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Neuroblastoma tumorigenesis is regulated through the Nm23-H1/h-Prune C-terminal interaction.

Carotenuto M, Pedone E, Diana D, de Antonellis P, Džeroski S, Marino N, Navas L, Di Dato V, Scoppettuolo MN, Cimmino F, Correale S, Pirone L, Monti SM, Bruder E, Zenko B, Slavkov I, Pastorino F, Ponzoni M, Schulte JH, Schramm A, Eggert A, Westermann F, Arrigoni G, Accordi B, Basso G, Saviano M, Fattorusso R, Zollo M - Sci Rep (2013)

Bottom Line: H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers.We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation.We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP.

View Article: PubMed Central - PubMed

Affiliation: Centro di Ingegneria Genetica e Biotecnologie Avanzate-CEINGE, Naples, Italy.

ABSTRACT
Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.

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A common molecular network for NBL aggressiveness, derived by meta-analysis, combining gene expression NBL databases from two different studies.(a) Two microarray datasets were analysed: Cologne 2-Color (251 samples) and Essen Affymetrix (76 samples). In the network, the NME1 gene is the most connected. Nodes represent genes; node size represents the degree of the node (i.e., the number of connections it has). Nodes are connected with lines, which represent interactions between genes. Line thickness represents the strength of interactions, as measured by mutual information. Statistical confidence of the interactions is represented by the opacity of the color of the lines (strong gray, most reliable interactions; light gray, least reliable interactions). (b) Proteins extracted from tumor tissues and SH-SY5Y cells were loaded on acrylamide gels to determine the protein expression levels of TRIM22 and PTPRA. β-Actin was used as the loading control. Action view of gene networks of genes that are highly correlated with Trim22 (c, green) and PTPRA (d, green). Modes of action are shown in different colors. Genes highlighted in yellow were further investigated. (e) Western blotting showing CPP effects on the Akt pathway in SH-SY5Y cells. β-Actin was used as the loading control. (f) Western blotting for activated β-Catenin, c-Myc, P-Ikbα, and Ikbα protein levels in SH-SY5Y cells after CPP overexpression. β-Actin was used as the loading control. (g) Following Ad-CPP infections in SH-SY5Y cells, protein levels of EGFR, Paxillin, Fak, Fak (Y397), Grb2 and Src (Y527) were analyzed by Western blotting. β-Actin was used as the loading control.
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f6: A common molecular network for NBL aggressiveness, derived by meta-analysis, combining gene expression NBL databases from two different studies.(a) Two microarray datasets were analysed: Cologne 2-Color (251 samples) and Essen Affymetrix (76 samples). In the network, the NME1 gene is the most connected. Nodes represent genes; node size represents the degree of the node (i.e., the number of connections it has). Nodes are connected with lines, which represent interactions between genes. Line thickness represents the strength of interactions, as measured by mutual information. Statistical confidence of the interactions is represented by the opacity of the color of the lines (strong gray, most reliable interactions; light gray, least reliable interactions). (b) Proteins extracted from tumor tissues and SH-SY5Y cells were loaded on acrylamide gels to determine the protein expression levels of TRIM22 and PTPRA. β-Actin was used as the loading control. Action view of gene networks of genes that are highly correlated with Trim22 (c, green) and PTPRA (d, green). Modes of action are shown in different colors. Genes highlighted in yellow were further investigated. (e) Western blotting showing CPP effects on the Akt pathway in SH-SY5Y cells. β-Actin was used as the loading control. (f) Western blotting for activated β-Catenin, c-Myc, P-Ikbα, and Ikbα protein levels in SH-SY5Y cells after CPP overexpression. β-Actin was used as the loading control. (g) Following Ad-CPP infections in SH-SY5Y cells, protein levels of EGFR, Paxillin, Fak, Fak (Y397), Grb2 and Src (Y527) were analyzed by Western blotting. β-Actin was used as the loading control.

Mentions: In order to discover the genes related to Myc activity, and to investigate how these genes might interact, we performed meta-analysis of data from two different datasets and produced a network (see Supplementary Information). In this network, NME1 and NME2 resulted the most connected genes to NBL aggressiveness (Fig. 6a). In addition to these genes, PTPRA, NTRK1, PHGDH, and LAPTM4B are also highly connected.


Neuroblastoma tumorigenesis is regulated through the Nm23-H1/h-Prune C-terminal interaction.

Carotenuto M, Pedone E, Diana D, de Antonellis P, Džeroski S, Marino N, Navas L, Di Dato V, Scoppettuolo MN, Cimmino F, Correale S, Pirone L, Monti SM, Bruder E, Zenko B, Slavkov I, Pastorino F, Ponzoni M, Schulte JH, Schramm A, Eggert A, Westermann F, Arrigoni G, Accordi B, Basso G, Saviano M, Fattorusso R, Zollo M - Sci Rep (2013)

A common molecular network for NBL aggressiveness, derived by meta-analysis, combining gene expression NBL databases from two different studies.(a) Two microarray datasets were analysed: Cologne 2-Color (251 samples) and Essen Affymetrix (76 samples). In the network, the NME1 gene is the most connected. Nodes represent genes; node size represents the degree of the node (i.e., the number of connections it has). Nodes are connected with lines, which represent interactions between genes. Line thickness represents the strength of interactions, as measured by mutual information. Statistical confidence of the interactions is represented by the opacity of the color of the lines (strong gray, most reliable interactions; light gray, least reliable interactions). (b) Proteins extracted from tumor tissues and SH-SY5Y cells were loaded on acrylamide gels to determine the protein expression levels of TRIM22 and PTPRA. β-Actin was used as the loading control. Action view of gene networks of genes that are highly correlated with Trim22 (c, green) and PTPRA (d, green). Modes of action are shown in different colors. Genes highlighted in yellow were further investigated. (e) Western blotting showing CPP effects on the Akt pathway in SH-SY5Y cells. β-Actin was used as the loading control. (f) Western blotting for activated β-Catenin, c-Myc, P-Ikbα, and Ikbα protein levels in SH-SY5Y cells after CPP overexpression. β-Actin was used as the loading control. (g) Following Ad-CPP infections in SH-SY5Y cells, protein levels of EGFR, Paxillin, Fak, Fak (Y397), Grb2 and Src (Y527) were analyzed by Western blotting. β-Actin was used as the loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3584926&req=5

f6: A common molecular network for NBL aggressiveness, derived by meta-analysis, combining gene expression NBL databases from two different studies.(a) Two microarray datasets were analysed: Cologne 2-Color (251 samples) and Essen Affymetrix (76 samples). In the network, the NME1 gene is the most connected. Nodes represent genes; node size represents the degree of the node (i.e., the number of connections it has). Nodes are connected with lines, which represent interactions between genes. Line thickness represents the strength of interactions, as measured by mutual information. Statistical confidence of the interactions is represented by the opacity of the color of the lines (strong gray, most reliable interactions; light gray, least reliable interactions). (b) Proteins extracted from tumor tissues and SH-SY5Y cells were loaded on acrylamide gels to determine the protein expression levels of TRIM22 and PTPRA. β-Actin was used as the loading control. Action view of gene networks of genes that are highly correlated with Trim22 (c, green) and PTPRA (d, green). Modes of action are shown in different colors. Genes highlighted in yellow were further investigated. (e) Western blotting showing CPP effects on the Akt pathway in SH-SY5Y cells. β-Actin was used as the loading control. (f) Western blotting for activated β-Catenin, c-Myc, P-Ikbα, and Ikbα protein levels in SH-SY5Y cells after CPP overexpression. β-Actin was used as the loading control. (g) Following Ad-CPP infections in SH-SY5Y cells, protein levels of EGFR, Paxillin, Fak, Fak (Y397), Grb2 and Src (Y527) were analyzed by Western blotting. β-Actin was used as the loading control.
Mentions: In order to discover the genes related to Myc activity, and to investigate how these genes might interact, we performed meta-analysis of data from two different datasets and produced a network (see Supplementary Information). In this network, NME1 and NME2 resulted the most connected genes to NBL aggressiveness (Fig. 6a). In addition to these genes, PTPRA, NTRK1, PHGDH, and LAPTM4B are also highly connected.

Bottom Line: H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers.We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation.We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP.

View Article: PubMed Central - PubMed

Affiliation: Centro di Ingegneria Genetica e Biotecnologie Avanzate-CEINGE, Naples, Italy.

ABSTRACT
Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.

Show MeSH
Related in: MedlinePlus