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Endocytosis represents a fundamental process in the biology of the cell, with roles spacing from proliferation and migration to cell fate determination. In particular endocytosis has been tightly connected to cell signal regulation. Ligand-mediated endocytosis is indeed typical of signaling receptors, such as receptor tyrosine kinases (RTKs).
Previous studies in Di Fiore laboratory have shown that different endocytic routes are associated with distinct receptor fates (e.g., degradation vs recycling) and consequently have an impact on the net signaling output. In details, they have established that ligand-induced internalization of the RTK Epidermal Growth Factor Receptor (EGFR) can be endocytosed through different entry routes depending on ligand concentration. At low doses of EGF, the receptor is not ubiquitinated and internalized exclusively through clathrin-mediated endocytosis (CME). At higher concentrations of ligand a substantial fraction of the receptor becomes ubiquitinated and is endocytosed through non-clathrin endocytosis (NCE). Importantly, the two pathways have distinct receptor functions: CME is mainly involved in receptor recycling and signalling, while NCE targets the majority of the receptors to degradation.
It is not surprising that an altered CME to NCE ratio could be related to aberrant pathological conditions, such as cancer.
A quantitative proteomic approach allowed the identification of a list of molecular components of NCE. I am involved in the functional validation of the selected players by large-scale RNA interference and colocalization analysis with the EGFR. The most promising candidates, able to selectively affect NCE, will be further investigated to highlight their mechanism of action in this process.
This approach could allow us to identify negative regulators of EGFR of potential interest for anti-cancer therapies.
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Cohesion is established in S phase by a well-conserved protein complex called cohesin (composed of SMC1, SMC3, SCC1, SCC3), deposited in S phase by SCC2 and SCC4, which function as a cohesin deposition complex. Experiments using diverse model organisms have provided compelling evidence for the critical function of cohesin-related proteins in genome integrity. In addition, the existence of human genetic disorders known as “cohesinopathies” clearly shows the critical relationship between cohesin and genome integrity. In this project, I use genetically amenable chicken DT40 cells as a model system to establish cohesinopathies resemble cell-lines and examine whether these mutations result in defects in specific DNA damage response pathway. This study will help us understand the genetic pathways that contribute to the chromosome aberrations of these cells. The present line of research has potential implications for cancer biology and therapy.
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Leukemias, as other tumors, are abnormal tissues organized in a hierarchical way, in which a small fraction of quiescent leukemic stem cells is able to maintain the entire tumor. Our goal is to find a therapy able to knock out cancer stem cells because with cancer stem cells gone the tumor can be eradicated.
Previous observations from our laboratory demonstrate that the expression of the cell-cycle inhibitor p21 is crucial to provide leukemia stem cells with an unlimited self-renewal capacity because p21 activation leads to reversible cell cycle arrest and DNA repair. Expression of leukemia-associated oncogenes in normal hematopoietic SCs (HSCs) induces DNA damage and activates a p21-dependent cellular response that, in turn, imposes cell-cycle restriction and triggers repair of the damaged DNA (Viale A. et al., Nature 51, 457, 2009). This effect of p21 prevents the physiological exhaustion of HSC self-renewal, which occurs in time owing to accumulation of DNA damage, and confers an advantage to HSCs when they hyper-proliferate, thus explaining the role of p21 and DDR (DNA Damage Repair) in the maintenance of the self-renewal potential of LSCs (Leukemia Stem Cells).
These findings imply that cell-cycle-restricted LSCs are critical for the initiation and/or maintenance of the leukemic clone, suggesting that targeting this compartment might be critical to disease eradication, and suggest that inhibition of DNA repair might be synthetic lethal with oncogene expression. Therefore our goal is to validate p21 and p21-mediated DNA-repair pathways as molecular targets for leukemia treatment.
In order to explore DNA-repair pathways as anti-leukaemic targets we study the effect of drugs inhibiting the DDR, administered as single agent or in combination with DNA-damage-inducing drugs, in leukaemia. We already obtained some encouraging data with a new combined therapy that specifically induces DNA damage and apoptosis in Acute Myeloid Leukemia via targeting S-phase.
It is envisaged that these studies will yield insights into the mechanisms of how DNA-repair- pathways underlie leukemia fate and the regulation of LSC-specific phenotypes providing the basis for therapeutic approaches based on LSC eradication.
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My current project focuses on the characterization of the structure/function relationships of the long pentraxin PTX3, an important component of the humoral arm of innate immunity that plays additional roles at the crossroad between matrix remodelling, vascular biology and inflammation. A major goal of my research is understanding how glycosylation affects PTX3 biology, with specific regard to the protein's function in innate immunity and inflammation. I also aim at elucidating the role that glycosaminoglycans (i.e., main components of the extracellular matrix) play in the interaction between PTX3 and the Fibroblast Growth Factor 2, a key event in PTX3 activity in vascular biology.
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A correlation between aberrant microRNA (miRNA, miR) expression and cancer has been frequently reported. The miR17-92 cluster (composed of miR17, miR18, miR19a, miR19b, miR20a and miR92) is located in a genomic region amplified in several human B-cell lymphomas. In a mouse B-cell lymphoma model, enforced expression of the miR17–92 cluster acts synergistically with cMyc to accelerate tumor development. Of the few targets identified so far, a significant portion is cancer-related. We employ a functional genomics approach combining SILAC-based quantitative proteomics and transcriptomics to extend the analysis of miR17-92 targets and thus shed light on its role in cMyc full-blown B-cell lymphoma.
We obtained a high-confidence quantitative proteome of about 4700 proteins, and a corresponding transcriptome of about 10000 transcripts. Comparative analysis of the proteome and transcriptome revealed that the cellular response is predominantly at the protein level, with significantly higher down-regulation in predicted protein targets of miR17/20a and miR19a/19b, as compared to non-targets. The intersection between predicted miR-targets and down-regulated proteins produced a list of 187 miR17-92 candidate targets. Validation of miR-engagement in observed down-regulation is ongoing by using RISC-IP coupled with qPCR. Interestingly, among them we found several proteins (e.g. RRM2, ATAD2) that participate in cell proliferation control, acting as either oncogenes or tumor suppressors depending on the context and on the expression level. Moreover, the emerging response from molecular targets largely coincides with phenotypic analyses: miR17-92 overexpression results in reduced cell proliferation and in slower G1/S transition, having as a consequence loss of cells which overexpress the cluster in competition assays performed in vitro and in vivo. This suggests that, in this context, the miR17-92 cluster has a protective role.
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