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Leukaemia Stem Cell and Cancer Cell survival and proliferation mechanisms

(Stem Cell selection and definition of drug resistance)

Dipartimento di Patologia e Oncologia Sperimentali dell'Università degli Studi di Firenze
viale G.B. Morgagni 50 - 50134 FIRENZE
Tel. 055-4598-900
Fax 055-4598-209
E-mail: persio@unifi.it

Principal Investigator: Persio Dello Sbarba (M.D., Ph.D., associate professor of General Pathology)

Staff

  • Maria Grazia Cipolleschi, M.D., ricercatore universitario confermato
  • Elisabetta Rovida, Ph.D., professore a contratto, Research Theme leader
  • Valentina Barbetti, Ph.D., assegnista di ricerca
  • Serena Giuntoli, Ph.D., assegnista di ricerca
  • Andrea Morandi, dottorando di ricerca
  • Michele Tanturli, dottorando di ricerca

Scientific background:
The Unit has been engaged in studies of the:

  • control of survival and proliferation in vitro of murine and human stem and progenitor cells; role of severe hypoxia in the control of maintenance and self-renewal of stem cells, as well as in the modulation of action of stem cell-active cytokines (first publication in the field: 1987);
  • role of the Macrophage Colony-Stimulating Factor (MCSF) in survival, proliferation, adhesion and activation of mononuclear phagocytes; interference of the raf and fes oncogene products with the macrophage response to MCSF; role of the Mitogen-Activated Protein Kinases (MAPK) in the response to MCSF; effects of macrophage activators on the expression of MCSF receptor (first publication in the field: 1991);
  • apoptotic and differentiative effects of inhibitors of histone deacetylases on acute myeloid leukaemia (AML) cells; signaling pathways involved in the epigenetic modulation of AML cell survival (first publication in the field: 2003);
  • response of leukaemia cells to hypoxia; selection of leukaemia stem cells in hypoxia; drug-resistance of hypoxia-selected chronic myeloid leukaemia (CML) stem cells (first publication in the field: 2006).

Main Research Themes

Research Theme 1 - Role of hypoxia in the selection and maintenance of leukaemia stem cells.
The main achievements in this field are:

  1. the demonstration that incubation in severe hypoxia selects normal haematopoietic stem cells (HSC) and enhances their maintenance in vitro and that hypoxia is a characteristic feature of the "stem cell niche" microenvironment (1993-2002);
  2. the development of an innovative method (the Culture-Repopulating Ability - CRA - assay) to measure in vitro the capacity of HSC to reconstitute haematopoiesis in vivo; the CRA assay is suitable to assess the effects of treatments on short-term repopulating(STR)-HSC and is particularly useful to study human HSC, enabling their simple, rapid and economic detection (2000);
  3. the adaptation of CRA assay to detect leukaemia stem cells (LSC) of the activated (standardCRA) or quiescent (extendedCRA) types; this allows, on one hand, to study LSC under conditions (hypoxia) where their metabolical peculiarity, hypoxia-adaptation, is boosted, on the other hand, to interfere with their maintenance (for instance, by drug treatment) while keeping control of their functional state and position within the haematopoietic hierarchy (2006);
  4. the demonstration that incubation in severe hypoxia selects LSC from different types of leukaemias, and in particular BCR/Abl-independent and Imatinib-resistant LSC of CML; the possibility of monitoring this selection by the CRA assay enables a simple and efficient testing of drugs as for their specific effects on LSC (2006-2007).

Current work

  1. We are engaged in understanding how the expression of BCR/Abl protein is down-regulated in hypoxia-adapted CML cells, determining in particular whether the effector mechanism of suppression is transcriptional or post-translational; current emphasis is on establishing whether proteolytic degradation of BCR/Abl is involved and, if this is the case, whether a treatment preventing this degradation interfere with LSC maintenance in hypoxia.

Future plans
The follow-up of this research theme includes:

  1. to establish whether the expression of BCR/Abl protein, which is necessary to confer autonomous growth on CML progenitors, is on the contrary detrimental to LSC maintenance in hypoxia; it will be determined in particular whether hypoxia induces BCR/Abl accumulation in the nucleus, thereby triggering apoptogenic mechanisms;
  2. to undertake the characterization of mechanisms enabling the BCR/Abl-independent maintenance of LSC in hypoxia, trying to establish first whether these mechanisms are triggered by cytokines sustaining maintenance of HSC and antagonised by cytokines, such as IL3, boosting clonal expansion.

Research Theme 2 - Role of MAPK in survival, proliferation and apoptosis of epithelial neoplasias.
The main achievements in this field are:

  1. the demonstration that the MAPK MEK and ERK mediate the mitogenic effect of the v-fes oncogene product in macrophages (Blood 95:3959, 2000);
  2. the demonstration of a critical modulation of MEK and ERK activation in MCSF-stimulated macrophages based on the MCSF dose; ERK activation was increased by MCSF doses capable to elicit a mitogenic response, while 100-fold lower doses reduced ERK phosphorylation (via the involvement of cytosolic phosphatases) and nuclear content as well as cell proliferation; this was the first report that the same growth factor, based on its dose, can exert opposite effects on cell proliferation by switching on or off ERK signaling (Oncogene 21:3670, 2002);
  3. the demonstration that ERK5 is involved in the proliferative response of macrophages to M-CSF and that kinases of the Src family connect the ligand-induced activation of MCSF-receptor (MCSF-R) to that of ERK5 (J. Immunol. 180:4166, 2008);
  4. the demonstration that ERK5 differentially regulates PDGF-induced proliferation and migration of hepatic stellate cells (J. Hepatol. 48:107, 2007).

Current work and future plans

  1. We are engaged in transferring the experience accumulated in the above-mentioned studies to tha characterisation of the role of MAPK in survival, proliferation and apoptosis of gastric and hepatic carcinomas.
    These studies will be carried out in cooperation with the Unit coordinated by Prof. Fabio Marra.

Research Theme 3 - Role of MCSF and MCSF-dependent signaling in survival, proliferation and adhesion of neoplastic cells.
The main achievements in this field are:

  1. the demonstration that MCSF induces tyrosine phosphorylation of FAK in macrophages, and thereby cell spreading and adhesion, in a Src-dependent fashion, while the MAPK ERK and JNK inhibit this effect (Biol. Chem. 386:919, 2005);
  2. those listed for the Research Theme 2.

Current work and future plans

  1. We are engaged in transferring the experience accumulated in the above-mentioned studies to tha characterisation of the role of MCSF and MCSF-dependent signaling in survival, proliferation and adhesion of mammary carcinoma cells.
    These studies will be carried out in cooperation with the Unit coordinated by Prof. Luigi Cataliotti.

Research Theme 4 - Epigenetic modulation of survival and proliferation of myeloid leukaemia cells.The main achievements in this field are:

  1. the demonstration that the treatment with histone-deacetylase (HDAC) inhibitors (sodium butyrate; D1) restores histone acetylation, inhibits proliferation and triggers apoptosis in AML cells, but induces terminal granulocytic maturation only in t(8;21) AML cells, a subset of the so-called Core Binding Factor (CBF)-AML; this points to the possibility of using HDAC inhibitors as single drug in CBF-AML cases to effectively remove the maturation block due to the constitutive HDAC recruitment (Cancer Res. 63:8955, 2003);
  2. the demonstration that the JNK inhibitor SP600125, but not inhibitors of other MAPK, enhances the D1-induced growth reduction and apoptosis in an additive fashion; in non-t(8;21) AML cell lines, where D1 is ineffective, SP600125 alone significantly induces apoptosis and synergizes with D1, indicating that the combined administration of the two drugs is a very promising tool for AML treatment (Br. J. Haematol. 135:653, 2006).
  3. the demonstration that ITF2357, a new HDAC inhibitor, is ten times more powerful on t(8;21) than on non-t(8;21) AML cells in blocking proliferation and inducing apoptosis, indicating that ITF2357 is a very potent anti-leukemic agent, to be used especially at low doses to treat AML subtypes involving an abnormal HDAC activity (Oncogene 27:1767, 2008).

Current work and Future plans

  1. Characterization of the effects of hypoxia as inducer of apoptosis and epigenetic modulator in t(8;21) AML cells, to establish in particular if this modulation, if any, contributes to maintenance of stemness or, conversely, induces commitment and differentiation.

Fonti di finanziamento:

  1. "Characterisation and drug sensitivity of BCR/Abl-independent, Imatinib-resistant CML cells selected in hypoxia", finanziato per il 2009 dall'Associazione Italiana per la Ricerca sul Cancro.
  2. "Inhibition of BCR/Abl expression and induction of alternative antiapoptotic mechanisms in hypoxia-adapted / Imatinib-insensitive CML stem cells", finanziato per il 2008-2009 dall'Istituto Toscano Tumori.
  3. "Role of hypoxia in the selection and maintenance of normal and neoplastic haematopoietic stem cells", finanziato per il 2003-2005 dall'Istituto Superiore di Sanità, nell'ambito del Programma Nazionale Cellule Staminali (Area Tematica 1A - Convenzione CS64);
  4. "Role of microenvironment and epigenetical events in the regulation of leukaemia stem cell compartment" finanziato per il 2008-2010 dalla Federazione Italiana per la Ricerca sul Cancro (FIRC).
  5. "Ruolo del microambiente e di eventi epigenetici nella regolazione del compartimento cellulare staminale leucemico" finanziato per il 2007-2008 dalla FIRC.
  6. "Caratterizzazione delle cellule staminali BCR-Abl-negative e resistenti all'Imatinib della leucemia mieloide cronica e saggio della loro sensibilità all'Interferon-?" finanziato per il 2008 dall'Associazione Italiana Leucemie (AIL).
  7. "Modulazione in ipossia della espressione di BCR/Abl e di altri caratteri fenotipici in cellule di leucemia mieloide cronica" finanziato per il 2007 dall'AIL.
  8. "Ruolo dell'ipossia nel controllo della crescita cellulare nelle leucemie mieloidi", finanziato per il 2003-2004 dall'AIL.
  9. "Ruolo dell'ipossia e dell'oncogene fes nel controllo della crescita cellulare nella Leucemia Mieloide Cronica", finanziato per il 2002-2003 dall'AIL.

Publications

  1. Rovida E., Paccagnini A., Del Rosso M., Peschon J., Dello Sbarba P. Tumor Necrosis Factor- -Converting Enzyme cleaves the Macrophage Colony-Stimulating Factor Receptor in macrophages undergoing activation. J. Immunol. 166 1583-1589, 2001 (I.F.: 6,4).
  2. Torcia M.G., De Chiara G., Nencioni L., Ammendola S., Labardi D., Lucibello M., Rosini P., Marlier L.N.J.L., Bonini P., Dello Sbarba P., Palamara A.T., Zambrano N., Russo T., Garaci E., Cozzolino F. NGF inhibits apoptosis in memory B lymphocytes via inactivation of p38 MAPK, prevention of Bcl-2 phosphorylation and cytochrome-C release. J. Biol. Chem. 276 39027-39036, 2001 (I.F.: 5,9).
  3. Rovida E., Baccarini M., Olivotto M., Dello Sbarba P. Opposite effects of different doses of MCSF on ERK phosphorylation and cell proliferation in macrophages. Oncogene 21 3670-3676, 2002 (I.F.: 6,9).
  4. Rigacci S., Rovida E., Dello Sbarba P., Berti A. Low M(r) phosphotyrosine protein phosphatase associates with and dephosphorylates p125 focal adhesion kinase. J. Biol. Chem. 277 41631-41636, 2002 (I.F.: 5,9).
  5. Ivanovic Z., Belloc F., Faucher J.-L., Cipolleschi M.-G., Praloran V., Dello Sbarba P. Hypoxia maintains and IL3 reduces the pre-CFC potential of dividing CD34+ murine bone marrow cells. Exp. Hematol. 30 67-73, 2002 (I.F.: 4,0).
  6. Desplat V., Faucher J.-L., Mahon F.X., Dello Sbarba P., Praloran V., Ivanovic Z. Hypoxia modifies proliferation, CD34 and PAF-R expression in CD34+ CML cells. Stem Cells 20 347-354, 2002 (I.F.: 6,1).
  7. Chiarugi A., Rovida E., Dello Sbarba P., Moroni F. Tryptophan availability selectively limits NO-synthase induction in macrophages. J. Leukocyte Biol. 73 172-177, 2003 (I.F.: 4,6).
  8. Gozzini A., Rovida E., Dello Sbarba P., Santini V. Butyrates as single drug induce histone acetylation and granulocytic maturation: possible selectivity on core binding factor-acute myeloid leukemia blasts. Cancer Res. 63 8955-8961, 2003 (I.F.: 7,6).
  9. Rovida E, Lugli B, Barbetti V., Giuntoli S., Olivotto M., Dello Sbarba P. Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF. Biol. Chem. 386 919-929, 2005 (I.F.: 2,6).
  10. Annunziato F., Cosmi L., Liotta F., Lazzeri E., Romagnani P., Angeli R., Lasagni L., Manetti R., Marra F., Gerard C., Petrai I., Dello Sbarba P., Tonelli F., Maggi E., Romagnani S. CXCR3 and E 7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intra-epithelial lymphocytes. Gut 55 961-968, 2006 (I.F.: 7,7).
  11. Rovida E., Gozzini A., Barbetti V., Giuntoli S., Santini V., Dello Sbarba P. The c-Jun-N-terminal-Kinase inhibitor SP600125 enhances the butyrate derivative D1-induced apoptosis via caspase 8 activation in Kasumi-1 t(8;21) AML cells. Br. J. Haematol., 135 653-659, 2006 (I.F.: 4,1). 12) Giuntoli S., Rovida E., Barbetti V., Cipolleschi M.G., Olivotto M., Dello Sbarba P. Hypoxia suppresses BCR/Abl and selects Imatinib-insensitive progenitors within clonal CML populations. Leukemia 20 1291-1293, 2006 (I.F.: 6,6).
  12. Giuntoli S., Rovida E., Gozzini A., Barbetti V., Cipolleschi M.G., Olivotto M., Dello Sbarba P. Severe hypoxia defines heterogeneity and selects highly immature progenitors within clonal erythroleukaemia cells. Stem Cells 25 1119-1125, 2007 (I.F.: 6,1).
  13. Rovida E., Navari N., Caligiuri A., Dello Sbarba P., Marra F. ERK5 differentially regulates PDGF-induced proliferation and migration of hepatic stellate cells. J. Hepatol. 48 107-115, 2007 (I.F.: 6,6).
  14. Barbetti V., Gozzini A., Rovida E., Morandi A., Spinelli E., Fossati G., Mascagni P., Lübbert M., Dello Sbarba P., Santini V. Selective anti-leukemic activity of low dose histone deacetylase inhibitor ITF2357 on AML1/ETO-positive cells. Oncogene 27 1767-1778, 2008 (I.F.: 6,4).
  15. Rovida E., Spinelli E., Sdelci S., Barbetti V., Morandi A., Giuntoli S., Dello Sbarba P. ERK5/BMK1 is indispensable for optimal CSF-1-induced proliferation in macrophages in a Src-dependent fashion. J. Immunol. 180 4166-4172, 2008 (I.F.: 6,1).
  16. Olivotto M, Dello Sbarba P. Environmental restrictions within tumor ecosystems select for a convergent, hypoxia-resistant phenotype of cancer stem cells. Cell Cycle 7 176-187, 2008 (I.F.: 3,3).
  17. Rovida E, Dello Sbarba P. and cancer: Yang gets Yin. Cancer Biol. Ther. 7 1241-1242, 2008 (I.F.: 2,9).

Collaborations

  • Prof. Massimo Olivotto, Dipartimento di Patologia e Oncologia Sperimentali, Università degli Studi di Firenze (Research Theme 1);
  • Prof. Vincent Praloran Unversité Victor Segalen, Research Group "Stem Cells" UMR-CNRS 5164, Bordeaux (Research Theme 1);
  • Prof. Zoran Ivanovic, Etablissement Français du Sang, Bordeaux (Research Theme 1);
  • Prof. Fabio Marra, Dipartimento di Medicina Interna, Università degli Studi di Firenze (Research Theme 2);
  • Prof. Luigi Cataliotti, U.O. Chirurgia Generale II, Dipartimento di Medicina e Chirurgia Generale, Azienda Ospedaliero - Universitaria Careggi (AOUC), Firenze (Research Theme 3);
  • Dr. Angelo Di Leo, U.O. Oncologia Medica, Ospedale "Misericordia e Dolce", Prato (Research Theme 3);
  • Prof. Valeria Santini, Divisione di Ematologia, Università degli Studi di Firenze, AOUC, Firenze (Research Theme 4).

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