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Ursolic acid promotes apoptosis and mediates transcriptional suppression of CT45A2 gene expression in non-small-cell lung carcinoma harbouring EGFR T790M mutations.

ABSTRACT: BACKGROUND AND PURPOSE:In non-small-cell lung carcinoma (NSCLC) patients, the L858R/T790M mutation of the epithelial growth factor receptor (EGFR) is a major cause of acquired resistance to EGFR-TKIs treatment that limits their therapeutic efficacy. Identification of drugs that can preferentially kill the NSCLC harbouring L858R/T790M mutation is therefore critical. Here, we have evaluated the effects of ursolic acid, an active component isolated from herbal sources, on erlotinib-resistant H1975 cells that harbour the L858R/T790M mutation. EXPERIMENTAL APPROACH:Gene expression omnibus (GEO) profiles analyses was applied to detect differentially expressed genes in NSCLC cells harbouring EGFR mutation. AnnexinV-FITC/PI, TUNEL staining, MTT, wound healing, RT-PCR, qRT-PCR, western blots, immunostaining, dual-luciferase reporters and ChIP-PCR were utilized to investigate the effects of ursolic acid in vitro and in vivo. KEY RESULTS:The cancer/testis antigen family 45 member A2 (CT45A2) was highly expressed in H1975 cells. Ectopic expression of CT45A2 in H1975 cells increased cell proliferation and motility in vitro. Silencing the CT45A2 expression strongly attenuated H1975 cells motility and growth. The anti-cancer effect of ursolic acid was critically dependent on CT45A2 expression in H1975 cells. Ursolic acid suppressed CT45A2 gene transcription mediated by transcriptional factor TCF4 and ?-catenin signalling. CONCLUSIONS AND IMPLICATIONS:CT45A2 is a novel oncogene for NSCLC with an EGFR T790 mutation. Ursolic acid induced apoptosis and inhibited proliferation of H1975 cells by negatively regulating the ?-catenin/TCF4/CT45A2 signalling pathway. Therefore, ursolic acid may be a potential candidate treatment for NSCLC harbouring the EGFR-L858R/T790M mutation.

SUBMITTER: Yang K

PROVIDER: S-EPMC6965687 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Ursolic acid promotes apoptosis and mediates transcriptional suppression of CT45A2 gene expression in non-small-cell lung carcinoma harbouring EGFR T790M mutations.

Yang Kaiyong K Chen Yan Y Zhou Jiaqian J Ma Lin L Shan Yating Y Cheng Xiaoying X Wang Yun Y Zhang Zhaoxin Z Ji Xiaojun X Chen Lili L Dai Hui H Zhu Biqing B Li Chen C Tao Zhonghua Z Hu Xichun X Yin Wu W

British journal of pharmacology 20191226 24

<h4>Background and purpose</h4>In non-small-cell lung carcinoma (NSCLC) patients, the L858R/T790M mutation of the epithelial growth factor receptor (EGFR) is a major cause of acquired resistance to EGFR-TKIs treatment that limits their therapeutic efficacy. Identification of drugs that can preferentially kill the NSCLC harbouring L858R/T790M mutation is therefore critical. Here, we have evaluated the effects of ursolic acid, an active component isolated from herbal sources, on erlotinib-resistan ...[more]

PMID: 31322286

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Project description:BackgroundEpidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.Methods and findingsTo study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.ConclusionsOur results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.

Project description:Activating double mutations L858R/T790M in the epidermal growth factor receptor (EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs). Third-generation EGFR-TKIs, such as osimertinib and rociletinib (CO-1686), was developed to target such resistance mutations. The detection of activating L858R/T790M mutations is necessary to select sensitive patients for therapy. Hence, we aimed to develop novel radiobromine-labeled CO-1686 as a positron emission tomography (PET) imaging probe for detecting EGFR L858R/T790M mutations. Nonradioactive brominated-CO1686 (BrCO1686) was synthesized by the condensation of N-(3-[{2-chloro-5-(trifluoromethyl)pyrimidin-4-yl}amino]-5-bromophenyl) acrylamide with the corresponding substituted 1-(4-[4-amino-3-methoxyphenyl]piperazine-1-yl)ethan-1-one. The radiobrominated [77Br]BrCO1686 was prepared through bromodestannylation of the corresponding tributylstannylated precursor with [77Br]bromide and N-chlorosuccinimide. Although we aimed to provide a novel PET imaging probe, 77Br was used as an alternative radionuclide for 76Br. We fundamentally evaluated the potency of [77Br]BrCO1686 as a molecular probe for detecting EGFR L858R/T790M using human non-small-cell lung cancer (NSCLC) cell lines: H1975 (EGFR L858R/T790M), H3255 (EGFR L858R), and H441 (wild-type EGFR). The BrCO1686 showed high cytotoxicity toward H1975 (IC50 0.18 ± 0.06 µM) comparable to that of CO-1686 (IC50 0.14 ± 0.05 µM). In cell uptake experiments, the level of accumulation of [77Br]BrCO1686 in H1975 was significantly higher than those in H3255 and H441 upon 4 h of incubation. The radioactivity of [77Br]BrCO1686 (136.3% dose/mg protein) was significantly reduced to 56.9% dose/mg protein by the pretreatment with an excess CO-1686. These results indicate that the binding site of the radiotracers should be identical to that of CO-1686. The in vivo accumulation of radioactivity of [77Br]BrCO1686 in H1975 tumor (4.51 ± 0.17) was higher than that in H441 tumor (3.71 ± 0.13) 1 h postinjection. Our results suggested that [77Br]BrCO1686 has specificity toward NSCLC cells with double mutations EGFR L858R/T790M compared to those in EGFR L858R and wild-type EGFR. However, the in vivo accumulation of radioactivity in the targeted tumor needs to be optimized by structural modification.

Dataset Information

Ursolic acid promotes apoptosis and mediates transcriptional suppression of CT45A2 gene expression in non-small-cell lung carcinoma harbouring EGFR T790M mutations.

Publications

Ursolic acid promotes apoptosis and mediates transcriptional suppression of CT45A2 gene expression in non-small-cell lung carcinoma harbouring EGFR T790M mutations.

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