Defective IGF-1 prohormone N-glycosylation and reduced IGF-1 receptor signaling activation in congenital disorders of glycosylation

none14sìThe insulin-like growth factor-1 (IGF-1) signaling pathway is crucial for the regulation of growth and development. The correct processing of the IGF-1Ea prohormone (proIGF-1Ea) and the IGF-1 receptor (IGF-1R) peptide precursor requires proper N-glycosylation. Deficiencies of N-linked glycosylation lead to a clinically heterogeneous group of inherited diseases called Congenital Disorders of Glycosylation (CDG). The impact of N-glycosylation defects on IGF-1/IGF-1R signaling components is largely unknown. In this study, using dermal fibroblasts from patients with different CDG [PMM2-CDG (n = 7); ALG3-CDG (n = 2); ALG8-CDG (n = 1); GMPPB-CDG (n = 1)], we analyzed the glycosylation pattern of the proIGF-1Ea, IGF-1 secretion efficiency and IGF-1R signaling activity. ALG3-CDG, ALG8-CDG, GMPPB-CDG and some PMM2-CDG fibroblasts showed hypoglycosylation of the proIGF-1Ea and lower IGF-1 secretion when compared with control (CTR). Lower IGF-1 serum concentration was observed in ALG3-CDG, ALG8-CDG and in some patients with PMM2-CDG, supporting our in vitro data. Furthermore, reduced IGF-1R expression level was observed in ALG3-CDG, ALG8-CDG and in some PMM2-CDG fibroblasts. IGF-1-induced IGF-1R activation was lower in most PMM2-CDG fibroblasts and was associated with decreased ERK1/2 phosphorylation as compared to CTR. In general, CDG fibroblasts showed a slight upregulation of Endoplasmic Reticulum (ER) stress genes compared with CTR, uncovering mild ER stress in CDG cells. ER-stress-related gene expression negatively correlated with fibroblasts IGF-1 secretion. This study provides new evidence of a direct link between N-glycosylation defects found in CDG and the impairment of IGF-1/IGF-1R signaling components. Further studies are warranted to determine the clinical consequences of reduced systemic IGF-1 availability and local activity in patients with CDG.openDi Patria, Laura; Annibalini, Giosuè; Morrone, Amelia; Ferri, Lorenzo; Saltarelli, Roberta; Galluzzi, Luca; Diotallevi, Aurora; Bocconcelli, Matteo; Donati, Maria Alice; Barone, Rita; Guerrini, Renzo; Jaeken, Jaak; Stocchi, Vilberto; Barbieri, ElenaDi Patria, Laura; Annibalini, Giosuè; Morrone, Amelia; Ferri, Lorenzo; Saltarelli, Roberta; Galluzzi, Luca; Diotallevi, Aurora; Bocconcelli, Matteo; Donati, Maria Alice; Barone, Rita; Guerrini, Renzo; Jaeken, Jaak; Stocchi, Vilberto; Barbieri, Elen

Effectiveness of cardiac resynchronization therapy in heart failure patients with valvular heart disease: comparison with patients affected by ischaemic heart disease or dilated cardiomyopathy. The InSync/InSync ICD Italian Registry

AimsTo analyse the effectiveness of cardiac resynchronization therapy (CRT) in patients with valvular heart disease (a subset not specifically investigated in randomized controlled trials) in comparison with ischaemic heart disease or dilated cardiomyopathy patients.Methods and resultsPatients enrolled in a national registry were evaluated during a median follow-up of 16 months after CRT implant. Patients with valvular heart disease treated with CRT (n = 108) in comparison with ischaemic heart disease (n = 737) and dilated cardiomyopathy (n = 635) patients presented: (i) a higher prevalence of chronic atrial fibrillation, with atrioventricular node ablation performed in around half of the cases; (ii) a similar clinical and echocardiographic profile at baseline; (iii) a similar improvement of LVEF and a similar reduction in ventricular volumes at 6-12 months; (iv) a favourable clinical response at 12 months with an improvement of the clinical composite score similar to that occurring in patients with dilated cardiomyopathy and more pronounced than that observed in patients with ischaemic heart disease; (v) a long-term outcome, in term of freedom from death or heart transplantation, similar to patients affected by ischaemic heart disease and basically more severe than that of patients affected by dilated cardiomyopathy.ConclusionIn 'real world' clinical practice, CRT appears to be effective also in patients with valvular heart disease. However, in this group of patients the outcome after CRT does not precisely overlap any of the two other groups of patients, for which much more data are currently available

The complexity of insulin-like growth factor 1 (IGF-1): development of innovative bio-molecular methods for detection and quantification of IGF-1 protein variants and study of their biological roles in physio-pathological conditions

The Insulin-like growth factor-1 (IGF-1) is a polypeptide growth factor with essential roles in physio-pathological conditions, including cellular growth and differentiation. Secretion of IGF-1 required proper post-translational modifications of IGF-1 prohormones (proIGF-1s), which include furin cleavage and N-glycosylation. This complex mechanism of IGF-1 production determines the presence of an IGF-1 pool composed of several IGF-1 protein variants: unglycosylated and glycosylated proIGF-1s, mature IGF-1, and E-peptides. Chapter I of the Thesis provides a description of the complex molecular mechanism regulating the production of the proIGF-1Es and the currently available methods for detecting and quantifying these IGF-1 protein variants. Particular attention was paid to the recent evidence showing that proIGF-1s are stable intermediate of IGF-1 processing and have specific biological activities. Subsequently, we describe a system to produce a relatively high amount of the glycosylated proIGF-1Ea (glyc_proIGF-1Ea) based on HEK-293 cells stably expressing the human IGF-1Ea isoform. Recombinant components of the IGF-1 pool, were characterized by High Resolution Mass Spectrometry (HRMS). Moreover, we used HRMS to detect the proGF-1Ea and Ea-peptide in the cell culture supernatants of HEK-293 cells overexpressing the IGF-1Ea isoform. The different strategies adopted to increase the glyc_proIGf-1Ea yield and the protein stability were also described. The synthesis of new protein standards corresponding to different IGF-1 isoforms, combined with the use of HRMS technology, may provide a useful tool to characterize and quantify the different components of the IGF-1 poll in complex biological matrices. The II chapter describes the development of a fluorescent- based Western Blot (WB) for the simultaneous detection of the three different proIGF-1s and E-peptides. Specifically, we described the use of two different primary antibodies, which recognize different epitopes of proIGF-1 sequences, and two secondary antibodies conjugated to different fluorophores to develop a multiplex fluorescent WB able to discriminate between the different IGF-1 protein variants. Our results demonstrate the feasibility of simultaneously detecting different isoforms of the same protein using different fluorescence filter combinations. Furthermore, combining antibodies to two different epitopes of the same target protein increased the specificity and reliability of protein detection. In the III chapter, we analyzed the molecular mechanisms regulating the 1 production of proIGF-1Ea and the biological effect of IGF-1 on 3D Breast Cancer (BC) spheroids formation. We describe different cell-culture conditions that favor the MCF-7 and MDA-2B-231 spheroids formation and the effects of IGF-1 on spheroids growth, compactness, viability, and gene expression patterns of metabolic and epithelial to mesenchymal transition (EMT) markers. Our results highlight the value of 3D spheroid models to better understand the role of IGF-1 on multiple aspects of tumor progression, including cancer dormancy and EMT. The IV chapter describes the impact of N-glycosylation inhibition on IGF-1 production and IGF-1 receptor (IGF1R) signalling pathway activation in the context of diseases associated with aberrant N-glycosylation such as Congenital Disorder of Glycosylation (CDG). Using muscle cellular models and mice models we demonstrated that N-glycosylation inhibition reduces myoblast fusion and impairs the early stage of the myogenic program in vitro and decreased myogenic markers in mice muscles. Finally, muscle gly_proIGF-1Ea production and IGF1R signalling pathway activation were markedly inhibited after N-glycosylation inhibition. Our results offer new insights that increase understanding of possible impairments of the myogenic differentiation capacity in the pathological context of disorders of N-glycosylation

The Role of miRNAs in Downregulation of PTEN for Glioblastoma Multiforme

In this paper we show, in silico, the role of miRNAs in the post-transcriptional regulation of the onco suppressor gene PTEN by means of a (stochastic) competitive model including interactions with miRNAs and other concurrent genes. The model also covers protein formation and the mechanism going from the protein back to the miRNAs. The numerical simulations confirm the biologically observed situation that the increase of miRNAs concentration within the cell results in the lower expression of PTEN, thus suggesting severe implications in brain tumorigenesis

Floating macro-litter pollution in the northern South China Sea

Marine litter pollution, particularly plastics pollution, is an increasing global concern. While various studies have contributed useful information on this topic, there has been a scarcity of data on floating marine macro-litter (FMML) in poorly monitored areas such as the South China Sea (SCS). This paper describes a large-scale FMML assessment research in the northern SCS. Our data indicated the ubiquitous presence, abundant quan-tity, spatiotemporal variability, complex composition, and potential sources of FMML in the investigated region during boreal spring-summer periods over multiple years. According to observer-based records, the average FMML density was estimated to be 131.0 +/- 91.8 items/km2 (mean +/- SD), with anthropogenic FMML density of 118.7 +/- 86.2 items/km(2). Anthropogenic and natural items accounted for 90.6% and 5.5% of the total, respec-tively. Plastics (72.0%) and styrofoam (9.3%) dominated the recorded items. The great majority of items (92.1%) were characterized by small size of <= 20 cm. Labels of plastic bottle/packaging litter indicated that identifiable sources included surrounding countries of the SCS. Fishing activities were recognized as key sources of FMML, with 15.3% of FMML items likely being fishing-related. Globally, known estimates of FMML densities could vary from 0.002 to 578 items/km(2), with plastics accounting for 34.8-99.0%. Therefore, marine pollution from anthropogenic FMML in our investigated area ranked at a medium-to-high level around the globe. To conclude, this study demonstrated that the SCS is one of the world's hotspot areas with FMML pollution and sheds light on marine litter pollution, especially plastics pollution, in this intensively human-exploited but poorly monitored region. In future research, FMML pollution in other sections of SCS and possible negative impacts of FMML on marine ecosystems and megafauna should be further examined

A home-based lifestyle intervention program reduces the tumorigenic potential of triple-negative breast cancer cells

Abstract Translational research for the evaluation of physical activity habits and lifestyle modifications based on nutrition and exercise has recently gained attention. In this study, we evaluated the effects of serum samples obtained before and after a 12-week home-based lifestyle intervention based on nutrition and exercise in breast cancer survivors in terms of modulation of the tumorigenic potential of breast cancer cells. The home-based lifestyle intervention proposed in this work consisted of educational counselling on exercise and nutritional behaviors and in 12 weeks of structured home-based exercise. Triple-negative breast cancer cell line MDA-MB-231 was cultured in semi-solid medium (3D culture) with sera collected before (PRE) and after (POST) the lifestyle intervention program. Spheroid formation was evaluated by counting cell colonies after 3 weeks of incubation. Results show a slight but significant reduction of spheroid formation induced by serum collected POST in comparison to those obtained PRE. Moreover, statistical analyses aimed to find physiologic and metabolic parameters associated with 3D cell proliferation revealed the proliferative inducer IGF-1 as the only predictor of cell tumorigenic potential. These results highlight the importance of lifestyle changes for cancer progression control in a tertiary prevention context. Translational research could offer a useful tool to identify metabolic and physiological changes induced by exercise and nutritional behaviors associated with cancer progression and recurrence risk

Synergism Through WEE1 and CHK1 Inhibition in Acute Lymphoblastic Leukemia

Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. Methods: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. Results: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. Conclusion: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL

Impact of mitral regurgitation on the outcome of patients treated with CRT-D: data from the InSync ICD Italian Registry.

Background: We assessed the influence of clinically significant mitral regurgitation (MR) on clinical-echocardiographic response and outcome in heart failure (HF) patients treated with a biventricular defibrillator (cardiac resynchronization therapy defibrillator [CRT-D]). Methods and Results: A total of 659 HF patients underwent successful implantation of CRT-D and were enrolled in a multicenter prospective registry (median follow-up of 15 months). Following baseline echocardiographic evaluation, patients were stratified into two groups according to the severity of MR: 232 patients with more than mild MR (Group MR+: grade 2, 3, and 4 MR) versus 427 patients with mild (grade 1) or no functional MR (Group MR−). On 6- and 12-month echocardiographic evaluation, MR was seen to have improved in the vast majority of MR+ patients, while it remained unchanged in most MR− patients. On 12-month follow-up evaluation, a comparable response to CRT was observed in the two groups, in terms of the extent of left ventricular reverse remodeling and combined clinical and echocardiographic response. During long-term follow-up, event-free survival did not differ between MR+ and MR− patients, even when subpopulations of patients with ischemic heart disease and with dilated cardiomyopathy were analyzed separately. On multivariate analysis, the only independent predictor of death from any cause was the lack of β-blocker use. Conclusions: This observational analysis supports the use of CRT-D in HF patients with clinically significant MR; MR had no major influence on patient outcom