Project description:Simple Summary: Neuroblastoma (NB) accounts for 15% of all cancer related deaths of children. While amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB, expression of the neurotrophin receptor, NTRK1/TrkA, has been shown to associate with an excellent outcome. MYCN down-regulates NTRK1 expression, but it is unknown if the molecular effects of NTRK1 signaling also affect MYCN-induced networks. The aim of this study was to decipher NTRK1 signaling using an unbiased proteome and phosphoproteome approach. To this end, we realized inducible ectopic NTRK1-expression in a NB cell line with MYCN amplification and analyzed the proteomic changes upon NTRK1-activation in a time-dependent manner. In line with phenotypes observed, NTRK1-activation induced markers of neuronal differentiation and cell cycle arrest. Most prominently, NTRK1 upregulated expression and phosphorylation of the nuclear lamina component Lamin A/C. Moreover, NTRK1 signaling also induced aggregation of LMNA within nucleic foci, which accompanies differentiation in other cell types. Abstract: Background: Neuroblastomas (NB) are the most common extracranial solid tumors of childhood. Amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB aggressiveness, while high expression of the neurotrophin receptor NTRK1/TrkA is associated with mild disease courses. The molecular effects of NTRK1 signaling in MYCN-amplified NB, however, are still poorly understood and require elucidation. Methods: Inducible NTRK1-expression was realized in four NB cell lines with (IMR5, NGP) or without MYCN amplification (SKNAS, SH-SY5Y). Proteome and phosphoproteome dynamics upon NTRK1-activation by its ligand, NGF, were analyzed in a time-dependent manner in IMR5 cells. Target validation by immunofluorescence staining and automated image processing was performed using the three other NB cell lines. Results: In total, 230 proteins and 134 single phosphorylated class I phosphosites were found to be significantly regulated upon NTRK1 activation. Among known NTRK1-targets, Stathmin and the neurosecretory protein VGF were recovered. Additionally, we observed upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. Phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells.

Project description:Presence of BM-infiltrating neuroblastoma (NB) cells occurs in about 50% of patients affected by this peculiar pediatric cancer. Children aged more than 18 months at diagnosis with metastatic NB are high-risk patients with 25-30% overall survival at 5 years, despite multimodal aggressive therapy. Little is known about the metastatic process in NB patients. Hereby, we focused on miRNA expression profiles of BM-infiltrating as compared to those of primary NB tumors. For this purpose, we analyzed 22 BM-infiltrating NB cells, 22 primary NB tumors, and 4 paired BM-infiltrating and primary tumor specimens, all from patients with metastatic disease and aged > 18 months at diagnosis. Statistical analysis indicated that in human BM-infiltrating cells miR-659-3p was down-modulated as compared to primary tumors, in all sets of samples. Functional studies in HTLA and SH-SY5Y NB cell lines by miR-659-3p mimic and inhibitors demonstrated that miR-659-3p regulates the expression of the transcription factor CNOT1, that in turn regulates the expression of AU-rich element (ARE)-containing target genes AKT3, BCL2, THSB2 and CYR61, all belonging to the focal adhesion pathway. Indeed, CNOT1 expression was found significantly higher, whereas that of the ARE-containing target genes was significantly lower in BM-infiltrating cells than in primary tumors. Our findings about a role of the focal adhesion pathway, regulated by miR-659-3p through CNOT1, in NB metastatic process are intriguing for the development of new therapeutic strategies aimed to interrupt the metastatic process. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results. HTLA-230 (kindly donated by Dr E. Bogenman, Los Angeles, USA) and SH-SY5Y (purchased from Banca Biologica and Cell Factory, Genoa, Italy) NB cell lines were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin and 2 mM glutamine (Sigma-Aldrich, Milan, Italy). The HTLA-230 cells present MYCN amplification and SH-SY5Y cells have normal MYCN status. Both cell lines were checked for MYCN amplification, morphology, proliferation rate and mycoplasma contamination, after thawing and within four passages in culture. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, 1x106 HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results.

Project description:The crizotinibâresistant ALKF1174L mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel ALK inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALKF1174L. Here we demonstrate acquired resistance to TAE684 and LDK378 in ALKF1174L-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated ERK signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, which also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALKF1174L-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance. In order to understand the molecular mechanisms driving resistance to ALK inhibition in ALK-mutated neuroblatoma, we established cell line models of resistance to TAE684, an ALK inhibitor, by treating SH-SY5Y cells (bearing the ALKF1174L mutation) with increasing concentration of this compound over time. We then performed an analysis of gene expression changes genome wide using Affymetrix U133 Plus 2 arrays, by comparing the TAE684-sensitive parental SH-SY5Y cells to the TAE684-resistant SH-SY5Y cells (named SY5Y-TR1). For that experiment, we analyzed gene expression variations by comparing the parental SH-SY5Y (control sample) to the resistant SY5Y-TR1 cells. So 2 samples were analyzed, with 3 replicates run for each.

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Innovative Thiosemicarbazones Down-Regulate N-Myc Expression in Neuroblastoma Cells via Transcriptional and Post-Transcriptional Mechanisms

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