Regulation of Tumor Suppression by Endocytosis in Drosophila
Thomas Vaccari, Ph.D.
c/o IFOM-IEO Campus
Via Adamello, 16 - 20139 Milan, Italy
Tel: +39 02574303823
Our lab studies how endocytosis modulates signaling to the effect of controlling tissue growth and architecture in Drosophila epithelial organs. This interest springs from the fact that control of tissue growth and architecture is tumor suppressive and its alteration contributes to pathological states including malignant transformation. We are convinced that the understanding of the endocytic mechanisms that control tissue growth and architecture will lead to development of new and more effective anticancer treatments.
Fig.1: ESCRT mutant eye disc cells (non-green) are neoplastic-like and trap Notch (red) in endosomes. [+zoom]
The development of multicellular organisms requires orchestrated proliferation, migration, polarization, differentiation, and programmed death of groups of individual cells. The fate of each cell is genetically instructed and relies on coordinated cell-cell communication pathways for its execution. Not surprisingly, tight control of these pathways is required to suppress the emergence of tumors1. Therefore, the investigation of how cells regulate signaling programs is critical to understanding tumor etiology. Unfortunately, difficulty to perform forward genetic analyses and functional redundancy hampers such studies in mammalian tissues. Drosophila is a simple and powerful system to study signaling and its relation to tumorigenesis, due to the conservation of cancer-associated genes and of mechanisms that ensure proper tissue architecture and growth, and to the ability to screen for new genes involved in a given biological pathway2-4.
Fig.2: Drosophila larvae containing WT epithelial disc monolayers or tumorous discs (left and right respectively; insets show disc sections). [+zoom]
Endocytosis, the process by which the cell internalizes and traffics diverse sets of molecules, is a potent regulator of signal transduction5. Indeed, we and others have found that in Drosophila, some endocytic genes act as neoplastic tumor suppressor genes (TSGs) by virtue of their ability to regulate signaling6. A specific class of conserved endocytic genes, the ESCRT genes, control trafficking and lysosomal degradation of ubiquitinated transmembrane proteins, including the signaling receptor Notch. In wild-type cells (WT), transduction of Notch signals is coupled to receptor trafficking and degradation, and requires ligand binding. However, inactivation of ESCRT function in epithelial cells leads to Notch accumulation in endosomes, resulting in ligand-independent activation of Notch that resembles a type of deregulation frequently associated with oncogenic Notch activity7-8. ESCRT mutant cells also lose growth control and epithelial architecture in a manner that recapitulates aspects of malignant transformation9-11 (Fig. 1). This leads to the formation of tumors, which display several hallmarks of vertebrate cancer (Fig. 2). The cellular and molecular mechanisms underlying ligand-independent Notch activation and the formation of endocytic TSG tumors remain largely unknown.
1. Endocytic Trafficking Routes and Notch Signaling Activation.
Following up on the study of endocytic trafficking and Notch signaling activation8, we found that mutants in genes that are thought to act at the same step of endocytosis can affect Notch signaling activation very differently. In addition, different forms of Notch appear to traffic differently. This preliminary evidence suggests a scenario in which distinct pools of Notch are trafficked via alternative internalization routes with different potential for physiological signaling activation. We are using trafficking assays, activation assays and biochemical cleavage assays to test the hypothesis that ligand-activated Notch follows a different internalization route compared to inactive Notch.
2. The Role of the Endosomal Environment in Notch Signaling.
We recently found that vacuolar ATPase (v-ATPase) function is essential for Notch activation and its proliferative effects in Drosophila. v-ATPases are required for acidification of the endosomal compartment, suggesting that this process potently modulates Notch signaling activation. Thus, we are currently characterizing the role of v-ATPases during signal transduction.
3. Pathological Notch Signaling and Tumorigenesis.
ESCRT mutant tissue displays ligand-independent Notch activation. This observation is relevant to human neoplasia, since some leukemia-associated Notch mutations display such pathological type of signaling activation7. Thus, the study of ligand-independent Notch activation in ESCRT mutant might uncover mechanisms of signaling potentially important to understand the etiology of Notch malignancies. To gain further insight into the cellular mechanisms underlying ligand-independent Notch activation, we are taking advantage of the ease of RNA interference approaches in Drosophila tissue culture cells. We are planning to use established dsRNA libraries to screen cells engineered to display ligand-independent Notch activation. This unbiased approach will identify molecules that mediate pathological Notch signaling.
4. Endocytosis as a master regulator of signaling pathways and tumor suppression.
Drosophila cells lacking ESCRT function are neoplastic and under certain conditions metastatic9-10. Considering that in Drosophila overactivation of Notch signaling leads to excess tissue growth but per se is not sufficient to generate a neoplastic phenotype, ESCRT mutants are likely to mistraffic other molecules, potentially leading to synergistic effects with Notch. Are other signaling pathways contributing to the ESCRT and, more generally, endocytic nTSG phenotypes? To test this hypothesis, we are assaying known signaling pathways for mistrafficking and either down- or up-regulation of signaling activity in endocytic TSG mutant tissue. As a complement to this approach, we are also taking an unbiased, forward genetic approach to identify modulators of ESCRT activity. Both these approaches will lead to the identification of novel pathways of endocytic tumor suppression.
5. Fly Tumor suppressors and human cancers.
Since Drosophila endocytic TSGs are evolutionarily conserved, do they also function as TSGs in mammals? As a long-term goal, we are interested testing whether the functions of mammalian homologs of fly endocytic TSGs are altered in human cancers.
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- Vaccari T, Bilder D. At the crossroads of polarity, proliferation and apoptosis: the use of Drosophila to unravel the multifaceted role of endocytosis in tumor suppression. Mol Oncol. 2009 Aug;3(4):354-65. Epub 2009 Jun 6.
- Malecki, M. et al. Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol Cell Biol 26, 4642-4651 (2006).
- Vaccari, T. et al. Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. The Journal of cell biology 180, 755-762 (2008).
- Thompson, B. et al. Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Dev Cell 9, 711-720 (2005).
- Vaccari, T. & Bilder, D. The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking. Dev Cell 9, 687-698 (2005).
- Vaccari, T. et al. Comparative genetic, cell biological and developmental analysis of metazoan ESCRT function by efficient isolation of Drosophila ESCRT-I, -II, -III mutants. J Cell Sci., Jul 15;122(Pt 14):2413-23 (2009).