Francesca Monteleone

Characterization of carbonic anhydrase IX interactome reveals proteins assisting its nuclear localization in hypoxic cells.

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface.

A novel community driven software for functional enrichment analysis of extracellular vesicles data

ABSTRACT Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture “what the community needs in a tool”. Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Interleukin 3- receptor targeted exosomes inhibit in vitro and in vivo chronic myelogenous Leukemia cell growth

Despite Imatinib (IM), a selective inhibitor of Bcr-Abl, having led to improved prognosis in Chronic Myeloid Leukemia (CML) patients, acquired resistance and long-term adverse effects is still being encountered. There is, therefore, urgent need to develop alternative strategies to overcome drug resistance. According to the molecules expressed on their surface, exosomes can target specific cells. Exosomes can also be loaded with a variety of molecules, thereby acting as a vehicle for the delivery of therapeutic agents. In this study, we engineered HEK293T cells to express the exosomal protein Lamp2b, fused to a fragment of Interleukin 3 (IL3). The IL3 receptor (IL3-R) is overexpressed in CML blasts compared to normal hematopoietic cells and thus is able to act as a receptor target in a cancer drug delivery system. Here we show that IL3L exosomes, loaded with Imatinib or with BCR-ABL siRNA, are able to target CML cells and inhibit in vitro and in vivo cancer cell growth.

Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody.

Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti‐EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab‐sensitive and ‐resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose‐6‐phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab‐resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia‐inducible factor 1‐associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti‐EGFR therapy in colorectal cancer.

Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype, via exosomal miR-21

Tumor derived exosomes are vesicles which contain proteins and microRNAs that mediate cell-cell communication and are involved in angiogenesis and tumor progression. Curcumin derived from the plant Curcuma longa, shows anticancer effects. Exosomes released by CML cells treated with Curcumin contain a high amount of miR-21 that is shuttled into the endothelial cells in a biologically active form. The treatment of HUVECs with CML Curcu-exosomes reduced RhoB expression and negatively modulated endothelial cells motility. We showed that the addition of CML control exosomes to HUVECs caused an increase in IL8 and VCAM1 levels, but Curcu-exosomes reversed these effects thus attenuating their angiogenic properties. This antiangiogenic effect was confirmed with in vitro and in vivo vascular network formation assays. SWATH analysis of the proteomic profile of Curcu-exosomes revealed that Curcumin treatment deeply changes their molecular properties, in particular, Curcumin induces a release of exosomes depleted in pro-angiogenic proteins and enriched in proteins endowed with anti-angiogenic activity. Among the proteins differential expressed we focused on MARCKS, since it was the most modulated protein and a target of miR-21. Taken together our data indicated that also Curcumin attenuates the exosomes ability to promote the angiogenic phenotype and to modulate the endothelial barrier organization.

In vitro and in vivo models for analysis of resistance to anticancer molecular therapies.

The efficacy of classical and molecular therapies in cancer is hampered by the occurrence of primary (intrinsic) and secondary (acquired) refractoriness of tumours to selected therapeutic regimens. Nevertheless, the increased knowledge of the genetic, molecular and metabolic mechanisms underlying cancer results in the generation of a correspondingly increasing number of druggable targets and molecular drugs. Thus, a current challenge in molecular oncology and medicinal chemistry is to cope with the increased need for modelling, both in cellular and animal systems, the genetic assets associated to cancer resistance to drugs. In this review, we summarize the current strategies for generation and analysis of in vitro and in vivo models, which may reveal useful to extract information on the molecular basis of intrinsic and acquired resistance to anticancer molecular agents.

Proteomic signatures in thapsigargin-treated hepatoma cells.

Thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium transporters, generates Ca(2+)-store depletion within the ER and simultaneously increases Ca(2+) level in the cytosol. Perturbation of Ca(2+) homeostasis leads cells to cope with stressful conditions, including ER stress, which affect the folding of newly synthesized proteins and induce the accumulation of unfolded polypeptides and eventually apoptosis, via activation of the unfolded protein response pathway. In the present work, we analyzed the proteome changes in human hepatoma cells following acute treatment with thapsigargin. We highlighted a peculiar pattern of protein expression, marked by altered expression of calcium-dependent proteins, and of proteins involved in secretory pathways or in cell survival. For specific deregulated proteins, the thapsigargin-induced proteomic signature was compared by Western blotting to that resulting from the treatment of hepatoma cells with reducing agents or with proteasome inhibitors, to elicit endoplasmic reticulum stress by additional means and to reveal novel, potential targets of the unfolded protein response pathway.

Exosomes from metastatic cancer cells transfer amoeboid phenotype to non-metastatic cells and increase endothelial permeability: their emerging role in tumor heterogeneity

The goal of this study was to understand if exosomes derived from high-metastatic cells may influence the behavior of less aggressive cancer cells and the properties of the endothelium. We found that metastatic colon cancer cells are able to transfer their amoeboid phenotype to isogenic primary cancer cells through exosomes, and that this morphological transition is associated with the acquisition of a more aggressive behavior. Moreover, exosomes from the metastatic line (SW620Exos) exhibited higher ability to cause endothelial hyperpermeability than exosomes from the non metastatic line (SW480Exos). SWATH-based quantitative proteomic analysis highlighted that SW620Exos are significantly enriched in cytoskeletal-associated proteins including proteins activating the RhoA/ROCK pathway, known to induce amoeboid properties and destabilization of endothelial junctions. In particular, thrombin was identified as a key mediator of the effects induced by SW620Exos in target cells, in which we also found a significant increase of RhoA activity. Overall, our results demonstrate that in a heterogeneous context exosomes released by aggressive sub-clones can contribute to accelerate tumor progression by spreading malignant properties that affect both the tumor cell plasticity and the endothelial cell behavior.

Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521.

The study of protein-protein interactions is increasingly relying on mass spectrometry (MS). The classical approach of separating immunoprecipitated proteins by SDS-PAGE followed by in-gel digestion is long and labor-intensive. Besides, it is difficult to integrate it with most quantitative MS-based workflows, except for stable isotopic labeling of amino acids in cell culture (SILAC). This work describes a fast, flexible and quantitative workflow for the discovery of novel protein-protein interactions. A cleavable cross-linker, dithiobis[succinimidyl propionate] (DSP), is utilized to stabilize protein complexes before immunoprecipitation. Protein complex detachment from the antibody is achieved by limited proteolysis. Finally, protein quantitation is performed via (18)O labeling. The workflow has been optimized concerning (i) DSP concentration and (ii) incubation times for limited proteolysis, using the stem cell-associated transcription cofactor ZNF521 as a model target. The interaction of ZNF521 with the core components of the nuclear remodelling and histone deacetylase (NuRD) complex, already reported in the literature, was confirmed. Additionally, interactions with newly discovered molecular partners of potentially relevant functional role, such as ZNF423, Spt16, Spt5, were discovered and validated by Western blotting.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.