Opposing forces fight over the same ground to regulate interferon signaling.

The current SARS-CoV-2 pandemic has spurred new interest in interferon signaling in response to viral pathogens. Much of what we know about the signaling molecules and associated signal transduction induced during the host cellular response to viral pathogens has been gained from research conducted from the 1990's to the present day, but certain intricacies of the mechanisms involved, still remain unclear. In a recent study by Vaughn et al. the authors examine one of the main mechanisms regulating interferon induction following viral infection, the RIG-I/MAVS/IRF3 pathway, and find that similar to PKR both DICER interacting proteins, PACT and TRBP, regulate RIG-I signaling in an opposing manner. More specifically, the reported findings demonstrate, like others, that PACT stimulates RIG-I-mediated signaling in a manner independent of PACT dsRNA-binding ability or phosphorylation at sites known to be important for PACT-dependent PKR activation. In contrast, they show for the first time that TRBP inhibits RIG-I-mediated signaling. RIG-I inhibition by TRBP did not require phosphorylation of sites shown to be important for inhibiting PKR, nor did it involve PACT or PKR, but it did require the dsRNA-binding ability of TRBP. These findings open the door to a complex co-regulation of RIG-I, PKR, MDA5, miRNA processing, and interferon induction.

Combined Treatment with PI3K Inhibitors BYL-719 and CAL-101 Is a Promising Antiproliferative Strategy in Human Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer arising from skeletal muscle myogenic progenitors. Recent studies have shown an important role for AKT signaling in RMS progression. Aberrant activation of the PI3K/AKT axis is one of the most frequent events occurring in human cancers and serves to disconnect the control of cell growth, survival, and metabolism from exogenous growth stimuli. In the study reported here, a panel of five compounds targeting the catalytic subunits of the four class I PI3K isoforms (p110α, BYL-719 inhibitor; p110ß, TGX-221 inhibitor; p110γ, CZC24832; p110δ, CAL-101 inhibitor) and the dual p110α/p110δ, AZD8835 inhibitor, were tested on the RMS cell lines RD, A204, and SJCRH30. Cytotoxicity, cell cycle, apoptosis, and the activation of downstream targets were analyzed. Of the individual inhibitors, BYL-719 demonstrated the most anti-tumorgenic properties. BYL-719 treatment resulted in G1/G0 phase cell cycle arrest and apoptosis. When combined with CAL-101, BYL-719 decreased cell viability and induced apoptosis in a synergistic manner, equaling or surpassing results achieved with AZD8835. In conclusion, our findings indicate that BYL-719, either alone or in combination with the p110δ inhibitor, CAL-101, could represent an efficient treatment for human rhabdomyosarcoma presenting with aberrant upregulation of the PI3K signaling pathway.

Expression of the double-stranded RNA-dependent kinase PKR influences osteosarcoma attachment independent growth, migration, and invasion.

Osteosarcoma (OS) is a rare, insidious tumor of mesenchymal origin that most often affects children, adolescents, and young adults. While the primary tumor can be controlled with chemotherapy and surgery, it is the lung metastases that are eventually fatal. Multiple studies into the initial drivers of OS development have been undertaken, but few of these have examined innate immune/inflammatory signaling. A central figure in inflammatory signaling is the innate immune/stress-activated kinase double-stranded RNA-dependent protein kinase (PKR). To characterize the role of PKR in OS, U2OS, and SaOS-2 osteosarcoma cell lines were stably transfected with wild-type or dominant-negative (DN) PKR. Overexpression of PKR enhanced colony formation in soft agar (U2OS and SaOS-2), enhanced cellular migration (U2OS), and invasive migration (SaOS-2). In contrast, overexpression of DN-PKR inhibited attachment-independent growth, migration and/or invasion. These data demonstrate a role for inflammatory signaling in OS formation and migration/invasion and suggest the status of PKR expression/activation may have prognostic value.

AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Murine thymoma viral oncogene homolog (AKT) kinases target both cytosolic and nuclear substrates for phosphorylation. Whereas the cytosolic substrates are known to be closely associated with the regulation of apoptosis and autophagy or metabolism and protein synthesis, the nuclear substrates are, for the most part, poorly understood. To better define the role of nuclear AKT, potential AKT substrates were isolated from the nuclear lysates of leukemic cell lines using a phosphorylated AKT substrate antibody and identified in tandem mass spectrometry. Among the proteins identified was adenosine deaminase acting on RNA (ADAR)1p110, the predominant nuclear isoform of the adenosine deaminase acting on double-stranded RNA. Coimmunoprecipitation studies and in vitro kinase assays revealed that AKT-1, -2, and -3 interact with both ADAR1p110 and ADAR2 and phosphorylate these RNA editases. Using site-directed mutagenesis of suspected AKT phosphorylation sites, AKT was found to primarily phosphorylate ADAR1p110 and ADAR2 on T738 and T553, respectively, and overexpression of the phosphomimic mutants ADAR1p110 (T738D) and ADAR2 (T553D) resulted in a 50-100% reduction in editase activity. Thus, activation of AKT has a direct and major impact on RNA editing.-Bavelloni, A., Focaccia, E., Piazzi, M., Raffini, M., Cesarini, V., Tomaselli, S., Orsini, A., Ratti, S., Faenza, I., Cocco, L., Gallo, A., Blalock, W. L. AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Therapeutic potential of nvp-bkm120 in human osteosarcomas cells.

Osteosarcoma (OS) is the most common pediatric malignant neoplasia of the skeletal system. It is characterized by a high degree of malignancy and a severe tendency to metastasize. In the past decade, many studies have provided evidence that the phosphoinositide 3-kinase (PI3K) signaling pathway is one of the most frequently altered pathways in human cancer, and has a critical role in driving tumor initiation and progression. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120, which has recently entered clinical Phase II for treatment of PI3K-dependent cancers on three OS cell lines. We observed a concentration- and time-dependent decrease of Ser473 p-Akt as well as reduced levels of Thr37/46 p-4E-BP1, an indicator of the mammalian target of rapamycin complex 1 activity. All OS cell lines used in this study responded to BKM120 treatment with an arrest of cell proliferation, an increase in cell mortality, and an increase in caspase-3 activity. MG-63 cells were the most responsive cell line, demonstrating a significant increase in sub-G1 cells, and a rapid induction of cell death. Furthermore, we demonstrate that BKM120 is more effective when used in combination with other standard chemotherapeutic drugs. Combining BKM120 with vincristine demonstrated a more synergistic effect than BKM120 with doxorubicin in all the lines. Hence, we suggest that BKM120 may be a novel therapy for the treatment of OS presenting with anomalous upregulation of the PI3K signaling pathway.

Signal Transduction in Ribosome Biogenesis: A Recipe to Avoid Disaster.

Energetically speaking, ribosome biogenesis is by far the most costly process of the cell and, therefore, must be highly regulated in order to avoid unnecessary energy expenditure. Not only must ribosomal RNA (rRNA) synthesis, ribosomal protein (RP) transcription, translation, and nuclear import, as well as ribosome assembly, be tightly controlled, these events must be coordinated with other cellular events, such as cell division and differentiation. In addition, ribosome biogenesis must respond rapidly to environmental cues mediated by internal and cell surface receptors, or stress (oxidative stress, DNA damage, amino acid depletion, etc.). This review examines some of the well-studied pathways known to control ribosome biogenesis (PI3K-AKT-mTOR, RB-p53, MYC) and how they may interact with some of the less well studied pathways (eIF2α kinase and RNA editing/splicing) in higher eukaryotes to regulate ribosome biogenesis, assembly, and protein translation in a dynamic manner.

BMP-2 Induced Expression of PLCß1 That is a Positive Regulator of Osteoblast Differentiation.

Bone morphogenetic protein 2 (BMP-2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide-phospholipase Cß 1 (PLCß1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCß1. Here, the function of PLCß1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP-2 we assist to a remarkable increase in PLCß1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCß1 promotes osteogenic differentiation by up-regulating alkaline phosphatase (ALP). Moreover, PLCß1 is present in the nuclear compartment of these cells and overexpression of a cytosolic-PLCß1mutant (cyt-PLCß1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR-214 is a regulator of Osterix (Osx) which is an osteoblast-specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCß1 could be a potential target of miR-214 in the control of osteogenic differentiation by gain- and loss- of function experiment. The results indicated that inhibition of miR-214 in C2C12 cells significantly enhances the protein level of PLCß1 and promotes C2C12 BMP-2-induced osteogenesis by targeting PLCß1.

PI-PLCß1b affects Akt activation, cyclin E expression, and caspase cleavage, promoting cell survival in pro-B-lymphoblastic cells exposed to oxidative stress.

The phosphoinositide-dependent signal transduction pathway has been implicated in the control of a variety of biologic processes, such as the regulation of cellular metabolism and homeostasis, cell proliferation and differentiation, and apoptosis. One of the key players in the regulation of inositol lipid signaling is the phospholipase Cß1 (PI-PLCß1), that hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtIns(4,5)P2], giving rise to the second messengers inositol triphosphate and diacylglicerol. PI-PLCß1 has been associated with the regulation of several cellular functions, some of which have not yet been fully understood. In particular, it has been reported that PI-PLCß1 protects murine fibroblasts from oxidative stress-induced cell death. The mediators of oxidative stress, reactive oxygen species (ROS), have been shown to regulate major epigenetic processes, causing the silencing of tumor suppressors and enhancing the proliferation of leukemic cells under oxidative stress. Investigation of the interplay between ROS, PI-PLCß1, and their signaling mediators in leukemia might therefore reveal innovative targets of pharmacological therapy in the treatment for leukemia. In this work, we demonstrate that in pro-B-lymphoblastic cells (Ba/F3), treated with H2O2, PI-PLCß1b conferred resistance to cell death, promoting cell cycle progression and cell proliferation and influencing the expression of cyclin A and E. Interestingly, we found that, expression of PI-PLCß1b affects the activity of caspase-3, caspase-7, and of several protein kinases induced by oxidative stress. In particular, PI-PLCß1b expression completely abolished the phosphorylation of Erk1/2 MAP kinases, down-regulated phosphatase and tensin homolog (PTEN), and up-regulated the phosphorylation of Akt, thereby sustaining cellular proliferation.

Intolerant contact lens wearers exhibit ocular surface impairment despite 3 months wear discontinuation.

PURPOSE: To evaluate ocular surface (OS) parameters recovery in intolerant contact lens (CL) wearers after a period of discontinuation. METHODS: This is a retrospective analysis of data from 87 intolerant CL wearers who had discontinued their use for an average period of 12 weeks because of associated discomfort and failure to successfully refit. Data were collected from clinical charts. Data from 50 matched healthy volunteers served as controls. Clinical tests included subjective discomfort symptoms questionnaire (Ocular Surface Disease Index, OSDI), Schirmer test, break-up time (BUT), corneal esthesiometry and corneo-conjunctival staining. Laboratory tests included scraping and imprint cytology. Tear protein analysis included dosage of total tear protein (TP), lysozyme-C (LYS-C), lactoferrin (LACTO), zinc-α2-glycoprotein (ZAG-2), IgA heavy chain bands (Ig-A), and serum albumin (ALB). Data were correlated to wear parameters. RESULTS: All values were significantly worse in intolerant CL wearers group (p always <0.001). In particular, lower values compared to controls were found for BUT, corneal esthesiometry, goblet cell density, LYS-C, LACTO, ZAG-2, and TP. On the contrary, higher values compared to controls were found for OSDI, staining, imprint cytology, scraping cytology, ALB, IgA-heavy chain. The IgA/LYS-C ratio calculated as an index of the increased activity of the IgA-producing cell was found significantly higher in the intolerant group and in correlation with discomfort symptoms. CONCLUSIONS: Ocular surface parameters were altered in intolerant CL wearers, even after a prolonged discontinuation period. Our data suggest that OS recovery necessary to successfully refit lenses may need a discontinuation time longer than 3 months.

PLCß1a and PLCß1b selective regulation and cyclin D3 modulation reduced by kinamycin F during k562 cell differentiation.

Here we report that both PLCß1a and PLCß1b are relevant regulators of erythropoiesis in that kinamycin F, a potent inducer of γ-globin production in K562 cells, caused a selectively reduction of both PLCß1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCß1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCß1b induced an increase in γ-globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCß1 signaling pathway. Namely the amplification of the expression of the PLCß1a, but not of PLCß1b, is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F. This could be due to their different distribution in the cell compartments since the amount of PLCß1b is mainly present in the nucleus in respect to PLCß1a. Our data indicate that the amplification of PLCß1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis.

Prohibitin 2: At a communications crossroads.

Prohibitins (PHBs) are a highly conserved class of proteins first discovered as inhibitors of cellular proliferation. Since then PHBs have been found to have a significant role in transcription, nuclear signaling, mitochondrial structural integrity, cell division, and cellular membrane metabolism, placing these proteins among the key regulators of pathologies such as cancer, neuromuscular degeneration, and other metabolic diseases. The human genome encodes two PHB proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2), which function not only as a heterodimeric complex, but also independently. While many previous reviews have focused on the better characterized prohibitin, PHB1, this review focuses on PHB2 and new data concerning its cellular functions both in complex with PHB1 and independent of PHB1.

Prohibitin 2 represents a novel nuclear AKT substrate during all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells.

The AKT/PKB kinase is essential for cell survival, proliferation, and differentiation; however, aberrant AKT activation leads to the aggressiveness and drug resistance of many human neoplasias. In the human acute promyelocytic leukemia cell line NB4, nuclear AKT activity increases during all-trans retinoic acid (ATRA)-mediated differentiation. As nuclear AKT activity is associated with differentiation, we sought to identify the nuclear substrates of AKT that were phosphorylated after ATRA treatment. A proteomics-based search for nuclear substrates of AKT in ATRA-treated NB4 cells was undertaken by using 2D-electrophoresis/mass spectrometry (MS) in combination with an anti-AKT phospho-substrate antibody. Western blot analysis, an in vitro kinase assay, and/or site-directed mutagenesis were performed to further characterize the MS findings. MS analysis revealed prohibitin (PHB)-2, a multifunctional protein involved in cell cycle progression and the suppression of oxidative stress, to be a putative nuclear substrate of AKT. Follow-up studies confirmed that AKT phosphorylates PHB2 on Ser-91 and that forced expression of the PHB2(S91A) mutant results in a rapid loss of viability and apoptotic cell death. Activation of nuclear AKT during ATRA-mediated differentiation results in the phosphorylation of several proteins, including PHB2, which may serve to coordinate nuclear-mitochondrial events during differentiation.

Phosphoinositide-specific phospholipase C ß 1b (PI-PLCß1b) interactome: affinity purification-mass spectrometry analysis of PI-PLCß1b with nuclear protein.

Two isoforms of inositide-dependent phospholipase C ß1 (PI-PLCß1) are generated by alternative splicing (PLCß1a and PLCß1b). Both isoforms are present within the nucleus, but in contrast to PLCß1a, the vast majority of PLCß1b is nuclear. In mouse erythroid leukemia cells, PI-PLCß1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCß1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCß1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCß1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule.

Identification of the PKR nuclear interactome reveals roles in ribosome biogenesis, mRNA processing and cell division.

The double-strand RNA-dependent protein kinase, PKR, plays a central role in inflammatory/chronic stress-mediated pathologies such as cancer, diabetes, and neuro/muscular degenerative diseases. Although a significant amount of research has been conducted to elucidate the role of PKR signaling in the cytosol, only recently has attention been paid to the role of PKR in the nuclear compartment. Previously our group reported that phosphorylated forms of PKR are present in the nucleus of acute leukemic cell lines, representing a reservoir of active kinase that responds to stress. Using the CCRF-CEM acute T-cell leukemia cell line, a PKR-specific inhibitor, co-immunoprecipitation and a proteomics approach, which included affinity purified mass spectrometry analysis (AP/MS), we identified the proteins present in active and inactive PKR nuclear complexes. Of the proteins identified in the PKR complexes, sixty-nine (69) were specific to the active complex, while thirty-eight (38) were specific to the inactive complex. An additional thirteen (13) proteins associated specifically with both complexes. The majority of the proteins identified are involved in, ribosome biogenesis, RNA splicing, mRNA stability, gene expression, cell cycle, or chromatin organization, including several with known significance to normal hematopoiesis and/or hematological disease. In agreement with the AP/MS data, basal- or over-expression of PKR under normal growth conditions favored cell proliferation in the tested cell lines, whereas pharmacological inhibition of PKR or shRNA-mediated knock-down did not. PKR was also found to influence the isoform and the level of expression of the proto-oncogene MYC.

A role for PLCß1 in myotonic dystrophies type 1 and 2.

Phosphoinositide-phospholipase C ß1 (PLCß1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. We previously demonstrated that nuclear PLCß1 activates the cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLCß1 is essential for cyclin D3 promoter activation and gene transcription, through c-jun/AP1. Myotonic dystrophy (DM) is the most prevalent form of muscular dystrophy in adults. DM type 1 (DM1) and type 2 (DM2) are dominantly inherited multisystem disorders. DM1 is triggered by the pathological expansion of a (CTG)(n) triplet repeat in the gene coding for DMPK, the dystrophia myotonica-protein kinase, whereas a (CCTG)(n) tetranucleotide repeat expansion in the ZNF9 gene, encoding a CCHC-type zinc finger protein, causes DM2. We found that, unlike in normal myotubes, the level of expression of PLCß1 in DM1 and DM2 cells was already elevated in proliferating cells. Treatment with insulin induced a dramatic decrease in the amount of PLCß1. During differentiation, cyclin D3 and myogenin were elevated in normal myotubes, whereas differentiating DM1 and DM2 cells did not increase these proteins. Forced expression of PLCß1 in DM1 and DM2 cells increased the expression of differentiation markers, myogenin and cyclin D3, and enhanced fusion of DM myoblasts. These results highlight again that PLCß1 expression is a key player in myoblast differentiation, functioning as a positive regulator in the correction of delayed differentiation of skeletal muscle in DM human myoblasts.

Alternative Splicing, RNA Editing, and the Current Limits of Next Generation Sequencing.

The advent of next generation sequencing (NGS) has fostered a shift in basic analytic strategies of a gene expression analysis in diverse pathologies for the purposes of research, pharmacology, and personalized medicine. What was once highly focused research on individual signaling pathways or pathway members has, from the time of gene expression arrays, become a global analysis of gene expression that has aided in identifying novel pathway interactions, the discovery of new therapeutic targets, and the establishment of disease-associated profiles for assessing progression, stratification, or a therapeutic response. But there are significant caveats to this analysis that do not allow for the construction of the full picture. The lack of timely updates to publicly available databases and the "hit and miss" deposition of scientific data to these databases relegate a large amount of potentially important data to "garbage", begging the question, "how much are we really missing?" This brief perspective aims to highlight some of the limitations that RNA binding/modifying proteins and RNA processing impose on our current usage of NGS technologies as relating to cancer and how not fully appreciating the limitations of current NGS technology may negatively affect therapeutic strategies in the long run.

The Cytotoxic Effect of Curcumin in Rhabdomyosarcoma Is Associated with the Modulation of AMPK, AKT/mTOR, STAT, and p53 Signaling.

Approximately 7% of cancers arising in children and 1% of those arising in adults are soft tissue sarcomas (STS). Of these malignancies, rhabdomyosarcoma (RMS) is the most common. RMS survival rates using current therapeutic protocols have remained largely unchanged in the past decade. Thus, it is imperative that the main molecular drivers in RMS tumorigenesis are defined so that more precise, effective, and less toxic therapies can be designed. Curcumin, a common herbal supplement derived from plants of the Curcuma longa species, has an exceptionally low dietary biotoxicity profile and has demonstrated anti-tumorigenic benefits in vitro. In this study, the anti-tumorigenic activity of curcumin was assessed in rhabdomyosarcoma cell lines and used to identify the major pathways responsible for curcumin's anti-tumorigenic effects. Curcumin treatment resulted in cell cycle arrest, inhibited cell migration and colony forming potential, and induced apoptotic cell death. Proteome profiler array analysis demonstrated that curcumin treatment primarily influenced flux through the AKT-mammalian target of rapamycin (mTOR), signal transducer and activator of transcription (STAT), AMP-dependent kinase (AMPK), and p53 associated pathways in a rhabdomyosarcoma subtype-specific manner. Thus, the strategic, combinational therapeutic targeting of these pathways may present the best option to treat this group of tumors.

Phospholipase Family Enzymes in Lung Cancer: Looking for Novel Therapeutic Approaches.

Lung cancer (LC) is the second most common neoplasm in men and the third most common in women. In the last decade, LC therapies have undergone significant improvements with the advent of immunotherapy. However, the effectiveness of the available treatments remains insufficient due to the presence of therapy-resistant cancer cells. For decades, chemotherapy and radiotherapy have dominated the treatment strategy for LC; however, relapses occur rapidly and result in poor survival. Malignant lung tumors are classified as either small- or non-small-cell lung carcinoma (SCLC and NSCLC). Despite improvements in the treatment of LC in recent decades, the benefits of surgery, radiotherapy, and chemotherapy are limited, although they have improved the prognosis of LC despite the persistent low survival rate due to distant metastasis in the late stage. The identification of novel prognostic molecular markers is crucial to understand the underlying mechanisms of LC initiation and progression. The potential role of phosphatidylinositol in tumor growth and the metastatic process has recently been suggested by some researchers. Phosphatidylinositols are lipid molecules and key players in the inositol signaling pathway that have a pivotal role in cell cycle regulation, proliferation, differentiation, membrane trafficking, and gene expression. In this review, we discuss the current understanding of phosphoinositide-specific phospholipase enzymes and their emerging roles in LC.

Extracellular vesicle-associated IGF2BP3 tunes Ewing sarcoma cell migration and affects PI3K/Akt pathway in neighboring cells.

Ewing sarcoma (EWS) is a challenging pediatric cancer characterized by vast intra-tumor heterogeneity. We evaluated the RNA-binding protein IGF2BP3, whose high expression correlates with a poor prognosis and an elevated tendency of metastases, as a possible soluble mediator of inter-cellular communication in EWS. Our data demonstrate that (i) IGF2BP3 is detected in cell supernatants, and it is released inside extracellular vesicles (EVs); (ii) EVs from IGF2BP3-positive or IGF2BP3-negative EWS cells reciprocally affect cell migration but not the proliferation of EWS recipient cells; (iii) EVs derived from IGF2BP3-silenced cells have a distinct miRNA cargo profile and inhibit the PI3K/Akt pathway in recipient cells; (iv) the 11 common differentially expressed miRNAs associated with IGF2BP3-positive and IGF2BP3-negative EVs correctly group IGF2BP3-positive and IGF2BP3-negative clinical tissue specimens. Overall, our data suggest that IGF2BP3 can participate in the modulation of phenotypic heterogeneity.

Mineralocorticoid Receptor Antagonism Prevents Type 2 Familial Partial Lipodystrophy Brown Adipocyte Dysfunction.

Type-2 Familial Partial Lipodystrophy (FPLD2), a rare lipodystrophy caused by LMNA mutations, is characterized by a loss of subcutaneous fat from the trunk and limbs and excess accumulation of adipose tissue in the neck and face. Several studies have reported that the mineralocorticoid receptor (MR) plays an essential role in adipose tissue differentiation and functionality. We previously showed that brown preadipocytes isolated from a FPLD2 patient's neck aberrantly differentiate towards the white lineage. As this condition may be related to MR activation, we suspected altered MR dynamics in FPLD2. Despite cytoplasmic MR localization in control brown adipocytes, retention of MR was observed in FPLD2 brown adipocyte nuclei. Moreover, overexpression of wild-type or mutated prelamin A caused GFP-MR recruitment to the nuclear envelope in HEK293 cells, while drug-induced prelamin A co-localized with endogenous MR in human preadipocytes. Based on in silico analysis and in situ protein ligation assays, we could suggest an interaction between prelamin A and MR, which appears to be inhibited by mineralocorticoid receptor antagonism. Importantly, the MR antagonist spironolactone redirected FPLD2 preadipocyte differentiation towards the brown lineage, avoiding the formation of enlarged and dysmorphic lipid droplets. Finally, beneficial effects on brown adipose tissue activity were observed in an FPLD2 patient undergoing spironolactone treatment. These findings identify MR as a new lamin A interactor and a new player in lamin A-linked lipodystrophies.