Ruggero Pardi: Training Module 2

Direct Supervisor: Ruggero Pardi, MD
Location: Unit of Leukocyte Biology, Department of Functional Genomics
Descriptive title of the research activity: Genetic Programs underlying the migratory phenotype

Overall goals:

Overall goal of this project is the dissection of the contribution of integrin-driven signals in controlling the onset of genetic programs underlying cell migration and invasiveness, both in normal and in transformed cells.

Rationale and Significance:

Directional migration typically ensues upon short-range stimulation of cells with chemotactic and/or pro-migratory stimuli. However, cells acquire a pro-migratory phenotype also as a result of a coordinate expression of genes whose products control motility as well as invasiveness in three-dimensional matrices. We have identified an integrin-driven signaling pathway in leukocytes, acting through a multi-molecular complex, the COP9 signalosome (CSN), which controls gene expression by regulating the ubiquitination and proteasome degradation of transcription factors (Bianchi E. et al, Nature, 2000). The CSN has recently been shown to be upstream of genetic programs involved in migration and invasiveness, and is frequently over-expressed in metastasizing cancer (Adler, AS. et al, Nature Genetics, 2006). We hypothesize that integrin signaling contributes to establishing a pro-migratory, pro-invasive phenotype through a CSN-dependent control of gene expression. A corollary of such hypothesis is that cancer cells should display a dysregulated control of such programs, which become independent of integrin-driven signals for their onset and maintenance.

Description of work:

Task 1: To establish the role of CSN5/JAB1 in controlling cell migration-associated genetic programs by gain of function and loss of function experiments in cellular models. A quantitative wound healing assay in MDCK cells will be used to assess the consequences on migration rates of inducibly down-regulating or overexpressing CSN5/JAB1.
Task 2: To establish the role of CSN5/JAB1 in controlling cell migration-associated genetic programs by gain of function and loss of function experiments in vivo. We have generated a mouse carrying a floxed allele of CSN5/JAB1 and are currently deleting the gene in several leukocyte subsets. In vivo assays, such as intravital microscopy (IVM), will be used to assess the consequence of CSN5/JAB1 deficiency on directional migration and tissue homing of leukocytes.
Task 3: To identify and characterize gene expression patterns controlled and involved in conferring the prom-migratory, pro-invasive phenotype. Gene expression profiling from cells as described in task 1 will be performed to identify gene clusters whose expression is controlled by the CSN.

Methodology:

Task 1: stable transfection of Tet-inducible vectors coding for CSN5/JAB1 or validated shRNAs promoting downregulation of endogenous CSN5/JAB1 will be carried out in epithelial cell lines amenable to be assessed in wound healing type of assays.
Task 2: conditional KO mice, already available in the lab, will be assessed for CK-induced migration by IVM in tissues perifused with selected chemokines.
Task 3: conventional Affimetrix microarray analysis will be performed using the house facilities.

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