Endoplasmic reticulum stress: A possible connection between intestinal inflammation and neurodegenerative disorders.

BACKGROUND: Different studies have shown the key role of endoplasmic reticulum (ER) stress in autoimmune and chronic inflammatory disorders, as well as in neurodegenerative diseases. ER stress leads to the formation of misfolded proteins which affect the secretion of different cell types that are crucial for the intestinal homeostasis. PURPOSE: In this review, we discuss the role of ER stress and its involvement in the development of inflammatory bowel diseases, chronic conditions that can cause severe damage of the gastrointestinal tract, focusing on the alteration of Paneth cells and goblet cells (the principal secretory phenotypes of the intestinal epithelial cells). ER stress is also discussed in the context of neurodegenerative diseases, in which protein misfolding represents the signature mechanism. ER stress in the bowel and consequent accumulation of misfolded proteins might represent a bridge between bowel inflammation and neurodegeneration along the gut-to-brain axis, affecting intestinal epithelial homeostasis and the equilibrium of the commensal microbiota. Targeting intestinal ER stress could foster future studies for designing new biomarkers and new therapeutic approaches for neurodegenerative disorders.

KRAS is a molecular determinant of platinum responsiveness in glioblastoma.

BACKGROUND: KRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis. METHODS: Long-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRASG12V plasmids. RESULTS: Here, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRASG12V rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype. CONCLUSION: These findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as 'actionable target' of cisplatin-based chemotherapy for glioblastoma.

Expression and role of cocaine-amphetamine regulated transcript (CART) in the proliferation of biliary epithelium.

Cholangiocytes, the epithelial cells that line the biliary tree, can proliferate under the stimulation of several factors through both autocrine and paracrine pathways. The cocaine-amphetamine-regulated-transcript (CART) peptide has several physiological functions, and it is widely expressed in several organs. CART increases the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor (BDNF), whose expression has been correlated to the proliferation rate of cholangiocytes. In the present study, we aimed to evaluate the expression of CART and its role in modulating cholangiocyte proliferation in healthy and bile duct ligated (BDL) rats in vivo, as well as in cultured normal rat cholangiocytes (NRC) in vitro. Liver samples from both healthy and BDL (1 week) rats, were analyzed by immunohistochemistry and immunofluorescence for CART, CK19, TrkB and p75NTR BDNF receptors. PCNA staining was used to evaluate the proliferation of the cholangiocytes, whereas TUNEL assay was used to evaluate biliary apoptosis. NRC treated or not with CART were used to confirm the role of CART on cholangiocytes proliferation and the secretion of BDNF. Cholangiocytes proliferation, apoptosis, CART and TrkB expression were increased in BDL rats, compared to control rats. We found a higher expression of TrkB and p75NTR, which could be correlated with the proliferation rate of biliary tree during BDL. The in vitro study demonstrated increased BDNF secretion by NRC after treatment with CART compared with control cells. As previously reported, proliferating cholangiocytes acquire a neuroendocrine phenotype, modulated by several factors, including neurotrophins. Accordingly, CART may play a key role in the remodeling of biliary epithelium during cholestasis by modulating the secretion of BDNF.

The clinically relevant CHK1 inhibitor MK-8776 induces the degradation of the oncogenic protein PML-RARα and overcomes ATRA resistance in acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is a hematological disease characterized by the expression of the oncogenic fusion protein PML-RARα. The current treatment approach for APL involves differentiation therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, the development of resistance to therapy, occurrence of differentiation syndrome, and relapses necessitate the exploration of new treatment options that induce differentiation of leukemic blasts with low toxicity. In this study, we investigated the cellular and molecular effects of MK-8776, a specific inhibitor of CHK1, in ATRA-resistant APL cells. Treatment of APL cells with MK-8776 resulted in a decrease in PML-RARα levels, increased expression of CD11b, and increased granulocytic activity consistent with differentiation. Interestingly, we showed that the MK-8776-induced differentiating effect resulted synergic with ATO. We found that the reduction of PML-RARα by MK-8776 was dependent on both proteasome and caspases. Specifically, both caspase-1 and caspase-3 were activated by CHK1 inhibition, with caspase-3 acting upstream of caspase-1. Activation of caspase-3 was necessary to activate caspase-1 and promote PML-RARα degradation. Transcriptomic analysis revealed significant modulation of pathways and upstream regulators involved in the inflammatory response and cell cycle control upon MK-8776 treatment. Overall, the ability of MK-8776 to induce PML-RARα degradation and stimulate differentiation of immature APL cancer cells into more mature forms recapitulates the concept of differentiation therapy. Considering the in vivo tolerability of MK-8776, it will be relevant to evaluate its potential clinical benefit in APL patients resistant to standard ATRA/ATO therapy, as well as in patients with other forms of acute leukemias.

A functional genetic screen for metabolic proteins unveils GART and the de novo purine biosynthetic pathway as novel targets for the treatment of luminal A ERα expressing primary and metastatic invasive ductal carcinoma.

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17ß-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs.

TERT Extra-Telomeric Roles: Antioxidant Activity and Mitochondrial Protection.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H2O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

Maternal Immune Activation Induced by Prenatal Lipopolysaccharide Exposure Leads to Long-Lasting Autistic-like Social, Cognitive and Immune Alterations in Male Wistar Rats.

Several studies have supported the association between maternal immune activation (MIA) caused by exposure to pathogens or inflammation during critical periods of gestation and an increased susceptibility to the development of various psychiatric and neurological disorders, including autism and other neurodevelopmental disorders (NDDs), in the offspring. In the present work, we aimed to provide extensive characterization of the short- and long-term consequences of MIA in the offspring, both at the behavioral and immunological level. To this end, we exposed Wistar rat dams to Lipopolysaccharide and tested the infant, adolescent and adult offspring across several behavioral domains relevant to human psychopathological traits. Furthermore, we also measured plasmatic inflammatory markers both at adolescence and adulthood. Our results support the hypothesis of a deleterious impact of MIA on the neurobehavioral development of the offspring: we found deficits in the communicative, social and cognitive domains, together with stereotypic-like behaviors and an altered inflammatory profile at the systemic level. Although the precise mechanisms underlying the role of neuroinflammatory states in neurodevelopment need to be clarified, this study contributes to a better understanding of the impact of MIA on the risk of developing behavioral deficits and psychiatric illness in the offspring.

The Emerging Role of Ferroptosis in Liver Cancers.

Liver cancer represents a global health challenge with worldwide growth. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Indeed, approximately 90% of HCC cases have a low survival rate. Moreover, cholangiocarcinoma (CC) is another malignant solid tumor originating from cholangiocytes, the epithelial cells of the biliary system. It is the second-most common primary liver tumor, with an increasing course in morbidity and mortality. Tumor cells always show high metabolic levels, antioxidant modifications, and an increased iron uptake to maintain unlimited growth. In recent years, alterations in iron metabolism have been shown to play an important role in the pathogenesis of HCC. Several findings show that a diet rich in iron can enhance HCC risk. Hence, elevated iron concentration inside the cell may promote the development of HCC. Growing evidence sustains that activating ferroptosis may potentially block the proliferation of HCC cells. Even in CC, it has been shown that ferroptosis plays a crucial role in the treatment of tumors. Several data confirmed the inhibitory effect in cell growth of photodynamic therapy (PDT) that can induce reactive oxygen species (ROS) in CC, leading to an increase in malondialdehyde (MDA) and a decrease in intracellular glutathione (GSH). MDA and GSH depletion/modulation are crucial in inducing ferroptosis, suggesting that PDT may have the potential to induce this kind of cell death through these ways. A selective induction of programmed cell death in cancer cells is one of the main treatments for malignant tumors; thus, ferroptosis may represent a novel therapeutic strategy against HCC and CC.

Extranuclear effects of thyroid hormones and analogs during development: An old mechanism with emerging roles.

Thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine), induce a variety of long-term effects on important physiological functions, ranging from development and growth to metabolism regulation, by interacting with specific nuclear or cytosolic receptors. Extranuclear or nongenomic effects of thyroid hormones are mediated by plasma membrane or cytoplasmic receptors, mainly by αvß3 integrin, and are independent of protein synthesis. A wide variety of nongenomic effects have now been recognized to be elicited through the binding of thyroid hormones to this receptor, which is mainly involved in angiogenesis, as well as in cell cancer proliferation. Several signal transduction pathways are modulated by thyroid hormone binding to αvß3 integrin: protein kinase C, protein kinase A, Src, or mitogen-activated kinases. Thyroid hormone-activated nongenomic effects are also involved in the regulation of Na+-dependent transport systems, such as glucose uptake, Na+/K+-ATPase, Na+/H+ exchanger, and amino acid transport System A. Of note, the modulation of these transport systems is cell-type and developmental stage-dependent. In particular, dysregulation of Na+/K+-ATPase activity is involved in several pathological situations, from viral infection to cancer. Therefore, this transport system represents a promising pharmacological tool in these pathologies.

The antiviral drug telaprevir induces cell death by reducing FOXA1 expression in estrogen receptor α (ERα)-positive breast cancer cells.

Previously, we found that telaprevir (Tel), the inhibitor of hepatitis C virus NS3/4A serine protease, reduces estrogen receptor α (ERα) content at the transcriptional level without binding to the receptor, prevents ERα transcriptional activity, and inhibits basal and 17ß-estradiol (E2)-dependent cell proliferation in different breast cancer (BC) cell lines. Here, we further characterize the Tel action mechanisms on ERα levels and function, identify a possible molecular target of Tel in BC cells, and evaluate Tel as an antiproliferative agent for BC treatment. Tel-dependent reduction in ERα levels and function depends on a Tel-dependent decrease in FOXA1 levels and activity. The effect of Tel is transduced by the IGF1-R/AKT/FOXA1 pathway, with the antiviral compound interacting with IGF1-R. Tel prevents the proliferation of several BC cell lines, while it does not affect the proliferation of normal nontransformed cell lines, and its antiproliferative effect is correlated with the ratio of FOXA1/IGF1-R expression. In conclusion, Tel interferes with the IGF1-R/AKT/FOXA1 pathway and induces cell death in ERα-expressing BC cells. Thus, we propose that this antiviral could be repurposed for the treatment of ERα-expressing BC.

Tailoring a Solvent-Assisted Method for Solid-Supported Hybrid Lipid-Polymer Membranes.

Combining amphiphilic block copolymers and phospholipids opens new opportunities for the preparation of artificial membranes. The chemical versatility and mechanical robustness of polymers together with the fluidity and biocompatibility of lipids afford hybrid membranes with unique properties that are of great interest in the field of bioengineering. Owing to its straightforwardness, the solvent-assisted method (SA) is particularly attractive for obtaining solid-supported membranes. While the SA method was first developed for lipids and very recently extended to amphiphilic block copolymers, its potential to develop hybrid membranes has not yet been explored. Here, we tailor the SA method to prepare solid-supported polymer-lipid hybrid membranes by combining a small library of amphiphilic diblock copolymers poly(dimethyl siloxane)-poly(2-methyl-2-oxazoline) and poly(butylene oxide)-block-poly(glycidol) with phospholipids commonly found in cell membranes including 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl). The optimization of the conditions under which the SA method was applied allowed for the formation of hybrid polymer-lipid solid-supported membranes. The real-time formation and morphology of these hybrid membranes were evaluated using a combination of quartz crystal microbalance and atomic force microscopy. Depending on the type of polymer-lipid combination, significant differences in membrane coverage, formation of domains, and quality of membranes were obtained. The use of the SA method for a rapid and controlled formation of solid-supported hybrid membranes provides the basis for developing customized artificial hybrid membranes.

Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ERα expression and proliferation in luminal primary and metastatic breast cancer cells.

BACKGROUND: Challenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs. METHODS: ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan-Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17ß-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed. RESULTS: A reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC. CONCLUSIONS: CHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs.

Protease Activated Receptor 1 and Its Ligands as Main Regulators of the Regeneration of Peripheral Nerves.

In contrast with the brain and spinal cord, peripheral nerves possess a striking ability to regenerate after damage. This characteristic of the peripheral nervous system is mainly due to a specific population of glial cells, the Schwann cells. Schwann cells promptly activate after nerve injury, dedifferentiate assuming a repair phenotype, and assist axon regrowth. In general, tissue injury determines the release of a variety of proteases which, in parallel with the degradation of their specific targets, also activate plasma membrane receptors known as protease-activated receptors (PARs). PAR1, the prototypical member of the PAR family, is also known as thrombin receptor and is present at the Schwann cell plasma membrane. This receptor is emerging as a possible regulator of the pro-regenerative capacity of Schwann cells. Here, we summarize the most recent literature data describing the possible contribution of PAR1 and PAR1-activating proteases in regulating the regeneration of peripheral nerves.

The Peculiar Estrogenicity of Diethyl Phthalate: Modulation of Estrogen Receptor α Activities in the Proliferation of Breast Cancer Cells.

Phthalates comprise a group of synthetic chemicals present in the environment because of their wide use as plasticizers and as additives in products for personal care. Among others, diethyl phthalate (DEP) is largely used in products for infants, children, and adults, in which its exposure has been correlated with an increased risk of breast cancer. The adverse health outcomes deriving from phthalate exposure have been associated with their activity as endocrine disruptors (EDCs) of the steroid and thyroid hormone signaling by affecting developmental and reproductive health, and even carcinogenicity. However, the estrogen disruptor activities of DEP are still controversial, and the mechanism at the root of the estrogenic-disrupting action of DEP remains to be clarified. Here, we evaluated the DEP mechanism of action on the activation status of estrogen receptor α (ERα) by analyzing the receptor's phosphorylation as well as both nuclear and extra-nuclear pathways triggered by the receptor to modulate the proliferation of breast cancer cells. Although DEP does not bind to ERα, our results suggest that this phthalate ester exerts multiple parallel interactions with ERα signaling and emphasize the importance to determine an appropriate battery of in vitro methods that will include specific molecular mechanisms involved in the endocrine disruption.

Expanding the Potential of the Solvent-Assisted Method to Create Bio-Interfaces from Amphiphilic Block Copolymers.

Artificial membranes, as materials with biomimetic properties, can be applied in various fields, such as drug screening or bio-sensing. The solvent-assisted method (SA) represents a straightforward method to prepare lipid solid-supported membranes. It overcomes the main limitations of established membrane preparation methods, such as Langmuir-Blodgett (LB) or vesicle fusion. However, it has not yet been applied to create artificial membranes based on amphiphilic block copolymers, despite their enhanced mechanical stability compared to lipid-based membranes and bio-compatible properties. Here, we applied the SA method on different amphiphilic di- and triblock poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) copolymers and optimized the conditions to prepare artificial membranes on a solid support. The real-time membrane formation, the morphology, and the mechanical properties have been evaluated by a combination of atomic force microscopy and quartz crystal microbalance. Then, selected biomolecules including complementary DNA strands and an artificial deallylase metalloenzyme (ADAse) were incorporated into these membranes relying on the biotin-streptavidin technology. DNA strands served to establish the capability of these synthetic membranes to interact with biomolecules by preserving their correct conformation. The catalytic activity of the ADAse following its membrane anchoring induced the functionality of the biomimetic platform. Polymer membranes on solid support as prepared by the SA method open new opportunities for the creation of artificial membranes with tailored biomimetic properties and functionality.

Neuroprotective Effects of Curcumin in Methamphetamine-Induced Toxicity.

Curcumin (CUR), a natural polyphenol extracted from rhizome of the Curcuma longa L, has received great attention for its multiple potential health benefits as well as disease prevention. For instance, CUR protects against toxic agents acting on the human body, including the nervous system. In detail, CUR possesses, among others, strong effects as an autophagy activator. The present study indicates that CUR counteracts methamphetamine (METH) toxicity. Such a drug of abuse is toxic by disturbing the autophagy machinery. We profited from an unbiased, low variable cell context by using rat pheochromocytoma PC12 cell line. In such a system, a strong protection was exerted by CUR against METH toxicity. This was associated with increased autophagy flux, merging of autophagosomes with lysosomes and replenishment of autophagy vacuoles with LC3, which instead is moved out from the vacuoles by METH. This is expected to enable the autophagy machinery. In fact, while in METH-treated cells the autophagy substrates α-synuclein accumulates in the cytosol, CUR speeds up α-synuclein clearance. Under the effects of CUR LC3 penetrate in autophagy vacuoles to commit them to cell clearance and promotes the autophagy flux. The present data provide evidence that CUR counteracts the neurotoxic effects induced by METH by promoting autophagy.

A New Anti-Estrogen Discovery Platform Identifies FDA-Approved Imidazole Anti-Fungal Drugs as Bioactive Compounds against ERα Expressing Breast Cancer Cells.

17ß-estradiol (E2) exerts its physiological effects through the estrogen receptor α (i.e., ERα). The E2:ERα signaling allows the regulation of cell proliferation. Indeed, E2 sustains the progression of ERα positive (ERα+) breast cancers (BCs). The presence of ERα at the BC diagnosis drives their therapeutic treatment with the endocrine therapy (ET), which restrains BC progression. Nonetheless, many patients develop metastatic BCs (MBC) for which a treatment is not available. Consequently, the actual challenge is to complement the drugs available to fight ERα+ primary and MBC. Here we exploited a novel anti-estrogen discovery platform to identify new Food and Drug Administration (FDA)-approved drugs inhibiting E2:ERα signaling to cell proliferation in cellular models of primary and MBC cells. We report that the anti-fungal drugs clotrimazole (Clo) and fenticonazole (Fenti) induce ERα degradation and prevent ERα transcriptional signaling and proliferation in cells modeling primary and metastatic BC. The anti-proliferative effects of Clo and Fenti occur also in 3D cancer models (i.e., tumor spheroids) and in a synergic manner with the CDK4/CDK6 inhibitors palbociclib and abemaciclib. Therefore, Clo and Fenti behave as "anti-estrogens"-like drugs. Remarkably, the present "anti-estrogen" discovery platform represents a valuable method to rapidly identify bioactive compounds with anti-estrogenic activity.

HIV-1 Nef Protein Affects Cytokine and Extracellular Vesicles Production in the GEN2.2 Plasmacytoid Dendritic Cell Line.

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon production, whose role in Human Immunodeficiency Virus (HIV) infection and pathogenesis is complex and not yet well defined. Considering the crucial role of the accessory protein Nef in HIV pathogenicity, possible alterations in intracellular signalling and extracellular vesicle (EV) release induced by exogenous Nef on uninfected pDCs have been investigated. As an experimental model system, a human plasmacytoid dendritic cell line, GEN2.2, stimulated with a myristoylated recombinant NefSF2 protein was employed. In GEN2.2 cells, Nef treatment induced the tyrosine phosphorylation of STAT-1 and STAT-2 and the production of a set of cytokines, chemokines and growth factors including IP-10, MIP-1ß, MCP-1, IL-8, TNF-α and G-CSF. The released factors differed both in type and amount from those released by macrophages treated with the same viral protein. Moreover, Nef treatment slightly reduces the production of small EVs, and the protein was found associated with the small (size < 200 nm) but not the medium/large vesicles (size > 200 nm) collected from GEN2.2 cells. These results add new information on the interactions between this virulence factor and uninfected pDCs, and may provide the basis for further studies on the interactions of Nef protein with primary pDCs.

Ouabain and Digoxin Activate the Proteasome and the Degradation of the ERα in Cells Modeling Primary and Metastatic Breast Cancer.

Estrogen receptor α expressing breast cancers (BC) are classically treated with endocrine therapy. Prolonged endocrine therapy often results in a metastatic disease (MBC), for which a standardized effective therapy is still lacking. Thus, new drugs are required for primary and metastatic BC treatment. Here, we report that the Food and Drug Administration (FDA)-approved drugs, ouabain and digoxin, induce ERα degradation and prevent proliferation in cells modeling primary and metastatic BC. Ouabain and digoxin activate the cellular proteasome, instigating ERα degradation, which causes the inhibition of 17ß-estradiol signaling, induces the cell cycle blockade in the G2 phase, and triggers apoptosis. Remarkably, these effects are independent of the inhibition of the Na/K pump. The antiproliferative effects of ouabain and digoxin occur also in diverse cancer models (i.e., tumor spheroids and xenografts). Additionally, gene profiling analysis reveals that these drugs downregulate the expression of genes related to endocrine therapy resistance. Therefore, ouabain and digoxin behave as 'anti-estrogen'-like drugs, and are appealing candidates for the treatment of primary and metastatic BCs.

Control of the Mechanical Adhesion of III-V Materials Grown on Layered h-BN.

Hexagonal boron nitride (h-BN) can be used as a p-doped material in wide-bandgap optoelectronic heterostructures or as a release layer to allow lift-off of grown three-dimensional (3D) GaN-based devices. To date, there have been no studies of factors that lead to or prevent lift-off and/or spontaneous delamination of layers. Here, we report a unique approach of controlling the adhesion of this layered material, which can result in both desired lift-off layered h-BN and mechanically inseparable robust h-BN layers. This is accomplished by controlling the diffusion of Al atoms into h-BN from AlN buffers grown on h-BN/sapphire. We present evidence of Al diffusion into h-BN for AlN buffers grown at high temperatures compared to conventional-temperature AlN buffers. Further evidence that the Al content in BN controls lift-off is provided by comparison of two alloys, Al0.03B0.97N/sapphire and Al0.17B0.83N/sapphire. Moreover, we tested that management of Al diffusion controls the mechanical adhesion of high-electron-mobility transistor (HEMT) devices grown on AlN/h-BN/sapphire. The results extend the control of two-dimensional (2D)/3D hetero-epitaxy and bring h-BN closer to industrial application in optoelectronics.