Project description:Lymphatic endothelial cells (LEC) were isolated from MT-ret tumor tissue 12h after treatment with an Ang2-blocking antibody or control-IgG
Project description:Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy. From a set of PTC patients whose tumor did not harbour any BRAF or RAS mutations, a 35 years old male patient’s normal, primary tumor and lymph node (LN) metastatic tissues were subjected to genomics and proteomics analysis. By RNA-seq analysis, we identified a novel RET rearrangement involving exons 1-4 from the 5’ end of the Trk fused Gene (TFG) fused to the 3’ end of RET tyrosine kinase leading to a TFG-RET fusion which transforms immortalized human thyroid cells in a kinase-dependent manner. Further, TFG-RET oncogene oligomerises in a PB1 domain-dependent manner and consistently, mutation of the oligomerisation interface led to the inhibition of RET-mediated oncogenic transformation. Quantitative proteomic analysis of the same samples revealed the upregulation of proteins involved in the ubiquitination machinery including E3 Ubiquitin ligase HUWE1 and DUBs like USP9X and UBP7 in both tumor and LN metastatic lesions. We further identified that expression of TFG-RET led to the upregulation of HUWE1. Further, in a cohort of PTC patients, we observed higher expression of HUWE1, USP9X and USP7 in the tumor and metastatic lesions, when compared to the matched normal tissue. Transient knockdown of HUWE1, USP9X and USP7 affected viability and proliferation of TFG-RET transformed cells. Consistently, inhibition of RET, HUWE1 and DUBs by small molecule inhibitors significantly reduced RET-mediated oncogenesis. Apart from unveiling a novel oncogenic RET fusion in PTCs, our data may open a novel avenue of targeting ubiquitin signaling machinery in human PTCs.
Project description:Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy. From a set of PTC patients whose tumor did not harbour any BRAF or RAS mutations, a 35 years old male patient’s normal, primary tumor and lymph node (LN) metastatic tissues was subjected to genomics and proteomics analysis. By RNA-seq analysis, we identified a novel RET rearrangement involving exons 1-4 from the 5’ end of the Trk fused Gene (TFG) fused to the 3’ end of RET tyrosine kinase leading to a TFG-RET fusion which transforms immortalized human thyroid cells in a kinase dependent manner. Further, TFG-RET oncogene oligomerises in a PB1 domain dependent manner and consistently, mutation of the oligomerisation interface led to the inhibition of RET-mediated oncogenic transformation. Quantitative proteomic analysis of the same samples revealed the upregulation of proteins involved in the ubiquitination machinery including HECT carrying E3 Ubiquitin ligase HUWE1 and DUBs like USP9X and UBP7 in the tumor and LN metastatic lesions. We further identify that expression of TFG-RET led to the upregulation of HUWE1. Further, in a cohort of PTC patients, we observed higher expression of HUWE1, USP9X and USP7 in the tumor and metastatic lesions, when compared to the matched normal tissue. Transient knockdown of HUWE1, USP9X and USP7 affected viability and proliferation of TFG-RET transformed cells. Consistently, inhibition of RET, HUWE1 and DUBs by small molecule inhibitors significantly reduced RET-mediated oncogenesis. Apart from unveiling a novel oncogenic RET fusion in PTCs, our data may open a novel avenue of targeting ubiquitin signaling machinery in human PTCs.
Project description:Blood (BEC) and lymphatic (LEC) endothelial cells were isolated from MT-ret tumors and 10x Genomics-based single-cell RNA-seq analysis was performed.
Project description:RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, in RET-rearranged LADC has not been reported. The aims of the study are to explore anti-tumor effects and mechanisms of acquired resistance of dovitinib in RET-rearranged LADC. Using structural modeling and in vitro analysis, we demonstrated that dovitinib induced cell cycle arrest at G0/G1 phase and apoptosis by selective inhibition of RET kinase activity and ERK1/2 signaling in RET-rearranged LC-2/ad cells. Strong anti-tumor effect of dovitinib was observed in LC-2/ad tumor xenograft model. To identify the acquired resistance mechanisms to dovitinib, LC-2/ad cells were exposed to increasing concentrations of dovitinib to generate LC-2/ad DR cells. Gene set enrichment analysis of gene expression and receptor tyrosine kinase assay revealed that Src, a central gene in focal adhesion , was activated in LC-2/ad DR cells. Saracatinib, a src kinase inhibitor, suppressed ERK1/2 phosphorylation and growth of LC-2/ad DR cells. Taken together, these findings suggest that dovitinib can be a potential therapeutic option for RET-rearranged LADC, in which acquired resistance to dovitinib can be overcome by targeting Src. To identify potential mechanisms of acquired resistance to dovitinib, we established LC-2/ad DR cells with acquired resistance to dovitinib by exposing LC-2/ad cells to increasing doses of dovitinib. LC-2/ad DR cells showed strong resistance to dovitinib (IC50> 3 μmol/L). Next, LC-2/ad and LC-2/ad DR cells were subjected to genome-wide gene expression profiling using cDNA microarray.
Project description:Genomic rearrangements leading to intragenic gene fusion are mainly found in some types of haematopoietic malignancies and sarcomas. Recently they have been described also in carcinomas such as the papillary thyroid histotype (60%-70%) and the Hürthle thyroid tumours (58%). The presence of junction oncogene constitutes an area of exciting research for emerging therapy as targeting the RET-PTC1 fusion oncogene by using small interfering RNA (siRNA) strategies since it is present only in the tumour cells and not in the surrounding normal cells. Therefore, we developed a siRNA against RET-PTC1 junction and assess its efficiency on the human papillary thyroid carcinoma cell line TPC-1 which spontaneously harbours the RET-PTC1 oncogene. The targeted genes are assessed by microarray analysis by comparing the regulated genes by the siRNA_RET-PTC1 vs a siRNA_RET developed on the RET part of the mRNA minus the siRNA_control that contain four mutation within the RET-PTC1 sequence. To test the targeted genes in the TPC-1 cell line that spontaneously harbours RET-PTC1 junction of two siRNAs developed: Within the RET-PTC1 junction, and in the mRNA RET part. A non-specific siRNA harbouring 4 mutations within the RET-PTC1 sequence was used as negative control (siRNA_control). By real-time PCR (Q-RT-PCR) we demonstrated that both siRNAs (siRNA_RET-PTC1 and siRNA_RET) significantly reduce RET mRNA levels of about 85 % in TPC-1 cells. The negative control did not show an effect of RET mRNA levels. Three independent transfections were performed on TPC-1 cells using 5 µl of Lipofectamine 2000 transfection reagent (Invitrogen, Cergy-Pontoise, France) and 50nM of i) siRNA_RET-PTC1 or ii) siRNA_RET or iii) siRNA_control that harbour 4 mutations within its sequence. Total RNAs of untreated cells and transfected cells were purified using the RNA cleanup and concentration kit (QIAGEN, Hilden, Germany) and gathered in 4 pools : 1) TPC-1 harbouring RET-PTC1 ; 2) TPC-1 silenced for RET-PTC1 with siRNA RET-PTC1 ; 3) TPC-1 silenced for siRNA RET ; 4) TPC-1 treated with the siRNA control.
Project description:RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, in RET-rearranged LADC has not been reported. The aims of the study are to explore anti-tumor effects and mechanisms of acquired resistance of dovitinib in RET-rearranged LADC. Using structural modeling and in vitro analysis, we demonstrated that dovitinib induced cell cycle arrest at G0/G1 phase and apoptosis by selective inhibition of RET kinase activity and ERK1/2 signaling in RET-rearranged LC-2/ad cells. Strong anti-tumor effect of dovitinib was observed in LC-2/ad tumor xenograft model. To identify the acquired resistance mechanisms to dovitinib, LC-2/ad cells were exposed to increasing concentrations of dovitinib to generate LC-2/ad DR cells. Gene set enrichment analysis of gene expression and receptor tyrosine kinase assay revealed that Src, a central gene in focal adhesion , was activated in LC-2/ad DR cells. Saracatinib, a src kinase inhibitor, suppressed ERK1/2 phosphorylation and growth of LC-2/ad DR cells. Taken together, these findings suggest that dovitinib can be a potential therapeutic option for RET-rearranged LADC, in which acquired resistance to dovitinib can be overcome by targeting Src.