Supplementary Figure Legends from Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors
Weicai ChenYe Chow KuangHaibo QiuZhifa CaoYuqing TuQuan Z. ShengGrant EilersQuan HeHailong LiMei‐Jun ZhuYuexiang WangRongqing ZhangYeqing WuFanguo MengJonathan A. FletcherWen‐Bin Ou
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<p>This file contains legends for figures supplied as supplementary data. Immunoblot evaluations of EGFR and phospho-EGFR in GIST cell lines and non-small cell lung cancer cell lines (A549 and PC-9). Actin stain is a loading control. Coordinated inhibition of IR and KIT has additive effect on cell viability in imatinib-resistant GIST430, as compared to either intervention alone, but not in imatinib-sensitive GIST882.</p><p>Fig. S4. Response of GIST KIT mutant cells to TKI inhibitors A-B. Cell viability of SDH-wildtype (or KIT mutant) GIST cells (GIST-T1 and GIST882 cells) after treatment with imatinib (A) and sunitinib (B) at 72 h as measured by MTT viability assay. Percentage viability, relative to the control wells is plotted.</p>
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Abstract Background: Gastrointestinal stromal tumor (GIST) is a common tumor originating from mesenchyme in the alimentary system. GISTs exhibit unique malignant behavior compared to typical carcinomas. Here, we explored genes that drive the progression and imatinib resistance of GISTs by bioinformatic tools and experiments. Methods: We analyzed gene expression profiles of GISTs and identified a key gene, AURKA, which regulated the progression of GISTs. We further determined the clinical significance of AURKA by immunohistochemical (IHC) staining assays and survival analysis. A series of in vitro experiments were performed to investigate in detail the effect of AURKA on the aggressiveness and imatinib resistance of GIST cells. Results: Our results showed that AURKA was higher in advanced GIST tissues than in non-advanced GIST tissues. AURKA mainly involved in cell cycle related biological processes as revealed by GSEA. AURKA was positively related with GIST progression and could be an independent prognostic marker of GISTs. It was also found to exhibit consistent expression patterns in several human malignancies. Additionally, AURKA influenced gene mutations associated with imatinib resistance. Overexpression of AURKA in GIST cells promoted cell proliferation and apoptosis, even after administration of imatinib. But imatinib significantly inhibited the effects of AURKA overexpression on the migration of GIST cells. Conclusions: Bioinformatics analyses and subsequent experiments identified AURKA as a key gene in GIST progression. AURKA showed a contribution to imatinib resistance and could be a candidate therapeutic target for GISTs.
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Gastrointestinal stromal tumour (GIST) is mainly initialised by receptor tyrosine kinase gene mutations. Although the tyrosine kinase inhibitor imatinib mesylate considerably improved the outcome of patients, imatinib resistance still remains a major therapeutic challenge in GIST therapy. Herein we evaluated the clinical impact of microRNAs in imatinib-treated GISTs.The expression levels of microRNAs were quantified using microarray and RT-qPCR in GIST specimens from patients treated with neoadjuvant imatinib. The functional roles of miR-125a-5p and PTPN18 were evaluated in GIST cells. PTPN18 expression was quantified by western blotting in GIST samples.We showed that overexpression levels of miR-125a-5p and miR-107 were associated with imatinib resistance in GIST specimens. Functionally, miR-125a-5p expression modulated imatinib sensitivity in GIST882 cells with a homozygous KIT mutation but not in GIST48 cells with double KIT mutations. Overexpression of miR-125a-5p suppressed PTPN18 expression, and silencing of PTPN18 expression increased cell viability in GIST882 cells upon imatinib treatment. PTPN18 protein levels were significantly lower in the imatinib-resistant GISTs and inversely correlated with miR-125a-5p. Furthermore, several microRNAs were significantly associated with metastasis, KIT mutational status and survival.Our findings highlight a novel functional role of miR-125a-5p on imatinib response through PTPN18 regulation in GIST.
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<p>Fig. S4. Response of GIST KIT mutant cells to TKI inhibitors A-B. Cell viability of SDH-wildtype (or KIT mutant) GIST cells (GIST-T1 and GIST882 cells) after treatment with imatinib (A) and sunitinib (B) at 72 h as measured by MTT viability assay. Percentage viability, relative to the control wells is plotted.</p>
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<p>Expression of FGFR1-4, FGF1-23, KIT, ETV1, PRKCQ, ANO1, EGFR, ERBB2-4, MET, IGF1R, HGF, EGF and NRG1 in multiple primary tumor types and cancer cell lines.</p>
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Gastrointestinal stromal tumors are the most frequent mesenchymal tumors in the gastrointestinal tract.They originate from the interstitial cells of Cajal and are characterized by an anomalous receptor for a growth factor with tyrosine-kinase activity (ckit).This anomaly causes a permanent activation of the receptor and uncontrolled cell growth.These tumors show a poor response to traditional chemotherapy drugs, and are thus associated with low survival in cases of advanced disease.Imatinib, a tyrosine kinase inhibitor, is an example of selective targeted oncologic therapy that induces improved survival in these patients.We discuss two cases of metastatic gastrointestinal stromal tumors with a good response to imatinib, and also review the pathophysiology and treatment-related outcome of this type of tumors.We include results from clinical phase-III studies.
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10552 Background: GISTs are relatively uncommon and predominantly sporadic tumors of the gastrointestinal tract. The high incidence of SPMs observed in patients (p) with GIST (13-20 %), has suggested a possible cause-and-effect relationship between treatment with imatinib and SPMs. Whether this concomitant occurrence is a causal association or a coincidence is not yet resolved. Methods: We have retrospectively analyzed the incidence of synchronous and metachronous SPMs, in all p diagnosed with GIST and treated at a single institution between 1997 and 2012. Results: A total of 95p were diagnosed with GIST, 18 (19%) of whom developed SPMs. For these 18p, the median age at GIST diagnosis was 61.7 years (range, 27-75). The GIST was located in the stomach in 10p (52.6%) and in the small bowel in 8 (47.4%). The SPMs were more frequent in the colon (4p), breast (4), kidney (3), esophagus (2), lymphoproliferative disease (2), bladder (2), adrenal gland (1), prostate (1), pancreas (1), vocal cord (1) and oligodendroglioma (1). 3p had more than one SPM and only one p had the diagnosis of Neurofibromatosis as a genetic sydrome. Of 14p with metachronous SPMs, GIST was the first tumor in 8p, with a median time of 53.9m (range, 4-169) between tumors. GIST was the second tumor in the remaining 6p, with a median time to diagnosis of 34.3m (range, 5-98). Only 4 of our 18p (22.2 %) received imatinib, two of whom were diagnosed with SPMs after having been treated with Imatinib. Conclusions: We have observed SPMs in 19% of p with GIST. The cause of this association is difficult to determine but it seems unrelated to imatinib treatment. The potential non-random association between GIST and other malignances merits further investigation.
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Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107-17. ©2017 AACR.
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<div>Abstract<p>Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of <i>KIT</i> secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or <i>IR</i> shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from <i>IR</i> amplification, <i>IR</i> mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. <i>Cancer Res; 77(18); 5107–17. ©2017 AACR</i>.</p></div>
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