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1. Caloric restriction leads to druggable LSD1-dependent cancer stem cells expansion

Author

Pallavi, R, Gatti, E, Durfort, T, Stendardo, M, Ravasio, R, Leonardi, T, Falvo, P, Duso, B, Punzi, S, Xieraili, A, Polazzi, A, Verrelli, D, Trastulli, D, Ronzoni, S, Frascolla, S, Perticari, G, Elgendy, M, Varasi, M, Colombo, E, Giorgio, M, Lanfrancone, L, Minucci, S, Mazzarella, L, Pelicci, P, Pallavi R., Gatti E., Durfort T., Stendardo M., Ravasio R., Leonardi T., Falvo P., Duso B. A., Punzi S., Xieraili A., Polazzi A., Verrelli D., Trastulli D., Ronzoni S., Frascolla S., Perticari G., Elgendy M., Varasi M., Colombo E., Giorgio M., Lanfrancone L., Minucci S., Mazzarella L., Pelicci P. G., Pallavi, R, Gatti, E, Durfort, T, Stendardo, M, Ravasio, R, Leonardi, T, Falvo, P, Duso, B, Punzi, S, Xieraili, A, Polazzi, A, Verrelli, D, Trastulli, D, Ronzoni, S, Frascolla, S, Perticari, G, Elgendy, M, Varasi, M, Colombo, E, Giorgio, M, Lanfrancone, L, Minucci, S, Mazzarella, L, Pelicci, P, Pallavi R., Gatti E., Durfort T., Stendardo M., Ravasio R., Leonardi T., Falvo P., Duso B. A., Punzi S., Xieraili A., Polazzi A., Verrelli D., Trastulli D., Ronzoni S., Frascolla S., Perticari G., Elgendy M., Varasi M., Colombo E., Giorgio M., Lanfrancone L., Minucci S., Mazzarella L., and Pelicci P. G.

Abstract

Caloric Restriction (CR) has established anti-cancer effects, but its clinical relevance and molecular mechanism remain largely undefined. Here, we investigate CR's impact on several mouse models of Acute Myeloid Leukemias, including Acute Promyelocytic Leukemia, a subtype strongly affected by obesity. After an initial marked anti-tumor effect, lethal disease invariably re-emerges. Initially, CR leads to cell-cycle restriction, apoptosis, and inhibition of TOR and insulin/IGF1 signaling. The relapse, instead, is associated with the non-genetic selection of Leukemia Initiating Cells and the downregulation of double-stranded RNA (dsRNA) sensing and Interferon (IFN) signaling genes. The CR-induced adaptive phenotype is highly sensitive to pharmacological or genetic ablation of LSD1, a lysine demethylase regulating both stem cells and dsRNA/ IFN signaling. CR + LSD1 inhibition leads to the re-activation of dsRNA/IFN signaling, massive RNASEL-dependent apoptosis, and complete leukemia eradication in similar to 90% of mice. Importantly, CR-LSD1 interaction can be modeled in vivo and in vitro by combining LSD1 ablation with pharmacological inhibitors of insulin/IGF1 or dual PI3K/MEK blockade. Mechanistically, insulin/IGF1 inhibition sensitizes blasts to LSD1-induced death by inhibiting the anti-apoptotic factor CFLAR. CR and LSD1 inhibition also synergize in patient-derived AML and triple-negative breast cancer xenografts. Our data provide a rationale for epi-metabolic pharmacologic combinations across multiple tumors.

Published
2024

3. Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts

Author

Woo, X.Y., Giordano, J., Srivastava, A., Zhao, Z.-., Lloyd, M.W., de Bruijn, R., Suh, Y.-., Patidar, R., Chen, L., Scherer, S., Bailey, M.H., Yang, C.-., Cortes-Sanchez, E., Xi, Y., Wang, J., Wickramasinghe, J., Kossenkov, A.V., Rebecca, V.W., Sun, H., Mashl, R.J., Davies, S.R., Jeon, R., Frech, C., Randjelovic, J., Rosains, J., Galimi, F., Bertotti, A., Lafferty, A., O'Farrell, A.C., Modave, E., Lambrechts, D., ter Brugge, P., Marangoni, E., El Botty, R., Kim, H., Kim, J.-., Yang, H.-., Lee, C., Dean, D.A., Davis-Dusenbery, B., Evrard, Y.A., Doroshow, J.H., Welm, A.L., Welm, B.E., Lewis, M.T., Fang, B., Roth, J.A., Meric-Bernstam, F., Herlyn, M., Davies, M.A., Ding, L., Li, S., Govindan, R., Isella, C., Moscow, J.A., Trusolino, L., Byrne, A.T., Jonkers, J., Bult, C.J., Medico, E., Chuang, J.H., Robinson, P.N., Sanderson, B.J., Neuhauser, S.B., Dobrolecki, L.E., Zheng, X., Majidi, M., Zhang, R., Zhang, X., Akcakanat, A., Evans, K.W., Yap, T.A., Li, D., Yucan, E., Lanier, C.D., Saridogan, T., Kirby, B.P., M. J., H., Chen, H., Kopetz, S., Menter, D.G., Zhang, J., Westin, S.N., Kim, M.P., Dai, B., Gibbons, D.L., Tapia, C., Jensen, V.B., Boning, G., Minna, J.D., Park, H., Timmons, B.C., Girard, L., Fingerman, D., Liu, Q., Somasundaram, R., Xiao, M., Yennu-Nanda, V.G., Tetzlaff, M.T., Xu, X., Nathanson, K.L., Cao, S., Chen, F., Dipersio, J.F., Lim, K.H., C. X., M., Rodriguez, F.M., Van Tine, B.A., Wang-Gillam, A., Wendl, M.C., Wu, Y., Wyczalkowski, M.A., Yao, L., Jayasinghe, R., Aft, R.L., Fields, R.C., Luo, J., Fuh, K.C., Chin, V., Digiovanna, J., Grover, J., Koc, S., Seepo, S., Wallace, T., Pan, C.-., Chen, M.S., Carvajal-Carmona, L.G., Kirane, A.R., Cho, M., Gandara, D.R., Riess, J.W., Le, T., deVere White, R.W., Tepper, C.G., Zhang, H., Coggins, N.B., Lott, P., Estrada, A., Toal, T., Arana, A.M., Polanco-Echeverry, G., Rocha, S., A. -H., M., Mitsiades, N., Kaochar, S., O'Malley, B.W., Ellis, M.J., Hilsenbeck, S.G., Ittmann, M., Corso, S., Fiori, A., Giordano, S., van de Ven, M., Peeper, D.S., Miller, I., Bernado, C., Morancho, B., Ramirez, L., Arribas, J., Palmer, H.G., Piris-Gimenez, A., Soucek, L., Dahmani, A., Montaudon, E., Nemati, F., Dangles-Marie, V., Decaudin, D., Roman-Roman, S., Alferez, D.G., Spence, K., Clarke, R.B., Bentires-Alj, M., Chang, D.K., Biankin, A.V., Bruna, A., O'Reilly, M., Caldas, C., Casanovas, O., Gonzalez-Suarez, E., Munoz, P., Villanueva, A., Conte, N., Mason, J., Thorne, R., Meehan, T.F., Parkinson, H., Dudova, Z., Krenek, A., Stuchlik, D., Elemento, O., Inghirami, G., Golebiewska, A., Niclou, S.P., Wisman, G.B.A., de Jong, S., Kralova, P., Sedlacek, R., Claeys, E., Leucci, E., Borsani, M., Lanfrancone, L., Pelicci, P.G., Maelandsmo, G.M., Norum, J.H., Vinolo, E., Serra, V., and Leucci, Eleonora

Subjects
endocrine system diseases, Microarray, gene amplification, Whole Exome Sequencing, Mice, 0302 clinical medicine, Gene expression, Databases, Genetic, Gene duplication, Neoplasm Metastasis, Exome sequencing, Cancer, Regulation of gene expression, 0303 health sciences, Manchester Cancer Research Centre, adult, adult, animal experiment, animal model, animal tissue, breast cancer, microarray, DNA sequencing engraftment, gene amplification, tumor, xenograft, tumor-related gene, Polymorphism, Single Nucleotide/genetics, 3. Good health, Gene Expression Regulation, Neoplastic, 030220 oncology & carcinogenesis, microarray, hormones, hormone substitutes, and hormone antagonists, tumor, endocrine system, DNA sequencing engraftment, animal experiment, DNA Copy Number Variations/genetics, Biology, digestive system, DNA sequencing, Article, animal tissue, 03 medical and health sciences, breast cancer, Breast cancer, Genetics, medicine, Animals, Humans, xenograft, Gene, 030304 developmental biology, Settore MED/04 - Patologia Generale, Microarray analysis techniques, animal model, ResearchInstitutes_Networks_Beacons/mcrc, tumor-related gene, medicine.disease, Xenograft Model Antitumor Assays, Computational biology and bioinformatics, Cancer research, Human cancer
Abstract

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host., Analysis of copy number alterations in 1,451 patient-derived xenografts (PDXs) and matched patient tumor samples shows strong conservation from patient tumors through late-passage PDXs and a lack of systematic copy number evolution driven by the mouse host.

Published
2021

4. Modeling cell proliferation in human acute myeloid leukemia xenografts

Author

Nobile, M, Vlachou, T, Spolaor, S, Bossi, D, Cazzaniga, P, Lanfrancone, L, Mauri, G, Pelicci, P, Besozzi, D, Nobile, MS, BOSSI, DANIELA, Pelicci, PG, Nobile, M, Vlachou, T, Spolaor, S, Bossi, D, Cazzaniga, P, Lanfrancone, L, Mauri, G, Pelicci, P, Besozzi, D, Nobile, MS, BOSSI, DANIELA, and Pelicci, PG

Abstract

Acute myeloid leukemia (AML) is one of the most common hematological malignancies, characterized by high relapse and mortality rates. The inherent intra-tumor heterogeneity in AML is thought to play an important role in disease recurrence and resistance to chemotherapy. Although experimental protocols for cell proliferation studies are well established and widespread, they are not easily applicable to in vivo contexts, and the analysis of related time-series data is often complex to achieve. To overcome these limitations, model-driven approaches can be exploited to investigate different aspects of cell population dynamics. Results: In this work, we present ProCell, a novel modeling and simulation framework to investigate cell proliferation dynamics that, differently from other approaches, takes into account the inherent stochasticity of cell division events. We apply ProCell to compare different models of cell proliferation in AML, notably leveraging experimental data derived from human xenografts in mice. ProCell is coupled with Fuzzy Self-Tuning Particle Swarm Optimization, a swarm-intelligence settings-free algorithm used to automatically infer the models parameterizations. Our results provide new insights on the intricate organization of AML cells with highly heterogeneous proliferative potential, highlighting the important role played by quiescent cells and proliferating cells characterized by different rates of division in the progression and evolution of the disease, thus hinting at the necessity to further characterize tumor cell subpopulations. Availability and implementation: The source code of ProCell and the experimental data used in this work are available under the GPL 2.0 license on GITHUB at the following URL: https://github.com/aresio/ProCell. Supplementary information: Supplementary data are available at Bioinformatics online.

Published
2019

6. The life span determinant p66Shc localizes to mitochondria where it associates with mtHsp70 and regulates trans-membrane potential

Author

ORSINI F., MIGLIACCIO E., MORONI M., CONTURSI C., RAKER V. A., PICCINI D., MARTIN PADURA I., PELLICCIA G., TRINEI M., BONO M., PURI C., TACCHETTI, CARLO, FERRINI M., MANNUCCI R., NICOLETTI I., LANFRANCONE L., GIORGIO M., PELICCI P., Orsini, F., Migliaccio, E., Moroni, M., Contursi, C., Raker, V. A., Piccini, D., MARTIN PADURA, I., Pelliccia, G., Trinei, M., Bono, M., Puri, C., Tacchetti, Carlo, Ferrini, M., Mannucci, R., Nicoletti, I., Lanfrancone, L., Giorgio, M., and Pelicci, P.

Published
2004

11. Retroviral gene transfer, rapid selection, and maintenance of the immature phenotype in mouse dendritic cells

Author

Gasperi, C, Rescigno, M, Granucci, F, Citterio, S, Matyszak, M, Sciurpi, M, Lanfrancone, L, Castagnoli, P, GRANUCCI, FRANCESCA, CITTERIO, STEFANIA, Matyszak, MK, Sciurpi, MT, CASTAGNOLI, PAOLA, Gasperi, C, Rescigno, M, Granucci, F, Citterio, S, Matyszak, M, Sciurpi, M, Lanfrancone, L, Castagnoli, P, GRANUCCI, FRANCESCA, CITTERIO, STEFANIA, Matyszak, MK, Sciurpi, MT, and CASTAGNOLI, PAOLA

Abstract

We used the retroviral vector PINCO [which expresses the green fluorescent protein (GFP) as a selectable marker], to infect growth factor-dependent immature D1 dendritic cells (DC), The efficiency of infection in different experiments tvas between 5 and 30%, but subsequent cell sorting led to a virtually homogeneous population of GFP-positive cells. Retroviral infection did not modify the immature DC phenotype, as shown by the low expression of major histocompatibility complex and co-stimulatory molecules. Furthermore, the GFP-positive D1 cells underwent full maturation after lipopolysaccharide treatment, as indicated by a high expression of cell-surface MHC and co-stimulatory molecules, and also by strong stimulatory activity in allogeneic mixed lymphocyte reaction. The high efficiency of this retroviral system, the rapidity of the technique, and the possibility to overcome in, vitro selection make this method very attractive for the stable introduction of heterologous genes into proliferating immature mouse D1 cells. Furthermore, this approach is suitable for functional studies of new DC-specific genes involved in DC maturation and survival

Published
1999

12. Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.

Author

Migliaccio, E, Mele, S, Salcini, A E, Pelicci, G, Lai, K M, Superti-Furga, G, Pawson, T, Di Fiore, P P, Lanfrancone, L, Pelicci, P G, Migliaccio, E, Mele, S, Salcini, A E, Pelicci, G, Lai, K M, Superti-Furga, G, Pawson, T, Di Fiore, P P, Lanfrancone, L, and Pelicci, P G

Abstract

Udgivelsesdato: 1997-Feb-17, Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.

Published
1997

14. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

Author

Lotti, L V, Lanfrancone, L, Migliaccio, E, Zompetta, C, Pelicci, G, Salcini, A E, Falini, B, Pelicci, P G, Torrisi, M R, Lotti, L V, Lanfrancone, L, Migliaccio, E, Zompetta, C, Pelicci, G, Salcini, A E, Falini, B, Pelicci, P G, and Torrisi, M R

Abstract

Udgivelsesdato: 1996-May, The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.

Published
1996

15. The motogenic and mitogenic responses to HGF are amplified by the Shc adaptor protein.

Author

Pelicci, G, Giordano, S, Zhen, Z, Salcini, A E, Lanfrancone, L, Bardelli, A, Panayotou, G, Waterfield, M D, Ponzetto, C, Pelicci, P G, Pelicci, G, Giordano, S, Zhen, Z, Salcini, A E, Lanfrancone, L, Bardelli, A, Panayotou, G, Waterfield, M D, Ponzetto, C, and Pelicci, P G

Abstract

Udgivelsesdato: 1995-Apr-20, The receptor of Hepatocyte Growth Factor-Scatter Factor (HGF) is a tyrosine kinase which regulates cell motility and growth. After ligand-induced tyrosine phosphorylation, the HGF receptor associates with the Shc adaptor, via the SH2 domain. Site-directed mutagenesis of the HGF receptor indicates that phosphotyrosines Y1349VHV and Y1356VNV can work as docking sites for Shc. The Kd of this interaction, measured in real time using synthetic phosphopeptides and recombinant Shc on a BIAcore biosensor, is 150 nm for both sites. After stimulation of the HGF receptor, Shc is phosphorylated on Y317VNV, generating an high affinity binding site for Grb2 (Kd = 15 nM). This duplicates the high affinity binding site for Grb2 present on the HGF receptor (Y1356VNV). Thus HGF stimulation can trigger the Ras pathway by recruiting Grb2 both directly through the receptor, and indirectly, through Shc. Overexpression of wild-type Shc, but not of the Y317-->F mutant, enhances cell migration and growth in response to HGF. These data show that Shc is a relevant substrate of the HGF receptor, and works as an 'amplifier' of the motogenic as well as of the mitogenic response.

Published
1995

16. Constitutive phosphorylation of Shc proteins in human tumors.

Author

Pelicci, G, Lanfrancone, L, Salcini, A E, Romano, A, Mele, S, Grazia Borrello, M, Segatto, O, Di Fiore, P P, Pelicci, P G, Pelicci, G, Lanfrancone, L, Salcini, A E, Romano, A, Mele, S, Grazia Borrello, M, Segatto, O, Di Fiore, P P, and Pelicci, P G

Abstract

Udgivelsesdato: 1995-Sep-7, The Shc gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are ubiquitously expressed and are downstream targets and effectors of activated tyrosine kinases (TK). We investigated tyrosine-phosphorylation of Shc proteins in normal and transformed cells. In tumor cells with known TK gene alterations Shc proteins were constitutively phosphorylated and complexed with the activated TK. No constitutive Shc phosphorylation was found in primary cell cultures and normal tissues. In 14 of 27 tumor cell lines with no reported TK alterations, Shc proteins were constitutively phosphorylated and formed stable complexes with novel tyrosine-phosphorylated polypeptides. Ten distinct Shc-associated phosphoproteins were identified with molecular weights ranging from 30 to 200 kDa. In a subset of carcinoma cell lines, phosphorylated Shc proteins complexed with a p175 phosphoprotein that was identified as the constitutively activated EGFR. In one glioblastoma cell line, a Shc-associated p190 was identified as the activated PDGFR. In 13 of 14 acute leukemia samples phosphorylated Shc proteins were constitutively complexed with a p140 phosphoprotein. Some of the Shc-associated phosphoproteins (EGFR, PDGFR, erbB-2, Met, bcr-abl, H4-ret) bound both the Shc- and Grb2-SH2 domains in vitro; others (p175; p70-p80) only the Shc-SH2 domain and yet others (p140) only the Grb2-SH3 domains. These results indicate that Shc proteins are common substrates of constitutively activated TKs and that the analysis of Shc phosphorylation allow the identification of tumors with constitutive TK activation.

Published
1995

17. Chromosome locations of genes encoding human signal transduction adapter proteins, Nck (NCK), Shc (SHC1), and Grb2 (GRB2).

Author

Huebner, K, Kastury, K, Druck, T, Salcini, A E, Lanfrancone, L, Pelicci, G, Lowenstein, E, Li, W, Park, S H, Cannizzaro, L, Huebner, K, Kastury, K, Druck, T, Salcini, A E, Lanfrancone, L, Pelicci, G, Lowenstein, E, Li, W, Park, S H, and Cannizzaro, L

Abstract

Udgivelsesdato: 1994-Jul-15, Abnormalities due to chromosomal aberration or point mutation in gene products of growth factor receptors or in ras gene products, which lie on the same signaling pathway, can cause disease in animals and humans. Thus, it can be important to determine chromosomal map positions of genes encoding "adapter" proteins, which are involved in transducing signals from receptor tyrosine kinases to downstream signal recipients such as ras, because adaptor protein genes could also, logically, serve as targets of mutation, rearrangement, or other aberration in disease. Therefore, DNAs from panels of rodent-human hybrids carrying defined complements of human chromosomes were assayed for the presence of the cognate genes for NCK, SHC, and GRB2, three SH2 or SH2/SH3 (Src homology 2 and 3) domain-containing adapter proteins. Additionally, NCK and SHC genes were more narrowly localized by chromosomal in situ hybridization. The NCK locus is at chromosome region 3q21, a region involved in neoplasia-associated changes; the SHC cognate locus, SHC1, is at 1q21, and the GRB2 locus is at 17q22-qter telomeric to the HOXB and NGFR loci. Both SHC1 and GRB2 are in chromosome regions that may be duplicated in some tumor types.

Published
1994

19. Structural and functional organization of the HF.10 human zinc finger gene (ZNF35) located on chromosome 3p21-p22.

Author

Lanfrancone, L, Pengue, G, Pandolfi, P P, Salcini, A E, Giacomucci, A, Longo, L, Donti, E, De Luca, P, La Mantia, G, Pelicci, P G, Lanfrancone, L, Pengue, G, Pandolfi, P P, Salcini, A E, Giacomucci, A, Longo, L, Donti, E, De Luca, P, La Mantia, G, and Pelicci, P G

Abstract

Udgivelsesdato: 1992-Apr, We report the structural and functional characterization of the HF.10 zinc finger gene (ZNF35) in normal human cells, as well as a processed pseudogene. The HF.10 gene spans about 13 kb and it is interrupted by three introns. All 11 zinc finger DNA-binding domains are contiguously encoded within the last 3' exon. The genomic region surrounding HF.10 exon 1 contains a CpG island and acts as a promoter in vitro. Using transient CAT assay in cotransfection experiments in cultured cells, we have determined that the HF.10 finger protein is a transcriptional transactivator. Restriction enzyme mapping and partial nucleotide sequencing of the HF.10 pseudogene indicated that it has arisen by retroposition of spliced HF.10 mRNA. In situ hybridization experiments revealed that both the functional locus and the pseudogene map to chromosome 3p21p22, a region that is frequently deleted in small cell lung and renal carcinomas. Hybridization of the HF.10 gene and the HF.10 pseudogene DNA probes to metaphases from a small cell lung carcinoma cell line with the 3p deletion revealed that both loci are part of the deleted chromosome region.

Published
1992

29. Tyrosine 474 of ZAP-70 is required for association with the Shc adaptor and for T-cell antigen receptor-dependent gene activation.

Author

Pacini, S, Ulivieri, C, Di Somma, M M, Isacchi, A, Lanfrancone, L, Pelicci, P G, Telford, J L, and Baldari, C T

Abstract

The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation. Following receptor engagement, ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor (TCR). This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins. Among these is the Shc adaptor, which couples the activated TCR to Ras. Shc interaction with ZAP-70 is mediated by the Shc PTB domain. The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Phe474) of the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta. Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR-dependent gene activation. ZAP-70 could effectively phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway.

Published
1998

30. Human leukemia cells synthesize and secrete proteins related to platelet-derived growth factor.

Author

Pantazis, P, Lanfrancone, L, Pelicci, P G, Dalla-Favera, R, and Antoniades, H N

Abstract

Human leukemia cells in culture (HL-60) synthesize and secrete proteins that are recognized by antiserum to human platelet-derived growth factor (PDGF). The molecular mass of the intracellular proteins immunoprecipitated by PDGF antiserum ranged from 34 kDa to 240 kDa. PDGF-related proteins were also identified in the conditioned medium of the cells. Several of these immunoprecipitated proteins were glycosylated. A single protein of 46 kDa was immunoprecipitated from the cell-free translation products of mRNA obtained from the leukemia cells. Antiserum to the C but not to the N terminus of the predicted amino acid sequence of the transforming protein p28sis/PDGF-2 also immunoprecipitated proteins secreted by the HL-60 cells. These findings provide a direct demonstration for the synthesis and secretion of PDGF-like proteins by leukemia cells in culture. These proteins do not appear to be coded by the known c-sis/PDGF-2 locus since no sis mRNA was detectable in the HL-60 cells.

Published
1986
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31. Evolutionary conservation in various mammalian species of the human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody.

Author

Falini, B, Flenghi, L, Fagioli, M, Stein, H, Schwarting, R, Riccardi, C, Manocchio, I, Pileri, S, Pelicci, P G, and Lanfrancone, L

Abstract

The human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody (MAb) was detected in proliferating normal and neoplastic cells of many mammalian species (lamb, calf, dog, rabbit, rat) besides human. In contrast, Ki-67 stained proliferating cells from other species weakly (mouse) or not at all (swine, cat, chicken, pigeon). The immunostaining pattern of Ki-67 in animal tissues was identical to that previously described in human: Ki-67 reacted only with cells known to proliferate (e.g., germinal center cells, cortical thymocytes) but not with resting cells (e.g., hepatocytes, brain cells, renal cells); this MAb produced a characteristic nuclear staining pattern (e.g., stronger labeling of nucleoli than of the rest of the nuclei and staining of chromosomes in mitotic figures); and Ki-67 crossreacted with the squamous epithelium in both animal and human tissues. In vitro studies showed that when quiescent (Ki-67-negative) NIH 3T3 fibroblasts or bovine peripheral blood lymphocytes were induced to proliferate, the appearance of Ki-67-positive cells paralleled the induction of cell proliferation caused by addition of fetal calf serum or PHA, respectively, to the cultures, and in both human and rat proliferating cells the Ki-67 expression closely paralleled the incorporation of [3H]-thymidine. These findings indicate that the epitope recognized by the Ki-67 MAb in human and animal species is the same. The widespread evolutionary conservation of the human proliferation-associated epitope recognized by the Ki-67 MAb suggests that it and/or its carrier molecule may play an important role in regulation of cell proliferation.

Published
1989
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32. The aminoterminal phosphotyrosine binding domain of Shc associates with ZAP-70 and mediates TCR dependent gene activation

Author

Milia, E., Di Somma, M. M., Baldoni, F., rita chiari, Lanfrancone, L., Pelicci, P. G., Telford, J. L., and Baldari, C. T.

Subjects
Transcriptional Activation, Shc, ZAP-70 Protein-Tyrosine Kinase, NFATC Transcription Factors, TCR, molecular adaptor, Ras, ZAP-70. T cell, signal transduction, Molecular Sequence Data, Receptors, Antigen, T-Cell, Nuclear Proteins, Proteins, Protein-Tyrosine Kinases, Cell Line, DNA-Binding Proteins, src Homology Domains, Gene Expression Regulation, Humans, Amino Acid Sequence, Phosphotyrosine, Protein Binding, Transcription Factors
Abstract

T-cell antigen receptor stimulation results in recruitment to the zeta chain and phosphorylation both of the syk family protein tyrosine kinase ZAP-70 and of the Shc adaptor protein, which transduces activating signals to Ras. Both ZAP-70 and Ras are required for T-cell activation. We have investigated the functional link between these two molecules in TCR signaling. She was found to associate with ZAP-70 in response to TCR triggering. This association was dependent on the presence of the aminoterminal phosphotyrosine binding (PTB) domain of She. The analysis of She binding to a potential PTB domain binding site on ZAP-70 confirmed the interaction of the She PTB domain with ZAP-70 and identified the ZAP-70 phosphotyrosine residue involved in this interaction. To test the role of the She PTB domain in transducing TCR derived signals we measured the effects of the isolated She PTB domain on the activation of the T-cell specific transcription factor NF-AT. The isolated She PTB domain was designed to compete non productively with endogenous She for binding to up-stream tyrosine phosphorylated proteins and thus interfere with coupling to regulators of Ras activation. A significant inhibition of NF-AT activation by TCR triggering was observed, showing a functional involvement of She in TCR signaling through its PTB domain and suggesting an important role for She association with ZAP-70.

33. Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches

Author

Viale Giuseppe, Giorgetti Luca, Zanardi Andrea, Luise Chiara, Licciulli Silvia, Lanfrancone Luisa, Carbone Roberta, and Alcalay Myriam

Subjects
Cytology, QH573-671
Abstract

Abstract Background Pirin (PIR) is a highly conserved nuclear protein originally isolated as an interactor of NFI/CTF1 transcription/replication factor. It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes. Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies. Results We performed immunohistochemical analysis of PIR expression in primary samples from normal human tissues and tumors and identified a dislocation of PIR to the cytoplasm in a subset of melanomas, and a positive correlation between cytoplasmic PIR levels and melanoma progression. PIR localization was subsequently analyzed in vitro in melanoma cell lines through a high content immunofluorescence based approach (ImmunoCell-Array). Conclusions The high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.

Published
2010
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35. Modeling cell proliferation in human acute myeloid leukemia xenografts

Author

Daniela Bossi, Marco S. Nobile, Simone Spolaor, Giancarlo Mauri, Pier Giuseppe Pelicci, Thalia Vlachou, Paolo Cazzaniga, Luisa Lanfrancone, Daniela Besozzi, Nobile, M, Vlachou, T, Spolaor, S, Bossi, D, Cazzaniga, P, Lanfrancone, L, Mauri, G, Pelicci, P, and Besozzi, D

Subjects
Myeloid, Statistics and Probability, Fuzzy Self-Tuning Particle Swarm Optimization, Cell division, Cell, Population, Computational biology, Acute, acute myeloid leukemia, Biology, Stochastic modeling, Biochemistry, ING-INF/05 - SISTEMI DI ELABORAZIONE DELLE INFORMAZIONI, Flow cytometry, 03 medical and health sciences, Mice, 0302 clinical medicine, medicine, Animals, Humans, Cell Division, Cell Proliferation, Heterografts, Leukemia, Myeloid, Acute, education, Molecular Biology, 030304 developmental biology, 0303 health sciences, education.field_of_study, Leukemia, medicine.diagnostic_test, Settore INF/01 - Informatica, Cell growth, Systems Biology, INF/01 - INFORMATICA, Myeloid leukemia, medicine.disease, Original Papers, Computer Science Applications, Computational Mathematics, medicine.anatomical_structure, Computational Theory and Mathematics, 030220 oncology & carcinogenesis, parameter estimation
Abstract

Motivation Acute myeloid leukemia (AML) is one of the most common hematological malignancies, characterized by high relapse and mortality rates. The inherent intra-tumor heterogeneity in AML is thought to play an important role in disease recurrence and resistance to chemotherapy. Although experimental protocols for cell proliferation studies are well established and widespread, they are not easily applicable to in vivo contexts, and the analysis of related time-series data is often complex to achieve. To overcome these limitations, model-driven approaches can be exploited to investigate different aspects of cell population dynamics. Results In this work, we present ProCell, a novel modeling and simulation framework to investigate cell proliferation dynamics that, differently from other approaches, takes into account the inherent stochasticity of cell division events. We apply ProCell to compare different models of cell proliferation in AML, notably leveraging experimental data derived from human xenografts in mice. ProCell is coupled with Fuzzy Self-Tuning Particle Swarm Optimization, a swarm-intelligence settings-free algorithm used to automatically infer the models parameterizations. Our results provide new insights on the intricate organization of AML cells with highly heterogeneous proliferative potential, highlighting the important role played by quiescent cells and proliferating cells characterized by different rates of division in the progression and evolution of the disease, thus hinting at the necessity to further characterize tumor cell subpopulations. Availability and implementation The source code of ProCell and the experimental data used in this work are available under the GPL 2.0 license on GITHUB at the following URL: https://github.com/aresio/ProCell. Supplementary information Supplementary data are available at Bioinformatics online.

Published
2019

36. Dual modulation of MCL-1 and mTOR determines the response to sunitinib

Author

Viviana Bornaghi, Daniela Bossi, Mohamed Elgendy, Jose Luis Perez-Gracia, Amal Kamal Abdel-Aziz, Salvatore Lorenzo Renne, Luisa Lanfrancone, Maurizio Colecchia, Ravindran Kanesvaran, Franco Nolè, Chee Keong Toh, Nicola Fazio, Isabella Pallavicini, Giuseppe Renne, Valeria Giandomenico, Bin Tean Teh, Maria D. Lozano, Giuseppe Procopio, Ciro Mercurio, Saverio Minucci, Elgendy, M, Abdel-Aziz, Ak, Renne, Sl, Bornaghi, V, Procopio, G, Colecchia, M, Kanesvaran, R, Toh, Ck, Bossi, D, Pallavicini, I, Perez-Gracia, Jl, Lozano, Md, Giandomenico, V, Mercurio, C, Lanfrancone, L, Fazio, N, Nole, F, Teh, Bt, Renne, G, and Minucci, S

Subjects
0301 basic medicine, Indoles, MAP Kinase Signaling System, Mice, Nude, mTORC1, Mechanistic Target of Rapamycin Complex 1, Pharmacology, urologic and male genital diseases, Mice, 03 medical and health sciences, 0302 clinical medicine, Mice, Inbred NOD, hemic and lymphatic diseases, Cell Line, Tumor, Neoplasms, Enzyme Stability, Sunitinib, Autophagy, Animals, Humans, Medicine, Pyrroles, GSK3B, PI3K/AKT/mTOR pathway, Mice nude, Glycogen Synthase Kinase 3 beta, business.industry, Kinase, TOR Serine-Threonine Kinases, General Medicine, Xenograft Model Antitumor Assays, female genital diseases and pregnancy complications, cell death, 030104 developmental biology, Drug Resistance, Neoplasm, Multiprotein Complexes, 030220 oncology & carcinogenesis, Commentary, mTOR, Cancer research, Myeloid Cell Leukemia Sequence 1 Protein, biological phenomena, cell phenomena, and immunity, MCL-1, business, medicine.drug, Research Article
Abstract

Most patients who initially respond to treatment with the multi-tyrosine kinase inhibitor sunitinib eventually relapse. Therefore, developing a deeper understanding of the contribution of sunitinib's numerous targets to the clinical response or to resistance is crucial. Here, we have shown that cancer cells respond to clinically relevant doses of sunitinib by enhancing the stability of the antiapoptotic protein MCL-1 and inducing mTORC1 signaling, thus evoking little cytotoxicity. Inhibition of MCL-1 or mTORC1 signaling sensitized cells to clinically relevant doses of sunitinib in vitro and was synergistic with sunitinib in impairing tumor growth in vivo, indicating that these responses are triggered as prosurvival mechanisms that enable cells to tolerate the cytotoxic effects of sunitinib. Furthermore, higher doses of sunitinib were cytotoxic, triggered a decline in MCL-1 levels, and inhibited mTORC1 signaling. Mechanistically, we determined that sunitinib modulates MCL-1 stability by affecting its proteasomal degradation. Dual modulation of MCL-1 stability at different dose ranges of sunitinib was due to differential effects on ERK and GSK3β activity, and the latter also accounted for dual modulation of mTORC1 activity. Finally, comparison of patient samples prior to and following sunitinib treatment suggested that increases in MCL-1 levels and mTORC1 activity correlate with resistance to sunitinib in patients.

Published
2016

37. Isolation of cDNAs encoding finger proteins and measurement of the corresponding mRNA levels during myeloid terminal differentiation

Author

Antonio Pannuti, Luisa Lanfrancone, G La Mantia, L. Lania, Anna Pascucci, Pier Giuseppe Pelicci, Pannuti, A, Lanfrancone, L, Pascucci, A, Pelicci, Pg, La Mantia, G, Lania, Luigi, Pannuti, A., Lanfrancone, L., Pascucci, A., Pelicci, P. G., and LA MANTIA, Girolama

Subjects
Genetics, Regulation of gene expression, Base Sequence, Transcription, Genetic, Cellular differentiation, Molecular Sequence Data, DNA, Biology, Ribosome, DNA-binding protein, Cell Line, DNA-Binding Proteins, Genes, Transcription (biology), Complementary DNA, Humans, Amino Acid Sequence, RNA, Messenger, Cloning, Molecular, Gene, Transcription factor
Abstract

The finger motif is a tandemly repeated DNA-binding domain recently identified in the primary structure of several eukaryotic transcriptional regulatory proteins. It was first described for Xenopus TFIIIA, a factor required for transcription of 5S ribosomal genes and subsequently identified in regulatory proteins from other eukaryotic organisms including yeast, Drosophila and mammals. In this paper we report the isolation and characterization of two human cDNA clones both encoding for multifingered protein products. Transcriptional studies indicate that the two finger genes are expressed in a variety of human tissues. Moreover, upon in vitro induced terminal differentiation of human HL-60 and THP-1 myeloid cell lines the expression of both genes is drastically reduced. These data provide support for the existence of a human multigene family coding for regulatory finger proteins which are likely involved in the processes of cell differentiation and/or proliferation.

Published
1988

38. Retroviral gene transfer, rapid selection, and maintenance of the immature phenotype in mouse dendritic cells

Author

Paola Ricciardi-Castagnoli, Luisa Lanfrancone, M. T. Sciurpi, Maria Rescigno, Francesca Granucci, Stefania Citterio, C. Gasperi, Malgosia K. Matyszak, Gasperi, C, Rescigno, M, Granucci, F, Citterio, S, Matyszak, M, Sciurpi, M, Lanfrancone, L, and Castagnoli, P

Subjects
green fluorescent protein, Genetic Vectors, Green Fluorescent Proteins, Immunology, Population, Major histocompatibility complex, Immunophenotyping, Viral vector, Green fluorescent protein, Mice, Animals, Humans, Immunology and Allergy, retroviral vector, education, Selectable marker, education.field_of_study, biology, Gene Transfer Techniques, Dendritic Cells, Cell Biology, Cell sorting, Mixed lymphocyte reaction, Phenotype, Molecular biology, Luminescent Proteins, Retroviridae, biology.protein
Abstract

We used the retroviral vector PINCO [which expresses the green fluorescent protein (GFP) as a selectable marker], to infect growth factor-dependent immature D1 dendritic cells (DC). The efficiency of infection in different experiments was between 5 and 30%, but subsequent cell sorting led to a virtually homogeneous population of GFP-positive cells. Retroviral infection did not modify the immature DC phenotype, as shown by the low expression of major histocompatibility complex and co-stimulatory molecules. Furthermore, the GFP-positive D1 cells underwent full maturation after lipopolysaccharide treatment, as indicated by a high expression of cell-surface MHC and co-stimulatory molecules, and also by strong stimulatory activity in allogeneic mixed lymphocyte reaction. The high efficiency of this retroviral system, the rapidity of the technique, and the possibility to overcome in vitro selection make this method very attractive for the stable introduction of heterologous genes into proliferating immature mouse D1 cells. Furthermore, this approach is suitable for functional studies of new DC-specific genes involved in DC maturation and survival. J. Leukoc. Biol. 66: 263–267; 1999.

Published
1999

39. Identification and characterization of novel human endogenous retroviral sequences prefentially expressed in undifferentiated embryonal carcinoma cells

Author

Girolama La Mantia, Gina Pengue, Luisa Lanfrancone, Antonio Simeone, Domenico Maglione, Antonio Di Cristofano, Luigi Lania, La Mantia, G, Maglione, D, Pengue, G, Di Cristofano, A, Simeone, A, Lanfrancone, L, and Lania, Luigi

Subjects
Embryonal Carcinoma Stem Cells, Genes, Viral, Sequence analysis, viruses, Molecular Sequence Data, Restriction Mapping, Biology, Embryonal carcinoma, Open Reading Frames, Restriction map, Complementary DNA, Tumor Cells, Cultured, Genetics, medicine, Humans, Amino Acid Sequence, RNA, Neoplasm, Northern blot, Promoter Regions, Genetic, Gene, Base Sequence, cDNA library, DNA, Neoplasm, medicine.disease, Molecular biology, Blotting, Southern, Retroviridae, DNA, Viral, Neoplastic Stem Cells, RNA, Viral, Sequence Alignment, Plasmids
Abstract

A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.

Published
1991

40. The RIalpha subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-receptor

Author

Giampaolo Tortora, Vincenzo Damiano, Caterina Bianco, Pier Giuseppe Pelicci, Gustavo Baldassarre, Luisa Lanfrancone, Fortunato Ciardiello, Ar Bianco, Tortora, G, Damiano, V, Bianco, C, Baldassarre, G, Bianco, Ar, Lanfrancone, L, Pelicci, Pg, and Ciardiello, Fortunato

Subjects
MAPK/ERK pathway, Cancer Research, Protein subunit, EGFR, Cyclic AMP-Dependent Protein Kinase Type II, src Homology Domains, Epidermal growth factor, Genetics, Grb2, Tumor Cells, Cultured, Humans, PKA, Protein kinase A, Molecular Biology, Adaptor Proteins, Signal Transducing, GRB2 Adaptor Protein, biology, Proteins, Cyclic AMP-Dependent Protein Kinases, Cell biology, Enzyme Activation, ErbB Receptors, Calcium-Calmodulin-Dependent Protein Kinases, biology.protein, Cancer research, GRB2, Signal transduction, hormones, hormone substitutes, and hormone antagonists, Transforming growth factor, Protein Binding, Signal Transduction
Abstract

Functional interactions between protein kinase A (PKA) and epidermal growth factor receptor (EGF-R) signalling pathways have been suggested. Unlike the type II isoform of PKA (PKAII), the type I (PKAI) and/or its regulatory subunit RIalpha are generally overexpressed in cancer cells and are induced following transforming growth factor alpha (TGF alpha)/EGF-R-dependent transformation. Downregulation of RIalpha/PKAI inhibits TGF alpha expression and EGF-R-dependent signalling. We have previously shown that addition of EGF to quiescent human normal epithelial MCF-10A cells determines PKAI expression and cell membrane translocation before cells enter S phase, while PKAI inhibition prevents S phase entry. Constitutive overexpression of PKAI confers the ability to grow in serum free medium, bypassing EGF requirement. Here we demonstrate a direct interaction of PKAI, but not of PKAII, with the activated EGF-R, that occurs within 5 min following EGF treatment of MCF-10A cells. Moreover, induction of mitogen-activated protein kinase (MAPK) activity following EGF-R activation is mimicked by PKAI overexpression and inhibited by downregulators of PKAI. Finally, the PKAI-EGF-R association occurs through the binding of RIalpha to the SH3 domain(s) of Grb2 adaptor protein, thus allowing the recruitment of the PKAI holoenzyme to the activated EGF-R. This is the first demonstration of a direct interaction of PKAI with the activated EGF-R macromolecular signalling complex.

Published
1997

41. cDNA isolation, expression analysis, and chromosomal localization of two human zinc finger genes

Author

Anna Pascucci, Gina Pengue, Pier Giuseppe Pelicci, Girolama La Mantia, Antonio Pannuti, Luisa Lanfrancone, Isidoro Feliciello, Luigi Lania, Emilio Donti, Lania, Luigi, Donti, E, Pannuti, A, Pascucci, A, Pengue, G, Feliciello, Isidoro, La Mantia, G, Lanfrancone, L, and Pelicci, Pg

Subjects
Transcription, Genetic, Cellular differentiation, Molecular Sequence Data, Chromosomes, Human, Pair 20, Kruppel-Like Transcription Factors, Gene Expression, Biology, Cell Line, Complementary DNA, Sequence Homology, Nucleic Acid, Gene expression, Metalloproteins, Genetics, Humans, Northern blot, Amino Acid Sequence, Cloning, Molecular, Gene, Zinc finger, Base Sequence, Chromosome Mapping, Cell Differentiation, DNA, Blotting, Northern, Molecular biology, Blot, DNA-Binding Proteins, Zinc, Genes, Female, Chromosome 20, Chromosomes, Human, Pair 8
Abstract

On the basis of sequence similarity in the repeated zinc finger domain, we have identified and characterized two human cDNA clones (ZNF7 and ZNF8), both encoding proteins containing potential finger-like nucleic acid binding motifs. Northern blot analysis indicates that both genes are expressed as multiple transcripts and they are ubiquitously present in many human cell lines of different embryological derivation. Moreover, their expression is modulated during in vitro induced terminal differentiation of human myeloid cell line HL-60. By in situ hybridization experiments, we have localized the ZNF7 gene to chromosome 8 (region q24) and the ZNF8 gene to the terminal band of the long arm of chromosome 20 (20q13).

Published
1990

43. Caloric restriction leads to druggable LSD1-dependent cancer stem cells expansion.

Author

Pallavi R, Gatti E, Durfort T, Stendardo M, Ravasio R, Leonardi T, Falvo P, Duso BA, Punzi S, Xieraili A, Polazzi A, Verrelli D, Trastulli D, Ronzoni S, Frascolla S, Perticari G, Elgendy M, Varasi M, Colombo E, Giorgio M, Lanfrancone L, Minucci S, Mazzarella L, and Pelicci PG

Subjects
Humans, Animals, Mice, Caloric Restriction, Histone Demethylases genetics, Neoplastic Stem Cells pathology, Cell Line, Tumor, Leukemia, Myeloid, Acute pathology, Insulins
Abstract

Caloric Restriction (CR) has established anti-cancer effects, but its clinical relevance and molecular mechanism remain largely undefined. Here, we investigate CR's impact on several mouse models of Acute Myeloid Leukemias, including Acute Promyelocytic Leukemia, a subtype strongly affected by obesity. After an initial marked anti-tumor effect, lethal disease invariably re-emerges. Initially, CR leads to cell-cycle restriction, apoptosis, and inhibition of TOR and insulin/IGF1 signaling. The relapse, instead, is associated with the non-genetic selection of Leukemia Initiating Cells and the downregulation of double-stranded RNA (dsRNA) sensing and Interferon (IFN) signaling genes. The CR-induced adaptive phenotype is highly sensitive to pharmacological or genetic ablation of LSD1, a lysine demethylase regulating both stem cells and dsRNA/ IFN signaling. CR + LSD1 inhibition leads to the re-activation of dsRNA/IFN signaling, massive RNASEL-dependent apoptosis, and complete leukemia eradication in ~90% of mice. Importantly, CR-LSD1 interaction can be modeled in vivo and in vitro by combining LSD1 ablation with pharmacological inhibitors of insulin/IGF1 or dual PI3K/MEK blockade. Mechanistically, insulin/IGF1 inhibition sensitizes blasts to LSD1-induced death by inhibiting the anti-apoptotic factor CFLAR. CR and LSD1 inhibition also synergize in patient-derived AML and triple-negative breast cancer xenografts. Our data provide a rationale for epi-metabolic pharmacologic combinations across multiple tumors., (© 2024. The Author(s).)

Published
2024
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44. Comprehensive genomic profiling on metastatic Melanoma: results from a network screening from 7 Italian Cancer Centres.

Author

Pallocca M, Molineris I, Berrino E, Marcozzi B, Betti M, Levati L, D'Atri S, Menin C, Madonna G, Ghiorzo P, Bulgarelli J, Ferraresi V, Venesio T, Rodolfo M, Rivoltini L, Lanfrancone L, Ascierto PA, Mazzarella L, Pelicci PG, De Maria R, Ciliberto G, Medico E, and Russo G

Subjects
Humans, Proto-Oncogene Proteins B-raf, Genomics, Italy, Early Detection of Cancer, Melanoma genetics
Abstract

Background: The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma., Methods: 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting., Results: The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan-Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints., Conclusions: The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research., (© 2023. The Author(s).)

Published
2024
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45. Actionable Genetic Screens Unveil Targeting of AURKA, MEK, and Fatty Acid Metabolism as an Alternative Therapeutic Approach for Advanced Melanoma.

Author

Marocchi F, Palluzzi F, Nicoli P, Melixetian M, Lovati G, Bertalot G, Pece S, Ferrucci PF, Bossi D, and Lanfrancone L

Subjects
Humans, Mice, Animals, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Pyrimidinones therapeutic use, Mitogen-Activated Protein Kinase Kinases, Fatty Acids, Proto-Oncogene Proteins B-raf genetics, Mutation, Aurora Kinase A genetics, Melanoma drug therapy, Melanoma genetics
Abstract

Despite the remarkable improvements achieved in the management of metastatic melanoma, there are still unmet clinical needs. A considerable fraction of patients does not respond to immune and/or targeted therapies owing to primary and acquired resistance, high-grade immune-related adverse events, and a lack of alternative treatment options. To design effective combination therapies, we set up a functional ex vivo preclinical assay on the basis of a drop-out genetic screen in metastatic melanoma patient-derived xenografts. We showed that this approach can be used to isolate actionable vulnerabilities predictive of drug efficacy. In particular, we highlighted that the dual targeting of AURKA and MAPK/extracellular signal-regulated kinase kinase employing the combination of alisertib and trametinib is highly effective in a cohort of metastatic melanoma patient-derived xenografts, both ex vivo and in vivo. Alisertib and trametinib combination therapy outperforms standard-of-care therapy in both BRAF-mutant patient-derived xenografts and targeted therapy-resistant models. Furthermore, alisertib and trametinib treatment modulates several critical cancer pathways, including an early metabolic reprogramming that leads to the transcriptional upregulation of the fatty acid oxidation pathway. This acquired trait unveiled an additional point of intervention for pharmacological targeting, and indeed, the triple combination of alisertib and trametinib with the fatty acid oxidation inhibitor etomoxir proved to be further beneficial, inducing tumor regression and remarkably prolonging the overall survival of the mice., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2023
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46. BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

Author

Sorbi C, Belluti S, Atene CG, Marocchi F, Linciano P, Roy N, Paradiso E, Casarini L, Ronsisvalle S, Zanocco-Marani T, Brasili L, Lanfrancone L, Imbriano C, Di Rocco G, and Franchini S

Subjects
Humans, Apoptosis, Signal Transduction, Endoplasmic Reticulum Stress, Transcription Factor CHOP metabolism, Activating Transcription Factor 4 metabolism, eIF-2 Kinase metabolism, Melanoma drug therapy, Melanoma genetics, Melanoma pathology, Receptors, sigma genetics
Abstract

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Published
2023
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47. TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.

Author

Di Malta C, Zampelli A, Granieri L, Vilardo C, De Cegli R, Cinque L, Nusco E, Pece S, Tosoni D, Sanguedolce F, Sorrentino NC, Merino MJ, Nielsen D, Srinivasan R, Ball MW, Ricketts CJ, Vocke CD, Lang M, Karim B, Lanfrancone L, Schmidt LS, Linehan WM, and Ballabio A

Subjects
Humans, Mice, Animals, Kidney pathology, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Transcription Factors, Carcinogenesis genetics, Kidney Neoplasms genetics, Kidney Neoplasms pathology, Birt-Hogg-Dube Syndrome genetics, Birt-Hogg-Dube Syndrome pathology, Cysts
Abstract

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Published
2023
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48. Epigenetically regulated PCDHB15 impairs aggressiveness of metastatic melanoma cells.

Author

Carrier A, Desjobert C, Lobjois V, Rigal L, Busato F, Tost J, Ensenyat-Mendez M, Marzese DM, Pradines A, Favre G, Lamant L, Lanfrancone L, Etievant C, Arimondo PB, and Riond J

Subjects
Humans, DNA Methylation, Signal Transduction, Exons, Melanoma genetics, Lung Neoplasms genetics
Abstract

The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients' metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHβ genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness., (© 2022. The Author(s).)

Published
2022
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49. Targeting the MITF/APAF-1 axis as salvage therapy for MAPK inhibitors in resistant melanoma.

Author

Carotenuto P, Romano A, Barbato A, Quadrano P, Brillante S, Volpe M, Ferrante L, Tammaro R, Morleo M, De Cegli R, Iuliano A, Testa M, Andreone F, Ciliberto G, Clery E, Troncone G, Palma G, Arra C, Barbieri A, Capone M, Madonna G, Ascierto PA, Lanfrancone L, Indrieri A, and Franco B

Subjects
Humans, Apoptosis, Cell Line, Tumor, Gene Expression Regulation, Neoplastic, Microphthalmia-Associated Transcription Factor metabolism, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Melanoma pathology, Salvage Therapy
Abstract

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma., Competing Interests: Declaration of interests P.A.A. reports grants and/or personal fees from BMS, Roche-Genentech and Array, MSD, Novartis, Merck Serono, Pierre Fabre, Incyte, Genmab, NewLink Genetics, Medimmune, AstraZeneca, Syndax, Sun Pharma, Sanofi, Idera, Ultimovacs, Sandoz, Immunocore, 4SC, Alkermes, and Nektar, Italfarmaco, outside the submitted work. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2022
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50. DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.

Author

Carrier A, Desjobert C, Ponger L, Lamant L, Bustos M, Torres-Ferreira J, Henrique R, Jeronimo C, Lanfrancone L, Delmas A, Favre G, Daunay A, Busato F, Hoon DSB, Tost J, Etievant C, Riond J, and Arimondo PB

Subjects
Animals, Chromosomes, CpG Islands, Cytosine, DNA Methylation, Epigenesis, Genetic, Epigenome, Gene Expression Regulation, Neoplastic, Guanine, Humans, Mice, Phosphates, Rats, Melanoma, Cutaneous Malignant, Melanoma genetics, Melanoma pathology, Skin Neoplasms genetics
Abstract

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations., Competing Interests: AC, CD, LP, LL, MB, JT, RH, CJ, LL, AD, GF, AD, FB, DH, JT, CE, JR, PA No competing interests declared, (© 2022, Carrier et al.)

Published
2022
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