Elena Sacco

First experimental identification of Ras-inhibitor binding interface using a water-soluble Ras ligand

By combining in the same molecule Ras-interacting aromatic moieties and a sugar, we prepared a water-soluble Ras ligand that binds Ras and inhibits guanine nucleotide exchange. With this compound it was possible to determine experimentally by a (15)N-edited HSQC NMR experiment the ligand-Ras binding interface.

Selective cytotoxicity of a bicyclic Ras inhibitor in cancer cells expressing K-RasG13D

Mutation of RAS genes is a critical event in the pathogenesis of different human tumors and in some developmental disorders. Here we present an arabinose-derived bicyclic compound displaying selective cytotoxicity in human colorectal cancer cells expressing K-Ras(G13D), that shows high intrinsic nucleotide exchange rate. We characterize binding of bicyclic compounds by docking and NMR experiments and their inhibitory activity on GEF-mediated nucleotide exchange on wild-type and mutant Ras proteins. We demonstrate that the in vitro inhibition of Ras nucleotide exchange depends on the molar ratio between Ras and its GEF activator, suggesting that the observed in vivo selective effect may depend on biochemical parameters and actual intracellular concentration of the Ras protein and its regulators.

Characterization and Properties of Dominant-negative Mutants of the Ras-specific Guanine Nucleotide Exchange Factor CDC25Mm

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25Mm, are described. In vitro, the mutant GEFW1056E and GEFT1184Eproteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21 ras , and strongly reduce affinity of the nucleotide-free ras·GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras·GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEFW1056Emutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.

5-Fluorouracil resistant colon cancer cells are addicted to OXPHOS to survive and enhance stem-like traits

Despite marked tumor shrinkage after 5-FU treatment, the frequency of colon cancer relapse indicates that a fraction of tumor cells survives treatment causing tumor recurrence. The majority of cancer cells divert metabolites into anabolic pathways through Warburg behavior giving an advantage in terms of tumor growth. Here, we report that treatment of colon cancer cell with 5-FU selects for cells with mesenchymal stem-like properties that undergo a metabolic reprogramming resulting in addiction to OXPHOS to meet energy demands. 5-FU treatment-resistant cells show a de novo expression of pyruvate kinase M1 (PKM1) and repression of PKM2, correlating with repression of the pentose phosphate pathway, decrease in NADPH level and in antioxidant defenses, promoting PKM2 oxidation and acquisition of stem-like phenotype. Response to 5-FU in a xenotransplantation model of human colon cancer confirms activation of mitochondrial function. Combined treatment with 5-FU and a pharmacological inhibitor of OXPHOS abolished the spherogenic potential of colon cancer cells and diminished the expression of stem-like markers. These findings suggest that inhibition of OXPHOS in combination with 5-FU is a rational combination strategy to achieve durable treatment response in colon cancer.

Comparative analysis of the molecular mechanisms controlling the initiation of chromosomal DNA replication in yeast and in mammalian cells.

In eukaryotes DNA replication takes place in the S phase of the cell cycle. It initiates from hundreds to thousands of replication origins in a coordinated manner, in order to efficiently duplicate the genome. The sequence of events leading to the onset of DNA replication is conventionally divided in two interdependent processes: licensing-a process during which replication origins acquire replication competence but are kept inactive- and firing-a process during which licensed origins are activated but not re-licensed. In this review we investigate the evolutionary conservation of the molecular machinery orchestrating DNA replication initiation both in yeast and in mammalian cells, highlighting a remarkable conservation of the general architecture of this central biological mechanism. Many steps are conserved down to molecular details and are performed by orthologous proteins with high sequence conservation, while differences in molecular structure of the performing proteins and their interactions are apparent in other steps. Tight regulation of initiation of DNA replication is achieved through protein phosphorylation, exerted mostly by Cyclin-dependent kinases in order to ensure that each chromosome is fully replicated once, and only once, during each cycle, and to avoid the formation of aberrant DNA structures and incorrect chromosomal duplication, that in mammalian cells are a prerequisite for genome instability and tumorigenesis. We then consider a molecular mathematical model of DNA replication, recently proposed by our group in a collaborative project, as a frame of reference to discuss similarities and differences observed in the regulatory program controlling DNA replication initiation in yeast and in mammalian cells and discuss whether they may be dependent upon different functional constraints. We conclude that a systems biology approach, integrating molecular analysis with modeling and computational investigations, is the best choice to investigate the control of DNA replication in mammalian cells.

Novel RasGRF1-derived Tat-fused peptides inhibiting Ras-dependent proliferation and migration in mouse and human cancer cells

Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.

Binding properties and biological characterization of new sugar-derived Ras ligands

Since mutations of Ras genes have a great incidence in human tumours, Ras oncoproteins are a major clinical target for the development of anticancer agents. We have developed synthetic molecules able to inhibit Ras activation. Here we present new, water-soluble Ras inhibitors composed by an aromatic pharmacofore moiety covalently linked to different sugars. New glycosylated compounds bind to Switch 2 region of Ras, also involved in effector binding, inhibit GEF-catalyzed nucleotide exchange on Ras in vitro, and reduce Ras-dependent proliferation of murine fibroblasts. The influence of the sugar unit on Ras binding affinity and on the biological activity of Ras inhibitors has been investigated.

A comparative study of Whi5 and retinoblastoma proteins: from sequence and structure analysis to intracellular networks

Cell growth and proliferation require a complex series of tight-regulated and well-orchestrated events. Accordingly, proteins governing such events are evolutionary conserved, even among distant organisms. By contrast, it is more singular the case of “core functions” exerted by functional analogous proteins that are not homologous and do not share any kind of structural similarity. This is the case of proteins regulating the G1/S transition in higher eukaryotes–i.e., the retinoblastoma (Rb) tumor suppressor Rb—and budding yeast, i.e., Whi5. The interaction landscape of Rb and Whi5 is quite large, with more than one hundred proteins interacting either genetically or physically with each protein. The Whi5 interactome has been used to construct a concept map of Whi5 function and regulation. Comparison of physical and genetic interactors of Rb and Whi5 allows highlighting a significant core of conserved, common functionalities associated with the interactors indicating that structure and function of the network—rather than individual proteins—are conserved during evolution. A combined bioinformatics and biochemical approach has shown that the whole Whi5 protein is highly disordered, except for a small region containing the protein family signature. The comparison with Whi5 homologs from Saccharomycetales has prompted the hypothesis of a modular organization of structural disorder, with most evolutionary conserved regions alternating with highly variable ones. The finding of a consensus sequence points to the conservation of a specific phosphorylation rhythm along with two disordered sequence motifs, probably acting as phosphorylation-dependent seeds in Whi5 folding/unfolding. Thus, the widely disordered Whi5 appears to act as a hierarchical, “date hub” that has evolutionary assayed an original way of modular organization before being supplanted by the globular, multi-domain structured Rb, more suitable to cover the role of a “party hub”.

Catalytic competence of the Ras–GEF domain of hSos1 requires intra-REM domain interactions mediated by Phenylalanine 577

The Ras‐specific guanine nucleotide exchange region of hSos1 consists of two consecutive domains: the catalytic core (residues 742–1024) contains all residues binding to Ras, including the catalytic hairpin, and an upstream REM domain (residues 553–741), so called because it contains an evolutionary conserved Ras Exchange Motif (REM). We functionally define the boundaries of the REM domain through a combination of in vivo and in vitro assays. We show that an intra‐REM domain interaction, mediated by phenylalanine 577, is required to allow interaction of the REM domain with the catalytic core, constraining it in the active conformation.

The isolated catalytic hairpin of the Ras-specific guanine nucleotide exchange factor Cdc25Mm retains nucleotide dissociation activity but has impaired nucleotide exchange activity

Cdc25Mm is a mammalian Ras‐specific guanine nucleotide exchange factor (GEF). By homology modeling we show that it shares with Sos‐GEF the structure of the putative catalytic HI hairpin where the dominant negative T1184E mutation is located. Similarly to Cdc25MmT1184E, the isolated wild‐type and mutant hairpins retain the ability to displace Ras‐bound nucleotide, originate a stable Ras/GEF complex and downregulate the Ras pathway in vivo. These results indicate that nucleotide re‐entry and Ras/GEF dissociation – final steps in the GEF catalytic cycle – require GEF regions different from the HI hairpin. GEF down‐sizing could lead to development of novel Ras inhibitors.