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Melanoma is the deadliest skin cancer. RACK1 (Receptor for activated protein kinase C) protein was proposed as a biological marker of melanoma in human and domestic animal species harboring spontaneous melanomas. As a scaffold protein, RACK1 is able to coordinate the interaction of key signaling molecules implicated in both physiological cellular functions and tumorigenesis. A role for RACK1 in rewiring ERK and JNK signaling pathways in melanoma cell lines had been proposed. Here, we used a genetic approach to test this hypothesis in vivo in the mouse. We show that Rack1 knock-down in the mouse melanoma cell line B16 reduces invasiveness and induces cell differentiation. We have developed the first mouse model for RACK1 gain of function, Tyr::Rack1-HA transgenic mice, targeting RACK1 to melanocytes in vivo. RACK1 overexpression was not sufficient to initiate melanomas despite activated ERK and AKT. However, in a context of melanoma predisposition, RACK1 overexpression reduced latency and increased incidence and metastatic rate. In primary melanoma cells from Tyr::Rack1-HA, Tyr::NRasQ61K mice, activated JNK (c-Jun N-terminal kinase) and activated STAT3 (signal transducer and activator of transcription 3) acted as RACK1 oncogenic partners in tumoral progression. A sequential and coordinated activation of ERK, JNK and STAT3 with RACK1 is shown to accelerate aggressive melanoma development in vivo.

The cluster of differentiation 4 (CD4) molecule functions as a co-receptor for MHC class II binding to TCR in T helper cells. A CD4 epitope deficiency was identified in the swine MeLiM (melanoblastoma-bearing Libechov minipig) strain, a model for spontaneous cutaneous melanoma development and regression. Extensive sequencing revealed a high genetic variability of CD4 and the existence of several haplotypes segregating in MeLiM. Forty polymorphisms were identified in the coding sequence, out of which 20 correspond to non-synonymous variants and 10 are located in the 3'UTR of CD4 transcripts. One of the haplotypes segregating in the MeLiM explained the epitope deficiency observed. An association analysis between CD4 genotype and several phenotypes related to tumor regression was performed in 267 animals. An association was evidenced between a MeLiM alternative CD4 haplotype and skin and eye depigmentation, as well as the extent of hair depigmentation. Also, seric IgG concentration was shown to be higher in pigs carrying the alternative haplotype at the homozygous state. In conclusion, the genetic variability of the CD4 gene is associated with immune response-related phenotypes in MeLiM minipigs.

Chantier qualité GA; International audience; Melanoma diagnosis in dogs can be challenging due to the variety of histological appearances of canine melanocytic neoplasms. Markers of malignancy are needed. Receptor for activated C-kinase 1 (RACK1) was found to characterize melanomas in other mammals. We investigated the value of RACK1 detection in the classification of 19 cutaneous and 5 mucosal melanocytic neoplasms in dogs. These tumors were categorized as melanocytomas or benign and melanomas or malignant after evaluation of their morphology, mitotic index, and Ki-67 growth fraction. Using immunofluorescence, we confirmed microphthalmia-associated transcription factor (MITF) as a marker of normal and transformed melanocytic cells in dog tissues. All control (n = 10) and tumoral (n = 24) samples stained positively for MITF (34/34, 100%). Whereas RACK1 was not detected in healthy skin melanocytes, melanocytic lesions were all positive for RACK1 signal (24/24, 100%). RACK1 cytoplasmic staining appeared with 2 distinct distribution patterns: strong, diffuse, and homogeneous or granular and heterogeneous. All melanoma samples (13/13, 100%) stained homogeneously for RACK1. All melanocytomas (11/11, 100%) stained heterogeneously for RACK1. Immunohistochemistry was less consistent than immunofluorescence for all labelings in melanocytic lesions, which were often very pigmented. Thus, the fluorescent RACK1-MITF labeling pattern helped to distinguish melanomas from melanocytomas. Furthermore, RACK1 labeling correlated with 2 of 11 morphological features linked to malignancy: cell and nuclear size. These results suggest that RACK1 may be used as a marker in dog melanomas.

Cutaneous melanoma : what if Laënnec was right ? Search for a common malignancy marker in mammals. Cutaneous melanomas are characterized by considerable inter and intraspecific heterogeneity. In 1806, René Laënnec used a single term to describe these malignant tumours believed to originate from melanocytes. Due to their high metastatic potential, the prognosis of melanomas remains unfavourable. To improve it, early diagnosis is essential since metastasis-free stages can be treated. This is why we sought malignancy markers for melanomas. We identified the RACK1 protein as a marker of malignancy in human melanomas. A common malignancy marker for different mammalian melanomas would support the use of the single term “melanoma”. In the present article, we review current knowledge on the molecular basis of melanocyte tumorigenesis to explain our interest in the RACK1 protein., Les mélanomes cutanés se caractérisent par une considérable hétérogénéité intra et interspécifique. Ces tumeurs malignes censées dériver des mélanocytes ont été décrites sous un terme unique par René Laënnec en 1806. Par leur forte capacité métastatique, elles sont toujours de sombre pronostic. Pour améliorer celui-ci, leur diagnostic précoce est indispensable car les stades sans métastase peuvent être traités efficacement. Aussi, des marqueurs de malignité du mélanome sont recherchés. Nous avons identifié la protéine RACK1 comme marqueur de malignité des mélanomes chez l’homme. Un marqueur de malignité commun des mélanomes de mammifères conforterait l’utilisation du terme unique «mélanome». Nous commentons les connaissances actuelles des bases moléculaires de la tumorigenèse des mélanocytes pour expliquer notre intérêt pour la protéine RACK1., Campagne Cécile, Jule Sophia, Estrada Mercedes, Bernex Florence, Egidy Giorgia. Le mélanome cutané: et si Laënnec avait raison ? Recherche d'un marqueur de malignité commun chez les mammifères. In: Bulletin de l'Académie Vétérinaire de France tome 163 n°1, 2010. pp. 55-60.

Resume Lors de la presentation de son Memoire sur la melanose, en 1806, Rene Laennec a decrit sous ce terme unique un ensemble de presentations cliniques tumorales. Aujourd’hui, l’analyse histologique rassemble sous le terme de melanome toute tumeur maligne derivee des melanocytes. Le melanome a un pronostic sombre de par sa forte capacite a metastaser chez le chien comme chez l’homme. Une identification precoce de ces tumeurs malignes constituerait un progres indeniable pour le pronostic. Dans ce but, des marqueurs de malignite du melanome sont recherches pour faciliter l’etablissement d’un pronostic par les praticiens. Nous avons identifie dans notre laboratoire que la proteine RACK1 est un marqueur de malignite des melanomes cutanes et metastatiques chez le porc de race MeLiM, et chez l’homme. Cette proteine, qui pourrait egalement etre un marqueur de malignite et de pronostic des melanomes cutaneomuqueux du chien, fait aujourd’hui l’objet d’analyses approfondies que nous vous proposons de decouvrir dans cet article. La participation a ce projet des praticiens veterinaires pourrait aboutir a de la recherche translationnelle. A l’instar des tests genetiques predictifs de caracteres morphologiques, morbides ou de sensibilites aux medicaments, ce type d’etudes prefigure un axe de l’evolution du diagnostic veterinaire.

International audience; The role of the Pax3 gene in embryonic development of pigment cells is well characterized. By contrast, the function of Pax3 in melanoma development is controversial. Indeed, data obtained from cultured cells suggest that PAX3 may contribute to melanomagenesis. PAX3 is found to be overexpressed in melanomas and also in nevi compared with normal skin samples. Pax3 homozygous loss of function is embryonic lethal. To assess the role of Pax3 in melanoma development in vivo, we analyzed Pax3 haploinsufficiency in a mouse model of melanoma predisposition. The Pax3(GFP/+) knock-in reporter system was combined with the Tyr::NRAS(Q61K); Cdkn2a(-/-) mouse melanoma model. Melanoma development was followed over 18 months. Histopathological, immunohistochemical, and molecular analyses of lesions at different stages of melanoma progression were carried out. Fluorescence-activated cell sorting on GFP of cells from primary or metastatic melanoma was followed by ex-vivo transformation tests and in-vivo passaging. We report here that Tyr::NRAS(Q61K); Cdkn2a(-/-); Pax3(GFP/+) mice developed metastasizing melanoma as their Tyr::NRAS(Q61K); Cdkn2a(-/-); littermates. Histopathology showed no differences between the two genotypes, although Pax3 mRNA and PAX3 protein levels in Pax3(GFP/+) lesions were reduced by half. The Pax3(GFP) allele proved to be a convenient marker to identify and directly sort heterogeneous populations of melanoma cells within the tumor bulk at each stage of melanoma progression. This new mouse model represents an accurate and reproducible means for identifying melanoma cells in vivo to study the mechanisms of melanoma development.

Several minipig strains develop spontaneous malignant melanoma. As a first step toward the analysis of genes involved in the tumoral progression of melanoma in these animal models, we developed culture conditions for pig melanocytes whereby melanocytes from normal epidermis can be isolated directly onto mitotically inactivated keratinocytes in Eagle's minimal essential medium supplemented with fetal calf serum, tetradecanoyl phorbol acetate (TPA) and cholera toxin. We also derived an immortal line of pigmented melanocytes from the epidermis of a healthy Meishan pig. This cell line, designated PigMel, retains differentiation function in culture, dependence on TPA and cholera toxin and a diploid chromosome number. PigMel melanocytes exhibit morphological and molecular characteristics common to normal mammalian skin melanocytes.

International audience; Objectives: Endothelin-1 (ET-1), plays an important role in the pathophysiology of CHF and the pulmonary endothelium is an early hemodynamic target in diastolic left ventricular dysfunction. Therefore we hypothesized that the lung is a main source of humoral endothelin in CHF and that its secretion is proportional to the degree of heart failure. Methods and results: We used rats with coronary artery ligation as an experimental model of either compensated or decompensated heart failure, depending on infarct size. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that in the lung, the expression of preproET-1 mRNA was higher in decompensated HF than in control and compensated HF rats (P

Endothelin (ET)-1 is a potent vasoconstrictor and mitogenic peptide that has a variety of biological effects in noncardiovascular tissues. The precise cellular distribution of the ET-1 system in the wall of the normal human colon was studied to identify the physiological role of ET in the gut. In situ hybridization revealed ET-converting enzyme-1 (ECE-1) mRNA in all vessels, the colon epithelium, and macrophages. Prepro-ET-1 (PPET-1) mRNA had a similar distribution except for a scattered signal in mucosal microvessels. ETAand ETBreceptor mRNAs were mainly in the lamina propria, pericryptal myofibroblasts, microvessels, and mononuclear cells, with ETAmRNA more abundant than ETBmRNA.125I-ET-1 binding showed ETBalong the crypts and in nerve fibers descending from the ganglionic plexus that contained PPET-1, ECE-1, and ETBtranscripts, whereas glia contained ETAreceptors. The finding of the entire ET system in the normal mucosa suggests its implication in some characteristic functions of the colon and its secretion as both a neuroactive and a vasoactive peptide.

Background Melanocytic proliferations are common in horses but the diagnosis of malignancy is not always straightforward. To improve diagnosis and prognosis, markers of malignancy are needed. Receptor for activated C kinase 1 (RACK1) protein may be such a marker. RACK1 was originally found to characterize malignant melanocytic lesions in the Melanoblastoma-bearing Libechov minipig (MeLiM) and, later, in human patients. Our purpose was to investigate the value of RACK1 in the classification of cutaneous melanocytic proliferations in horses. Results Using immunofluorescence, we report here that both MITF (Microphthalmia-associated transcription factor) and PAX3 (Paired box 3) allow the identification of melanocytic cells in horse skin samples. Importantly, RACK1 was detected in melanocytic lesions but not in healthy skin melanocytes. Finally, we found that RACK1 labeling can be used in horses to distinguish benign melanocytic tumors from melanomas. Indeed, RACK1 labeling appeared more informative to assess malignancy than individual histomorphological features. Conclusions This study confirms that horses provide an interesting model for melanoma genesis studies. It establishes MITF and PAX3 as markers of horse melanocytic cells. RACK1 emerges as an important marker of malignancy which may contribute to progress in the diagnosis of melanomas in both human and veterinary medicine.

An imbalance between proliferation and apoptosis is important in tumor progression. Endothelin-1 (ET-1) has vasoconstricting and mitogenic activities and may be involved in apoptosis regulation. We found that ET-1 and FasL systems were colocalized in human colon tumors and that ET-1 was secreted by human (HT-29, SW480) and rat (PROb, REGb) colon carcinoma cell lines. Bosentan, a mixed endothelin-A- and -B- (ET(A)/ET(B)) receptor antagonist, potentiated FasL- (APO-1, CD95) induced apoptosis in these cells. The specific inhibition of enzymes involved in ceramide production did not restore survival of cells exposed to FasL and bosentan. Inhibition of PKC with bisindolylmaleimide IX enhanced FasL-induced apoptosis in HT-29, PROb and REGb cells in the absence of bosentan. These results suggest that ET-1 is an autocrine survival factor able to protect colon carcinoma cells against FasL-induced apoptosis, involving the protein kinase C (PKC) but not the sphingomyelin-ceramide signaling transduction pathways.

International audience; Incubation of Candida albicans yeast cells with human luteinizing hormone (hLH), human chorionic gonadotrophin (hCG) or glucagon produced a significant rise in cAMP total levels. The effect of these hormones in permeabilized cells of the fungus produced a 2-3 fold increase in the Mg2+, GTP-dependent adenylyl cyclase activity as well as full activation of the cAMP-dependent protein kinase (PKA) activity. These results indicate that the interaction of the mammalian hormones with the fungus triggered the cAMP activation cascade in a similar way to that found in higher eukaryotic organisms.

The proposed feedback between angiotensin II AT2 and AT1 receptors prompted us to study AT1 receptor expression in kidneys of male AT2 receptor-gene disrupted mice (agtr2 −/y). In wild-type (agtr2 +/y) mice, AT1 receptor binding and mRNA is abundant in glomeruli, and AT1 receptor binding is also high in the inner stripe of the outer medulla. AT2 receptors are scarce, primarily associated to cortical vascular structures. In agtr2 −/y mice, AT1 receptor binding and mRNA were increased in the kidney glomeruli, and AT1 receptor binding was higher in the rest of the cortex and outer stripe of the outer medulla, but not in its inner stripe, indicating different cellular regulation. Although AT2 receptor expression is very low in male agtr 2 +/y mice, their gene disruption alters AT1 receptor expression. AT1 upregulation alone may explain the AT2 gene-disrupted mice phenotype such as increased blood pressure, higher sensitivity to angiotensin II, and altered renal function. The indirect AT1/AT2 receptor feedback could have clinical significance because AT1antagonists are widely used in medical practice. Fil: Saavedra, Juan M.. National Institute of Mental Health; Estados Unidos Fil: Häuser, Walter. National Institute of Mental Health; Estados Unidos Fil: Ciuffo, Gladys Maria. National Institute of Mental Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Egidy, Giorgia. National Institute of Mental Health; Estados Unidos Fil: Hoe, Kwang Lae. National Institute of Mental Health; Estados Unidos Fil: Jöhren, Olaf. National Institute of Mental Health; Estados Unidos Fil: Sembonmatsu, Takaaki. Vanderbilt University; Estados Unidos Fil: Inagami, Tadashi. Vanderbilt University; Estados Unidos Fil: Armando, Inés. National Institute of Mental Health; Estados Unidos

Tumor neovascularization is considered to be a critical step in the development of a malignant tumor. Endothelin (ET)-1 is a powerful vasoconstrictor and mitogenic peptide that is produced by many cancer cell lines. The cellular distribution of the ET components was evaluated in human colon tumors and compared to normal colon. There was more of the ET components (preproET-1, endothelin-converting enzyme-1, and ETA and ETB receptors) in adenomas and adenocarcinomas than in the normal colon. There was overproduction of preproET-1 and endothelin-converting enzyme-1 in carcinoma cells and stromal vessels, suggesting that they are a local source of ET-1. ETA receptors were present in stromal myofibroblasts of neoplastic tissue, and there were large amounts of ETB receptors in the endothelium and myofibroblasts. There was also a redistribution of alpha-smooth muscle actin-positive cells in the vascular structures of tumors. An experimental rat model of induced colon cancer treated for 30 days with bosentan, a mixed antagonist of both ET receptors, confirmed the morphological changes observed during the tumor vascularization. Our data suggest that ET-1 and its receptor play a role in colon cancer progression, with ET-1 functioning as a negative modulator of the stromal response.

Brain angiotensin II (Ang II) inhibits pituitary prolactin release by an indirect mechanism requiring stimulation of dopamine formation and release. We report that [125I]Sar1–Ang II binding to AT1receptors and AT1Areceptor mRNA expression increase selectively in the dorsomedial arcuate nucleus of 17β-estradiol-primed ovariectomized rats after treatment with progesterone. In hormone-treated rats, arcuate nucleus AT1Areceptor mRNA expression is associated with tyrosine hydroxylase-positive neurons. No AT1Areceptor mRNA was detected in tyrosine hydroxylase-positive cells of the arcuate nucleus of intact male rats. Conversely, in the anterior pituitary, where local or circulating Ang II stimulates prolactin release, [125I]Sar1–Ang II binding to AT1receptors and AT1Breceptor mRNA expression are decreased in 17β-estradiol/progesterone-treated ovariectomized rats.Thus, AT1Areceptors in the dorsal arcuate nucleus and AT1Breceptors in the anterior pituitary are regulated inversely by estrogen/progesterone treatment, supporting the hypothesis of a dual role for brain and pituitary Ang II on prolactin release. The colocalization of AT1Areceptor mRNA and tyrosine hydroxylase in neurons of the arcuate nucleus furthermore indicates that within this area central Ang II acts directly on dopaminergic neurons. These results support the hypothesis that central Ang II inhibits pituitary prolactin release indirectly via modulation of dopaminergic activity in the arcuate nucleus.

Angiotensin II (Ang II) plays a key role in cardiovascular homeostasis in developing animals as well as in adults by regulating blood pressure, fluid electrolite balance and renal hemodynamics(1–5).

Endothelin-converting-enzyme-1 (ECE-1) belongs to the family of zinc metallopeptidases and is responsible for generating endothelin (ET) peptides from their inactive precursors the big endothelins (bigET). The enzyme is a type II integral membrane protein consisting of a short amino-terminal cytosolic domain of 56 amino acids, a single transmembrane domain and a large putative extracellular domain containing the catalytic site. Recombinant and native ECE-1 are expressed as a dimer. We have constructed a soluble form of ECE, named sECE*, by fusing the cleavable signal peptide of pro-opiomelanocortin in frame to the complete extracellular domain of human ECE-1. Stable expression of this construct in CHO cells resulted in the secretion of a fully active enzyme. In contrast to membrane-bound ECE, sECE* was expressed as a monomer, highly glycosylated, as assessed by gel filtration and Western blot. However, recombinant sECE* converted bigET-1 with similar specific activity as ECE-1a. This activity was completely inhibited by phosphoramidon, but not by thiorphan and captopril. sECE* was active in a broad range of pH, showing an optimum of 6.6–6.8 for bigET-1. Thus, the extracellular domain alone is sufficient for conferring full ECE-1 activity, inhibitors recognition and substrate specificity.

Reference EPFL-ARTICLE-188431doi:10.1111/pcmr.12115View record in Web of Science Record created on 2013-09-09, modified on 2017-05-12

Background Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses. Methods Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting. Results We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses. Conclusions These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis. These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research.

Background Metastatic melanoma is a severe disease. Few experimental animal models of metastatic melanoma exist. MeLiM minipigs exhibit spontaneous melanoma. Cutaneous and metastatic lesions are histologically similar to human's. However, most of them eventually spontaneously regress. Our purpose was to investigate whether the MeLiM model could reveal markers of malignancy in human melanocytic proliferations. Results We compared the serial analysis of gene expression (SAGE) between normal pig skin melanocytes and melanoma cells from an early pulmonary metastasis of MeLiM minipigs. Tag identification revealed 55 regulated genes, including GNB2L1 which was found upregulated in the melanoma library. In situ hybridisation confirmed GNB2L1 overexpression in MeLiM melanocytic lesions. GNB2L1 encodes the adaptor protein RACK1, recently shown to influence melanoma cell lines tumorigenicity. We studied the expression of RACK1 by immunofluorescence and confocal microscopy in tissues specimens of normal skin, in cutaneous and metastatic melanoma developped in MeLiM minipigs and in human patients. In pig and human samples, the results were similar. RACK1 protein was not detected in normal epidermal melanocytes. By contrast, RACK1 signal was highly increased in the cytoplasm of all melanocytic cells of superficial spreading melanoma, recurrent dermal lesions and metastatic melanoma. RACK1 partially colocalised with activated PKCαβ. In pig metastases, additional nuclear RACK1 did not associate to BDNF expression. In human nevi, the RACK1 signal was low. Conclusion RACK1 overexpression detected in situ in human melanoma specimens characterized cutaneous and metastatic melanoma raising the possibility that RACK1 can be a potential marker of malignancy in human melanoma. The MeLiM strain provides a relevant model for exploring mechanisms of melanocytic malignant transformation in humans. This study may contribute to a better understanding of melanoma pathophysiology and to progress in diagnosis.