Endocytosis, Signalling and Cancer
Pier Paolo Di Fiore
c/o IFOM-IEO Campus
Via Adamello, 16 - 20139 Milan, Italy
Tel: +39 02 574303257 - Fax: +39 02 94375991
Our lab is performing both basic and translational cancer research in the fields of
endocytosis, stem cells and functional genomics.
A long-standing goal of our group is to elucidate the role of endocytosis in cell signalling and cancer. Our contributions to this field have helped to change the way endocytosis is perceived, from being simply a mechanism for signal attenuation to being a vital component of the cell signalling machinery that provides spatial and temporal dimensions to signalling events (1). Currently, our ongoing endocytosis projects are aimed at elucidating the molecular mechanisms governing endocytosis of the EGFR, mapping the endocytic interactome and understanding the functional significance of endocytosis in normal physiological processes and in disease, particularly in cancer.
More recently, our studies on the endocytic protein, Numb, have revealed that it is a tumour suppressor in breast cancer (2). 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, ranging from transcriptional profiling analysis of normal and cancer stem cells, to the in-depth characterisation of a number of critical signalling/endocytic pathways involving Numb/Notch/p53 (2, 3).
Finally, our functional genomics-based projects exploit a range of high-throughput technologies to identify new cancer-specific signatures and druggable targets that can be employed in the clinic as diagnostic/prognostic/therapeutic tools.
ENDOCYTOSIS IN SIGNALLING AND CANCER
Endocytosis has traditionally been viewed as a mechanism for achieving signal attenuation: activated cell surface receptors are internalised and trafficked through endocytic vesicles to lysosomes, where they are degraded. This rather simplistic view is now changing with emerging evidence that endocytosis is an integral component of the cell signalling machinery, regulating aspects such as the duration, intensity, integration and spatial distribution of signals. We have several ongoing projects designed to uncover the molecular mechanisms that regulate endocytosis, and to determine its involvement in normal cellular processes and cancer.
Receptor-mediated endocytosis. Cell surface receptors can be endocytosed by both clathrin-dependent and clathrin-independent mechanisms, but, as yet, there is no clear understanding of why alternative endocytic routes exist or how receptors are committed to a particular route. We are using the epidermal growth factor receptor (EGFR) as a model 'endocytic system' to address these issues. This kinase receptor has important clinical relevance, as EGFR deregulated signalling is strongly associated with cancer.
Figure 1. The entry route of the EGFR depends on its Ub status, which is determined by the intensity of EGF stimulation. [+zoom]
The EGFR has long been known to be internalised by clathrin-mediated endocytosis (CME), upon its activation by ligand. However, we have recently discovered that, depending on the concentration of ligand, the EGFR may also be internalised via an alternative clathrin-independent/lipid raft-dependent endocytic route: non-clathrin endocytosis (NCE) (4). Internalisation by NCE is regulated by receptor ubiquitination and commits the EGFR to degradation. In contrast, internalisation by CME results in recycling of EGFR to the cell surface and sustained EGFR signalling (5) [Fig. 1]. Thus, the endocytic machinery appears to act as a 'sensor gauge' capable of triggering diverse biological processes depending on the intensity of the stimulus received by the cell. This allows cells to respond dynamically to the constantly fluctuating environment that surrounds them under physiological conditions. Current projects are designed to obtain a complete molecular characterisation of these endocytic pathways.
We are also investigating endocytosis of the cell surface receptor, Notch, a protein involved in cell fate specification (6). Notch activity is antagonised by the cell fate determinant Numb, which asymmetrically partitions at mitosis and 'switches off' Notch signalling in one of the daughter cells (7). Because Numb is an endocytic protein, we hypothesize that Notch antagonism may be mediated by Numb-dependent endocytic events (8). We are currently defining the mechanistic role of Numb in the endocytosis of Notch.
Figure 2. EH-mediated interactions are involved in many different biological processes - Polo et al. (2003) Sci STKE 2003, re17 [+zoom]
Mapping the physical and functional interactions of the endocytic machinery. Our group has discovered two protein-protein interaction networks that sit at the heart of numerous endocytic and trafficking processes: the EH (Eps15 Homology) and monoUb networks (9-13). The EH network relies on the interaction between an EH domain in one protein and an EH binding motif, such as NPF, in another protein [Fig. 2]. The monoUb network, on the other hand, is based on the interaction of monoubiquitinated proteins with monoUb-binding domains, such as the Ub-interacting motif (UIM). We are using high-throughput approaches in combination with classical high resolution genetic and biochemical studies in various model organisms, to map the physical and functional interactions of the endocytic machinery via these networks. These studies should shed light on the interplay between the endocytic machinery and other cellular components and help us to understand the role of endocytosis in diverse cellular processes. We already have evidence that endocytosis is involved in the spatial restriction of motogenic signals, leading to localised actin remodelling in response to growth factor stimulation (14). These results are relevant to our understanding of cell migration and metastasis.
Endocytosis and cancer. We are investigating whether defects in the endocytic machinery play a causative role in cancer (15). We are performing screening experiments at the genomic and protein level to determine if endocytic proteins, or their regulators (e.g. kinases, E3 ligases, deubiquitinases), are aberrantly expressed in cancer. We use tissue microarrays that contain normal and tumour tissue samples for high-throughput screenings and we have identified various endocytic proteins that are deregulated in cancer (16). We are characterising these through high-resolution studies.
Figure 3 . Numb expression is clearly visible in normal mammary ducts (N). Expression is lost upon tumorigenesis (T). [+zoom]
One such protein is Numb (9).
We observed that Numb expression is lost in ~50 % of breast cancers, due to its increased ubiquitination/degradation (2) [Fig.
3]. Numb-negative breast cancers display enhanced Notch signalling and an overall poorer prognosis when compared with Numb-positive cancers; hence, we have suggested that Numb is a novel tumour suppressor. Recently, we discovered that loss of Numb also causes decreased p53 activity in breast cancers (3). Therefore, Numb controls both an oncogenic pathway (the Numb:Notch axis) and a tumour suppressor pathway (the Numb:p53 axis). Ongoing projects aim to define the involvement of these two signalling axes in cancer and to investigate whether the endocytic function of Numb is relevant to its tumour suppressor function.
Finally, we are undertaking a vast mutational analysis of endocytic proteins in cancer, to define genetic alterations affecting the endocytic machinery.
CANCER STEM CELLS
The stem cell theory of cancer hypothesizes that 'stem-like' cancer cells are responsible for the generation of tumours and for sustaining tumour growth (reviewed in ref.17). This theory predicts the existence of cancer stem cells (CSCs) that possess properties characteristic of normal stem cells, such as self-renewal, multipotency and quiescence. We are investigating the molecular mechanisms governing maintenance of the stem cell compartment in normal tissues and how these mechanisms are subverted in cancer. Both Notch and p53 signalling are implicated in stem cell self-renewal (18, 19), and the endocytic protein Numb is connected to both these signalling pathways. Therefore, we are investigating the involvement of the Numb/Notch/p53 interaction and endocytic pathways in stem cell self-renewal.
Figure 4. The cartoon depicts a mammosphere, containing a single labelled breast stem cell (red). Both normal and cancer breast stem cells give rise to 3D mammospheres in suspension culture. [+zoom]
The existence of cancer stem cells has been amply demonstrated for a number of cancer types, including breast cancer (20, 21). However, the breast stem cell compartment remains poorly characterized due to the lack of reliable techniques for their identification and isolation. We have developed a new technique to specifically label and purify breast stem cells from mammary gland tissue. This technique exploits both the propensity of breast stem cells to generate mammospheres (3D clusters of cells) in suspension culture (21), and the relative quiescence of stem cells compared to other breast cell types, when propagated in vitro [Fig. 4]. We are using purified normal and cancer stem cells to isolate a "stemness" signature from which we can extract diagnostic, prognostic and therapeutic markers that will then be evaluated for testing in clinical trials. We are also studying the mechanisms of asymmetric cell division. In particular, we are investigating whether, and how, Numb and endocytosis-based mechanisms are involved in the regulation of asymmetric cell division of human breast stem cells.
In recent years, many labs have dedicated enormous resources to identifying new molecular cancer markers through 'unbiased' direct profiling of human cancers using DNA microarray approaches. However, these approaches have not yet delivered all that they promised, partly due to the inherent genetic variability between patient samples, but also because the diagnostic/prognostic accuracy of the cancer signatures identified tends to be linked to the patient cohorts from which those signatures were obtained. When these signatures are applied to other independent cohorts, their accuracy can decrease dramatically, which limits their clinical usefulness (reviewed in ref. 22). 'Biased' profiling of experimental model systems of cancer yields cleaner signatures as a result of the homogeneous genetic background. These systems are, however, limited to a single oncogenic event and therefore fail to capture the complex repertoire of alterations of real tumours.
Figure 5. An integrated approach to identify novel cancer signatures [+zoom]
We have developed a novel 'integrated' approach for gene expression profiling analysis that combines both 'unbiased' and 'biased' profiling (22) [Fig. 5]. Our ‘biased’ profiling is based on an in vitro model of mouse terminally differentiated myotubes, induced to re-enter the cell cycle by the E1A oncogene (23). E1A is the sole oncogene capable of overcoming the stringent proliferative block connected with terminal differentiation, likely due to E1A-induced mimicry of important proliferation and dedifferentiation pathways, which are prime candidates for cancer pathways (24). In the past, we have identified a candidate cancer signature in this model system, and validated it against "real" human cancers (23). Recently, we have successfully applied our integrated approach to identify a 10-gene prognostic signature for early stage lung cancer (25). This signature allows the identification of those patients who have a high risk of developing metastasis and who, therefore, might benefit from adjuvant chemotherapy. We are currently planning a clinical trial to evaluate the clinical validity of our signature.
- Polo, S., and Di Fiore, P. P. (2006) Endocytosis conducts the cell signaling orchestra. Cell 124, 897-900 [PubMed]
- Pece, S., Serresi, M., Santolini, E., Capra, M., Hulleman, E., Galimberti, V., Zurrida, S., Maisonneuve, P., Viale, G., and Di Fiore, P. P. (2004) Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. J Cell Biol 167, 215-221 [PubMed] [pdf]
- Colaluca, I. N., Tosoni, D., Nuciforo, P., Senic-Matuglia, F., Galimberti, V., Viale, G., Pece, S., and Di Fiore, P. P. (2008) NUMB controls p53 tumour suppressor activity. Nature 451, 76-80 [PubMed]
- Sigismund, S., Woelk, T., Puri, C., Maspero, E., Tacchetti, C., Transidico, P., Di Fiore, P. P., and Polo, S. (2005) Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A 102, 2760-2765 [PubMed] [pdf]
- Sigismund, S., Argenzio, E., Tosoni, D., Cavallaro, E., Polo, S., and Di Fiore, P. P. (2008) Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev Cell 15, 209-219 [PubMed]
- Lai, E. C. (2004) Notch signaling: control of cell communication and cell fate. Development 131, 965-973
- Roegiers, F., and Jan, Y. N. (2004) Asymmetric cell division. Curr Opin Cell Biol 16, 195-205
- Santolini, E., Puri, C., Salcini, A. E., Gagliani, M. C., Pelicci, P. G., Tacchetti, C., and Di Fiore, P. P. (2000) Numb is an endocytic protein. J Cell Biol 151, 1345-1352 [PubMed] [pdf]
- Santolini, E., Salcini, A. E., Kay, B. K., Yamabhai, M., and Di Fiore, P. P. (1999) The EH network. Exp Cell Res 253, 186-209 [PubMed]
- Confalonieri, S., and Di Fiore, P. P. (2002) The Eps15 homology (EH) domain. FEBS Lett 513, 24-29 [PubMed]
- Di Fiore, P. P., Polo, S., and Hofmann, K. (2003) When ubiquitin meets ubiquitin receptors: a signalling connection. Nat Rev Mol Cell Biol 4, 491-497 [PubMed]
- Polo, S., Confalonieri, S., Salcini, A. E., and Di Fiore, P. P. (2003) EH and UIM: endocytosis and more. Sci STKE 2003, re17 [PubMed]
- Sigismund, S., Polo, S., and Di Fiore, P. P. (2004) Signaling through monoubiquitination. Curr Top Microbiol Immunol 286, 149-185 [PubMed]
- Palamidessi, A., Frittoli, E., Garre, M., Faretta, M., Mione, M., Testa, I., Diaspro, A., Lanzetti, L., Scita, G., and Di Fiore, P. P. (2008) Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 134, 135-147 [PubMed]
- Polo, S., Pece, S., and Di Fiore, P. P. (2004) Endocytosis and cancer.Curr Opin Cell Biol 16, 156-161 [PubMed]
- Capra, M., Nuciforo, P. G., Confalonieri, S., Quarto, M., Bianchi, M., Nebuloni, M., Boldorini, R., Pallotti, F., Viale, G., Gishizky, M. L., Draetta, G. F., and Di Fiore, P. P. (2006) Frequent alterations in the expression of serine/threonine kinases in human cancers. Cancer Res 66, 8147-8154 [PubMed] [pdf]
- Wicha, M. S., Liu, S., and Dontu, G. (2006) Cancer stem cells: an old idea--a paradigm shift. Cancer Res 66, 1883-1890; discussion 1895-1886
- Farnie, G., and Clarke, R. B. (2006) Breast stem cells and cancer. Ernst Schering Found Symp Proc, 141-153
- Korkaya, H., and Wicha, M. S. (2007) Selective targeting of cancer stem cells: a new concept in cancer therapeutics. BioDrugs 21, 299-310
- Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., and Clarke, M. F. (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100, 3983-3988
- Dontu, G., Abdallah, W. M., Foley, J. M., Jackson, K. W., Clarke, M. F., Kawamura, M. J., and Wicha, M. S. (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17, 1253-1270
- Bianchi, F., Nicassio, F., and Di Fiore, P. P. (2008) Unbiased vs. biased approaches to the identification of cancer signatures: the case of lung cancer. Cell Cycle 7, 729-734 [PubMed]
- Nicassio, F., Bianchi, F., Capra, M., Vecchi, M., Confalonieri, S., Bianchi, M., Pajalunga, D., Crescenzi, M., Bonapace, I. M., and Di Fiore, P. P. (2005) A cancer-specific transcriptional signature in human neoplasia. J Clin Invest 115, 3015-3025 [PubMed] [pdf]
- Crescenzi, M., Soddu, S., Sacchi, A., and Tato, F. (1995) Adenovirus infection induces reentry into the cell cycle of terminally differentiated skeletal muscle cells. Ann N Y Acad Sci 752, 9-18
- Bianchi, F., Nuciforo, P., Vecchi, M., Bernard, L., Tizzoni, L., Marchetti, A., Buttitta, F., Felicioni, L., Nicassio, F., and Di Fiore, P. P. (2007) Survival prediction of stage I lung adenocarcinomas by expression of 10 genes. J Clin Invest 117, 3436-3444 [PubMed] [pdf]