Pharmacological and In Silico Analysis of Oat Avenanthramides as EGFR Inhibitors: Effects on EGF-Induced Lung Cancer Cell Growth and Migration.

Avena sativa L. is a wholegrain cereal and an important edible crop. Oats possesses high nutritional and health promoting values and contains high levels of bioactive compounds, including a group of phenolic amides, named avenanthramides (Avns), exerting antioxidant, anti-inflammatory, and anticancer activities. Epidermal growth factor receptor (EGFR) represents one of the most known oncogenes and it is frequently up-regulated or mutated in human cancers. The oncogenic effects of EGFR include enhanced cell growth, angiogenesis, and metastasis, and down-regulation or inhibition of EGFR signaling has therapeutic benefit. Front-line EGFR tyrosine kinase inhibitor therapy is the standard therapy for patients with EGFR-mutated lung cancer. However, the clinical effects of EGFR inhibition may be lost after a few months of treatment due to the onset of resistance. Here, we showed the anticancer activity of Avns, focusing on EGFR activation and signaling pathway. Lung cancer cellular models have been used to evaluate the activity of Avns on tumor growth, migration, EMT, and anoikis induced by EGF. In addition, docking and molecular dynamics simulations showed that the Avns bind with high affinity to a region in the vicinity of αC-helix and the DGF motif of EGFR, jeopardizing the target biological function. Altogether, our results reveal a new pharmacological activity of Avns as EGFR tyrosine kinase inhibitors.

KRIT1 as a possible new player in melanoma aggressiveness.

Krev interaction trapped protein 1 (KRIT1) is a scaffold protein known to form functional complexes with distinct proteins, including Malcavernin, PDCD10, Rap1 and others. It appears involved in several cellular signaling pathways and exerts a protective role against inflammation and oxidative stress. KRIT1 has been studied as a regulator of endothelial cell functions and represents a determinant in the pathogenesis of Cerebral Cavernous Malformation (CCM), a cerebrovascular disease characterized by the formation of clusters of abnormally dilated and leaky blood capillaries, which predispose to seizures, neurological deficits and intracerebral hemorrhage. Although KRIT1 is ubiquitously expressed, few studies have described its involvement in pathologies other than CCM including cancer. Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic propensity. Despite the numerous efforts made to define the signaling pathways activated during melanoma progression, the molecular mechanisms at the basis of melanoma growth, phenotype plasticity and resistance to therapies are still under investigation.

Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells.

Inflammatory prostaglandin E-2 (PGE-2) favors cancer progression in epithelial tumors characterized by persistent oncogene input. However, its effects on tumor cell stemness are poorly understood at molecular level. Here we describe two epithelial tumor cells A431 and A459, originating from human lung and skin tumors, in which epithelial growth factor (EGF) induces sequential up-regulation of mPGES-1 and iNOS enzymes, producing an inflammatory intracellular milieu. We demonstrated that concerted action of EGF, mPGES-1 and iNOS causes sharp changes in cell phenotype demonstrated by acquisition of stem-cell features and activation of the epithelial-mesenchymal transition (EMT). When primed with EGF, epithelial tumor cells transfected with mPGES-1 or iNOS to ensure steady enzyme levels display major stem-like and EMT markers, such as reduction in E-cadherin with a concomitant rise in vimentin, ALDH-1, CD133 and ALDH activity. Tumorsphere studies with these cells show increased sphere number and size, enhanced migratory and clonogenic capacity and sharp changes in EMT markers, indicating activation of this process. The concerted action of the enzymes forms a well-orchestrated cascade where expression of iNOS depends on overexpression of mPGES-1. Indeed, we show that through its downstream effectors (PGE-2, PKA, PI3K/Akt), mPGES-1 recruits non-canonical transcription factors, thus facilitating iNOS production. In conclusion, we propose that the initial event leading to tumor stem-cell activation may be a leveraged intrinsic mechanism in which all players are either inherent constituents (EGF) or highly inducible proteins (mPGES-1, iNOS) of tumor cells. We suggest that incipient tumor aggressiveness may be moderated by reducing pivotal input of mPGES-1.

Formulation of liposomes functionalized with Lotus lectin and effective in targeting highly proliferative cells.

BACKGROUND: Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. METHODS: We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. RESULTS: We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. CONCLUSIONS: Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. GENERAL SIGNIFICANCE: Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.

The syndecan-4/protein kinase Cα pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo.

Prostaglandin E2 (PGE2) is regarded as the main mediator of inflammatory symptoms. In addition, it also plays an important role in tumor growth and angiogenesis. In this study, we examined the mechanism of PGE2-induced angiogenic response. We show that in the absence of proteoglycan syndecan-4 (Sdc4), PGE2-induced ERK activation is decreased significantly, as is endothelial cell migration and cord formation in a two-dimensional Matrigel assay. In vivo, PGE2-induced angiogenesis is reduced dramatically in Sdc4(-/-) mice. The mechanism was traced to Sdc4-dependent activation of protein kinase Cα (PKCα). Transduction of an Sdc4 S183E mutant (a cytoplasmic domain mutation that blocks Sdc4-dependent PKCα activation) into Sdc4(-/-) endothelial cells was not able to rescue the loss of PGE2-induced ERK activation, whereas a transduction with full-length Sdc4 resulted in full rescue. Furthermore, PGE2-induced angiogenesis was also reduced in PKCα(-/-) mice. Taken together, these results demonstrate that PGE2-induced activation of angiogenesis is mediated via syndecan-4-dependent activation of PKCα.

Potential Human Health Benefits of Phaseolus vulgaris L. var Venanzio: Effects on Cancer Cell Growth and Inflammation.

It is widely recognized that foods, biodiversity, and human health are strongly interconnected, and many efforts have been made to understand the nutraceutical value of diet. In particular, diet can affect the progression of intestinal diseases, including inflammatory bowel disease (IBD) and intestinal cancer. In this context, we studied the anti-inflammatory and antioxidant activities of extracts obtained from a local endangered variety of Phaseolus vulgaris L. (Fagiola di Venanzio, FV). Using in vitro intestinal cell models, we evaluated the activity of three different extracts: soaking water, cooking water, and the bioaccessible fraction obtained after mimicking the traditional cooking procedure and gastrointestinal digestion. We demonstrated that FV extracts reduce inflammation and oxidative stress prompted by interleukin 1ß through the inhibition of cyclooxygenase 2 expression and prostaglandin E2 production and through the reduction in reactive oxygen species production and NOX1 levels. The reported data outline the importance of diet in the prevention of human inflammatory diseases. Moreover, they strongly support the necessity to safeguard local biodiversity as a source of bioactive compounds.

A novel bioresponsive self-immolative spacer based on aza-quinone methide reactivity for the controlled release of thiols, phenols, amines, sulfonamides or amides.

A stimuli-sensitive linker is one of the indispensable components of prodrugs for cancer therapy as it covalently binds the drug and releases it upon external stimulation at the tumour site. Quinone methide elimination has been widely used as the key transformation to release drugs based on their nucleofugacity. The usual approach is to bind the drug to the linker as a carbamate and release it as a free amine after a self-immolative 1,6-elimination. Although this approach is very efficient, it is limited to amines (as carbamates), alcohols or phenols (as carbonates) or other acidic functional groups. We report here a self-immolative spacer capable of directly linking and releasing amines, phenols, thiols, sulfonamides and carboxyamides after a reductive stimulus. The spacer is based on the structure of (5-nitro-2-pyrrolyl)methanol (NPYM-OH), which was used for the direct alkylation of the functional groups mentioned above. The spacer is metabolically stable and has three indispensable sites for bioconjugation: the bioresponsive trigger, the conjugated 1,6 self-immolative system and a third arm suitable for conjugation with a carrier or other modifiers. Release was achieved by selective reduction of the nitro group over Fe/Pd nanoparticles (NPs) in a micellar aqueous environment (H2O/TPGS-750-M), or by NADH mediated nitroreductase activation. A DFT study demonstrates that, during the 1,6 elimination, the transition state formed from 5-aminopyrrole has a lower activation energy compared to other 5-membered heterocycles or p-aminobenzyl derivatives. The NPYM scaffold was validated by late-stage functionalisation of approved drugs such as celecoxib, colchicine, vorinostat or ciprofloxacin. A hypoxia-activated NPYM-based prodrug (HAP) derived from HDAC inhibitor ST7612AA1 was also produced, which was active in cancer cells under hypoxic conditions.

In vitro Digestion of Phaseolus vulgaris L. Cooked Beans Induces Autophagy in Colon Cancer Cells.

Phaseolus vulgaris L. (common bean) contains high levels of proteins, unsaturated fatty acids, minerals, fibers, and vitamins, and for this reason, it represents an essential component of the diet. More than 40,000 varieties of beans have been recognized and are staple foods in the traditional cuisine of many countries. In addition to its high nutritional value, P. vulgaris is also characterized by its nutraceutical properties and favors environmental sustainability. In this manuscript, we studied two different varieties of P. vulgaris, Cannellino and Piattellino. We investigated the effects of traditional processing (soaking and cooking) and in vitro gastrointestinal digestion of beans on their phytochemical composition and anticancer activity. Using HT29 and HCT116 colon cancer cell lines, we showed that the extract obtained after gastrointestinal digestion of cooked beans (the bioaccessible fraction, BF) induces cell death through the induction of the autophagic process. We demonstrated that the BF of Cannellino and Piattellino beans at the concentration of 100 µg/mL reduces cell vitality, measured by MMT assay, of both HT29 (88.41% ± 5.79 and 94.38% ± 0.47) and HCT116 (86.29% ± 4.3 and 91.23% ± 0.52) cell lines. Consistently, the treatment of HT29 cells with 100 µg/mL of Cannellino and Piattellino BFs reduced clonogenicity by 95% ± 2.14 and 96% ± 0.49, respectively. Moreover, the activity of extracts appeared to be selective for colon cancer cells. The data shown in this work further confirm P. vulgaris to be among foods with beneficial effects for human health.

Cooperation between Prostaglandin E2 and Epidermal Growth Factor Receptor in Cancer Progression: A Dual Target for Cancer Therapy.

It is recognized that prostaglandin E2 (PGE2) is one key lipid mediator involved in chronic inflammation, and it is directly implicated in tumor development by regulating cancer cell growth and migration, apoptosis, epithelial-mesenchymal transition, angiogenesis, and immune escape. In addition, the expression of the enzymes involved in PGE2 synthesis, cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES1), positively correlates with tumor progression and aggressiveness, clearly indicating the crucial role of the entire pathway in cancer. Moreover, several lines of evidence suggest that the COX2/mPGES1/PGE2 inflammatory axis is involved in the modulation of epidermal growth factor receptor (EGFR) signaling to reinforce the oncogenic drive of EGFR activation. Similarly, EGFR activation promotes the induction of COX2/mPGES1 expression and PGE2 production. In this review, we describe the interplay between COX2/mPGES1/PGE2 and EGFR in cancer, and new therapeutic strategies that target this signaling pathway, to outline the importance of the modulation of the inflammatory process in cancer fighting.

A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

A pH-responsive crosslinker platform for antibody-drug conjugate (ADC) targeting delivery.

We report a new 1-6 self-immolative, traceless crosslinker derived from the natural product gallic acid. The linker acts through a pH-dependent mechanism for drug release. This 5-(hydroxymethyl)pyrogallol orthoester derivative (HMPO) was stable for 24 hours at pH values of 7.4 and 6.6 and in plasma, releasing molecules bound to the hydroxymethyl moiety under acid-dependent stimuli at pH 5.5. The linker was non-toxic and was used for the conjugation of Doxorubicin (Doxo) or Combretastatin A4 with Cetuximab. The ADCs formed showed their pH responsivity reducing cell viability of A431 and A549 cancer cells better than Cetuximab alone.

Prostaglandin E(2) primes the angiogenic switch via a synergic interaction with the fibroblast growth factor-2 pathway.

RATIONALE: Prostaglandin (PG)E(2) exerts temporally distinct actions on blood vessels, immediate vasodilatation, and long-term activation of angiogenesis. OBJECTIVE: To study the mechanism of PGE(2) induction of angiogenesis, we characterized its effect on fibroblast growth factor (FGF)-2 signaling in cultured endothelial cells and in ex vivo and in vivo assays of blood vessel formation. METHODS AND RESULTS: Using Western blotting assay, we demonstrated that PGE(2) induced upregulation of components of the FGF-2 pathway: FGF-2 protein, phosphorylation of FGF receptor type 1 (FGFR1), activation of FRS2alpha (FGFR substrate 2alpha), phospholipase Cgamma, endothelial nitric oxide synthase, extracellular signal-regulated kinase 1/2, and the transcription factor STAT-3. Synergism between PGE(2) and FGF-2 promoted endothelial cell proliferation and robust angiogenesis in vivo, in rabbit cornea and Matrigel assays. The magnitude of the angiogenic response to PGE(2) was directly related to FGF-2 availability which determined the extent of FGFR1 activation. In fact, PGE(2) induction of angiogenesis in vitro was impaired in FGF-2(-/-) endothelial cells and FGFR1 blockade abrogated PGE(2) action on the endothelium, preventing the activation of FGF-2 signaling. CONCLUSION: We propose a model for the angiogenic switch based on the autocrine/paracrine FGF-2/FGFR1 activation by PGE(2) and FGF-2 synergistic interaction. The synergism between the PGE(2) and FGF-2 signaling pathways here described may explain the mechanism of action of drug combinations, the most notable being cyclooxygenase inhibitors with growth factors or growth factor receptor inhibitors.

Non-peptide NK1 receptor ligands based on the 4-phenylpyridine moiety.

The quinoline nucleus of the previously described 4-phenylquinoline-3-carboxamides NK(1) receptor ligands 7 has been transformed into either substituted or azole-(i.e., triazole or tetrazole) fused pyridine moieties of compounds 9 and 10, respectively, in order to obtain NK(1) receptor ligands showing lower molecular weight or higher hydrophilicity. The program of molecular manipulations produced NK(1) receptor ligands showing affinity in the nanomolar range. In particular, 4-methyl-1-piperazinyl derivative 9j showed an IC(50) value of 4.8 nM and was proved to behave as a NK(1) antagonist blocking Sar(9)-SP-sulfone induced proliferation and migration of microvascular endothelial cells. Therefore, compound 9j has been labeled with [(11)C]CH(3)I (t(1/2)=20.4 min, ß(+)=99.8%) starting from the corresponding des-methyl precursor 9i using with a radiochemical yield of about 10% (not decay corrected) and a specific radioactivity>1 Ci/µmol in order to be used as a radiotracer in next PET studies.

RNA-mediated gene silencing of FUT1 and FUT2 influences expression and activities of bovine and human fucosylated nucleolin and inhibits cell adhesion and proliferation.

In a previous article, we demonstrated the existence of fucosyl-containing O-glycans forms of nucleolin in bovine post-capillary venular endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody found to interact strongly and exclusively with nucleolin in total protein extracts. The antibody was originally raised against a mollusc glycoprotein and was demonstrated to be directed against its O-glycans, recently found to belong prevalently to the blood group H-antigen type with fucose linked in alpha1, 2 to galactose. Here, we show that si-RNA induced down-regulation of the expression of FUT1 and FUT2, the fucosyltransferases required for the biosynthesis of the terminal glycan motif Fucalpha-2-Galbeta-R, reduced expression of the fucosylated nucleolin glycoforms and their exposure at the cell surface in CVEC. Treatment of the cells with FUT1/2 siRNA also reduced their ability to bind and internalize endostatin and their adhesion efficiency and inhibited cell growth. Expression of FUT1, FUT2, and FUT6 was also analyzed in serum-stimulated versus serum-starved cells and in cells treated with FUT1 and FUT2 siRNA. A reduced expression of fucosylated nucleolin and inhibition of cell growth by suppressing FUT1/2 expression was also tested and shown to be exhibited in human A431 cells.

Study of Molecular Interactions of CCM Proteins by Using a GAL4-Based Yeast Two-Hybrid Screening.

The yeast two-hybrid system was originally designed to detect protein-protein interactions using yeast transcriptional activators. Since its original description, this technique has been extensively used to identify protein-protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the known interaction between two proteins. Nowadays, the yeast two-hybrid screen remains one of the leading molecular tools to study protein-protein interactions in native intracellular conditions. In these years, the technique has improved to overcome the limitations of the original assay, and many efforts have been made to scale up the technique and to adapt it to large-scale studies. In addition, variations have been introduced to enlarge the range of proteins and interactors that can be assayed by hybrid-based approaches.Several groups studying molecular mechanisms underlying the Cerebral Cavernous Malformation disease have successfully used the yeast two-hybrid system or related methods to isolate, identify, and characterize molecular interactions involved in the onset and progression of the pathology.Here we describe general principles, strengths, and limits of the yeast two-hybrid technology, and the basic protocol for a yeast two-hybrid library screening and for a small-scale yeast two-hybrid assay by using a GAL4-based system.

Bidimentional In Vitro Angiogenic Assays to Study CCM Pathogenesis: Endothelial Cell Proliferation and Migration.

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3-0.5% in the general population. Genetic studies have identified three genes associated to CCMs: KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3), which account for about 50%, 20%, and 10% of the cases, respectively. The great advances in the knowledge of the physiopathological functions of CCM genes, such as their involvement in the angiogenic process, have allowed to propose distinct putative therapeutic compounds, which showed to be effective at least in limiting some pathological phenotypes in cellular and animal models of the disease. However, despite numerous efforts, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking.Here we describe simply and low-cost assays as in vitro endothelial cell proliferation and migration assays that can be used to better understand the role of CCM genes on endothelial cell functions and to screen potential new compounds for CCM therapy.

From Genes and Mechanisms to Molecular-Targeted Therapies: The Long Climb to the Cure of Cerebral Cavernous Malformation (CCM) Disease.

Cerebral cavernous malformation (CCM) is a rare cerebrovascular disorder of genetic origin consisting of closely clustered, abnormally dilated and leaky capillaries (CCM lesions), which occur predominantly in the central nervous system. CCM lesions can be single or multiple and may result in severe clinical symptoms, including focal neurological deficits, seizures, and intracerebral hemorrhage. Early human genetic studies demonstrated that CCM disease is linked to three chromosomal loci and can be inherited as autosomal dominant condition with incomplete penetrance and highly variable expressivity, eventually leading to the identification of three disease genes, CCM1/KRIT1, CCM2, and CCM3/PDCD10, which encode for structurally unrelated intracellular proteins that lack catalytic domains. Biochemical, molecular, and cellular studies then showed that these proteins are involved in endothelial cell-cell junction and blood-brain barrier stability maintenance through the regulation of major cellular structures and mechanisms, including endothelial cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, autophagy, and endothelial-to-mesenchymal transition, suggesting that they act as pleiotropic regulators of cellular homeostasis, and opening novel therapeutic perspectives. Indeed, accumulated evidence in cellular and animal models has eventually revealed that the emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses.In this introductory review, we present a general overview of 20 years of amazing progress in the identification of genetic culprits and molecular mechanisms underlying CCM disease pathogenesis, and the development of targeted therapeutic strategies.

Phaseolus vulgaris L. var. Venanzio Grown in Tuscany: Chemical Composition and In Vitro Investigation of Potential Effects on Colorectal Cancer.

Phaseolus vulgaris L. (common bean) is a leguminous species that is an important dietary component due to its high content of proteins, unsaturated fatty acids, minerals, dietary fibers and vitamins. Due to the high content of polyphenols, several biological activities have been described for bean extracts, making it possible to include P. vulgaris among food with beneficial effects for human health. Moreover, more than 40,000 varieties of beans have been recognised with different nutraceutical properties, pointing out the importance of food biodiversity. In this work, we describe for the first time the chemical composition and biological activity of a newly recognized Italian variety of P. vulgaris grown in a restricted area of the Tuscany region and named "Fagiola di Venanzio". Fagiola di Venanzio water extract is rich in proteins, sugars and polyphenols and displays antioxidant, anti-inflammatory and antiproliferative activities in in vitro assays on colon cancer cellular models. Our data indicate that this variety of P. vulgaris appears to be a promising source of bioactive compounds and encourage more in-depth studies to better elucidate the implications of its consumption for public health.

Prostaglandin E2 and Cancer: Insight into Tumor Progression and Immunity.

The involvement of inflammation in cancer progression has been the subject of research for many years. Inflammatory milieu and immune response are associated with cancer progression and recurrence. In different types of tumors, growth and metastatic phenotype characterized by the epithelial mesenchymal transition (EMT) process, stemness, and angiogenesis, are increasingly associated with intrinsic or extrinsic inflammation. Among the inflammatory mediators, prostaglandin E2 (PGE2) supports epithelial tumor aggressiveness by several mechanisms, including growth promotion, escape from apoptosis, transactivation of tyrosine kinase growth factor receptors, and induction of angiogenesis. Moreover, PGE2 is an important player in the tumor microenvironment, where it suppresses antitumor immunity and regulates tumor immune evasion, leading to increased tumoral progression. In this review, we describe the current knowledge on the pro-tumoral activity of PGE2 focusing on its role in cancer progression and in the regulation of the tumor microenvironment.

KRIT1 loss-mediated upregulation of NOX1 in stromal cells promotes paracrine pro-angiogenic responses.

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3-0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function. In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells. Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.