Enrique Zudaire

The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers

The aryl hydrocarbon receptor repressor (AHRR) is a bHLH/Per-ARNT-Sim transcription factor located in a region of chromosome 5 (5p15.3) that has been proposed to contain one or more tumor suppressor genes. We report here consistent downregulation of AHRR mRNA in human malignant tissue from different anatomical origins, including colon, breast, lung, stomach, cervix, and ovary, and demonstrate DNA hypermethylation as the regulatory mechanism of AHRR gene silencing. Knockdown of AHRR gene expression in a human lung cancer cell line using siRNA significantly enhanced in vitro anchorage-dependent and -independent cell growth as well as cell growth after transplantation into immunocompromised mice. In addition, knockdown of AHRR in non-clonable normal human mammary epithelial cells enabled them to grow in an anchorage-independent manner. Further, downregulation of AHRR expression in the human lung cancer cell line conferred resistance to apoptotic signals and enhanced motility and invasion in vitro and angiogenic potential in vivo. Ectopic expression of AHRR in tumor cells resulted in diminished anchorage-dependent and -independent cell growth and reduced angiogenic potential. These results therefore demonstrate that AHRR is a putative new tumor suppressor gene in multiple types of human cancers.

Adrenomedullin and cancer.

Adrenomedullin (AM) is a pluripotent hormone with structural similarities to calcitonin gene-related peptide (CGRP), which is expressed by many tissues in the body and shows a remarkable range of effects mediated by paracrine/autocrine and possibly endocrine mechanisms. AM has been implicated as a mediator of several pathologies such as cardiovascular and renal disorders, sepsis, inflammation, diabetes and cancer, among others. AM is expressed in a variety of tumors where it aggravates several of the molecular and physiological features of malignant cells. AM has been shown to be a mitogenic factor stimulating growth in several cancer types and to encourage a more aggressive tumor phenotype. In addition, AM is an apoptosis survival factor for cancer cells and an indirect suppressor of the immune response through its binding protein, complement factor H, and regulation in expression of cytokines. AM plays an important role in environments subjected to low oxygen tensions, which is a typical feature in the proximity of solid tumors. Under these conditions, AM is upregulated through a hypoxia-inducible factor 1 (HIF-1)-dependent pathway and acts as a potent angiogenic factor promoting neovascularization. The collective findings brought together over the last years place AM as a major regulator of carcinogenesis-tumor progression and identifies its autocrine loop as a putative target for developing new strategies against human cancers.

GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood–brain barrier

Every organ in the body requires blood vessels for efficient delivery of oxygen and nutrients, but independent vascular beds are highly specialized to meet the individual needs of specific organs. The vasculature of the brain is tightly sealed, with blood–brain barrier (BBB) properties developing coincident with neural vascularization. G protein-coupled receptor 124 (GPR124) (tumor endothelial marker 5, TEM5), an orphan member of the adhesion family of G protein-coupled receptors, was previously identified on the basis of its overexpression in tumor vasculature. Here, we show that global deletion or endothelial-specific deletion of GPR124 in mice results in embryonic lethality associated with abnormal angiogenesis of the forebrain and spinal cord. Expression of GPR124 was found to be required for invasion and migration of blood vessels into neuroepithelium, establishment of BBB properties, and expansion of the cerebral cortex. Thus, GPR124 is an important regulator of neurovasculature development and a potential drug target for cerebrovascular diseases.

Inhibitors of the Arachidonic Acid Pathway and Peroxisome Proliferator–Activated Receptor Ligands Have Superadditive Effects on Lung Cancer Growth Inhibition

Arachidonic acid (AA) metabolizing enzymes and peroxisome proliferator-activated receptors (PPARs) have been shown to regulate the growth of epithelial cells. We have previously reported that exposure to the 5-lipoxygenase activating protein-directed inhibitor MK886 but not the cyclooxygenase inhibitor, indomethacin, reduced growth, increased apoptosis, and up-regulated PPARalpha and gamma expression in breast cancer cell lines. In the present study, we explore approaches to maximizing the proapoptotic effects of PPARgamma on lung cancer cell lines. Non-small-cell cancer cell line A549 revealed dose-dependent PPARgamma reporter activity after treatment with MK886. The addition of indomethacin in combination with MK886 further increases reporter activity. We also show increased growth inhibition and up-regulation of apoptosis after exposure to MK886 alone, or in combination with indomethacin and the PPAR ligand, 15-deoxy-Delta12,14-prostaglandin J2 compared with single drug exposures on the adenocarcinoma cell line A549 and small-cell cancer cell lines H345, N417, and H510. Real-time PCR analyses showed increased PPAR mRNA and retinoid X receptor (RXR)alpha mRNA expression after exposure to MK886 and indomethacin in a time-dependent fashion. The results suggest that the principal proapoptotic effect of these drugs may be mediated through the known antiproliferative effects of the PPARgamma-RXR interaction. We therefore explored a three-drug approach to attempt to maximize this effect. The combination of low-dose MK886, ciglitazone, and 13-cis-retinoic acid interacted at least in a superadditive fashion to inhibit the growth of lung cancer cell lines A549 and H1299, suggesting that targeting PPARgamma and AA action is a promising approach to lung cancer growth with a favorable therapeutic index.

Expression of Complement Factor H by Lung Cancer Cells Effects on the Activation of the Alternative Pathway of Complement

The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complement-mediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the potential effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphylatoxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the expression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement activation.

The central role of adrenomedullin in host defense

Thirteen years after the isolation of adrenomedullin (AM) from a human pheochromocytoma, the literature is awash with reports describing its implication in countless physiological and disease mechanisms ranging from vasodilatation to cancer promotion. A growing body of evidence illustrates AM as a pivotal component in normal physiology and disease with marked beneficial effects in the host defense mechanism. Exogenous administration of AM as well as its ectopic overexpression and the use of drugs, which potentiates its activity, are promising strategies for treatment of septic shock and several other pathogen‐related disorders. Although major progress toward this end has been achieved over the past few years, our further understanding of the pleiotropic mechanisms involved with AM as a protective peptide is paramount to maximize its clinical application.

GPR124 Functions as a WNT7-Specific Coactivator of Canonical β-Catenin Signaling

G protein-coupled receptor 124 (GPR124) is an orphan receptor in the adhesion family of GPCRs, and previous global or endothelial-specific disruption of Gpr124 in mice led to defective CNS angiogenesis and blood-brain barriergenesis. Similar developmental defects were observed following dual deletion of Wnt7a/Wnt7b or deletion of β-catenin in endothelial cells, suggesting a possible relationship between GPR124 and canonical WNT signaling. Here, we show using in vitro reporter assays, mutation analysis, and genetic interaction studies in vivo that GPR124 functions as a WNT7A/WNT7B-specific costimulator of β-catenin signaling in brain endothelium. WNT7-stimulated β-catenin signaling was dependent upon GPR124s intracellular PDZ binding motif and a set of leucine-rich repeats in its extracellular domain. This study reveals a vital role for GPR124 in potentiation of WNT7-induced canonical β-catenin signaling with important implications for understanding and manipulating CNS-specific angiogenesis and blood-brain barrier-genesis.

hnRNP A2/B1 Modulates Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been reported to be overexpressed in lung cancer and in other cancers such as breast, pancreas, and liver. However, a mechanism linking hnRNP A2/B1 overexpression and progression to cancer has not yet been definitively established. To elucidate this mechanism, we have silenced hnRNPA2/B1 mRNA in non-small-cell lung cancer cell lines A549, H1703, and H358. These cell lines present different levels of expression of epithelial-to-mesenchymal transition (EMT) markers such as E-cadherin, fibronectin, and vimentin. Microarray expression analysis was performed to evaluate the effect of silencing hnRNP A2/B1 in A549 cells. We identified a list of target genes, affected by silencing of hnRNP A2/B1, that are involved in regulation of migration, proliferation, survival, and apoptosis. Silencing hnRNP A2/B1 induced formation of cell clusters and increased proliferation. In the anchorage-independent assay, silencing hnRNP A2/B1 increased colony formation by 794% in A549 and 174% in H1703 compared with a 25% increase in proliferation, in both cell lines, in a two-dimensional proliferation assay. Silencing hnRNP A2/B1 decreased migration in intermediate cell line A549 and mesenchymal cell line H1703; however, no changes in proliferation were observed in epithelial cell line H358. Silencing hnRNP A2/B1 in A549 and H1703 cells correlated with an increase of E-cadherin expression and downregulation of the E-cadherin inhibitors Twist1 and Snai1. These data suggest that expression of hnRNP A2/B1 may play a role in EMT, in nonepithelial lung cancer cell lines A549 and H1703, through the regulation of E-cadherin expression.

Proadrenomedullin NH2-Terminal 20 Peptide Is a Potent Angiogenic Factor, and Its Inhibition Results in Reduction of Tumor Growth

We have found through ex vivo and in vivo angiogenesis models that the adrenomedullin gene-related peptide, proadrenomedullin NH2-terminal 20 peptide (PAMP), exhibits a potent angiogenic potential at femtomolar concentrations, whereas classic angiogenic factors such as vascular endothelial growth factor and adrenomedullin mediate a comparable effect at nanomolar concentrations. We found that human microvascular endothelial cells express PAMP receptors and respond to exogenous addition of PAMP by increasing migration and cord formation. Exposure of endothelial cells to PAMP increases gene expression of other angiogenic factors such as adrenomedullin, vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor C. In addition, the peptide fragment PAMP(12-20) inhibits tumor cell–induced angiogenesis in vivo and reduces tumor growth in xenograft models. Together, our data demonstrate PAMP to be an extremely potent angiogenic factor and implicate this peptide as an attractive molecular target for angiogenesis-based antitumor therapy.

Gastrin-releasing peptide (GRP) induces angiogenesis and the specific GRP blocker 77427 inhibits tumor growth in vitro and in vivo

Angiogenesis is becoming a major target for antitumor therapies, and identifying new angiogenic factors and their specific inhibitors may provide new avenues for tumor management. Here we identify gastrin-releasing peptide (GRP) as a new angiogenic molecule that is secreted by tumors and acts directly upon GRP receptors in the endothelial cells. Addition of GRP increases endothelial cell migration and cord formation in vitro, and induces angiogenesis in an in vivo assay. We have recently identified a small molecule GRP blocker, compound 77427. This inhibitor significantly reduced endothelial cell cord formation in vitro and angiogenesis in vivo. Conversely, when applied to VEGF-induced angiogenesis, the small molecule did not have any effect, demonstrating its specificity. Furthermore, this GRP blocker was able to reduce lung tumor cell growth in vitro as demonstrated by MTT and clonogenic assays. When applied to a xenograft model with lung cancer cells, compound 77427 reduced tumor volume to undetectable sizes, although when the treatment was suspended, tumors began to grow again at normal rates. Our collective observations indicate that GRP is a new angiogenic peptide and that its inhibition offers an attractive tool to reduce tumor burden.