Small RNA expression signatures of gastrointestinal stromal tumors: associations to imatinib resistance and patient outcome
ABSTRACT: We performed miRNA expression profiling in a series of fresh-frozen neoadjuvantly imatinib treated gastrointestinal stromal tumors (GIST), using a microarray approach. Significant differentially expressed miRNAs among imatinib-resistant and imatinib-sensitive groups were identified using SAM analysis. Agilent microarray platform with probes matching 903 human miRNAs was used to determine miRNA expression profiles in 17 GISTs (imatinib-sensitive and 7 imatinib-resistant). To validate the microarray platform, the expression levels of selected miRNAs were evaluated using qRT-PCR.
Project description:We performed microRNA expression profiling in a series of fresh-frozen neoadjuvantly imatinib-treated and non-treated gastrointestinal stromal tumors (GIST), using a microarray approach. Significant differentially expressed microRNAs among imatinib-treated and non-treated groups were identified using SAM analysis. Agilent microarray platform with probes matching 903 human microRNAs was used to determine miRNA expression profiles in 34 GISTs (19 imatinib-treated and 15 non-treated). Re-analysis of GSE45901 for 17 imatinib-treated GISTs. To validate the microarray platform, the expression levels of selected microRNAs were evaluated using qRT-PCR.
Project description:We performed miRNA expression profiling in a series of de novo DLBCLs, transformed DLBCLs and non-neoplastic lymph nodes using a microarray approach. Significant differentially expressed miRNAs among groups were identified using SAM analysis. Agilent microarray platform containing 470 miRNAs was used to determine miRNA expression profiles in 45 DLBCL cases (32 de novo and 13 transformed) and 10 lymph nodes. To validate the microarray platform, the expression levels of selected miRNAs were evaluated using qRT-PCR.
Project description:In order to improve our understanding of microRNA (miRNA) deregulation in melanoma development and possible consequences for patient survival, miRNA expression profiles were determined, using an array based approach, in melanoma tumors, melanoma cell lines and normal melanocytes. Differentially expressed miRNAs were evaluated in relation to clinical characteristics, patient prognosis in terms of melanoma-specific survival, and mutational status for BRAF and NRAS. Agilent microarray platform containing 470 miRNAs was used to determine miRNA expression profiles in 3 normal melanocytes (as non-neoplastic control), 21 melanoma cell lines and 16 clinical samples from fresh frozen regional lymph node metastases. To validate the microarray platform, the expression levels of some miRNAs were evaluated using RT-PCR and the correlation between the two platforms was assessed using Pearson Correlation analysis. The results obtained were further verified and confirmed by RT-PCR in an independent set of melanoma samples. Association between deregulated miRNAs and survival was determined by Univariate Cox proportional hazards model and log rank test.
Project description:We performed miRNA expression profiling in a series of adrenocortical carcinomas, adrenocortical adenomas and normal adreno cortex using a microarray approach. Significant differentially expressed miRNAs among groups were identified using SAM analysis. Agilent microarray platform containing 903 miRNAs was used to determine miRNA expression profiles in 4 normal cortices, 26 adenomas and 22 adrenocortical carcinomas. SAM analysis was adopted to identify significant differentially expressed miRNAs between groups. SAM survival analysis was used to determine the association between miRNA expression and survival among carcinoma cases. The expression levels of candidate prognostic miRNAs were evaluated using qRT-PCR in the same cohort of adrenocortical carcinomas and association with survival was evaluated using Kaplan Meier curves and log rank tests.
Project description:The association of microRNA alterations with progression and treatment outcome has been revealed in different types of cancers. To find miRNAs involved in imatinib response we performed miRNA microarray followed by RT-qPCR verification of 9 available diagnostic bone marrow core biopsies from 9 CML patients including 4 imatinib-resistant and 5 imatinib-responder patients. Only one differentially expressed miRNA, miR-181c, was found when the imatinib-resistant group was compared with imatinib-responders. Significant down-regulation of miR-181c in imatinib-resistant versus imatinib-responders was confirmed by qRT-PCR. Some miR-181c target genes such as PBX3, HSP90B1, NMT2 and RAD21 have been associated with drug response.
Project description:Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1?, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.
Project description:The majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that HSP90 inhibition might be a valuable treatment option for GIST, which would be equally effective on imatinib-sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently being evaluated in clinical trials, in both in vitro and in vivo GIST models. AT13387 inhibited the proliferation of imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to the geldanamycin analogue HSP90 inhibitor, 17-AAG. Treatment with AT13387 resulted in depletion of HSP90 client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines, irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW, xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70, depletion of phospho-KIT and inhibition of KIT signaling were seen in tumors from both models after treatment with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model significantly enhanced tumor growth inhibition over either of the monotherapies. Importantly, the combination of AT13387 and imatinib was well tolerated. These results suggest AT13387 is an excellent candidate for clinical testing in GIST in combination with imatinib.
Project description:Despite the success of imatinib in advanced gastrointestinal stromal tumor (GIST) patients, 50% of the patients experience resistance within two years of treatment underscoring the need to get better insight into the mechanisms conferring imatinib resistance. Here the microRNA and mRNA expression profiles in primary (imatinib-naïve) and imatinib-resistant GIST were examined. Fifty-three GIST samples harboring primary KIT mutations (exon 9; n = 11/exon 11; n = 41/exon 17; n = 1) and comprising imatinib-naïve (IM-n) (n = 33) and imatinib-resistant (IM-r) (n = 20) tumors, were analyzed. The microRNA expression profiles were determined and from a subset (IM-n, n = 14; IM-r, n = 15) the mRNA expression profile was established. Ingenuity pathway analyses were used to unravel biochemical pathways and gene networks in IM-r GIST. Thirty-five differentially expressed miRNAs between IM-n and IM-r GIST samples were identified. Additionally, miRNAs distinguished IM-r samples with and without secondary KIT mutations. Furthermore 352 aberrantly expressed genes were found in IM-r samples. Pathway and network analyses revealed an association of differentially expressed genes with cell cycle progression and cellular proliferation, thereby implicating genes and pathways involved in imatinib resistance in GIST. Differentially expressed miRNAs and mRNAs between IM-n and IM-r GIST were identified. Bioinformatic analyses provided insight into the genes and biochemical pathways involved in imatinib-resistance and highlighted key genes that may be putative treatment targets.
Project description:Both imatinib-sensitive and imatinib-resistant cells were compared in the absence of imatinib. The resistant cells in the presence of imatinib were compared to the sample in the absence of imatinib. The sample from the resistant cells in the presence of imatinib was labelled in duplicate to assess false-positive rates.
Project description:Genome-scale CRISPR-Cas9 Knockout Screening was applied to investigate novel targets in imatinib-resistant gastrointestinal stromal tumor (GIST). 20 genes and 2 miRNAs have been selected by total reads of sgRNA and sgRNA diversity, which has been further validated in imatinib-resistant GIST cells by CCK8 and qPCR analysis. Our study has finally revealed 9 genes (DBP, NR3C1, TCF12, TP53, ZNF12, SOCS6, ZFP36, ACYP1, and DRD1) involved in imatinib-resistant GIST-T1 cells. TP53 and SOCS6 may be the most promising candidate genes for imatinib-resistance due to the possible signaling pathway, such as apoptosis pathway and Wnt signaling pathway, JAK-STAT signaling pathway. It is necessary to perform more studies to discover novel targets in imatinib-resistant GIST, including DBP, NR3C1, TCF12, ZNF12, ZFP36, ACYP1 and DRD1.