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Martino-Echarri E.,University of Granada | Fernandez-Rodriguez R.,University of Granada | Rodriguez-Baena F.J.,University of Granada | Barrientos-Duran A.,University of Granada | And 7 more authors.
International Journal of Cancer | Year: 2013

The extracellular protease ADAMTS1 (A disintegrin and metalloprotease with thrombospondin repeats 1) has been described as an anti-angiogenic molecule and its role as a putative tumor protective molecule has also been suggested. Here, we have used a tumor xenograft model to determine the role of ADAMTS1 in tumor growth and angiogenesis. Increasing levels of the protease led to the complete inhibition of tumor growth. In an attempt to elucidate the mechanism of action of this protease, we focused our attention on its proteolytic activity on nidogens, one of the main components of the vascular basement membrane. The increased expression of ADAMTS1 was accompanied by increased proteolysis of nidogen-1 and -2 and their almost complete removal from vascular structures, together with major morphological alterations of tumor blood vessels and a decreased vessel density. The clinical relevance of this work is supported by our observations that ADAMTS1 expression is decreased in breast tumor specimens when compared with healthy tissue. Our studies also reveal that the cleavage of nidogen-1 and -2 is partially inhibited in human tumor samples. Moreover, the deposition of both nidogens surrounding vascular structures is drastically altered, implying a possible reduction in the maintenance of vessel integrity. Our studies reflect the requirement to explore the functional interactions between proteases and specific substrates in cancer biology. What's new? The extracellular milieu is a complex and dynamic environment, and certain proteases within it may play a role in tumor suppression. Of particular interest is the protease ADAMTS1, which this study indicates is a key tumor inhibitor in the extracellular matrix. In mice, increased ADAMTS1 was correlated with increased proteolysis of nidogens in the vascular basement membrane and decreased vessel density in tumors. Conversely, the protease was downregulated and nidogen proteolysis partially inhibited, with implications for vessel integrity, in human breast cancer specimens. The findings suggest that protease interactions with specific substrates are of functional importance in the tumor microenvironment. Copyright © 2013 UICC.

Barbachano A.,Autonomous University of Madrid | Ordoez-Moran P.,Autonomous University of Madrid | Ordoez-Moran P.,Ecole Polytechnique Federale de Lausanne | Garcia J.M.,Hospital Universitario Puerta Of Hierro | And 10 more authors.
Oncogene | Year: 2010

SPROUTY-2 (SPRY2) regulates receptor tyrosine kinase signalling and therefore cell growth and differentiation. In this study, we show that SPRY2 expression in colon cancer cells is inhibited by the active vitamin D metabolite 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3) through E-cadherin-dependent and-independent mechanisms. In turn, SPRY2 represses both basal and 1,25(OH) 2 D 3-induced E-cadherin expression. In line with this, SPRY2 induces ZEB1 RNA and protein, but not that of other epithelial-to-mesenchymal transition inducers that repress the CDH1/E-cadherin promoter. Consistently, SPRY2 and E-cadherin protein levels inversely correlate in colon cancer cell lines and xenografted tumours. Moreover, SPRY2 knockdown by small hairpin RNA increases CDH1/E-cadherin expression and, reciprocally, CDH1/E-cadherin knockdown increases that of SPRY2. In colon cancer patients, SPRY2 is upregulated in undifferentiated high-grade tumours and at the invasive front of low-grade carcinomas. Quantification of protein expression in 34 tumours confirmed an inverse correlation between SPRY2 and E-cadherin. Our data demonstrate a tumourigenic action of SPRY2 that is based on the repression of E-cadherin, probably by the induction of ZEB1, and a reciprocal regulation of SPRY2 and E-cadherin that dictates cell phenotype. We propose SPRY2 as a candidate novel marker for high-grade tumours and a target of therapeutic intervention in colon cancer. © 2010 Macmillan Publishers Limited All rights reserved.

Serra V.,Vall dHebron Institute dOncologia | Vivancos A.,Vall dHebron Institute dOncologia | Puente X.S.,University of Oviedo | Felip E.,Vall dHebron Institute dOncologia | And 15 more authors.
Cancer Discovery | Year: 2013

Genomic characterization of recurrent breast and lung tumors developed over the course of 10 years in a 29-year-old patient with a germline TP53 mutation (Li- Fraumeni Syndrome) identified oncogenic alterations in the HER2 and EGFR genes across all tumors, including HER2 amplifications, an EGFR -exon 20 insertion, and the first-in-humans HER2V659E mutation showing a phenotypic convergent evolution toward HER2 and EGFR alterations. Following the identification of HER2-activating events in the most recent lung carcinoma and in circulating tumor cells, we treated the reminiscent metastatic lesions with a lapatinib-based therapy. A symptomatic and radiologic clinical response was achieved. HER2V659E sensitivity to lapatinib was confirmed in the laboratory. SIGNIFICANCE: The precise knowledge of the genomic alterations present in tumors is critical to selecting the optimal treatment for each patient. Here, we report the molecular characterization and clinical response to a lapatinib-based therapy for the tumors of a Li-Fraumeni patient showing prevalence of HER2 and EGFR genomic alterations. © 2013 American Association for Cancer Research.

Lambertini C.,Novartis | Barzaghi-Rinaudo P.,Novartis | D'Amato L.,Novartis | Schulz S.,Friedrich - Schiller University of Jena | And 2 more authors.
Regulatory Peptides | Year: 2013

Introduction: The expression and reliable detection of somatostatin receptor subtypes (SSTR1-5) is a prerequisite for the successful use of somatostatin analogs in neuroendocrine tumors (NETs). Two sets of monoclonal antibodies (mAbs) against human SSTR1, 2A, 3 and 5 have recently been developed by two independent laboratories using rabbit and mouse hybridomas. Our aim was to evaluate the usefulness of both sets of mAbs for detection of SSTRs in NET samples as they are routinely collected in clinical practice. Methods: Mouse and rabbit mAbs were characterized in SSTR1, 2A, 3 and 5-transfected HEK293 cells and human archival samples of pancreatic tissue and NET. Comparative analysis of mAbs was also conducted by immunostaining of a tissue microarray composed of 75 cores of NET. Results: Immunohistochemical analysis of HEK293 cells showed that both rabbit and mouse mAbs specifically detect their cognate receptor subtype, with mild cytoplasmic cross-reactivity observed for rabbit mAbs. Both sets of mAbs labeled normal pancreatic islets and showed similar patterns of immunoreactivity in NET controls. Direct comparison of mAb sets using a NET tissue microarray revealed strong correlation between rabbit and mouse mAbs against SSTR1 and 5, and moderate correlation for SSTR3. The rabbit mAb against SSTR2A showed higher affinity for its cognate receptor than the corresponding mouse mAb, resulting in a more reliable detection of this SSTR. Conclusions: mAbs from both sets are reliable tools for the detection of SSTR1, 3 and 5, whereas the rabbit mAb against SSTR2A is recommended for use in routine clinical testing due to its superior binding affinity. © 2013 Elsevier B.V.

Vila-Rico M.,Ramon Llull University | Colome-Calls N.,Vall dHebron Institute dOncologia | Martin-Castel L.,Vall dHebron Institute dOncologia | Gay M.,Barcelona Institute for Research in Biomedicine | And 4 more authors.
Journal of Proteomics | Year: 2015

Transthyretin (TTR) is an amyloidogenic tetrameric protein, present in human plasma, associated with several familial amyloidoses. Variability of TTR is not only due to point mutations in the encoding gene but also to post-translational modifications (PTMs) at Cys10, being the most common PTMs the S-sulfonation, S-glycinylcysteinylation, S-cysteinylation and S-glutathionylation. It is thought that PTMs at Cys10 may play an important biological role in the onset and pathological process of the amyloidosis. We report here the development of a methodology for quantification of PTMs in serum samples, as well as for the determination of serum TTR levels, from healthy (wt) and TTR-amyloidotic (V30M mutation) individuals. It involves an enrichment step by immunoprecipitation followed by mass spectrometry analysis of (i) the intact TTR protein and (ii) targeted LC-MS analysis of peptides carrying the PTMs of interest. Analysis of serum samples by the combination of the two methods affords complementary information on the relative and absolute amounts of the selected TTR PTM forms. It is shown that methods based on intact protein are biased for specific PTMs since they assume constant response factors, whereas the novel targeted LC-MS method provides absolute quantification of PTMs and total TTR variants. Biological significance: The study of TTR has a high clinical relevance since it is responsible for diverse familial polyneuropathies. In particular, more than 80 point mutations have been described through genetic studies. However, genetic heterogeneity alone fails to explain the diverse onset and pathological process of the TTR related amyloidosis. The use of proteomic characterization is required to gather information about the PTMs variants present in serum, which have been suggested to be relevant for the amyloidotic pathology. This article is part of a Special Issue entitled: HUPO 2014. © 2015 Elsevier B.V.

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