Loss of ADAM9 Leads to Modifications of the Extracellular Matrix Modulating Tumor Growth.

ADAM9 is a metalloproteinase strongly expressed at the tumor-stroma border by both tumor and stromal cells. We previously showed that the host deletion of ADAM9 leads to enhanced growth of grafted B16F1 melanoma cells by a mechanism mediated by TIMP1 and the TNF-α/sTNFR1 pathway. This study aimed to dissect the structural modifications in the tumor microenvironment due to the stromal expression of ADAM9 during melanoma progression. We performed proteomic analysis of peritumoral areas of ADAM9 deleted mice and identified the altered expression of several matrix proteins. These include decorin, collagen type XIV, fibronectin, and collagen type I. Analysis of these matrices in the matrix producing cells of the dermis, fibroblasts, showed that ADAM9-/- and wild type fibroblasts synthesize and secreted almost comparable amounts of decorin. Conversely, collagen type I expression was moderately, but not significantly, decreased at the transcriptional level, and the protein increased in ADAM9-/- fibroblast mono- and co-cultures with melanoma media. We show here for the first time that ADAM9 can release a collagen fragment. Still, it is not able to degrade collagen type I. However, the deletion of ADAM9 in fibroblasts resulted in reduced MMP-13 and -14 expression that may account for the reduced processing of collagen type I. Altogether, the data show that the ablation of ADAM9 in the host leads to the altered expression of peritumoral extracellular matrix proteins that generate a more favorable environment for melanoma cell growth. These data underscore the suppressive role of stromal expression of ADAM9 in tumor growth and call for a better understanding of how protease activities function in a cellular context for improved targeting.

Connectivity maps for biosimilar drug discovery in venoms: the case of Gila monster venom and the anti-diabetes drug Byetta®.

Like most natural product libraries animal venoms have long been recognized as potentially rich source of biologically active molecules with the potential to be mined for the discovery of drugs, drug leads and/or biosimilars. In this work we demonstrate as a proof of concept a novel approach to explore venoms for potential biosimilarity to other drugs based on their ability to alter the transcriptomes of test cell lines followed by informatic searches and Connectivity Mapping to match the action of the venom on the cell gene expression to that of other drugs in the Connectivity Map (C-Map) database. As our test animal venom we chose Heloderma suspectum venom (Gila monster) since exendin-4, a glucagon-like peptide 1 receptor agonist, isolated from the venom is currently on the market to treat type 2 diabetes. The action of Byetta(®) (exentide, synthetic exendin-4), was also used in transcriptome studies. Analysis of transcriptomes from cells treated with the venom or the drug showed similarities as well as differences. The former case was primarily attributed to the fact that Gila monster venom likely contains a variety of biologically active molecules that could alter the MCF7 cell transcriptome compared to that of the single perturbant Byetta(®). Using Ingenuity Pathway Analysis software, insulin-like growth factor 1 signaling was identified in the category of "Top Canonical Pathways" for both the venom and Byetta(®). In the category of "Top Molecules" up-regulated, both venom and Byetta(®) shared IL-8, cyclic AMP-dependent transcription factor 3 (ATF-3), neuron-derived orphan receptor 1 (NR4A3), dexamethasone-induced Ras-related protein 1 (RASD1) and early growth response protein 1, (EGR-1) all with potential relevance in diabetes. Using Connectivity Mapping, Gila monster venom showed positive correlation with 1732 instances and negative correlation with 793 instances in the Connectivity database whereas Byetta(®) showed positive correlation with 1692 instances and negative correlation with 868 instances. Interestingly, the Gila monster venom and Byetta(®) both showed positive correlation with the anti-diabetic drugs troglitazone, of the thiazolidinedione class, and metformin, of the biguanide class, although Byetta(®) as a glucagon-like peptide-1 (GLP-1) agonist functions in a different manner than either of these two classes of anti-diabetic drugs. In summary, despite the fact that Gila monster venom contains a mixture of biologically active molecules, similarities in terms of perturbation of gene expression profiles on MCF7 cells were observed between the venom and the drug Byetta(®). Furthermore, using Connectivity Mapping the Gila monster venom was demonstrated to have nodes of positive correlation to several anti-diabetic drugs two of which were the same as observed with Byetta(®). Therefore, this study suggests that by using this approach novel drug activities heretofore unconsidered may be discovered in venoms using informatic tools and Connectivity Mapping.

Proteomic anatomy of human skin.

The extracellular matrix is composed of a variety of proteins which are essential for growth, wound healing, and fibrosis. It provides both structural support as well as contributing to the regulation of the local microenvironment. To further characterize the molecular composition of human skin we have undertaken a proteomic approach to identify proteins in three skin regions from two locations. Using laser microdissection, extracellular matrix was obtained from three distinct regions (basement membrane, papillary dermis, and reticular dermis) of formalin-fixed, paraffin embedded tissue from normal human leg and breast skin. The proteome of these regions was determined by mass spectrometric analysis. This study provides a relative quantitative assessment, including protein composition and relative abundance, of the proteins found in different skin regions giving rise to a "proteomic anatomy" of skin. Our findings indicate that there was little difference detected in the subproteomes of the dermal and papillary regions and little difference between the proteomes of leg skin compared to breast skin. One finding of interest is the identification of tenascin-X only in the breast dermis and serum amyloid P-component in the leg dermis. The results of this proteomic analysis corroborate much of the information on the protein composition identified by other methodologies found in the literature but provide additional insight as to localization of skin proteins in the various regions of skin. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments. BIOLOGICAL SIGNIFICANCE: This investigation underscores the power of proteomics to bring semiquantitative, non-presumptive molecular characterization to the field of histological anatomy. Traditionally, anatomy relied on visual or histochemical structural characterization which generally involved some level of understanding of the area of interest. With the advent of laser microdissection or laser capture microscopy localization of anatomical structures of interest can be correlated to molecular composition by virtue of mass spectrometric determination of the proteome of that structure. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments.

Endogenous concentrations of ouabain act as a cofactor to stimulate fluid secretion and cyst growth of in vitro ADPKD models via cAMP and EGFR-Src-MEK pathways.

In autosomal-dominant polycystic kidney disease (ADPKD), renal cysts develop by aberrant epithelial cell proliferation and transepithelial fluid secretion. We previously showed that ouabain increases proliferation of cultured human ADPKD cells via stimulation of the EGF receptor (EGFR)-Src-MEK/ERK signaling pathway. We examined whether ouabain affects fluid secretion and in vitro cyst growth of human ADPKD cell monolayers, ADPKD cell microcysts cultured in a three-dimensional collagen matrix, and metanephric organ cultures from Pkd1(m1Bei) mice. Physiological concentrations of ouabain alone did not affect net transepithelial basal-to-apical fluid transport in ADPKD monolayers or growth of cultured ADPKD microcysts. In contrast, in the presence of forskolin or 8-bromo-cAMP, ouabain significantly enhanced ADPKD fluid secretion and microcyst expansion. Ouabain exerted this effect by enhancing cAMP-dependent Cl(-) secretion via the CFTR. Similarly, ouabain accelerated cAMP-dependent cyst enlargement in Pkd1(m1Bei) mice metanephroi, with a more prominent response in homozygous than heterozygous mice. Ouabain had no effect on fluid secretion and cystogenesis of normal human kidney cells and caused only slight cystic dilations in wild-type mouse kidneys. The effects of ouabain in ADPKD cells and Pkd1(m1Bei) metanephroi were prevented by inhibitors of EGFR (AG1478), Src (PP2), and MEK (U0126). Together, our results show that ouabain, used in physiological concentrations, has synergistic effects on cAMP-mediated fluid secretion and cyst growth, via activation of the EGFR-Src-MEK pathway. These data provide important evidence for the role of ouabain as an endogenous hormone that exacerbates ADPKD cyst progression.