‑G6pc−/−) mice as an alternative animal model for studying the long-term pathophysiology of the liver and the potential treatment strategies, such as cell therapy. LS‑G6pc−/− mice were viable and exhibited normal glucose profiles in the fed state, but showed significantly lower blood glucose levels than their control littermates after 6 hours of fasting. LS‑G6pc−/− mice developed hepatomegaly with glycogen accumulation and hepatic steatosis, and progressive hepatic degeneration. Ninety percent of the mice analyzed developed amyloidosis by 12 months of age. Finally, 25% of the mice sacrificed at age 10–20 months showed the presence of multiple HCAs and in one case late development of hepatocellular carcinoma (HCC). In conclusion, LS‑G6pc−/− mice manifest hepatic symptoms similar to those of human GSD-1a and, therefore, represent a valid model to evaluate long-term liver pathogenesis of GSD-1a

A Proteomic Analysis of GSD-1a in Mouse Livers: Evidence for Metabolic Reprogramming, Inflammation, and Macrophage Polarization

Glycogen storage disease type 1a (GSD-1a) is a rare genetic disease caused by mutations in the catalytic subunit of the enzyme glucose-6-phosphatase-alpha (G6Pase-\u3b1). The majority of patients develop long-term complications including renal failure and hepatocellular adenoma/carcinoma. The purpose of this study was to ascertain the proteomic changes in the liver of LS-G6pc-/- mice, a murine model of GSD-1a, in comparison with wild type mice to identify potential biomarkers of the pathophysiology of the affected liver. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze liver lysates from a total of 20 LS-G6pc-/- and 18 wild type (WT) mice. We compared the proteomic expression profile of LS-G6pc-/- and WT mice. We identified 4138 significantly expressed proteins, 1243 of which were differentially represented. Network and pathway analyses indicate that LS-G6pc-/- livers display an age-dependent modulation of the expression of proteins involved in specific biological processes associated with increased progression of liver disease. Moreover, we found upregulation of proteins involved in the process of tissue inflammation and macrophage polarization toward the M2 phenotype in LS-G6pc-/- mice with adenomas. Our results identify a metabolic reprogramming of glucose-6-P and a pathologic environment in the liver compatible with tumor development and progression

Exosomal MicroRNAs as Potential Biomarkers of Hepatic Injury and Kidney Disease in Glycogen Storage Disease Type Ia Patients

Glycogen storage disease type Ia (GSDIa) is an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-alpha (G6Pase-alpha). Affected individuals develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma and kidney failure. The purpose of this study was to identify potential biomarkers of the evolution of the disease in GSDIa patients. To this end, we analyzed the expression of exosomal microRNAs (Exo-miRs) in the plasma exosomes of 45 patients aged 6 to 63 years. Plasma from age-matched normal individuals were used as controls. We found that the altered expression of several Exo-miRs correlates with the pathologic state of the patients and might help to monitor the progression of the disease and the development of late GSDIa-associated complications

The SGLT2-inhibitor dapagliflozin improves neutropenia and neutrophil dysfunction in a mouse model of the inherited metabolic disorder GSDIb

Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells

Development and characterization of an inducible mouse model for glycogen storage disease type Ib

Background and aims: Glycogen storage disease type Ib (GSD1b) is a rare metabolic and immune disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT) and characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenomas. Despite maximal therapy, based on a strict diet and on granulocyte colony-stimulating factor treatment, long-term severe complications still develop. Understanding the pathophysiology of GSD1b is a prerequisite to develop new therapeutic strategies and depends on the availability of animal models. The G6PT-KO mouse mimics the human disease but is very fragile and rarely survives weaning. We generated a conditional G6PT-deficient mouse as an alternative model for studying the long-term pathophysiology of the disease. We utilized this conditional mouse to develop an inducible G6PT-KO model to allow temporally regulated G6PT deletion by the administration of tamoxifen (TM). Methods: We generated a conditional G6PT-deficient mouse utilizing the CRElox strategy. Histology, histochemistry, and phenotype analyses were performed at different times after TM-induced G6PT inactivation. Neutrophils and monocytes were isolated and analyzed for functional activity with standard techniques. Results: The G6PT-inducible KO mice display the expected disturbances of G6P metabolism and myeloid dysfunctions of the human disorder, even though with a milder intensity. Conclusions: TM-induced inactivation of G6PT in these mice leads to a phenotype which mimics that of human GSD1b patients. The conditional mice we have generated represent an excellent tool to study the tissue-specific role of the G6PT gene and the mechanism of long-term complications in GSD1b