Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases.

Tolerogenic dendritic cells play a critical role in promoting antigen-specific tolerance via dampening of T cell responses, induction of pathogenic T cell exhaustion and antigen-specific regulatory T cells. Here we efficiently generate tolerogenic dendritic cells by genetic engineering of monocytes with lentiviral vectors co-encoding for immunodominant antigen-derived peptides and IL-10. These transduced dendritic cells (designated DCIL-10/Ag) secrete IL-10 and efficiently downregulate antigen-specific CD4+ and CD8+ T cell responses from healthy subjects and celiac disease patients in vitro. In addition, DCIL-10/Ag induce antigen-specific CD49b+LAG-3+ T cells, which display the T regulatory type 1 (Tr1) cell gene signature. Administration of DCIL-10/Ag resulted in the induction of antigen-specific Tr1 cells in chimeric transplanted mice and the prevention of type 1 diabetes in pre-clinical disease models. Subsequent transfer of these antigen-specific T cells completely prevented type 1 diabetes development. Collectively these data indicate that DCIL-10/Ag represent a platform to induce stable antigen-specific tolerance to control T-cell mediated diseases.

An interdomain helix in IRE1α mediates the conformational change required for the sensor's activation.

The unfolded protein response plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1α is a major transducer that conveys endoplasmic reticulum stress via biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that, upon sensing unfolded proteins via its endoplasmic reticulum luminal domain, IRE1α dimerizes and then oligomerizes (often visualized as clustering). Once assembled, the kinase domain trans-autophosphorylates a neighboring IRE1α, inducing a conformational change that activates the RNase effector domain. However, the full details of how the signal is transmitted are not known. Here, we describe a previously unrecognized role for helix αK, located between the kinase and RNase domains of IRE1α, in conveying this critical conformational change. Using constructs containing mutations within this interdomain helix, we show that distinct substitutions affect oligomerization, kinase activity, and the RNase activity of IRE1α differentially. Furthermore, using both biochemical and computational methods, we found that different residues at position 827 specify distinct conformations at distal sites of the protein, such as in the RNase domain. Of importance, an RNase-inactive mutant, L827P, can still dimerize with wildtype monomers, but this mutation inactivates the wildtype molecule and renders leukemic cells more susceptible to stress. We surmise that helix αK is a conduit for the activation of IRE1α in response to stress.

ERp57 chaperon protein protects neuronal cells from Aß-induced toxicity.

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid-ß peptide (Aß) in the form of senile plaques. Aß can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aß peptides, suggesting that it may be a carrier protein which prevents aggregation of Aß. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aß with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aß25-35 fragment in vitro with high affinity via two in silico-predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short-term Aß25-35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aß25-35 and restores cellular viability, by preventing Aß25-35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aß toxicity and highlights it as a possible therapeutic tool in the treatment of AD.

Roles for growth factors and mutations in metastatic dissemination.

Cancer is initiated largely by specific cohorts of genetic aberrations, which are generated by mutagens and often mimic active growth factor receptors, or downstream effectors. Once initiated cells outgrow and attract blood vessels, a multi-step process, called metastasis, disseminates cancer cells primarily through vascular routes. The major steps of the metastatic cascade comprise intravasation into blood vessels, circulation as single or collectives of cells, and eventual colonization of distant organs. Herein, we consider metastasis as a multi-step process that seized principles and molecular players employed by physiological processes, such as tissue regeneration and migration of neural crest progenitors. Our discussion contrasts the irreversible nature of mutagenesis, which establishes primary tumors, and the reversible epigenetic processes (e.g. epithelial-mesenchymal transition) underlying the establishment of micro-metastases and secondary tumors. Interestingly, analyses of sequencing data from untreated metastases inferred depletion of putative driver mutations among metastases, in line with the pivotal role played by growth factors and epigenetic processes in metastasis. Conceivably, driver mutations may not confer the same advantage in the microenvironment of the primary tumor and of the colonization site, hence phenotypic plasticity rather than rigid cellular states hardwired by mutations becomes advantageous during metastasis. We review the latest reported examples of growth factors harnessed by the metastatic cascade, with the goal of identifying opportunities for anti-metastasis interventions. In summary, because the overwhelming majority of cancer-associated deaths are caused by metastatic disease, understanding the complexity of metastasis, especially the roles played by growth factors, is vital for preventing, diagnosing and treating metastasis.

Clustering of IRE1α depends on sensing ER stress but not on its RNase activity.

The sensors of the unfolded protein response react to endoplasmic reticulum (ER) stress by transient activation of their enzymatic activities, which initiate various signaling cascades. In addition, the sensor IRE1α exhibits stress-induced clustering in a transient time frame similar to activation of its endoRNase activity. Previous work had suggested that the clustering response and RNase activity of IRE1α are functionally linked, but here we show that they are independent of each other and have different behaviors and modes of activation. Although both clustering and the RNase activity are responsive to luminal stress conditions and to depletion of the ER chaperone binding protein, RNase-inactive IRE1α still clusters and, conversely, full RNase activity can be accomplished without clustering. The clusters formed by RNase-inactive IRE1α are much larger and persist longer than those induced by ER stress. Clustering requires autophosphorylation, and an IRE1α mutant whose RNase domain is responsive to ligands that bind the kinase domain forms yet a third type of stress-independent cluster, with distinct physical properties and half-lives. These data suggest that IRE1α clustering can follow distinct pathways upon activation of the sensor.-Ricci, D., Marrocco, I., Blumenthal, D., Dibos, M., Eletto, D., Vargas, J., Boyle, S., Iwamoto, Y., Chomistek, S., Paton, J. C., Paton, A. W., Argon, Y. Clustering of IRE1α depends on sensing ER stress but not on its RNase activity.

Inhibition of a pancreatic cancer model by cooperative pairs of clinically approved and experimental antibodies.

By year 2025 pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer related death. However, other than improved chemotherapy and a small molecule inhibitor of the epidermal growth factor receptor (EGFR), no targeted drugs are currently available. Repurposing of approved drugs might offer a rapid solution. We employed an animal PDAC model, expressing a mutant and a wild type form of p53 and KRAS, respectively. Cetuximab, a clinically approved anti-EGFR monoclonal antibody (mAb) weakly inhibited PDAC xenografts, similar to trastuzumab, a mAb against HER2, a co-receptor of EGFR. Because the combination of cetuximab and trastuzumab only moderately enhanced the anti-tumor effects, we combined each with a home-made mAb to the same receptor and identified two cooperative pairs. The pair of trastuzumab and a murine anti-HER2 mAb better than the anti-EGFR pair inhibited PDAC xenografts, although HER2's abundance in our model is 15-fold lower than the level of EGFR. In vitro studies attribute cooperation to forced receptor endocytosis/degradation and inhibition of both DNA synthesis and cell migration. Taken together, our results identify cooperative pairs of anti-PDAC antibodies and highlight potential mechanisms of anti-tumor effects.

Increased intake of energy-dense diet and negative energy balance in a mouse model of chronic psychosocial defeat.

PURPOSE: Chronic exposure to stress may represent a risk factor for developing metabolic and eating disorders, mostly driven by the overconsumption of easily accessible energy-dense palatable food, although the mechanisms involved remain still unclear. In this study, we used an ethologically oriented murine model of chronic stress caused by chronic psychosocial defeat (CPD) to investigate the effects of unrestricted access to a palatable high fat diet (HFD) on food intake, body weight, energy homeostasis, and expression of different brain neuropeptides. Our aim was to shed light on the mechanisms responsible for body weight and body composition changes due to chronic social stress. METHODS: In our model of subordinate (defeated), mice (CPD) cohabitated in constant sensory contact with dominants, being forced to interact on daily basis, and were offered ad libitum access either to an HFD or to a control diet (CD). Control mice (of the same strain as CPD mice) were housed in pairs and left unstressed in their home cage (UN). In all these mice, we evaluated body weight, different adipose depots, energy metabolism, caloric intake, and neuropeptide expression. RESULTS: CPD mice increased the intake of HFD and reduced body weight in the presence of enhanced lipid oxidation. Resting energy expenditure and interscapular brown adipose tissue (iBAT) were increased in CPD mice, whereas epididymal adipose tissue increased only in HFD-fed unstressed mice. Propiomelanocortin mRNA levels in hypothalamic arcuate nucleus increased only in HFD-fed unstressed mice. Oxytocin mRNA levels in the paraventricular nucleus and neuropeptide Y mRNA levels within the arcuate were increased only in CD-fed CPD mice. In the arcuate, CART was increased in HFD-fed UN mice and in CD-fed CPD mice, while HFD intake suppressed CART increase in defeated animals. In the basolateral amygdala, CART expression was increased only in CPD animals on HFD. CONCLUSIONS: CPD appears to uncouple the intake of HFD from energy homeostasis causing higher HFD intake, larger iBAT accumulation, increased energy expenditure and lipid oxidation, and lower body weight. Overall, the present study confirms the notion that the chronic activation of the stress response can be associated with metabolic disorders, altered energy homeostasis, and changes of orexigenic and anorexigenic signaling. These changes might be relevant to better understand the etiology of stress-induced obesity and eating disorders and might represent a valid therapeutic approach for the development of new therapies in this field.

A bispecific antibody targeting EGFR and AXL delays resistance to osimertinib.

Activating EGFR (epidermal growth factor receptor) mutations can be inhibited by specific tyrosine kinase inhibitors (TKIs), which have changed the landscape of lung cancer therapy. However, due to secondary mutations and bypass receptors, such as AXL (AXL receptor tyrosine kinase), drug resistance eventually emerges in most patients treated with the first-, second-, or third-generation TKIs (e.g., osimertinib). To inhibit AXL and resistance to osimertinib, we compare two anti-AXL drugs, an antibody (mAb654) and a TKI (bemcentinib). While no pair of osimertinib and an anti-AXL drug is able to prevent relapses, triplets combining osimertinib, cetuximab (an anti-EGFR antibody), and either anti-AXL drug are initially effective. However, longer monitoring uncovers superiority of the mAb654-containing triplet, possibly due to induction of receptor endocytosis, activation of immune mechanisms, or disabling intrinsic mutators. Hence, we constructed a bispecific antibody that engages both AXL and EGFR. When combined with osimertinib, the bispecific antibody consistently inhibits tumor relapses, which warrants clinical trials.

Resistance of Lung Cancer to EGFR-Specific Kinase Inhibitors: Activation of Bypass Pathways and Endogenous Mutators.

Epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitors (TKIs) have changed the landscape of lung cancer therapy. For patients who are treated with the new TKIs, the current median survival exceeds 3 years, substantially better than the average 20 month survival rate only a decade ago. Unfortunately, despite initial efficacy, nearly all treated patients evolve drug resistance due to the emergence of either new mutations or rewired signaling pathways that engage other receptor tyrosine kinases (RTKs), such as MET, HER3 and AXL. Apparently, the emergence of mutations is preceded by a phase of epigenetic alterations that finely regulate the cell cycle, bias a mesenchymal phenotype and activate antioxidants. Concomitantly, cells that evade TKI-induced apoptosis (i.e., drug-tolerant persister cells) activate an intrinsic mutagenic program reminiscent of the SOS system deployed when bacteria are exposed to antibiotics. This mammalian system imbalances the purine-to-pyrimidine ratio, inhibits DNA repair and boosts expression of mutation-prone DNA polymerases. Thus, the net outcome of the SOS response is a greater probability to evolve new mutations. Deeper understanding of the persister-to-resister transformation, along with the development of next-generation TKIs, EGFR-specific proteolysis targeting chimeras (PROTACs), as well as bispecific antibodies, will permit delaying the onset of relapses and prolonging survival of patients with EGFR+ lung cancer.

L858R emerges as a potential biomarker predicting response of lung cancer models to anti-EGFR antibodies: Comparison of osimertinib vs. cetuximab.

EGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer.

EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired Resistance.

The epidermal growth factor receptor (EGFR) has served as the founding member of the large family of growth factor receptors harboring intrinsic tyrosine kinase function. High abundance of EGFR and large internal deletions are frequently observed in brain tumors, whereas point mutations and small insertions within the kinase domain are common in lung cancer. For these reasons EGFR and its preferred heterodimer partner, HER2/ERBB2, became popular targets of anti-cancer therapies. Nevertheless, EGFR research keeps revealing unexpected observations, which are reviewed herein. Once activated by a ligand, EGFR initiates a time-dependent series of molecular switches comprising downregulation of a large cohort of microRNAs, up-regulation of newly synthesized mRNAs, and covalent protein modifications, collectively controlling phenotype-determining genes. In addition to microRNAs, long non-coding RNAs and circular RNAs play critical roles in EGFR signaling. Along with driver mutations, EGFR drives metastasis in many ways. Paracrine loops comprising tumor and stromal cells enable EGFR to fuel invasion across tissue barriers, survival of clusters of circulating tumor cells, as well as colonization of distant organs. We conclude by listing all clinically approved anti-cancer drugs targeting either EGFR or HER2. Because emergence of drug resistance is nearly inevitable, we discuss the major evasion mechanisms.

Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models.

Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anti-cancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first-, second-, and third-generation drugs. To explore alternatives to such whack-a-mole strategies, we simulated in patient-derived xenograft models the situation of patients receiving first-line TKIs. Monotherapies comprising approved first-line TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drug-drug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistance-conferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing next-generation TKIs.

Host-Dependent Phenotypic Resistance to EGFR Tyrosine Kinase Inhibitors.

Lung cancers driven by mutant forms of EGFR invariably develop resistance to kinase inhibitors, often due to secondary mutations. Here we describe an unconventional mechanism of resistance to dacomitinib, a newly approved covalent EGFR kinase inhibitor, and uncover a previously unknown step of resistance acquisition. Dacomitinib-resistant (DR) derivatives of lung cancer cells were established by means of gradually increasing dacomitinib concentrations. These DR cells acquired no secondary mutations in the kinase or other domains of EGFR. Along with resistance to other EGFR inhibitors, DR cells acquired features characteristic to epithelial-mesenchymal transition, including an expanded population of aldehyde dehydrogenase-positive cells and upregulation of AXL, a receptor previously implicated in drug resistance. Unexpectedly, when implanted in animals, DR cells reverted to a dacomitinib-sensitive state. Nevertheless, cell lines derived from regressing tumors displayed renewed resistance when cultured in vitro. Three-dimensional and cocultures along with additional analyses indicated lack of involvement of hypoxia, fibroblasts, and immune cells in phenotype reversal, implying that other host-dependent mechanisms might nullify nonmutational modes of resistance. Thus, similar to the phenotypic resistance of bacteria treated with antibiotics, the reversible resisters described here likely evolve from drug-tolerant persisters and give rise to the irreversible, secondary mutation-driven nonreversible resister state. SIGNIFICANCE: This study reports that stepwise acquisition of kinase inhibitor resistance in lung cancers driven by mutant EGFR comprises a nonmutational, reversible resister state. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3862/F1.large.jpg.

TSHZ2 is an EGF-regulated tumor suppressor that binds to the cytokinesis regulator PRC1 and inhibits metastasis.

Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.

Roles for receptor tyrosine kinases in tumor progression and implications for cancer treatment.

Growth factors and their receptor tyrosine kinases (RTKs), a group of transmembrane molecules harboring cytoplasm-facing tyrosine-specific kinase functions, play essential roles in migration of multipotent cell populations and rapid proliferation of stem cells' descendants, transit amplifying cells, during embryogenesis and tissue repair. These intrinsic functions are aberrantly harnessed when cancer cells undergo intertwined phases of cell migration and proliferation during cancer progression. For example, by means of clonal expansion growth factors fixate the rarely occurring driver mutations, which initiate tumors. Likewise, autocrine and stromal growth factors propel angiogenesis and penetration into the newly sprouted vessels, which enable seeding micro-metastases at distant organs. We review genetic and other mechanisms that preempt ligand-mediated activation of RTKs, thereby supporting sustained cancer progression. The widespread occurrence of aberrant RTKs and downstream signaling pathways in cancer, identifies molecular targets suitable for pharmacological intervention. We list all clinically approved cancer drugs that specifically intercept oncogenic RTKs. These are mainly tyrosine kinase inhibitors and monoclonal antibodies, which can inhibit cancer but inevitably become progressively less effective due to adaptive rewiring processes or emergence of new mutations, processes we overview. Similarly important are patient treatments making use of radiation, chemotherapeutic agents and immune checkpoint inhibitors. The many interfaces linking RTK-targeted therapies and these systemic or local regimens are described in details because of the great promise offered by combining pharmacological modalities.

Targeting HER3, a Catalytically Defective Receptor Tyrosine Kinase, Prevents Resistance of Lung Cancer to a Third-Generation EGFR Kinase Inhibitor.

Although two growth factor receptors, EGFR and HER2, are amongst the best targets for cancer treatment, no agents targeting HER3, their kinase-defective family member, have so far been approved. Because emergence of resistance of lung tumors to EGFR kinase inhibitors (EGFRi) associates with compensatory up-regulation of HER3 and several secreted forms, we anticipated that blocking HER3 would prevent resistance. As demonstrated herein, a neutralizing anti-HER3 antibody we generated can clear HER3 from the cell surface, as well as reduce HER3 cleavage by ADAM10, a surface metalloproteinase. When combined with a kinase inhibitor and an anti-EGFR antibody, the antibody completely blocked patient-derived xenograft models that acquired resistance to EGFRi. We found that the underlying mechanism involves posttranslational downregulation of HER3, suppression of MET and AXL upregulation, as well as concomitant inhibition of AKT signaling and upregulation of BIM, which mediates apoptosis. Thus, although HER3 is nearly devoid of kinase activity, it can still serve as an effective drug target in the context of acquired resistance. Because this study simulated in animals the situation of patients who develop resistance to EGFRi and remain with no obvious treatment options, the observations presented herein may warrant clinical testing.

Cancer Immunotherapy: The Dawn of Antibody Cocktails.

Since the approval of the first monoclonal antibody (mAb), rituximab, for hematological malignancies, almost 30 additional mAbs have been approved in oncology. Despite remarkable advances, relatively weak responses and resistance to antibody monotherapy remain major open issue. Overcoming resistance might require combinations of drugs blocking both the major target and the emerging secondary target. We review clinically approved combinations of antibodies and either cytotoxic regimens (chemotherapy and irradiation) or kinase inhibitors. Thereafter, we focus on the most promising and currently very active arena that combines mAbs inhibiting immune checkpoints or growth factor receptors. Clinically approved and experimental oligoclonal mixtures of mAbs targeting different antigens (hetero-combinations) or different epitopes of the same antigen (homo-combinations) are described. Effective oligoclonal mixtures of antibodies that mimic the polyclonal immune response will likely become a mainstay of cancer therapy.

Shmt2: A Stat3 Signaling New Player in Prostate Cancer Energy Metabolism.

Prostate cancer (PCa) is a multifactorial disease characterized by the aberrant activity of different regulatory pathways. STAT3 protein mediates some of these pathways and its activation is implicated in the modulation of several metabolic enzymes. A bioinformatic analysis indicated a STAT3 binding site in the upstream region of SHMT2 gene. We demonstrated that in LNCaP, PCa cells' SHMT2 expression is upregulated by the JAK2/STAT3 canonical pathway upon IL-6 stimulation. Activation of SHTM2 leads to a decrease in serine levels, pushing PKM2 towards the nuclear compartment where it can activate STAT3 in a non-canonical fashion that in turn promotes a transient shift toward anaerobic metabolism. These results were also confirmed on FFPE prostate tissue sections at different Gleason scores. STAT3/SHMT2/PKM2 loop in LNCaP cells can modulate a metabolic shift in response to inflammation at early stages of cancer progression, whereas a non-canonical STAT3 activation involving the STAT3/HIF-1α/PKM2 loop is responsible for the maintenance of Warburg effect distinctive of more aggressive PCa cells. Chronic inflammation might thus prime the transition of PCa cells towards more advanced stages, and SHMT2 could represent a missing factor to further understand the molecular mechanisms responsible for the transition of prostate cancer towards a more aggressive phenotype.

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma.

The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR's transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.

Punicalagin, an active pomegranate component, is a new inhibitor of PDIA3 reductase activity.

BACKGROUND: Polyphenolic compounds isolated from pomegranate fruit possess several pharmacological activities including anti-inflammatory, hepatoprotective, antigenotoxic and anticoagulant activities. The present work focuses the attention on PDIA3 interaction with punicalagin and ellagic acid, the most predominant components of pomegranate extracts. PDIA3, a member of the protein disulfide isomerase family involved in several cellular functions, is associated with different human diseases and it has the potential to be a pharmacological target. METHODS: The interaction of polyphenols with PDIA3 purified protein was explored by fluorescence quenching and calorimetric techniques and their effect on PDIA3 activity was investigated. RESULTS: A higher affinity was observed for punicalagin which also strongly affects PDIA3 reductase activity in vitro as a non-competitive inhibitor. Isothermal titration calorimetry confirmed the high affinity of punicalagin for PDIA3. Considering the PDIA3 involvement in oxidative cellular stress response observed in neuroblastoma cells after treatment with hydrogen peroxide, a comparative study was conducted to evaluate the effect of punicalagin on wild type and PDIA3-silenced cells. Punicalagin increases the cell sensitivity to hydrogen peroxide in neuroblastoma cells, but this effect is drastically reduced in PDIA3-silenced cells treated in the same experimental conditions. CONCLUSIONS: Punicalagin binds PDIA3 and inhibits its redox activity. Comparative experiments conducted on unsilenced and PDIA3-silenced neuroblastoma cells suggest the potential of punicalagin to modulate PDIA3 reductase activity also in a biological model. GENERAL SIGNIFICANCE: Punicalagin can be used as a new PDIA3 inhibitor and this can provide information on the molecular mechanisms underlying the biological activities of PDIA3 and punicalagin.