Major recent scientific achievements

1) In order to characterize the role played by STAT3 and its isoforms alpha or beta in mediating the signalling of different cytokines, we have generated conditional STAT3 mutant mice using the Cre-lox system (Alonzi et al., 2001). In addition, we also generated by a knock-in approach mice that can only produce STAT3 or STAT3 (STAT3 or  KO), showing that the two isoforms play non-redundant roles in inflammation and that Stat3beta is able to activate transcription of a subset of Stat3alpha target genes. Therefore, Stat3beta is not a dominant negative form, and the results of experiments aiming at blocking Stat3 activity using Stat3beta should be reconsidered (Maritano et al., 2004).

2) We have demonstrated that the absence of STAT3 in murine embryonal fibroblasts (MEFs) profoundly modifies STATs activation profiles and biological responses to gp130 cytokines, causing prolonged STAT1 activation and triggering an IFN--like response, including the induction of multiple IFN--inducible genes, expression of Class II MHC antigens and an antiviral state (Costa Pereira et al., 2004). Normal cells exposed to IL-6 thus require a STAT3-dependent function(s) to down regulate STAT1 activity and prevent an IFN--like response. We found that STAT3, but not STAT3, is required for this function.

3) We developed a computational method for the genome-wide identification of functional transcription factor binding sites based on positional weight matrices, comparative genomics and gene expression profiling (Vallania et al, 2009) and applied it to Stat3. We generated a new Positional Weight Matrix which allowed us to assign highly specific affinity scores to predicted Stat3 binding sites (Stat3 BSs). We then used phylogenetic conservation with seven vertebrate species to select the Stat3 BSs most likely to be functional and validated our prediction by testing in vivo binding of Stat3 to a subset of predicted sites obtaining a high validation rate. This method, coupled to the availability of large transcription factor-dependent gene expression datasets obtained under diverse experimental conditions, appears to be a valid alternative to high-throughput experimental assays for the discovery of novel direct targets of transcription factors.

Main ongoing research projects

1) Oncogenic potential of the constitutively active Stat3
In order to test the capacity of aberrantly active STAT3 to elicit tumoral transformation on itself or in cooperation with other oncogenes, we have generated mice where a constitutively active form of STAT3 (Stat3C, Bromberg et al., 1999, Cell 98:295-303) has been inserted into the STAT3 endogenous allele by a knock-in approach. Mice heterozygous for the STAT3C allele (STAT3C/WT) are normal and fertile while STAT3C/C homozygous mice invariably die at about four weeks of age. We are using this model to analyze spontaneous tumor onset as well as cooperativity with known oncogenes in an experimental set up similar to that found in primary tumors, characterized by continuous but low STAT3 activity. In particular, we could show that Stat3C can cooperate with the Neu oncogene in MMTV-Neu;Stat3C transgenic mice. Stat3C confers more aggressive growth to the Neu mammary tumors, with earlier onset, reduced spontaneous apoptosis and enhanced invasion of the surrounding fat pad. Analysis of the pathways and target genes involved is under way. In parallel, we are testing the requirements for Stat3 in the development of NeuT tumors by generating NeuT;MMTV-Cre;STAT3fl/fl mice, where Stat3 is inactivated in the mammary epithelium.
In addition, we are using primary mouse embryonal fibroblasts (MEF cells) derived from STAT3C/C or STAT3WT/WT embyos to investigate the general functions of Stat3 in oncogenesis. STAT3C/C MEFs show an increased proliferation potential and are significantly more resistant to both apoptosis and senescence, and represent an ideallly simple model to test multiple hypethesis concernong both canonical (i.e. nuclear and transcriptional) and non-canonical fucntions of Stat3, either acutely induced or constitutively active.

2) Role of Stat3 in tumor stem cells
It has been recently shown that many, if not all, cancers contain a minor population of stem cells, the so-called "cancer stem cells", a group of slowly-replicating, self-maintaining cells responsible for relapse and resistant to most chemotherapics. Mutations occurring in the mammary stem cells have been proposed to be the source of many types of breast cancer, and several distinct populations of stem/progenitor cells that display different degrees of commitment have been described. Stat3 is essential for the maintainance of the undifferentiated status of Embryonal Stem (ES) cells and was proposed to play a role in normal and tumor stem cells as well. We are taking advantage of our mouse models to dissect the role of Stat3 in the maintenance and characteristics of the tumor stem cells compartment, in particular downstream of Neu over-expression.

3) Functional analysis of newly discovered Stat3 target genes
We have identified a number of new Stat3 target genes by gene expression profiling of either MMTV-Neu;Stat3C tumor-derived cell lines or STAT3C/C and STAT3 MEFs. The validation of true transcriptional targets is being carried out using the bioinformatics method that we have recently developed (Vallania et al., 2009), followed by functional validation through ectopic expression and/or silencing of the different targets.

4) Role of Stat3 in orchestrating immune and inflammatory responses
Stat3 is able to elicit both pro-inflammatory and anti-inflammatory responses, depending on the cell type and the nature and duration of the stimulus. Specific, and sometimes contrasting, functions have been proposed in antigen presenting cells, B and T helper lymphocytes. Making use of the different mouse models generated we are attempting to characterize some of these specific functions in the context of immunity/auto-immunity, of chronic inflammation and of the interactions between tumor and the immune system.

Alonzi T., Maritano D., Gorgoni B., Rizzuto G., Libert C., Poli V. Essential role of STAT3 in the control of the acute phase response as revealed by inducible gene inactivation in the liver. (2001) Mol Cell Biol. 21, 1621-1632.

Gorgoni, B., Caivano, M., Arizmendi, C. and Poli, V. The Transcription Factor C/EBPbeta Is Essential for Inducible Expression of the cox-2 Gene in Macrophages but Not in Fibroblasts. (2001) J Biol Chem. 276, 40769-40777.

Caivano, M, Gorgoni, B, Cohen, P and Poli, V. The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both C/EBP{beta} and C/EBP{delta} transcription factors. (2001) J Biol Chem. 276, 48693-48701.

Gorgoni, B., Maritano, D., Marthyn, P., Righi, M. and Poli, V. C/EBPb gene inactivation causes both impaired and enhanced gene expression and inverse regulation of IL-12 p40 and p35 mRNAs in macrophages. (2002) J. Immunol. 168, 4055-4062.

Costa-Pereira, A.P.,Tininini, S., Strobl, B. Alonzi, T., Schlaak, J.F., Is’harc, H., Gesualdo, I. Newman, S.J., Kerr, I.M. and Poli, V. An Interferon--like response through IL-6 in the absence of STAT3. (2002) Proc. Natl. Acad. Sci. USA 99, 8043-8047

Poli, V. and Maritano, D. IL-6 knockout mice. (2003) In: G. Fantuzzi ed., "Contemporary Immunology: Cytokine Knockouts", 2nd Edition, Humana Press Inc., Totowa, NJ, pp 213-235.

Poli, V. and Alonzi, T. STAT3 function in vivo. (2003) In: P.B. Shegal, D.E.Levy, T.Hirano (eds.) “Signal Transducers and Activators of Transcription (STATs): Activation and Biology”, Kluwer Academic Publishers, The Netherlands; pp 493-512.

Maritano, D., Sugrue, M.L., Tininini, S., Dewilde, S., Strobl, B., Fu, XP, Murray-Tait, V., Chiarle, R. and Poli. V. The STAT3 isoforms, a and b, play unique and specific roles. (2004) Nat. Immunol. 5, 401-409.

Alonzi, T., Newton, I.P., Bryce, P. J. Di Carlo, E., Lattanzio, G., Musiani, P. and Poli, V. Induced somatic inactivation of STAT3 in mice triggers the development of a fulminant form of enterocolitis. (2004) Cytokine 26, 45-56.

Dewilde S, Vercelli A, Chiarle R and Poli V. Of alphas and betas: distinct and overlapping functions of STAT3 isoforms. (2008) Frontiers in Bioscience 13, 6501-6514.

Regis G, Pensa S, Boselli D, Novelli F, Poli V. Ups and downs: The STAT1:STAT3 seesaw of Interferon and gp130 receptor signalling. Seminars in Cell and Developmental Biology 19 (2008), pp. 351-359

Pensa S, Regis G, Boselli D, Novelli F, Poli V. STAT1 and STAT3 in Tumorigenesis: Two Sides of the Same Coin? (2008) in Stephanou ed, “JAK-STAT Pathway in Disease”, Landes Bioscience, in press. ISBN: TBA

Vallania, F., Schiavone, D., Dewilde, S., Pupo, E., Garbay, S., Calogero, R., Pontoglio, M., Provero, P. and Poli, V. Genome-Wide Discovery of Functional Transcription Factor Binding Sites by Comparative Genomics: the Case of Stat3. (2009) Proc. Natl. Acad. Sci. USA 106:5117-22.