ALDH1A1 overexpression in melanoma cells promotes tumor angiogenesis by activating the IL­8/Notch signaling cascade.

ALDH1A1 is a cytosolic enzyme upregulated in tumor cells, involved in detoxifying cells from reactive aldehydes and in acquiring resistance to chemotherapeutic drugs. Its expression correlates with poor clinical outcomes in a number of cancers, including melanoma. The present study hypothesized that the increased ALDH1A1 expression and activity upregulated the release of proangiogenic factors from melanoma cells, which regulate angiogenic features in endothelial cells (ECs) through a rearrangement of the Notch pathway. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing melanoma cells displayed a higher microvessel density. In a 3D multicellular system, obtained co­culturing melanoma cancer cells with stromal cells, including ECs, melanoma ALDH1A1 overexpression induced the recruitment of ECs into the core of the tumorspheres. By using a genes array, overexpression of ALDH1A1 in tumor cells also promoted modulation of Notch cascade gene expression in ECs, suggesting an interaction between tumor cells and ECs mediated by enrichment of angiogenic factors in the tumor microenvironment. To confirm this hypothesis, inactivation of ALDH1A1 by the pharmacological inhibitor CM037 significantly affected the release of angiogenic factors, including IL­8, from melanoma cells. High levels of ALDH1A1, through the retinoic acid pathway, regulated the activation of NF­kB­p65 and IL­8. Further, in a 2D co­culture system, the addition of an IL­8 neutralizing antibody to ECs co­cultured with melanoma cells forced to express ALDH1A1 dampened endothelial angiogenic features, both at the molecular (in terms of gene and protein expression of mediators of the Notch pathway) and at the functional level (proliferation, scratch assay, tube formation and permeability). In conclusion, these findings demonstrated the existence of a link between melanoma ALDH1A1 expression and EC Notch signaling modification that results in a pro­angiogenic phenotype. Based on the crucial role of ALDH1A1 in melanoma control of the tumor microenvironment, the enzyme seems a promising target for the development of novel drugs able to interrupt the cross­talk between cancer (stem) cells and endothelial cells.

Targeting endothelial-to-mesenchymal transition: the protective role of hydroxytyrosol sulfate metabolite.

PURPOSE: Endothelial-to-mesenchymal transition (EndMT) plays an important role in pathogenesis of a number of inflammatory diseases. Hydroxytyrosol (HT) and, particularly, its major plasma metabolite HT-3O sulfate (HT-3Os) are known olive oil antioxidant and anti-inflammatory polyphenols which exert benefits against vascular diseases by improving endothelial function. However, to date the HT-3Os role in EndMT is not well known. METHODS: To investigate the HT-3Os effects on EndMT in the inflamed endothelium, we used an in vitro model of endothelial dysfunction, challenging endothelial cells (EC), human umbilical EC (HUVEC) and human retinal EC (HREC) with Interleukin-1ß (IL-1ß), an inflammatory agent. HREC were used as a specific model to investigate HT-3Os effects on vascular retinal diseases. RESULTS: We found that IL-1ß treatment-induced EndMT phenotype in both cell models, also changing cell morphology. HT-3Os protected EC against IL-1ß effects, recovering cell morphology and phenotype. Mechanistically, HT-3Os targeting fibroblast growth factor receptor 1 FGFR1 expression and let-7 miRNA, controlled transforming growth factor beta (TGF-ß) signalling in EC, downregulating transcription factors expression (SNAI1 and ZEB2) and gene expression of late EndMT markers (FN1, VIM, NOTCH3, CNN1, MMP2 and MMP9). CONCLUSION: These results demonstrate that HT-3Os blunts pathological EndMT in inflamed EC, maintaining high let-7 miRNA expression and preventing activation of TGF-ß signalling.

ALDH3A1 Overexpression in Melanoma and Lung Tumors Drives Cancer Stem Cell Expansion, Impairing Immune Surveillance through Enhanced PD-L1 Output.

Melanoma and non-small-cell lung carcinoma (NSCLC) cell lines are characterized by an intrinsic population of cancer stem-like cells (CSC), and high expression of detoxifying isozymes, the aldehyde dehydrogenases (ALDHs), regulating the redox state. In this study, using melanoma and NSCLC cells, we demonstrate that ALDH3A1 isozyme overexpression and activity is closely associated with a highly aggressive mesenchymal and immunosuppressive profile. The contribution of ALDH3A1 to the stemness and immunogenic status of melanoma and NSCLC cells was evaluated by their ability to grow in 3D forming tumorspheres, and by the expression of markers for stemness, epithelial to mesenchymal transition (EMT), and inflammation. Furthermore, in specimens from melanoma and NSCLC patients, we investigated the expression of ALDH3A1, PD-L1, and cyclooxygenase-2 (COX-2) by immunohistochemistry. We show that cells engineered to overexpress the ALDH3A1 enzyme enriched the CSCs population in melanoma and NSCLC cultures, changing their transcriptome. In fact, we found increased expression of EMT markers, such as vimentin, fibronectin, and Zeb1, and of pro-inflammatory and immunosuppressive mediators, such as NFkB, prostaglandin E2, and interleukin-6 and -13. ALDH3A1 overexpression enhanced PD-L1 output in tumor cells and resulted in reduced proliferation of peripheral blood mononuclear cells when co-cultured with tumor cells. Furthermore, in tumor specimens from melanoma and NSCLC patients, ALDH3A1 expression was invariably correlated with PD-L1 and the pro-inflammatory marker COX-2. These findings link ALDH3A1 expression to tumor stemness, EMT and PD-L1 expression, and suggest that aldehyde detoxification is a redox metabolic pathway that tunes the immunological output of tumors.

mPGES-1 as a new target to overcome acquired resistance to gefitinib in non-small cell lung cancer cell lines.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) as gefitinib are standard treatment of non-small cell lung cancer (NSCLC), but resistance often occurs. This study demonstrates that NSCLC cells resistant to gefitinib (GR cells) displayed a significantly higher microsomal prostaglandin E synthase-1 (mPGES-1) expression and activity than parental cells. Overexpression of mPGES-1/prostaglandin E-2 (PGE-2) signaling in GR cells was associated with acquisition of mesenchymal and stem-like cell properties, nuclear EGFR translocation and tolerance to cisplatin. mPGES-1 inhibition reduced mesenchymal and stem-like properties, and nuclear EGFR translocation in GR cells. Consistently, inhibition of mPGES-1 activity enhanced sensitivity to cisplatin and responsiveness to gefitinib in GR cells. We propose the mPGES-1/PGE-2 signaling as a potential target for treating aggressive and resistant lung cancers.

Pharmacological Tools for the Study of H2S Contribution to Angiogenesis.

Recently, hydrogen sulfide (H2S) has been characterized as an endogenous mediator able to control a series of cellular and tissue functions relevant for tissue homeostasis and repair such as angiogenesis. This chapter describes the tools and their use in a set of angiogenesis assays performed by using cultured endothelial cells in order to study the relevance of exogenous or endogenous H2S production and release during the occurrence of angiogenesis.

miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1ß and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

Correction to: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells.

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Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells.

BACKGROUND: Aldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models. METHODS: Stemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD). RESULTS: In MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1α (HIF-1α) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD. CONCLUSION: In breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.

Use of Nutraceuticals in Angiogenesis-Dependent Disorders.

The term of angiogenesis refers to the growth of new vessels from pre-existing capillaries. The phenomenon is necessary for physiological growth, repair and functioning of our organs. When occurring in a not regulated manner, it concurs to pathological conditions as tumors, eye diseases, chronic degenerative disorders. On the contrary insufficient neovascularization or endothelial disfunction accompanies ischemic and metabolic disorders. In both the cases an inflammatory and oxidative condition exists in supporting angiogenesis deregulation and endothelial dysfunction. The use of nutraceuticals with antioxidant and anti-inflammatory activities can be a therapeutic option to maintain an adequate vascularization and endothelial cell proper functioning or to blunt aberrant angiogenesis. A revision of the updated literature reports on nutraceuticals to guide endothelial cell wellness and to restore physiological tissue vascularization is the objective of this paper. The critical aspects as well as lacking data for human use will be explored from a pharmacological perspective.

Bradykinin B2 Receptor Contributes to Inflammatory Responses in Human Endothelial Cells by the Transactivation of the Fibroblast Growth Factor Receptor FGFR-1.

Elevated levels of bradykinin (BK) and fibroblast growth factor-2 (FGF-2) have been implicated in the pathogenesis of inflammatory and angiogenic disorders. In angiogenesis, both stimuli induce a pro-inflammatory signature in endothelial cells, activating an autocrine/paracrine amplification loop that sustains the neovascularization process. Here we investigated the contribution of the FGF-2 pathway in the BK-mediated human endothelial cell permeability and migration, and the role of the B2 receptor (B2R) of BK in this cross-talk. BK (1 µM) upregulated the FGF-2 expression and promoted the FGF-2 signaling, both in human umbilical vein endothelial cells (HUVEC) and in retinal capillary endothelial cells (HREC) by the activation of Fibroblast growth factor receptor-1 (FGFR-1) and its downstream signaling (fibroblast growth factor receptor substrate: FRSα, extracellular signal⁻regulated kinases1/2: ERK1/2, and signal transducer and activator of transcription 3: STAT3 phosphorylation). FGFR-1 phosphorylation triggered by BK was c-Src mediated and independent from FGF-2 upregulation. Either HUVEC and HREC exposed to BK showed increased permeability, disassembly of adherens and tight-junction, and increased cell migration. B2R blockade by the selective antagonist, fasitibant, significantly inhibited FGF-2/FGFR-1 signaling, and in turn, BK-mediated endothelial cell permeability and migration. Similarly, the FGFR-1 inhibitor, SU5402, and the knock-down of the receptor prevented the BK/B2R inflammatory response in endothelial cells. In conclusion, this work demonstrates the existence of a BK/B2R/FGFR-1/FGF-2 axis in endothelial cells that might be implicated in propagation of angiogenic/inflammatory responses. A B2R blockade, by abolishing the initial BK stimulus, strongly attenuated FGFR-1-driven cell permeability and migration.

Involvement of Bradykinin B2 Receptor in Pathological Vascularization in Oxygen-Induced Retinopathy in Mice and Rabbit Cornea.

The identification of components of the kallikrein-kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic effects promoted by BK. Here, we investigated the role of BK/B2R signaling in the retinal neovascularization in the oxygen-induced retinopathy (OIR) model. Blockade of B2R signaling by the antagonist fasitibant delayed retinal vascularization in mouse pups, indicating that the retinal endothelium is a target of the BK/B2R system. In the rabbit cornea assay, a model of pathological neoangiogenesis, the B2 agonist kallidin induced vessel sprouting and promoted cornea opacity, a sign of edema and tissue inflammation. In agreement with these results, in the OIR model, a blockade of B2R signaling significantly reduced retinal neovascularization, as determined by the area of retinal tufts, and, in the retinal vessel, it also reduced vascular endothelial growth factor and fibroblast growth factor-2 expression. All together, these findings show that B2R blockade reduces retinal neovascularization and inhibits the expression of proangiogenic and pro-inflammatory cytokines, suggesting that targeting B2R signaling may be an effective strategy for treating ischemic retinopathy.

Inhibition of cell cycle progression by the hydroxytyrosol-cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells.

Hydroxytyrosol (HT), a polyphenol of olive oil, downregulates epidermal growth factor (EGFR) expression and inhibits cell proliferation in colon cancer (CC) cells, with mechanisms similar to that activated by the EGFR inhibitor, cetuximab. Here, we studied whether HT treatment would enhance the cetuximab inhibitory effects on cell growth in CC cells. HT-cetuximab combination showed greater efficacy in reducing cell growth in HT-29 and WiDr cells at concentrations 10 times lower than when used as single agents. This reduction was clearly linked to cell cycle blockade, occurring at G2/M phase. The cell cycle arrest in response to combination treatment is related to cyclins B, D1, and E, and cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 down-regulation, and to the concomitant over-expression of CDK inhibitors p21 and p27. HT and cetuximab stimulated a caspase-independent cell death cascade, promotedtranslocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus and activated the autophagy process. Notably, normal colon cells and keratinocytes were less susceptible to combo-induced cell death and EGFR downregulation. These results suggest a potential role of diet, containing olive oil, during cetuximab chemotherapy of colon tumor. HT may be a competent therapeutic agent in CC enhancing the effects of EGFR inhibitors.

Development of Phenol-Enriched Olive Oil with Phenolic Compounds Extracted from Wastewater Produced by Physical Refining.

While in the last few years the use of olive cake and mill wastewater as natural sources of phenolic compounds has been widely considered and several studies have focused on the development of new extraction methods and on the production of functional foods enriched with natural antioxidants, no data has been available on the production of a phenol-enriched refined olive oil with its own phenolic compounds extracted from wastewater produced during physical refining. In this study; we aimed to: (i) verify the effectiveness of a multi-step extraction process to recover the high-added-value phenolic compounds contained in wastewater derived from the preliminary washing degumming step of the physical refining of vegetal oils; (ii) evaluate their potential application for the stabilization of olive oil obtained with refined olive oils; and (iii) evaluate their antioxidant activity in an in vitro model of endothelial cells. The results obtained demonstrate the potential of using the refining wastewater as a source of bioactive compounds to improve the nutraceutical value as well as the antioxidant capacity of commercial olive oils. In the conditions adopted, the phenolic content significantly increased in the prototypes of phenol-enriched olive oils when compared with the control oil.

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.

Monitoring Endothelial and Tissue Responses to Cobalt Ferrite Nanoparticles and Hybrid Hydrogels.

Iron oxide nanoparticles (NPs) have been proposed for many biomedical applications as in vivo imaging and drug delivery in cancer treatment, but their toxicity is an ongoing concern. When NPs are intravenously administered, the endothelium represents the first barrier to tissue diffusion/penetration. However, there is little information about the biological effects of NPs on endothelial cells. In this work we showed that cobalt-ferrite (CoFe2O4) NPs affect endothelial cell integrity by increasing permeability, oxidative stress, inflammatory profile and by inducing cytoskeletal modifications. To overcome these problems, NPs have be loaded into biocompatible gels to form nanocomposite hybrid material (polysaccharide hydrogels containing magnetic NPs) that can be further conjugated with anticancer drugs to allow their release close to the target. The organic part of hybrid biomaterials is a carboxymethylcellulose (CMC) polymer, while the inorganic part consists of CoFe2O4 NPs coated with (3-aminopropyl)trimethoxysilane. The biological activity of these hybrid hydrogels was evaluated in vitro and in vivo. Our findings showed that hybrid hydrogels, instead of NPs alone, were not toxic on endothelial, stromal and epithelial cells, safe and biodegradable in vivo. In conclusion, biohydrogels with paramagnetic NPs as cross-linkers can be further exploited for antitumor drug loading and delivery systems.

H2S dependent and independent anti-inflammatory activity of zofenoprilat in cells of the vascular wall.

Cardiovascular diseases as atherosclerosis are associated to an inflammatory state of the vessel wall which is accompanied by endothelial dysfunction, and adherence and activation of circulating inflammatory cells. Hydrogen sulfide, a novel cardiovascular protective gaseous mediator, has been reported to exert anti-inflammatory activity. We have recently demonstrated that the SH containing ACE inhibitor zofenoprilat, the active metabolite of zofenopril, controls the angiogenic features of vascular endothelium through H2S enzymatic production by cystathionine gamma lyase (CSE). Based on H2S donor/generator property of zofenoprilat, the objective of this study was to evaluate whether zofenoprilat exerts anti-inflammatory activity in vascular cells through its ability to increase H2S availability. Here we found that zofenoprilat, in a CSE/H2S-mediated manner, abolished all the inflammatory features induced by interlukin-1beta (IL-1ß) in human umbilical vein endothelial cells (HUVEC), especially the NF-κB/cyclooxygenase-2 (COX-2)/prostanoid biochemical pathway. The pre-incubation with zofenoprilat/CSE dependent H2S prevented IL-1ß induced paracellular hyperpermeability through the control of expression and localization of cell-cell junctional markers ZO-1 and VE-cadherin. Moreover, zofenoprilat/CSE dependent H2S reduced the expression of the endothelial markers CD40 and CD31, involved in the recruitment of circulating mononuclear cells and platelets. Interestingly, this anti-inflammatory activity was also confirmed in vascular smooth muscle cells and fibroblasts as zofenoprilat reduced, in both cell lines, proliferation, migration and COX-2 expression induced by IL-1ß, but independently from the SH moiety and H2S availability. These in vitro data document the anti-inflammatory activity of zofenoprilat on vascular cells, reinforcing the cardiovascular protective effect of this multitasking drug.

Linking microsomal prostaglandin E Synthase-1/PGE-2 pathway with miR-15a and -186 expression: Novel mechanism of VEGF modulation in prostate cancer.

Prostaglandin E-2 (PGE-2) promotes tumor angiogenesis via paracrine secretion of pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF). Since miRNAs regulate several cell processes, including angiogenesis, we sought to determine whether they would influence PGE-2-induced VEGF. We compared DU145 and PC3 prostate cancer cells bearing the mPGES-1 enzyme (mPGES-1+/+) and producing PGE-2, with those in which the enzyme was silenced or deleted (mPGES-1-/-). We demonstrated that mPGES-1/PGE-2 signaling decreased Dicer expression and miRNA biogenesis. Genome-wide sequencing of miRNAs revealed that miR-15a and miR-186, associated with expression of VEGF and hypoxia inducible factor-1α (HIF-1α), were down-regulated in mPGES-1+/+ cells. As a consequence, mPGES-1+/+ tumor cells expressed high levels of VEGF and HIF-1α, induced endothelial cells activation and formed highly vascularized tumors. Mir-186 mimic inhibited VEGF expression in mPGES-1+/+ tumor xenografts and reduced tumor growth. In human prostate cancer specimens, mPGES-1 was over-expressed in tumors with high Gleason score, elevated expression of VEGF and HIF-1α, high microvessel density and decreased expression of Dicer, miR15a and miR-186. Thus, clear evidence for regulating miRNA processing and VEGF output by intrinsic PGE-2 production provides a means to distinguish between aggressive and indolent prostate tumors and suggests a potential target for controlling tumor progression.

Hydroxytyrosol, a product from olive oil, reduces colon cancer growth by enhancing epidermal growth factor receptor degradation.

SCOPE: We studied the effects and mechanism of 2-(3,4-dihydroxyphenil)ethanol (or hydroxytyrosol, HT), a polyphenol from extra virgin olive oil, investigating the regulation of epidermal growth factor receptor (EGFR) expression in colon tumour cells. METHODS AND RESULTS: We demonstrate that HT significantly downregulates EGFR expression in human colorectal adenocarcinoma cells HT-29, CaCo2, and WiDr, and in HT-29 xenografts. HT accelerates EGFR degradation by reducing its half-life. Specifically, HT induces EGFR ubiquitination that is mediated by phosphorylation at pY1045, the docking site for Cbl, thereby enabling receptor ubiquitination and degradation. Pretreatment with either the lysosomal inhibitor chloroquine, or the proteasomal inhibitor MG132 blocks HT-induced EGFR downregulation. In colon cancer cells, EGFR downregulation by HT is associated with reduced cell proliferation. Tumour growth and EGFR expression levels are also decreased by HT treatment in HT-29 xenograft. CONCLUSION: We conclude that HT downregulates EGFR expression via lysosomal and proteasomal degradation, activated by HT-induced EGFR phosphorylation at pY1045 and increased Cbl activity. Cbl activation induces, in turn, EGFR ubiquitination. Our results reveal a new mechanism for HT's antitumour effects that may be important for colon tumour prevention and treatment.

Nitric Oxide and PGE-2 Cross-Talk in EGFR-Driven Epithelial Tumor Cells.

Nitric oxide (NO) exerts physiopathological effects based mainly on its concentration. Thus, it facilitates or inhibits cancer-promoting characteristics. This review discusses the role of NO and its network of partners in tumor progression and angiogenesis: prostaglandin E 2 (PGE-2) and its producing enzymes, cyclooxigenase 2 (COX-2) and microsomal PGE synthase 1 (mPGES-1), and epidermal growth factor receptor (EGFR) signaling. Understanding the molecular mechanisms and cross-talk modulating NO effects by PGE-2 and EGFR and vice versa allows us to develop better therapeutic strategies for cancer treatment.

mPGES-1 in prostate cancer controls stemness and amplifies epidermal growth factor receptor-driven oncogenicity.

There is evidence that an inflammatory microenvironment is associated with the development and progression of prostate cancer (PCa), although the determinants of intrinsic inflammation in PCa cells are not completely understood. Here we investigated whether expression of intrinsic microsomal PGE synthase-1 (mPGES-1) enhanced aggressiveness of PCa cells and might be critical for epidermal growth factor receptor (EGFR)-mediated tumour progression. In PCa, overexpression of EGFR promotes metastatic invasion and correlates with a high Gleason score, while prostaglandin E2 (PGE2) has been reported to modulate oncogenic EGFR-driven oncogenicity. Immunohistochemical studies revealed that mPGES-1 in human prostate tissues is correlated with EGFR expression in advanced tumours. In DU145 and PC-3 cell lines expressing mPGES-1 (mPGES-1(SC) cells), we demonstrate that silencing or 'knock down' of mPGES-1 (mPGES-1(KD)) or pharmacological inhibition by MF63 strongly attenuates overall oncogenic drive. Indeed, mPGES-1(SC) cells express stem-cell-like features (high CD44, ß1-integrin, Nanog and Oct4 and low CD24 and α6-integrin) as well as mesenchymal transition markers (high vimentin, high fibronectin, low E-cadherin). They also show increased capacity to survive irrespective of anchorage condition, and overexpress EGFR compared to mPGES-1(KD) cells. mPGES-1 expression correlates with increased in vivo tumour growth and metastasis. Although EGFR inhibition reduces mPGES-1(SC) and mPGES-1(KD) cell xenograft tumour growth, we show that mPGES-1/PGE2 signalling sensitizes tumour cells to EGFR inhibitors. We propose mPGES-1 as a possible new marker of tumour aggressiveness in PCa.