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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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In vivo functional effect of CPP expression.(a) Representative explanted tumors. Tumor weight data are presented as means ± standard deviation. (b) Proteins extracted from tumor tissues were loaded onto acrylamide gels to evaluate the protein expression levels of phospho-Nm23-H1, Nm23-H1 and h-Prune. β-Actin was used as the loading control. (c, d) Two NOD/SCID mice groups were given intra-adrenal gland injections of SH-SY5Y-luc cells previously infected with Ad-Mock (7 mice) or Ad-CPP (7 mice). Tumorigenesis was followed by in-vivo bioluminescence photon emissions signals (IVIS Imaging System). Cells pre-infected with Ad-CPP showed impaired tumor growth, with respect to controls. (e) Photograph of resected tumors and representative images of the immunostaining for Ha-Tag, Nm23-H1, h-Prune, Tuj1 and Caspase 3.
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f5: In vivo functional effect of CPP expression.(a) Representative explanted tumors. Tumor weight data are presented as means ± standard deviation. (b) Proteins extracted from tumor tissues were loaded onto acrylamide gels to evaluate the protein expression levels of phospho-Nm23-H1, Nm23-H1 and h-Prune. β-Actin was used as the loading control. (c, d) Two NOD/SCID mice groups were given intra-adrenal gland injections of SH-SY5Y-luc cells previously infected with Ad-Mock (7 mice) or Ad-CPP (7 mice). Tumorigenesis was followed by in-vivo bioluminescence photon emissions signals (IVIS Imaging System). Cells pre-infected with Ad-CPP showed impaired tumor growth, with respect to controls. (e) Photograph of resected tumors and representative images of the immunostaining for Ha-Tag, Nm23-H1, h-Prune, Tuj1 and Caspase 3.

Mentions: CPP function was then investigated in NBL xenograft animal model. Ad-treated SH-SY5Y cells were injected into the flanks of athymic nude mice. After 4 weeks, the tumors were explanted and the inhibition of tumor growth by CPP reached significance (p = 0.00098), as compared to mock-treated SH-SY5Y mice (Fig. 5a). Moreover, tumors obtained from the Ad-CPP-treated SH-SY5Y mice showed a reduction in the expression of the phosphorylated Nm23-H1 protein, while the levels of the Nm23-H1 and h-Prune proteins did not changed, confirming the in vitro data (Fig. 5b). We also assayed two groups of heterotopic xenograft mice that were injected with SH-SY5Y-Luc cells (expressing the luciferase gene) previously infected with Ad-CPP or the control Ad-Mock; tumorigenesis was followed using in vivo biolumuniscence imaging (BLI) technology, over four weeks. The mice receiving Ad-CPP-treated SH-SY5Y-Luc cells (7 mice) showed significant reduction of tumor burden (Fig. 5c) compared to the control treated group (Ad-Mock, 7 mice). This result was confirmed in the quantified data that were obtained by counting the total photon emission (BLI) through whole-body analyses (Fig. 5d and Supplementary Table 1). The tumors generated from Ad-CPP-treated cells showed reduced size and reduced positive staining for both Nm23-H1 and h-Prune compared to Ad-Mock mice. In the Ad-CPP-treated tissues, we also observed positive staining for the neuronal marker, Tuj1, as a sign of benign neuronal differentiation processes, and minimal Caspase3 activation (Fig. 5e). These data illustrate the therapeutic benefit of the use of CPP in vivo.


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)

In vivo functional effect of CPP expression.(a) Representative explanted tumors. Tumor weight data are presented as means ± standard deviation. (b) Proteins extracted from tumor tissues were loaded onto acrylamide gels to evaluate the protein expression levels of phospho-Nm23-H1, Nm23-H1 and h-Prune. β-Actin was used as the loading control. (c, d) Two NOD/SCID mice groups were given intra-adrenal gland injections of SH-SY5Y-luc cells previously infected with Ad-Mock (7 mice) or Ad-CPP (7 mice). Tumorigenesis was followed by in-vivo bioluminescence photon emissions signals (IVIS Imaging System). Cells pre-infected with Ad-CPP showed impaired tumor growth, with respect to controls. (e) Photograph of resected tumors and representative images of the immunostaining for Ha-Tag, Nm23-H1, h-Prune, Tuj1 and Caspase 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: In vivo functional effect of CPP expression.(a) Representative explanted tumors. Tumor weight data are presented as means ± standard deviation. (b) Proteins extracted from tumor tissues were loaded onto acrylamide gels to evaluate the protein expression levels of phospho-Nm23-H1, Nm23-H1 and h-Prune. β-Actin was used as the loading control. (c, d) Two NOD/SCID mice groups were given intra-adrenal gland injections of SH-SY5Y-luc cells previously infected with Ad-Mock (7 mice) or Ad-CPP (7 mice). Tumorigenesis was followed by in-vivo bioluminescence photon emissions signals (IVIS Imaging System). Cells pre-infected with Ad-CPP showed impaired tumor growth, with respect to controls. (e) Photograph of resected tumors and representative images of the immunostaining for Ha-Tag, Nm23-H1, h-Prune, Tuj1 and Caspase 3.
Mentions: CPP function was then investigated in NBL xenograft animal model. Ad-treated SH-SY5Y cells were injected into the flanks of athymic nude mice. After 4 weeks, the tumors were explanted and the inhibition of tumor growth by CPP reached significance (p = 0.00098), as compared to mock-treated SH-SY5Y mice (Fig. 5a). Moreover, tumors obtained from the Ad-CPP-treated SH-SY5Y mice showed a reduction in the expression of the phosphorylated Nm23-H1 protein, while the levels of the Nm23-H1 and h-Prune proteins did not changed, confirming the in vitro data (Fig. 5b). We also assayed two groups of heterotopic xenograft mice that were injected with SH-SY5Y-Luc cells (expressing the luciferase gene) previously infected with Ad-CPP or the control Ad-Mock; tumorigenesis was followed using in vivo biolumuniscence imaging (BLI) technology, over four weeks. The mice receiving Ad-CPP-treated SH-SY5Y-Luc cells (7 mice) showed significant reduction of tumor burden (Fig. 5c) compared to the control treated group (Ad-Mock, 7 mice). This result was confirmed in the quantified data that were obtained by counting the total photon emission (BLI) through whole-body analyses (Fig. 5d and Supplementary Table 1). The tumors generated from Ad-CPP-treated cells showed reduced size and reduced positive staining for both Nm23-H1 and h-Prune compared to Ad-Mock mice. In the Ad-CPP-treated tissues, we also observed positive staining for the neuronal marker, Tuj1, as a sign of benign neuronal differentiation processes, and minimal Caspase3 activation (Fig. 5e). These data illustrate the therapeutic benefit of the use of CPP in vivo.

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