Proteomics Laboratory

Francesc Canals graduated in organic chemistry at the Institut Químic de Sarrià, Barcelona, in 1982, where he also obtained his PhD in 1989 in the field of organic photochemistry. He also obtained a degree in biochemistry at the Universitat Autònoma, Barcelona, in 1987.

He worked as a postdoctoral fellow in the laboratory of Prof. Jack Kyte, at the University of California San Diego (USA) where he performed protein chemistry studies on the signaling mechanism of the epidermal growth factor receptor, showing that the tyrosine kinase of the receptor is activated after its dimerization.In 2003 he joined the Medical Oncology Research program as head of the Proteomics Laboratory. Since then, he has set up the different separation and mass-spectrometry based proteomic technologies provided by the facility. His research focuses on the development and application of proteomic strategies to characterize the substrate repertoire - degradome - of metalloproteases of the ADAM and ADAMTS families, involved in tumor progression.

PRINCIPAL INVESTIGATOR

RESEARCH LINES

The importance of the interaction between tumor cells and their microenvironment in malignant progression has been recently highlighted.

Different metalloproteases play a crucial role in the regulation of the tumor microenvironment by mediating the remodeling of the extracellular matrix and the processing of extracellular and membrane proteins.

Knowledge of the substrate repertoire (degradome) of these proteases is needed to elucidate their role in tumor growth and metastasis, in order to evaluate their potential use as therapeutic targets. We have demonstrated the utility of proteomic techniques to explore these degradomes. The present focus of our research is to extend these studies, incorporating new proteomic analysis techniques, to study metalloproteases known to play key roles in tumor progression. Metalloproteases of the ADAM family (ADAM10, ADAM17/TACE) and of the ADAMTS family (ADAMTS1), will be the object of study.

TACE and ADAM10 are involved in the proteolytic cleavage of the transmembrane forms of EGFR ligands (shedding), required for the activation of the receptor, and are also involved in regulation of cell migration and adhesion. Several metalloproteases of this family are overexpressed in different types of tumors. The thrombospondin-domain containing protease ADAMTS1 has been recently found to be highly overexpressed in highly invasive mammary tumor cells, suggesting a major role for this protease in metastatic processes. The proposed proteomic studies aim to the identification and characterization of new substrates of these proteases in the context of cancer cells. The putative substrates identified will then be validated through characterization in vitro. The importance of the newly identified substrates in tumor development will be analyzed in preclinical models and their expression and shedding will be determined in mammary tumor samples.

Identification of substrates of ADAM10 and ADAM17 proteases using SILAC analysis.

In order to search for substrates of the ADAM10 and ADAM17 metalloproteases, we have used model breast cancer cells in which conditional expression has been introduced, using the tet-off system, of either the protease or siRNA to knock down specifically the protease of interest. The comparison between conditions where the protease is either inactive or active can be then carried out in the same cell clone. Differential proteomic analysis of the conditioned culture media of these cells has been performed using a SILAC (stable isotope labeling through aminoacids in culture) approach, and an analytical workflow comprising 1D-SDS-PAGE fractionation followed by liquid-chromatography coupled to electrospray mass spectrometry (LC-MS). Labeling is accomplished supplying specific labeled amino acids in the cell culture medium, thus allowing the labeling of all the proteins in the cell through its own metabolic processes. In this way, quantitative information can be obtained for all the proteins detected in the analysis.

A number of known substrates of both proteases were identified as such in the analysis, showing the expected decrease of the shed extracellular domain abundance in the medium upon knock down of the protease. In addition, several new candidate substrates of both proteases were identified.

Among them, the GPI-anchored protein C4.4A, was identified and further validated as substrate of both ADAM10 and ADAM17 proteases. According to the identified peptides, both proteases cleave this protein close to the juxtamembrane region, releasing a soluble form devoid of the GPI-anchor. C4.4A protein, homologous to the urokinase-type plasminogen activator receptor, has been related to tumor invasion and metastasis. Cleavage of this protein by ADAMs constitutes a previously unknown level of regulation of its function. Work is in progress to validate and further characterize other proteins identified as potential substrates of these proteases.

Identification of ADAMTS1 substrates using DIGE and SILAC proteomic analysis.

We have applied two complementary proteomic approaches, 2D electrophoresis DIGE and SILAC LC-MS analysis, to the search of substrates of the metalloprotease ADAMTS1, in a model breast cancer cell line were conditional overexpression of the protease was introduced.

Glycoproteins of the conditioned media from parental and ADAMTS1 overexpressing cells were purified and analyzed using a 2D-DIGE electrophoresis approach (Figure 2) and a SILAC methodology similar to the one described above for the ADAM10 and ADAM17 experiments.

Both approaches led to the identification of trombospondin-1 as a substrate of ADAMTS1, which has been reported recently by others, and shown to play a role in modulating angiogenesis.

Semaphorin 3C was also identified by both methodologies, and further validated as a substrate of both ADAMTS1 and ADAM17. This family of extracellular matrix proteins plays different roles in cell axon guidance in the nervous system, as well as in angiogenesis and tumor progression. The role of Semaphorin 3C in cancer cell migration is currently being investigated.

Other collaborative projects with VHIO groups

- Signaling through C-terminal fragments of HER-2 in breast cancer (with J. Arribas Lab.):

SILAC proteomic analysis was used to analyze protein-protein interaction partners of HER-2. Several potential mediators of HER-2 signaling, as well as previously unreported phosphorylation sites have been identified.

- Screening for surface marker proteins of glioma-initiating stem-cells (with J. Seoane Lab.): Several candidate proteins have been identified has putative surface markers of glioma neurosphere forming cells through cell-surface proteome analysis.

- Biomarkers to monitor response to Hsp-90 inhibitor IPI-504 treatment (with M. Scaltriti- J.Baselga Lab.): Several candidate biomarkers of IPI-504 action have been identified by SILAC proteomic analysis of model cells.

Facility and Collaborative Work

The Proteomics Laboratory has continued providing services as a member of the Instituto de Salud Carlos III Cancer Research Network, and of the Instituto Nacional de Proteómica ProteoRed, funded by Fundación Genoma España. This year, the laboratory has provided services to more than 30 research groups, not only from the Vall Hebron Hospital, but also from the main hospitals, research centers and universities in the area. In summary, the analysis performed include 40 2D-DIGE gels, 20 quantitative ICPL or SILAC experiments, representing a total of more than 450 LC-MS runs, and around 500 protein identifications by peptide mass fingerprint.

In conjunction with the services provided, the laboratory has actively participated in several projects involving proteomic analysis. The main contributions have been: in collaboration with Dr. R. Simó, of the Endocrinology Unit at the IR-HUVH we have continued to apply DIGE technology to study alterations in the protein contents of vitreous fluid of proliferative diabetic retinopathy patients subjected to vitrectomy.

Together with the Department of Immunology at the Universitat Autònoma, Barcelona, led by Dr. Dolores Jaraquemada, we have been working on the analysis of repertoires of HLA associated peptides of cell lines related to autoimmune diseases or cancer.

In the framework of the ProteoRed network, the laboratory has coordinated a multicentric study to evaluate reproducibility of a 2D-DIGE differential proteomic experiment. The results of the study show the robustness of the methodology used, and demonstrate the feasibility of across-lab validation schemes, pointing towards development of inter-lab QC strategies for proteomics research. The results were presented at the ABRF'09 Meeting, the 3rd SEPROT-LAHUPO Congress, and on the HUPO special meeting on reproducibility studies as preliminary requirements to launch HUPO Human Proteome Project.

PUBLICATIONS

Álvarez I, Collado J, Daura X, Colomé N, Rodríguez-García M, Gallart T, Canals F, Jaraquemada D. The rheumatoid arthritis–associated allele HLA–DR10 (DRB1*1001) shares part of its repertoire with HLA–DR1 (DRB1*0101) and HLA–DR4 (DRB*0401). Arthritis & Rheumatism. 2008;58:1630-1639.
(IF: 7.677, 1 cuartil, rheumatology)

Esselens C, Malapeira J, Colomé N, Canals F, Arribas, J. Metastasis-associated C4.4A, a GPI-anchored protein cleaved by ADAM10 and ADAM17. Biological Chemistry. 2008 Aug;389(8):1075-84.
(IF: 2.840, 2 cuartil, biochemistry & molecular biology)

Simó R, Higuera M, García-Ramírez M, Canals F, García-Arumí J, Hernández C. Elevation of apolipoprotein A-I and apolipoprotein H levels in the vitreous fluid and overexpression in the retina of diabetic patients. Arch Ophthalmol. 2008 Aug;126(8):1076-81.
(IF: 2.984, 1 cuartil, ophthalmology)

Esteve-Puig R, Canals F, Colomé N, Merlino G, Recio JA. Uncoupling of the LKB1-AMPK- energy sensor pathway by growth factors and oncogenic BRAFV600E. PLoS ONE. In press.

Doll A, Abal M, Rigau M, Monge M, González M, Demajo S, Colás E, Llauradó M, Alazzouzi H, Planagumá J, Lohmann MA, Garcia J, Castellvi J, Ramón y Cajal S, Gil-Moreno A, Xercavins J, Alameda F, Reventós J. Novel molecular profiles of endometrial cancer-new light through old windows. J Steroid Biochem Mol Biol. 2008 Feb;108(3-5):221-9. Epub 2007 Sep 15. Review.
(IF: 2.799, 2 cuartil, biochemistry molecular biology science)

RESEARCH PROJECTS

Funding Agency: ISCIII (FIS)

Title:

Identificación mediante análisis proteómico de nuevos sustratos de metaloproteasas implicadas en cáncer y caracterización de su papel funcional

PI: Dr. Francesc Canals

Duration: January 2008/December 2010

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