Nadia Dani

Physiological relevance of the endogenous mono(ADP‐ribosyl)ation of cellular proteins

The mono(ADP‐ribosyl)ation reaction is a post‐translational modification that is catalysed by both bacterial toxins and eukaryotic enzymes, and that results in the transfer of ADP‐ribose from βNAD+ to various acceptor proteins. In mammals, both intracellular and extracellular reactions have been described; the latter are due to glycosylphosphatidylinositol‐anchored or secreted enzymes that are able to modify their targets, which include the purinergic receptor P2X7, the defensins and the integrins. Intracellular mono(ADP‐ribosyl)ation modifies proteins that have roles in cell signalling and metabolism, such as the chaperone GRP78/BiP, the β‐subunit of heterotrimeric G‐proteins and glutamate dehydrogenase. The molecular identification of the intracellular enzymes, however, is still missing. A better molecular understanding of this reaction will help in the full definition of its role in cell physiology and pathology.

Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome

Mono-ADP-ribosylation is a reversible posttranslational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. For the latter, both the enzymes and their targets have largely remained elusive, mainly due to the lack of specific techniques to study this reaction. The recent discovery of the macro domain, a protein module that interacts selectively with ADP-ribose, prompted us to investigate whether this interaction can be extended to the identification of ADP-ribosylated proteins. Here, we report that macro domains can indeed be used as selective baits for high-affinity purification of mono-ADP-ribosylated proteins, which can then be identified by mass spectrometry. Using this approach, we have identified a series of cellular targets of ADP-ribosylation reactions catalyzed by cellular ADP-ribosyltransferases and toxins. These proteins include most of the known targets of ADP-ribosylation, indicating the validity of this method, and a large number of other proteins, which now need to be individually validated. This represents an important step toward the discovery of new ADP-ribosyltransferase targets and an understanding of the physiological role and the pharmacological potential of this protein modification.

Endogenous mono-ADP-ribosylation of the free Gbetagamma prevents stimulation of phosphoinositide 3-kinase-gamma and phospholipase C-beta2 and is activated by G-protein-coupled receptors.

We have recently demonstrated that the beta subunit of the heterotrimeric G-proteins is endogenously mono-ADP-ribosylated in intact cells. The modified betagamma heterodimer loses its ability to inhibit calmodulin-stimulated type 1 adenylate cyclase and, remarkably, is de-ADP-ribosylated by a cytosolic hydrolase that completes an ADP-/de-ADP-ribosylation cycle of potential physiological relevance. In the present study, we show that this ADP-ribosylation might indeed be a general mechanism for termination of betagamma signalling, since the ADP-ribosylated betagamma subunit is also unable to activate both phosphoinositide 3-kinase-gamma and phospholipase C-beta2. Moreover, we show that beta subunit ADP-ribosylation is induced by G-protein-coupled receptor activation, since hormone stimulation of Chinese-hamster ovary plasma membranes leads to increases in beta subunit labelling. This occurs when betagamma is in its active heterodimeric conformation, since full inhibition of this modification can be achieved by binding of GDP-alphai3 to the betagamma heterodimer. Taken together, these findings delineate a pathway that arises from the activation of a G-protein-coupled receptor and leads to the inhibition of betagamma activity through its reversible mono-ADP-ribosylation.

The MET oncogene transforms human primary bone‐derived cells into osteosarcomas by targeting committed osteo‐progenitors

The MET oncogene is aberrantly overexpressed in human osteosarcomas. We have previously converted primary cultures of human bone‐derived cells into osteosarcoma cells by overexpressing MET. To determine whether MET transforms mesenchymal stem cells or committed progenitor cells, here we characterize distinct MET overexpressing osteosarcoma (MET‐OS) clones using genome‐wide expression profiling, cytometric analysis, and functional assays. All the MET‐OS clones consistently display mesenchymal and stemness markers, but not most of the mesenchymal–stem cell‐specific markers. Conversely, the MET‐OS clones express genes characteristic of early osteoblastic differentiation phases, but not those of late phases. Profiling of mesenchymal stem cells induced to differentiate along osteoblast, adipocyte, and chondrocyte lineages confirms that MET‐OS cells are similar to cells at an initial phase of osteoblastic differentiation. Accordingly, MET‐OS cells cannot differentiate into adipocytes or chondrocytes, but can partially differentiate into osteogenic‐matrix‐producing cells. Moreover, in vitro MET‐OS cells form self‐renewing spheres enriched in cells that can initiate tumors in vivo. MET kinase inhibition abrogates the self‐renewal capacity of MET‐OS cells and allows them to progress toward osteoblastic differentiation. These data show that MET initiates the transformation of a cell population that has features of osteo‐progenitors and suggest that MET regulates self‐renewal and lineage differentiation of osteosarcoma cells.

The cellular apoptosis susceptibility CAS/CSE1L gene protects ovarian cancer cells from death by suppressing RASSF1C

The cellular apoptosis susceptibility gene CAS/CSE1L is overexpressed in cancer, although it was originally identified as a gene that renders cells vulnerable to apoptotic stimuli. CAS/CSE1L has roles in the nucleocytoplasmic recycling of importin‐α and in the regulation of gene expression, cell migration, and secretion. We identified CAS/CSE1L as a survival factor for ovarian cancer cells in vitro and in vivo. In 3/3 ovarian cancer cell lines, CAS/CSE1L was down‐modulated by the unorthodox proapoptotic signaling of the MET receptor. CAS/CSE1L knockdown with RNA interference committed the ovarian cancer cells to death, but not immortalized normal cells and breast and colon cancer cells. In 70 and 95% of these latter cells, respectively, CAS/CSE1L was localized in the cytoplasm, while it accumulated in the nucleus in >90% of ovarian cancer cells. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells. In the nucleus, CAS/CSE1L regulated the expression of the proapoptotic Ras‐association domain family 1 gene products RASSF1C and RASSF1A, which mediated death signals evoked by depletion of CAS/CSE1L. Our data show that CAS/CSE1L protects ovarian cancer cells from death through transcriptional suppression of a proapoptotic gene and suggest that the localization of CAS/CSE1L dictates its function.—Lorenzato, A., Martino, C., Dani, N., Oligschläger, Y., Ferrero, A. M., Biglia, N., Calogero, R., Olivero, M., Di Renzo, M. F. The cellular apoptosis susceptibility CAS/CSE1L gene protects ovarian cancer cells from death by suppressing RASSF1C. FASEB J. 26, 2446‐2456 (2012). www.fasebj.org

ADP-Ribosylated Proteins as Old and New Drug Targets for Anticancer Therapy: The Example of ARF6

Post-translational modifications of cellular proteins by mono- or poly-ADP-ribosylation are associated with numerous cellular processes. ADP-ribosylation reactions are important in the nucleus, and in mitochondrial activity, stress response signaling, intracellular trafficking, and cell senescence and apoptosis decisions. These reversible reactions add ADP-ribose to target proteins via specific enzymes to form the ADP-ribosylated protein; the cleaveage of this covalent bond is performed via hydrolases. Deficiencies in these enzymatic activities lead to cell death or tumor formation, thus defining their functional roles and impact on human disease. Unlike mono- ADP-ribosyltransferases, poly-ADP-ribose polymerases (PARPs) have been at the frontline of drug discovery since the 1980s. PARP1 is a valuable therapeutic target, with a central role in responses to DNA damage. With mono-ADP-ribosylation now linked to human diseases, such as inflammation, diabetes, neurodegeneration and cancer metastasis, novel and equally important functions of mono-ADPribosylation in cell signaling pathways can now be defined. Recently, we reported mono-ADP-ribosylation of ADP-ribosylation factor 6 (ARF6), a small G-protein of the Ras superfamily. In addition to its involvement in actin remodeling, plasma membrane reorganization and vesicular transport, ARF6 contributes to cancer progression through activation of cell motility and invasion. Consequently, targeting this modification will counteract the pro-invasive effects of ARF6, providing innovative anti-tumor therapy. This review summarizes our present knowledge of the enzymes and targets involved in ADP-ribosylation reactions, and describes in silico approaches to visualize their site of interaction and to identify the precise site for ADP-ribosylation. This should ultimately improve pharmacological strategies to enhance both anti-tumor efficacy and treatment of a number of inflammatory and neurodegenerative disorders.

Mono-ADP-ribosylation of the G Protein βγ Dimer Is Modulated by Hormones and Inhibited by Arf6

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the β subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the βγ dimer is inactive toward its effector enzymes. To better understand the physiological relevance of this post-translational modification, we have studied its hormonal regulation. Here, we report that Gβ subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces Gβ subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits Gβ subunit mono-ADP-ribosylation. We also provide the first demonstration that activation of the GnRHR can activate the ADP-ribosylation factor Arf6, which in turn inhibits Gβ subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of Gβ subunit mono-ADP-ribosylation, which is fully restored by re-addition of purified, myristoylated Arf6. We show that Arf6 acts as a competitive inhibitor of the endogenous ADP-ribosyltransferase and is itself modified by this enzyme. These data provide further understanding of the mechanisms that regulate endogenous ADP-ribosylation of the Gβ subunit, and they demonstrate a novel role for Arf6 in hormone regulation of Gβ subunit mono-ADP-ribosylation.

Characterisation of a novel glycosylphosphatidylinositol-anchored mono-ADP-ribosyltransferase isoform in ovary cells

The mammalian mono-ADP-ribosyltransferases are a family of enzymes related to bacterial toxins that can catalyse both intracellular and extracellular mono-ADP-ribosylation of target proteins involved in different cellular processes, such as cell migration, signalling and inflammation. Here, we report the molecular cloning and functional characterisation of a novel glycosylphosphatidylinositol (GPI)-anchored mono-ADP-ribosyltransferase isoform from Chinese hamster ovary (CHO) cells (cARTC2.1) that has both NAD-glycohydrolase and arginine-specific ADP-ribosyltransferase activities. cARTC2.1 has the R-S-EXE active-site motif that is typical of arginine-specific ADP-ribosyltransferases, with Glu209 as the predicted catalytic amino acid. When over-expressed in CHO cells, the E209G single point mutant of cARTC2.1 cannot hydrolyse NAD(+), although it retains low arginine-specific ADP-ribosyltransferase activity. This ADP-ribosyltransferase activity was abolished only with an additional mutation in the R-S-EXE active-site motif, with both of the glutamate residues of the EKE sequence of cARTC2.1 mutated to glycine (E207/209G). These glutamate-mutated proteins localise to the plasma membrane, as does wild-type cARTC2.1. Thus, the partial or total loss of enzymatic activity of cARTC2.1 that arises from these mutations does not affect its cellular localisation. Importantly, an endogenous ADP-ribosyltransferase is indeed expressed and active in a subset of CHO cells, while a similar activity cannot be detected in ovarian cancer cells. With respect to this endogenous ecto-ART activity, we have identified two cell populations: ART-positive and ART-negative CHO cells. The subset of ART-positive cells, which represented 5% of the total cells, is tightly maintained in the CHO cell population.

Abstract 215: CSE1L as a potential target to sensitize ovarian cancer cells to cisplatin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Human ovarian cancer is frequently (70% of cases) at advanced stage at presentation and requires multimodality treatment, consisting of cytoreductive surgery and chemotherapy based on platinum-drugs and taxanes. Patients are initially responsive to this treatment, but, in most cases, experience disease relapse because tumour cells acquire drug resistance. We demonstrated that the Hepatocyte Growth Factor (HGF) sensitizes to cisplatin (CDDP) and taxol an ovarian carcinoma cell line known to be resistant to platinum drugs: SK-OV-3, leading to an increased cancer cell death at very low drug doses both in vitro and in an animal model (Bardella et al., 2007; Rasola et al., 2004). This was an unexpected finding as HGF and its tyrosine kinase receptor, encoded by the MET oncogene, drives cell proliferation, survival and invasiveness during development and tumorigenesis. We also demonstrated that this effect is mediated by p38MAPK, which overwhelms the concomitant HGF activation of survival pathways, as the expression of a dominant negative form of p38MAPK abrogates the sensitization effect of HGF (Coltella et al., 2006). Using oligonucleotide microarrays, we studied the transcriptional responses of cell treated with CDDP in the presence or in the absence of HGF, and found that HGF pre-treatment modifies the transcriptional response to CDDP not only in SK-OV-3, but also in NIH-OVCAR-3, and TOV-21G ovarian cancer cells. The up- or down-modulation of the most differentially expressed genes found in common within these cell lines was reverted when the cells expressed a dominant negative form of p38MAPK, upholding once again the involvement of p38MAPK in this phenomenon. Among the top-ranked differentially expressed genes, which we identified, we focused functional studies on CSE1L/CAS. This gene is a nuclear transporter involved in both proliferation and apoptosis found to be amplified and over-expressed in ovarian cancer. Knocking down of CSE1L/CAS made ovarian cancer cells sensitized to low doses of cisplatin and triggered apoptosis while had no effect on other cancer and normal cell lines. In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or to revert chemotherapy resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 215.

Abstract LB-259: MET-oncogene transforms human bone-derived cells by targeting committed osteo-progenitors

Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults. Despite advances in medical and surgical management, survival rates for OS remain low. The biology of osteosarcomagenesis is poorly understood and the OS initiating cell remains elusive. We generated a model of osteosarcomagenesis by overexpressing MET oncogene in primary cultured human osteoblasts (HOB), since aberrant MET expression is found in high percentage of human OS. MET encodes for the receptor of HGF. It is normally expressed in epithelia, while HGF is secreted by cells of mesenchymal origin. MET activation elicits a physiologic program known as invasive growth. If deregulated, this program contributes to cell transformation and tumor progression. Using a LV-mediated approach, we overexpressed MET in HOB at level similar to human OS. We obtained transformed and tumorigenic clones derived from single cells, as shown by patterns of transgene integration. Interestingly, the number of susceptible cells was very small. A candidate for this minor population is the still elusive target cell of osteosarcomagenesis. Notably, MET-transformed clones are maintained by a self renewing cell population able to form bona fide sarcospheres. To characterize the target cell of MET-induced transformation, we used genome-wide expression profiling with Illumina platform. We compared the transformed clones to parental HOB and primary human mesenchymal stem cells (MSC). Transformed clones resulted phenotypically identical, supporting their common origin. They showed 584 genes differentially expressed with respect to parental HOB. The expression levels of MSC markers are down regulated in clones, while markers of stemness (POU5F1, ABCB1) are up regulated. Moreover, markers of early phase of osteoblastic differentiation (CDCA2, RUNX2, ALPL, MATN2) are up regulated, while that associated to late phase (FN1, SPARC, COL1A1, SPP1, BGLAP) are down-regulated. These results suggest that the target cell of MET-induced transformation is an osteoprogenitor rather than a MSC. To identify this progenitor along the osteoblastic differentiation pathway, we compared the expression profiles of transformed clones with that of MSC differentiated towards osteoblasts, chondrocytes and adipocytes at defined time points (7-14-21 days). MET-transformed clones clustered with 7 days osteoblasts, with some transcripts shared with other 7-days differentiated cells. These data suggest that clones originate from a committed osteoprogenitor cell, which retains self renewal, but not multi-lineage potentials. This assumption is supported by the fact that transformed clones are able to fully differentiate along the osteoblastic, but not the adipocytic or chondrocytic lineages. Altogether, these results indicate that MET overexpression sustains the amplification of premalignant population with an osteoprogenitor-defined gene signature. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-259.