Common Oncogene Mutations and Novel SND1-BRAF Transcript Fusion in Lung Adenocarcinoma from Never Smokers

Abstract

Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%), and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1-9 of SND1 and exons 2 to 3' end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation, and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy.

Figure 1. SND1-BRAF transcript fusion in a never smoker lung adenocarcinoma.

(A). Chromosomal rearrangement at 7q32 and 7q34 results in exons 1–9 of SND1 fused to the exon 2 to 3′ end of an inverted BRAF. SND, Staphylococcal nuclease domain; Ex, exon; RBD, Ras-binding domain; CRD, cysteine-rich domain; S/T, serine-threonine. (B). Sequencing traces of the RT-PCR product at fusion junction spanning exon 9 of SND1 (orange) and exon 2 of BRAF (red). The dashed lines show the the reading frame.

Figure 2. Validation and identification of additional SND1-BRAF fusions in lung adenocarcinomas.

(A). SND1-BRAF fusion genes in 3 of 89 samples by qRT-PCR using Fluidigm Dynamic Array. Samples are loaded on each row and the customized assays from ABI or IDT (*) are loaded in each column as quadruplets. The targets amplified are shown at the top and the positives are indicated by boxes. (B). FISH split-apart probe flanking BRAF in a control and Lu-5 samples. Arrows indicate the break-apart probe adjacent to the BRAF gene. Magnification ×1,000. (C). IHC using anti-BRAF^wt antibody in normal lung and indicated tumor samples. Overexpression of BRAF in tumor cells (red arrows) was observed when comparing staining intensity with those in the adjacent bronchial epithelium cells having a basal expression level of the protein (black arrows). Magnification ×200.

Figure 3. Functional Analyses of SND1-BRAF.

(A). Western blots of lysates from H1299 cells transfected with pCMV6-AC-GFP vector, pCMV6-AC-GFP-BRAF^wt, pCMV6-AC-GFP-BRAF^V600E, or pCMV6-AC-GFP-SND1-BRAF were probed with antibodies to C-terminus of BRAF, phospho-MEK1/2 (pMEK), total MEK (MEK), phospho-ERK1/2(pERK), or total ERK (ERK). (B). Proliferation assay of H1299 cells transfected as in A. Error bars represent the average ratio from triplicates. (C). Spheroid formation assay of H1299 cell transfected with indicated constructs. Error bars represent the standard error of the mean (SEM). Statistical significances are as indicated.