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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Journal of Hematology & Oncology 1/2018

Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2018
Autoren:
Zhentao Liu, Zuhua Chen, Jingyuan Wang, Mengqi Zhang, Zhongwu Li, Shubin Wang, Bin Dong, Cheng Zhang, Jing Gao, Lin Shen
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s13045-018-0651-z) contains supplementary material, which is available to authorized users.
Zhentao Liu and Zuhua Chen contributed equally to this work.

Abstract

Background

No approved targeted agents are available for esophageal squamous cell carcinoma (ESCC). Informative genomic analysis and mouse patient-derived xenografts (PDX) also called mouse avatar can greatly expedite drug discovery.

Methods

Six ESCC cell lines and 7 out of 25 PDX models derived from 188 biopsies with clear molecular features were employed to evaluate the sensitivity of several EGFR blockers in vitro and in vivo, as well as the underlying antitumor mechanisms of the most promising EGFR-TKI afatinib. Mechanisms involved in acquired resistance of afatinib were explored based on established resistant cell lines and PDX models followed by an attempt to reverse resistance.

Results

Compared with other EGFR blockers, the second-generation EGFR-TKI afatinib exerted superior antitumor effects in ESCC, and EGFR copy number gain (CNG) or overexpression was proposed to be predictive biomarkers. Afatinib played its antitumor effects by inhibiting EGFR downstream pathways, as well as inducing apoptosis and cell cycle arrest at G1. It was increased phosphorylation of Src family kinases (SFKs), rather than MET upregulation, that conferred to acquired resistance of afatinib. Dual blockade of EGFR and SFKs could overcome afatinib resistance and warrants validation in clinical practice.

Conclusion

Both ESCC cell lines and PDXs with EGFR CNG or overexpression are potential candidates for afatinib, and concomitant EGFR/SFKs inhibition could reverse afatinib-acquired resistance caused by SFKs activation in ESCC.

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Zusatzmaterial
Additional file 1: Materials and Methods. (DOCX 22 kb)
13045_2018_651_MOESM1_ESM.docx
Additional file 2: Table S1. Efficacy of different EGFR blockers on ESCC cell lines. (DOCX 17 kb)
13045_2018_651_MOESM2_ESM.docx
Additional file 3: Figure S1. Efficacy of different EGFR blockers on ESCC cell lines and PDX models. (A) Six ESCC cell lines were treated with different EGFR blockers (gefitinib, afatinib, osimertinib, cetuximab, and nimotuzumab) at the indicated concentrations (from 0 to 10 μM for TKIs and 0–1000 μg/mL for mAbs). After treatment for 72 h, cell growth inhibition was detected using CCK-8 assays. Cell viability at different doses relative to vehicle-treated controls is shown (means ± SD; three independent assays). (B) Curves plots the growth of PDX03 and PDX06 treated with vehicle control, gefitinib (50 mg/kg/day, oral gavage), afatinib (15 mg/kg/day, oral gavage), osimertinib (15 mg/kg/day, oral gavage), cetuximab (0.5 mg per mouse, twice a week, i.p.), or nimotuzumab (0.5 mg per mouse, twice a week, i.p.). Mice were sacrificed after 21 days of treatment and xenografts were isolated. Pictures of the xenografts are shown and the corresponding TGI is listed in the tables. Data are presented as means ± SDs; n = 5. (DOCX 1320 kb)
13045_2018_651_MOESM3_ESM.docx
Additional file 4: Figure S2. Effects of afatinib on cell cycle and apoptosis in EC109 cells. EC109 cells were treated with 0, 10 nM, 100 nM, and 1 μM afatinib after serum-starvation for 12 h. After 48 h treatment, the cells were harvested and assayed as described below. The effects of afatinib on cell cycle distribution were assessed using flow cytometry after PI/RNase staining (A). The distribution of cells in the cell cycle is depicted (B). G1 phase-associated proteins (P21, P27, CDK4, CDK6, and CCND1) were assessed using western blotting (C).Flow cytometry showed the apoptosis induced by afatinib treatment using PE-annexin V and 7-AAD staining (D). The percentage of cells in early apoptosis (Q3) and late apoptosis (Q2) was calculated as the total apoptosis ratio (E). Apoptosis-related proteins (c-PARP, c-caspase8, BCL2, and BAX) were measured by western blotting after afatinib treatment (F). Data are presented as means ± SDs of three independent assays. P values were calculated using one-way ANOVA or unpaired two-tailed t-tests.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant. (DOCX 570 kb)
13045_2018_651_MOESM4_ESM.docx
Additional file 5: Figure S3. Pathway enrichment by RNA-Seq. Dot plots showing the enrichment results of KEGG pathway analysis for KYSE450-R versus KYSE450-P (A), EC109-R versus EC109-P (B), and PDX03-R versus PDX03-P (C), as detected by RNA-seq. The size of the dots indicates the number of genes enriched in the corresponding pathways. The color of the dots indicates the significance level of the enriched pathways, as represented by the value of Padj. (DOCX 549 kb)
13045_2018_651_MOESM5_ESM.docx
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