Figure 1.
Identification of the EZR-ROS1 fusion.
(A) Junction reads representing EZR-ROS1 fusion transcripts in LCY66T sample (left). Sanger sequencing of the RT-PCR product validated tumor-specific in-frame fusion transcript (right). m: molecular marker. (B) Expression profiles of EZR and ROS1 in LCY66T. Active expression of the ROS1 gene was observed after the fusion point. (C) SNP array CGH analysis of the LCY66T. Copy number throughout chromosome 6 is plotted as the log2 ratio.
Figure 2.
Oncogenic activity of the EZR-ROS1 fusion gene.
(A) Schematic representation of EZR, ROS1, EZR-ROS1, and deletions/mutations of EZR-ROS1 genes. The domain organization is shown. C-C: coiled-coil domain; TM: transmembrane; C-ERMAD: C-terminal ERM associated domain. (B) ROS1 phosphorylation in wild-type and mutant EZR-ROS1 (E/R)-expressing NIH3T3 clones. Cell lysates from each clone were immunoblotted with anti-V5-tag (top) and anti-phosphorylated ROS1 (Tyr-2274, bottom) antibodies. (C) Suppression of ROS 1 kinase activity of EZR-ROS1 by crizotinib inhibits STAT3 activation. NIH3T3 cells transfected with 1: empty vector, 2: wild-type EZR-ROS1, 3: KD 4: DL1, 5: DL3 were serum starved and treated for 2 hr with DMSO or 1 µM of crizotinib, and immunoblotted with the relevant antibodies. β-actin was used as a loading control. E/R: EZR-ROS1, p-E/R: phosphorylated EZR-ROS1 detected with an anti-phosphotyrosine-2274 antibody of ROS1. (D) Soft agar colony formation of wild-type and mutant EZR-ROS1 expressing NIH3T3 clones. A representative picture of colony formation for each clone is plotted at the top (scale bar, 100 µm). The number of colonies obtained for each clone is plotted at the bottom. *P<0.05. (E) Crizotinib-induced suppression of anchorage-independent growth of NIH3T3 cells expressing EZR-ROS1. Bar graph showing the percentage of NIH3T3 colonies induced by EZR-ROS1 or CCDC6-RET after treatment with 200 nM of crizotinib or vandetanib with respect to those formed by DMSO-treated cells. EZ-ROS: EZR-ROS1, C6-RET: CCDC6-RET. *P<0.05. (F) Representative pictures of mice subcutaneously transplanted with NIH3T3 cells expressing wild-type, kinase domain-mutated, or amino-terminal-deleted EZR-ROS1. An EML4-ALK-expressing NIH3T3 clone was used as a positive control. The number of tumors per injection in each transfectant is shown below the photographs.
Figure 3.
Alveolar epithelium-specific EZR-ROS1 expression generates lung adenocarcinoma in vivo.
(A) Schematic presentation of the SP-C/EZR-ROS1/polyA transgene. (B) Expression of the exogenous EZR-ROS1 gene in transgenic mice. RT-PCR (top) and immunoblot analysis (bottom) of mouse tissues revealed that EZR-ROS1 was specifically expressed in the lungs of two transgenic mice (TgA21 and TgA25). HT: heart, LV: liver, ST: stomach, SP: spleen, KD: kidney, LG: lung (C) Representative histological analysis of lung lesions in transgenic mice. Hematoxylin-eosin staining shows wide-spread lesions in both 4-week-old and 15-week-old fusion-positive mice. Tg: fusion-positive, CR: fusion-negative. Scale bar, 100 µm. (D) Computed tomography (left) of lungs in TgA04 mouse at week 19. Enhanced lesions in both lungs were detected. Multiple nodular lesions (right) were observed on the pleural surface of the lung in TgC01 mouse at necropsy. (E) Survival curves for transgenic and control mice generated using the Kaplan-Meier method.