Our multidisciplinary lab hosts projects in the fields of endocytosis, stem cells and functional genomics.
Our long-standing goal is to clarify the role of endocytosis in cell signaling and to understand its impact in cancer. Over recent years, the perception of endocytosis has changed from that of a simple mechanism for signal attenuation to that of a vital component of the cell signaling machinery, providing spatial and temporal dimensions to signaling events [1, 2]. Our endocytosis projects span from mapping EGFR endocytic pathways biologically and biochemically, to understanding the functional significance of endocytosis in normal physiological processes and in disease through a systems biology approach. These projects have catalyzed a number of high-resolution studies of endocytic proteins that appear to be particularly relevant to tumorigenesis, such as Eps15 and Numb, a known tumor suppressor in breast cancer [3, 4].
Numb is also a known regulator of Notch, a protein implicated in stem cell maintenance, and together, these observations have driven the development of new stem cell research projects, spanning the transcriptional profiling of normal and cancer stem cells , the in-depth characterization of critical signaling/endocytic pathways involving Numb/Notch/p53 , and the study of mechanisms of stem cell division and their relevance to cancer.
Finally, we are applying various state-of-the-art whole-genome approaches (coding and non-coding RNA profiling, next generation sequencing, proteomics) to identify new cancer-specific profiles and druggable targets that can be employed in the clinic as diagnostic/prognostic/therapeutic tools. This last group of studies has fed a number of translational projects that were transferred to the IEO Molecular Medicine for Care Program, also under the directorship of Pier Paolo Di Fiore.
Endocytosis, signaling and cancer
Signaling by the EGFR family of receptors is commonly deregulated in cancer. The internalization route of the EGFR is decisive in determining receptor fate and signaling outcomes. Depending on ligand concentration, EGFR internalization may occur either via clathrin-mediated endocytosis (CME) or via the non-clathrin mediated endocytosis (NCE) more recently identified by us [7-9]. CME is involved in receptor recycling and signal amplification, while the NCE pathway targets the EGFR for degradation, causing signal suppression. So NCE could potentially be a novel tumor suppressor pathway, while CME, which sustains EGFR signaling, might be an oncogenic pathway.
To establish the true pathological relevance of endocytosis we are using classical biochemistry and cell biology approaches to define the full molecular details and functional significance of the novel NCE pathway. However, the integration of the NCE and CME networks is impossible to understand without the help of a systems level approach. Therefore, we also have projects that combine mathematical modeling and wet-lab experiments, to define the parameters that trigger the switch between the two EGFR endocytic pathways. Finally, thanks to the Molecular Medicine for Care Program we have access to the necessary resources for preclinical research into the pathological relevance of EGFR endocytic pathways in cancer.
Stem cells and cancer
There is a general consensus that cancer stem cells (CSCs) are the driving force of some (if not many) tumors, including breast cancer. Only a minor fraction of the cells within a tumor are CSCs, and these possess stem cell (SC)-like properties including self-renewal, quiescence and unlimited replication potential. While the SC compartment is tightly controlled in normal tissues, the CSC compartment appears to be expanded in breast cancers: more aggressive, poorly differentiated tumors have a larger proportion of CSCs than less aggressive, more differentiated tumors . The mechanisms governing CSC amplification remain unclear, but SC homeostasis depends on the continued asymmetric division of SCs to produce a single quiescent SC and a differentiating progenitor. This guarantees a constant population of SCs over time. Symmetric SC division, instead, produces two stem daughter cells, and increases SC numbers. Notch, p53 and the endocytic protein Numb are implicated in stem cell self-renewal and asymmetric cell division. We have shown that Numb controls p53 ubiquitination and degradation  and that Notch signaling is increased in Numb-deficient tumors . Various projects in the lab aim to characterize the involvement of these three players in stem cell self-renewal mechanisms and in tumorigenesis.
Numb is asymmetrically distributed during normal breast SC division, but its dynamics of distribution are altered in CSCs. By analogy with Numb, the discovery of other asymmetrically distributed proteins may shed light on molecular mechanisms for normal SC homeostasis. We are therefore characterizing the proteomic profile of asymmetrically partitioned proteins during normal SC division to identify new cancer-relevant candidates for subsequent high-resolution studies.
A simple search of the scientific literature highlights innumerable expression profiles describing any number of normal and pathological states. While these gene signatures have given an invaluable contribution to our scientific body of knowledge, their direct clinical applications have been few and far between. The studies published by others and by us [5, 10-18], have driven a number of high-resolution studies currently conducted in our lab on individual markers that potentially have significant clinical relevance as prognostic or therapeutic targets. Furthermore, some of our studies, in particular a lung cancer diagnostic signature and a stem cell expression profile, have been transferred to the Molecular Medicine for Care Program, for the development of new clinical applications. The know-how acquired through this work is also allowing us to perform new, independent studies on miRNA profiles in breast and ovarian cancers, to develop blood tests for their early diagnosis.
Associazione Italiana per la Ricerca sul Cancro – AIRC
Fondazione Italiana per la Ricerca sul Cancro – FIRC
Ministero dell’Istruzione, dell’Università e della Ricerca –MIUR (Italian Ministry of Education, University and Research)
Ministero della Salute (Italian Ministry of Health)
European Community Framework Programme 7 (FP7)
Fondazione Istituto Europeo di Oncologia – FIEO
Fondazione Umberto Veronesi – FUV
1. Scita, G. and P.P. Di Fiore, The endocytic matrix. Nature, 2010. 463(7280): p. 464-73.
2. Sigismund, S., S. Confalonieri, A. Ciliberto, S. Polo, G. Scita, and P.P. Di Fiore, Endocytosis and signaling: cell logistics shape the eukaryotic cell plan. Physiological reviews, 2012. 92(1): p. 273-366.
3. Pece, S., S. Confalonieri, R.R. P, and P.P. Di Fiore, NUMB-ing down cancer by more than just a NOTCH. Biochimica et biophysica acta, 2011. 1815(1): p. 26-43.
4. Pece, S., M. Serresi, E. Santolini, M. Capra, E. Hulleman, V. Galimberti, S. Zurrida, P. Maisonneuve, G. Viale, and P.P. Di Fiore, Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. The Journal of cell biology, 2004. 167(2): p. 215-21.
5. Pece, S., D. Tosoni, S. Confalonieri, G. Mazzarol, M. Vecchi, S. Ronzoni, L. Bernard, G. Viale, P.G. Pelicci, and P.P. Di Fiore, Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell, 2010. 140(1): p. 62-73.
6. Colaluca, I.N., D. Tosoni, P. Nuciforo, F. Senic-Matuglia, V. Galimberti, G. Viale, S. Pece, and P.P. Di Fiore, NUMB controls p53 tumour suppressor activity. Nature, 2008. 451(7174): p. 76-80.
7. Sigismund, S., V. Algisi, G. Nappo, A. Conte, R. Pascolutti, A. Cuomo, T. Bonaldi, E. Argenzio, L.G. Verhoef, E. Maspero, F. Bianchi, F. Capuani, A. Ciliberto, S. Polo, and P.P. Di Fiore, Threshold-controlled ubiquitination of the EGFR directs receptor fate. EMBO J, 2013.
8. Sigismund, S., E. Argenzio, D. Tosoni, E. Cavallaro, S. Polo, and P.P. Di Fiore, Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Developmental cell, 2008. 15(2): p. 209-19.
9. Sigismund, S., T. Woelk, C. Puri, E. Maspero, C. Tacchetti, P. Transidico, P.P. Di Fiore, and S. Polo, Clathrin-independent endocytosis of ubiquitinated cargos. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(8): p. 2760-5.
10. Bianchi, F., J. Hu, G. Pelosi, R. Cirincione, M. Ferguson, C. Ratcliffe, P.P. Di Fiore, K. Gatter, F. Pezzella, and U. Pastorino, Lung cancers detected by screening with spiral computed tomography have a malignant phenotype when analyzed by cDNA microarray. Clinical cancer research : an official journal of the American Association for Cancer Research, 2004. 10(18 Pt 1): p. 6023-8.
11. Bianchi, F., F. Nicassio, M. Marzi, E. Belloni, V. Dall'olio, L. Bernard, G. Pelosi, P. Maisonneuve, G. Veronesi, and P.P. Di Fiore, A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer. EMBO molecular medicine, 2011. 3(8): p. 495-503.
12. Bianchi, F., P. Nuciforo, M. Vecchi, L. Bernard, L. Tizzoni, A. Marchetti, F. Buttitta, L. Felicioni, F. Nicassio, and P.P. Di Fiore, Survival prediction of stage I lung adenocarcinomas by expression of 10 genes. The Journal of clinical investigation, 2007. 117(11): p. 3436-44.
13. Capra, M., P.G. Nuciforo, S. Confalonieri, M. Quarto, M. Bianchi, M. Nebuloni, R. Boldorini, F. Pallotti, G. Viale, M.L. Gishizky, G.F. Draetta, and P.P. Di Fiore, Frequent alterations in the expression of serine/threonine kinases in human cancers. Cancer research, 2006. 66(16): p. 8147-54.
14. Confalonieri, S., M. Quarto, G. Goisis, P. Nuciforo, M. Donzelli, G. Jodice, G. Pelosi, G. Viale, S. Pece, and P.P. Di Fiore, Alterations of ubiquitin ligases in human cancer and their association with the natural history of the tumor. Oncogene, 2009. 28(33): p. 2959-68.
15. Luise, C., M. Capra, M. Donzelli, G. Mazzarol, M.G. Jodice, P. Nuciforo, G. Viale, P.P. Di Fiore, and S. Confalonieri, An atlas of altered expression of deubiquitinating enzymes in human cancer. PloS one, 2011. 6(1): p. e15891.
16. Nicassio, F., F. Bianchi, M. Capra, M. Vecchi, S. Confalonieri, M. Bianchi, D. Pajalunga, M. Crescenzi, I.M. Bonapace, and P.P. Di Fiore, A cancer-specific transcriptional signature in human neoplasia. The Journal of clinical investigation, 2005. 115(11): p. 3015-25.
17. Vecchi, M., S. Confalonieri, P. Nuciforo, M.A. Vigano, M. Capra, M. Bianchi, D. Nicosia, F. Bianchi, V. Galimberti, G. Viale, G. Palermo, A. Riccardi, R. Campanini, M.G. Daidone, M.A. Pierotti, S. Pece, and P.P. Di Fiore, Breast cancer metastases are molecularly distinct from their primary tumors. Oncogene, 2008. 27(15): p. 2148-58.
18. Vecchi, M., P. Nuciforo, S. Romagnoli, S. Confalonieri, C. Pellegrini, G. Serio, M. Quarto, M. Capra, G.C. Roviaro, E. Contessini Avesani, C. Corsi, G. Coggi, P.P. Di Fiore, and S. Bosari, Gene expression analysis of early and advanced gastric cancers. Oncogene, 2007. 26(29): p. 4284-94.