Implicating the mechanisms of ADP-ribosylation factor activation in the resistance of invasive breast cancer cells to EGFR tyrosine kinase inhibitors

ADP-ribosylation factor-1 (ARF1) est une petite GTPase principalement connue pour son rôle dans la formation de vésicules au niveau de l’appareil de Golgi. Récemment, dans des cellules de cancer du sein, nous avons démontré qu’ARF1 est aussi un médiateur important de la signalisation du récepteur du facteur de croissance épidermique (EGFR) contrôlant la prolifération, la migration et l'invasion cellulaire. Cependant, le mécanisme par lequel l’EGFR active la GTPase ainsi que le rôle de cette dernière dans la régulation de la fonction du récepteur demeure inconnue. Dans cette thèse, nous avions comme objectifs de définir le mécanisme d'activation de ARF1 dans les cellules de cancer du sein hautement invasif et démontrer que l’activation de cette isoforme de ARF joue un rôle essentiel dans la résistance de ces cellules aux inhibiteurs de l'EGFR. Nos études démontrent que les protéines d’adaptatrices Grb2 et p66Shc jouent un rôle important dans l'activation de ARF1. Alors que Grb2 favorise le recrutement d’ARF1 à l'EGFR ainsi que l'activation de cette petite GTPase, p66Shc inhibe le recrutement du complexe Grb2-ARF1 au récepteur et donc contribue à limiter l’activation d’ARF1. De plus, nous démontrons que ARF1 favorise la résistance aux inhibiteurs des tyrosines kinases dans les cellules de cancer du sein hautement invasif. En effet, une diminution de l’expression de ARF1 a augmenté la sensibilité descellules aux inhibiteurs de l'EGFR. Nous montrons également que de hauts niveaux de ARF1 contribuent à la résistance des cellules à ces médicaments en améliorant la survie et les signaux prolifératifs à travers ERK1/2, Src et AKT, tout en bloquant les voies apoptotiques (p38MAPK et JNK). Enfin, nous mettons en évidence le rôle de la protéine ARF1 dans l’apoptose en réponse aux traitements des inhibiteurs de l’EGFR. Nos résultats indiquent que la dépletion d’ARF1 promeut la mort cellulaire induite par gefitinib, en augmentant l'expression de facteurs pro-apoptotiques (p66shc, Bax), en altérant le potentiel de la membrane mitochondriale et la libération du cytochrome C. Ensemble, nos résultats délimitent un nouveau mécanisme d'activation de ARF1 dans les cellules du cancer du sein hautement invasif et impliquent l’activité d’ARF1 comme un médiateur important de la résistance aux inhibiteurs EGFR.

Stage-Specific Plasticity in Ovary Size Is Regulated by Insulin/Insulin-Like Growth Factor and Ecdysone Signaling in Drosophila

Animals from flies to humans adjust their development in response to environmental conditions through a series of developmental checkpoints, which alter the sensitivity of organs to environmental perturbation. Despite their importance, we know little about the molecular mechanisms through which this change in sensitivity occurs. Here we identify two phases of sensitivity to larval nutrition that contribute to plasticity in ovariole number, an important determinant of fecundity, in Drosophila melanogaster. These two phases of sensitivity are separated by the developmental checkpoint called "critical weight"; poor nutrition has greater effects on ovariole number in larvae before critical weight than after. We find that this switch in sensitivity results from distinct developmental processes. In precritical weight larvae, poor nutrition delays the onset of terminal filament cell differentiation, the starting point for ovariole development, and strongly suppresses the rate of terminal filament addition and the rate of increase in ovary volume. Conversely, in postcritical weight larvae, poor nutrition affects only the rate of increase in ovary volume. Our results further indicate that two hormonal pathways, the insulin/insulin-like growth factor and the ecdysone-signaling pathways, modulate the timing and rates of all three developmental processes. The change in sensitivity in the ovary results from changes in the relative contribution of each pathway to the rates of terminal filament addition and increase in ovary volume before and after critical weight. Our work deepens our understanding of how hormones act to modify the sensitivity of organs to environmental conditions, thereby affecting their plasticity.

Vascular endothelial growth factor-B and retinal vascular development in the mouse

Purpose: Vascular endothelial growth factor-A (VEGF-A) is crucial to retinal vascular growth, both normal and pathological. VEGF-B, recently characterized, is reported to be expressed in retinal tissues, but the importance of VEGF-B to retinal vascular development remained unknown. The aim of this study was to analyse retinal vascular growth in the Vegfb (-/-) knockout mouse. Methods: Retinal vascular growth was measured in Vegfb (-/-) knockout mice raised under normal conditions, and Vegfb (-/-) knockout mice with an oxygen-induced proliferative retinopathy. Wild type Vegfb (+/+) mice served as controls. Vessels were perfused with ink and retinal flatmounts secondarily labelled with FITC-lectin (BS-1, Griffonia simplicifolia ). Area and diameter of retinal growth and retinal vascular growth were recorded over days 0-20, and capillary density and mean diameter recorded from day 17 pups. Results: A variety of techniques confirmed that Vegfb (+/+) mice expressed VEGF-B and that VEGF-B expression was absent in Vegfb (-/-) mice. Vegfb (-/-) mice raised in room air showed no significant differences from Vegfb (+/+) controls. No differences were found in oxygen-induced retinopathy between Vegfb (-/-) and Vegfb (+/+) pups in either the extent of the initial oxygen-induced ablation, or in the regrowth of retinal vessels or vitreal (neovascular) sprouts; vitreal sprouts are important markers of the abnormal proliferative response, and are maximally expressed on day 17 in this model of oxygen-induced retinopathy. Conclusions: These results indicate that a lack of VEGF-B does not significantly affect development of the retinal vasculature under normal conditions, nor does it appear to affect the proliferative retinal responses seen in oxygen-induced retinopathy.

CSF-1 as a regulator of macrophage activation and immune responses

Macrophage activation is a key determinant of susceptibility and pathology in a variety of inflammatory diseases. The extent of macrophage activation is tightly regulated by a number of pro-inflammatory cytokines (e.g. IFN-gamma, IL-2, GM-CSF, IL-3) and anti-inflammatory cytokines (e.g. IL-4, IL-10, TGF-beta). Macrophage colony-stimulating factor (CSF-1/M-CSF) is a key differentiation, growth and survival factor for monocytes/macrophages and osteoclasts. The role of this factor in regulating macrophage activation is often overlooked. This review will summarize our current understanding of the effects of CSF-1 on the activation state of mature macrophages and its role in regulating immune responses.

Growth hormone and epidermal growth factor in salivary glands of giant and dwarf transgenic mice

Epidermal growth factor (EGF) in rat salivary glands is regulated by testosterone, thyroxin, and growth hormone (GH). Salivary glands of 45-day-old giant and dwarf male and female transgenic mice were examined histologically and by immunohistochemistry (IHC) for EGF. Male giants showed no significant differences from wild-type (WT) parotid and submandibular glands. However, their sublingual glands expressed EGF diffusely and strongly in granular cells within the striated ducts, where they were not found in WT mice. Submandibular gland ducts of female WT were different, having individual granular cells strongly positive for EGF and distributed sporadically along the striated duct walls. Neither female GH-antagonist dwarf mice nor GH-receptor knockout mice had any granular cells expressing EGF in any gland. Obvious presence of granular duct cells in the sublingual glands of giant male mice suggests GH-upregulated granular cell EGF expression. Furthermore, absence of granular duct cells from all glands in female GH-antagonist and GH-receptor knockout transgenic mice suggests that GH is necessary for the differentiation of the granular cell phenotype in female salivary glands.

Epidermal Growth Factor Gene (EGF) Polymorphism and Risk of Melanocytic Neoplasiay

A common single nucleotide polymorphism (SNP) in the 5' untranslated region (5'UTR) of the epidermal growth factor (EGF) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case-control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGIF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.

Crystallization and preliminary x-ray diffraction analysis of the unliganded human growth hormone receptor

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1 - 238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 Angstrom and diffract to 2.5 Angstrom resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

Regulation of adult olfactory neurogenesis by insulin-like growth factor-I

Insulin-like growth factor-I (IGF-I) has multiple effects within the developing nervous system but its role in neurogenesis in the adult nervous system is less clear. The adult olfactory mucosa is a site of continuing neurogenesis that expresses IGF-I, its receptor and its binding proteins. The aim of the present study was to investigate the roles of IGF-I in regulating proliferation and differentiation in the olfactory mucosa. The action of IGF-I was assayed in serum-free culture combined with bromodeoxyuridine-labelling of proliferating cells and immunochemistry for specific cell types. IGF-I and its receptor were expressed by globose basal cells (the neuronal precursor) and by olfactory neurons. IGF-I reduced the numbers of proliferating neuronal precursors, induced their differentiation into neurons and promoted morphological differentiation of neurons. The evidence suggests that IGF-I is an autocrine and/or paracrine signal that induces neuronal precursors to differentiate into olfactory sensory neurons. These effects appear to be similar to the cellular effects of IGF-I in the developing nervous system.

Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappa B site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.

In vivo analysis of growth hormone receptor signaling domains and their associated transcripts

The growth hormone receptor (GHR) is a critical regulator of postnatal growth and metabolism. However, the GHR signaling domains and pathways that regulate these processes in vivo are not defined. We report the first knock-in mouse models with deletions of specific domains of the receptor that are required for its in vivo actions. Mice expressing truncations at residue m569 (plus Y539/545-F) and at residue m391 displayed a progressive impairment of postnatal growth with receptor truncation. Moreover, after 4 months of age, marked male obesity was observed in both mutant 569 and mutant 391 and was associated with hyperglycemia. Both mutants activated hepatic JAK2 and ERK2, whereas STAT5 phosphorylation was substantially decreased for mutant 569 and absent from mutant 391, correlating with loss of IGF-1 expression and reduction in growth. Microarray analysis of these and GHR(-/-) mice demonstrated that particular signaling domains are responsible for the regulation of different target genes and revealed novel actions of growth hormone. These mice represent the first step in delineating the domains of the GHR regulating body growth and composition and the transcripts associated with these domains.

Heterozygote effects in mice with partial truncations in the growth hormone receptor cytoplasmic domain: Assessment of growth parameters and phenotype

The GH receptor (GHR) is essential for normal postnatal growth and development, and the molecular basis of GHR action has been studied intensively. Clinical case studies and more recently mouse models have revealed the extensive phenotype of impaired GH action. We recently reported two new mouse models, possessing cytoplasmic truncations at position 569 (plus Y539/545-F) and 391, which were created to identify functional subdomains within the cytoplasmic signaling domain. In the homozygous state, these animals show progressively impaired postnatal growth coupled with complex changes in gene expression. We describe here an extended phenotype analysis encompassing the heterozygote state to identify whether single copies of these mutant receptors bring about partial or dominant-negative phenotypes. It appears that the retention of the ubiquitin-dependent endocytosis motif the N-terminal cytoplasmic domain permits turnover of these mutant receptors because no dominant-negative phenotype is seen. Nonetheless, we do observe partial impairment of postnatal growth in heterozygotes supporting limited haploinsufficiency. Reproductive function is impaired in these models in a progressive manner, in parallel with loss of signal transducer and activator of transcription-5 activation ability. In summary, we describe a more comprehensive phenotypic analysis of these mouse models, encompassing overall and longitudinal body growth, reproductive function, and hormonal status in both the heterozygote and homozygote state. Our results suggest that patients expressing single copies of similarly mutated GHRs would not display an obvious clinical phenotype.

FGFR1 emerges as a potential therapeutic target for lobular breast carcinomas

Purpose: Classic lobular carcinomas (CLC) account for 10% to 15% of all breast cancers. At the genetic level, CLCs show recurrent physical loss of chromosome16q coupled with the lack of E-cadherin (CDH1 gene) expression. However, little is known about the putative therapeutic targets for these tumors. The aim of this study was to characterize CLCs at the molecular genetic level and identify putative therapeutic targets. Experimental Design: We subjected 13 cases of CLC to a comprehensive molecular analysis including immunohistochemistry for E-cadherin, estrogen and progesterone receptors, HER2/ neu and p53; high-resolution comparative genomic hybridization (HR-CGH); microarray-based CGH (aCGH); and fluorescent and chromogenic in situ hybridization for CCND1 and FGFR1. Results: All cases lacked the expression of E-cadherin, p53, and HER2, and all but one case was positive for estrogen receptors. HR-CGH revealed recurrent gains on 1q and losses on 16q (both, 85%). aCGH showed a good agreement with but higher resolution and sensitivity than HR-CGH. Recurrent, high level gains at 11q13 (CCND1) and 8p12-p11.2 were identified in seven and six cases, respectively, and were validated with in situ hybridization. Examination of aCGH and the gene expression profile data of the cell lines, MDA-MB-134 and ZR-75-1, which harbor distinct gains of 8p12-p11.2, identified FGFR1 as a putative amplicon driver of 8p12-p11.2 amplification in MDA-MB-134. Inhibition of FGFR1 expression using small interfering RNA or a small-molecule chemical inhibitor showed that FGFR1 signaling contributes to the survival of MDA-MB-134 cells. Conclusions: Our findings suggest that receptor FGFR1 inhibitors may be useful as therapeutics in a subset of CLCs.

Overexpression of transforming growth factor-beta is associated with increased hyaluronan content and impairment of repair in Marfan syndrome aortic aneurysm

Background-Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGF beta 1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results-MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGF beta 1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGF beta 1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects = 5, versus control 8%, n = 3, P < 0.05; AF: 28%, n = 5 versus 7%, n = 5, P < 0.05; SF: 29%, n = 3 versus 4%, n = 3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and beta-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGF beta 1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or beta-3 integrin compared with controls. Conclusions-There was overexpression of TGF-beta in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

The use of heparan sulfate to augment fracture repair in a rat fracture model

Fracture healing is a complex process regulated by numerous growth and adhesive factors expressed at specific stages during healing. The naturally occurring, cell surface-expressed sugar, heparan sulfate (HS), is known to bind to and potentiate the effects of many classes of growth factors, and as such, may be a potential candidate therapy for enhancing bone repair. This study investigated the local application of bone-derived HS in the repair of rat femoral fractures. After 2 weeks, there was a significant increase in the callus size of rats administered with 5 mu g HS compared to the control and 50 mu g HS groups, presumably due to increased trabecular bone volume rather than increased cartilage production. In addition, 5 mu g HS increased the expression of ALP, Runx2, FGF-1, IGF-II, TGF-beta 1, and VEGF. It is hypothesized that these increases resulted from changes in HS-mediated receptor/ligand interactions that increase local growth factor production to augment bone formation. The findings of this study demonstrate the anabolic potential of HS in bone repair by recruiting and enhancing the production of endogenous growth factors at the site of injury. (c) 2006 Orthopaedic Research Society.