Differentially expressed genes in certinib-resistant NB-1 xenografts
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE82143 | GEO | 2016/09/01
SECONDARY ACCESSION(S): PRJNA324126
REPOSITORIES: GEO
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Project description:The discovery of either missense mutations or amplification of ALK (Anaplastic Lymphoma Kinase) gene identified this receptor tyrosine kinase as a therapeutic target in neuroblastoma (NB). Moreover, a high ALK expression has been associated with cases of metastatic NB and with a worse prognosis. In order to identify miRNAs involved in the regulation of ALK expression in NB we therefore analyzed their expression profile in 16 NB cell lines and in 22 patients by qPCR. We then identified miRNAs which were differentially expressed between two groups of samples showing high (ALK+) or low (ALK-) expression levels of ALK by using microarrays containing 866 human miRNA probes (Agilent). Next, the differential expression analysis between the two groups (ALK+ and ALK-) was performed using R. Our analysis showed a higher expression of 30 and 23 miRNAs (p-value <0.05) in NB lines and samples, respectively, both belonging to the ALK- group. Validation analysis and filtering according to in silico suggested miR-424-5p as a potential ALK regulator candidate. Dual luciferase assay indicated a direct binding of this miRNA to ALK-3’UTR. Moreover, we observed a down-regulation of ALK protein expression following transfection of miR-424-5p mimic as well as inhibition of cell viability in ALK+ NB cell lines. In conclusion, our data suggest miR-424-5p as involved in the regulation of ALK expression in NB.
2017-04-30 | GSE86652 | GEO
Project description:High-risk neuroblastoma (NB) often involves amplification of the neural MYC (MYCN) oncogene as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes such as MYCN and ALK result in increased replication stress in cancer cells, offering one such therapeutically exploitable option. Here, we followed up on earlier phosphoproteomic analyses that identified ATR activity in ALK-driven NB cell lines. We tested several ATR inhibitors, identifying BAY 1895344 as the most potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterized the response of NB cells and tumours to ATR inhibition, identifying key components of the DNA damage response (DDR) as well as ATRX, MYCN, E2F and DCK among other ATR targets in NB cells. ATR inhibition with BAY 1895344 also produced robust responses in mouse NB models. Remarkably, a 2 week protocol combining ATR and ALK inhibition led to complete regression of NB tumours in two independent NB genetically modified mouse tumour models. These results suggest that NB patients, particularly in high-risk groups with oncogene induced replication stress, may benefit from inhibition of ATR as therapeutic intervention.
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Project description:The discovery of either missense mutations or amplification of ALK (Anaplastic Lymphoma Kinase) gene identified this receptor tyrosine kinase as a therapeutic target in neuroblastoma (NB). Moreover, a high ALK expression has been associated with cases of metastatic NB and with a worse prognosis. In order to identify miRNAs involved in the regulation of ALK expression in NB we therefore analyzed their expression profile in 16 NB cell lines and in 22 patients by qPCR. We then identified miRNAs which were differentially expressed between two groups of samples showing high (ALK+) or low (ALK-) expression levels of ALK by using microarrays containing 866 human miRNA probes (Agilent). Next, the differential expression analysis between the two groups (ALK+ and ALK-) was performed using R. Our analysis showed a higher expression of 30 and 23 miRNAs (p-value <0.05) in NB lines and samples, respectively, both belonging to the ALK- group. Validation analysis and filtering according to in silico suggested miR-424-5p as a potential ALK regulator candidate. Dual luciferase assay indicated a direct binding of this miRNA to ALK-3’UTR. Moreover, we observed a down-regulation of ALK protein expression following transfection of miR-424-5p mimic as well as inhibition of cell viability in ALK+ NB cell lines. In conclusion, our data suggest miR-424-5p as involved in the regulation of ALK expression in NB.
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Project description:The rearrangement of anaplastic lymphoma kinase (ALK) occurs in 3%-5% of patients with non-small cell lung cancer (NSCLC) and confers sensitivity to ALK-tyrosine kinase inhibitors (TKIs). For the treatment of patients with ALK-rearranged NSCLC, various additional ALK-TKIs have been developed. Ceritinib is a second-generation ALK-TKI and has shown great efficacy in the treatment of patients with both newly diagnosed and crizotinib, a first-generation ALK-TKI, refractory ALK-rearranged NSCLC. However, tumors can also develop ceritinib resistance. This may result from secondary ALK mutations, but other mechanisms responsible for this have not been fully elucidated. In this study, we explored the mechanisms of ceritinib resistance by establishing ceritinib-resistant, echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive H3122 cells and ceritinib-resistant patient-derived cells. We identified a mechanism of ceritinib resistance induced by bypass signals that is mediated by the overexpression and activation of fibroblast growth factor receptor 3 (FGFR3). FGFR3 knockdown by small hairpin RNA or treatment with FGFR inhibitors was found to resensitize the resistant cells to ceritinib in vitro and in vivo. FGFR ligands from either human serum or fetal bovine serum were able to activate FGFR3 and induce ceritinib resistance. A detailed analysis of ceritinib-resistant patient-derived specimens confirmed that tyrosine-protein kinase Met (cMET) amplification induces ceritinib resistance. Amplified cMET counter-activated EGFR and/or Her3, and induced ceritinib resistance. These results reveal multiple ceritinib resistance mechanisms and suggest that ceritinib resistance might be able to overcome by identifying precise resistance mechanisms.
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Project description:Treatment with ALK tyrosine kinase inhibitors often elicits profound initial antitumor responses in ALK fusion-positive patients with lung adenocarcinoma. However, patients invariably develop acquired resistance to ALK inhibitors. In this study, we aimed to identify molecular events that limit the durable response to ALK inhibition using genetic and epigenetic approaches. To identify novel mechanisms of acquired resistance to ALK inhibitors, we established in vivo and in vitro models of acquired resistance to ceritinib and crizotinib using H3122 and H2228 cells. For in vivo model, mice with established H3122-derived tumors were treated with four doses of ceritinib (50mg/kg, 75mg/kg, 87.5mg/kg, 100mg/kg) to derive ceritinib-resistant tumors.
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Project description:Treatment with ALK tyrosine kinase inhibitors often elicits profound initial antitumor responses in ALK fusion-positive patients with lung adenocarcinoma. However, patients invariably develop acquired resistance to ALK inhibitors. In this study, we aimed to identify molecular events that limit the durable response to ALK inhibition using genetic and epigenetic approaches. To identify novel mechanisms of acquired resistance to ALK inhibitors, we established in vivo and in vitro models of acquired resistance to ceritinib and crizotinib using H3122 and H2228 cells. For in vivo model, mice with established H3122-derived tumors were treated with four doses of ceritinib (control, 75mg/kg, 87.5mg/kg, 100mg/kg) to derive ceritinib-resistant tumors.
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Project description:Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Metastatic NB cells were freshly isolated from patients’ BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Unselected BM samples from patients with metastatic NB were also included. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.
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