Somatic Mutations and Splicing Variants of Focal Adhesion Kinase in Non–Small Cell Lung Cancer

Somatic Mutations and Splicing Variants of Focal Adhesion Kinase in Non–Small Cell Lung Cancer Abstract Background Overexpression of focal adhesion kinase (FAK) has been reported in lung cancer, but the somatic mutations and alternative splicing variants of this nonreceptor tyrosine kinase remain to be investigated. Methods FAK in 91 lung cancer patients was sequenced using genomic DNA and cDNA samples of tumor and paired normal lung tissues as templates, and the RNA-seq data of The Cancer Genome Atlas (TCGA) data set were assessed. The biological functions of abnormal FAK transcripts and their response to FAK inhibitors were analyzed in eight cell lines using tyrosine kinase activity assay, trypan blue exclusion assay, MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide) assay, and transwell assay. Results We identified an internal tandem duplication (ITD), an A1004S point mutation, an exon 5–27 deletion (ΔE5-27) truncation variant, and four FAK6,7 splicing variants (containing exons for Boxes 6 and 7) in seven (7.7%) patients. Smokers had more FAK abnormalities than nonsmokers. In FAK-ITD, the sequence encoding the C-terminal of the FERM domain and kinase domain was duplicated in-frame and produced a protein product with elevated autophosphorylation and sensitivity to FAK inhibitors. FAK6,7 was detected in the tumor but not counterpart normal lung tissues of four (4.4%) patients. In TCGA RNA-seq data, Box 6 and/or Box 7 (Box 6/7)–containing FAK variants were positive in 42 (8.3%) of 508 lung adenocarcinomas (LUADs) and 37 (7.4%) of 501 lung squamous cell carcinomas, and smokers had higher expression of Box 6/7 (+) FAK than reformed or nonsmokers with LUAD. FAK6,7 promoted cell proliferation and migration, exhibited increased autophosphorylation, and was more sensitive to FAK inhibitor compared with wild-type FAK. Conclusions Somatic mutations and splicing variants of FAK may have a role in lung carcinogenesis and represent potential biomarkers for FAK-targeted therapies. Focal adhesion kinase (FAK), a cytoplasmic nonreceptor tyrosine kinase encoded by PTK2 (or FAK) (1,2), is overexpressed in a variety of cancers and associated with poor clinical outcome (3–6). While mutations are rarely reported in cancer genome sequencing projects, FAK splice variants are detected in leukemia (7) and breast cancer (8). FAK promotes cancer cell proliferation, survival, invasion, and stem cell activities (5). Inhibition of FAK renders pancreatic cancer responsiveness to checkpoint immunotherapy (9) and exerts beneficial effects in selected solid tumors (5,10–12). However, the underlying mechanism of FAK hyperactivation in most cancers remains obscure, and biomarkers (13) for FAK-targeting therapies are desired. Lung cancer, the most common cause of cancer-related mortality worldwide (14), is comprised of small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC); the latter type consists of lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large cell carcinoma (15). About 90% of lung cancer deaths are caused by cigarette smoke (16), which activates the c-Src/FAK loop to promote cancer cell migration and invasion (17). FAK promotes cancer development (18) and induces drug resistance (19,20) in lung cancer and is required for the maintenance of KRAS-driven LUAD (21). FAK is overexpressed and inversely associates with prognosis of patients (22–25). Inhibition of FAK suppresses cancer cells (21,26) and enhances the antitumor activity of erlotinib (27). Notably, beneficial effects were seen in one of the five patients receiving FAK inhibitor PF-562271 (11, 28). However, whether lung cancer cells harbor FAK mutations or structural variations and how to precisely select patients for FAK inhibitor treatment warrant further investigation. To uncover FAK somatic variations in lung cancer, here we analyzed the sequence of the whole FAK gene in tumor and paired normal lung tissues of 91 patients. The biological functions and sensitivity to FAK inhibitors PF-562271 and PF-573228 (29) of the identified mutants were investigated. Methods Patient Samples The study was approved by the research ethics committees of all participating sites; all samples were collected with written informed consent from the patient’s family. Tumor and adjacent normal lung tissues were immediately frozen in liquid nitrogen after surgical resection. Genomic DNA and total RNAs were extracted with an AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), and RNAs were reverse-transcribed (RT) into complementary DNA (cDNA) using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Basingstoke, UK) and random primers (Takara Biotechnology, Dalian, China). Polymerase chain reaction (PCR) was performed using DNA or cDNA templets and the primers listed in Supplementary Table 1 (available online), and the products were sequenced. FAK transcripts were cloned for functional studies. RNA Fluorescence In Situ Hybridization and Immunohistochemical Staining Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned at 5 µm intervals. Fluorescently labeled probes (Supplementary Table 1, available online) were designed at the exon junction of the duplicated region for FAK- internal tandem duplication (ITD) and at the Box 6/7 locus for FAK6,7 (refers to the FAK splicing variant containing exons for Boxes 6 and 7, respectively). Fluorescence in situ hybridization (FISH) was conducted as described (30). Immunohistochemical assay was conducted and scored (22) using FFPE tissue specimens and anti-FAK (Cell Signaling, Beverly, MA) and anti-p-FAK (Y397; Thermo Fisher Scientific, Basingstoke, UK) antibodies. The Cancer Genome Atlas RNA-Seq Data The Cancer Genome Atlas (TCGA) level 3 IlluminaHiseq RNAseqV2 data were downloaded from the Broad GDAC Firehose 2015_11_01 run. The percent spliced-in (PSI) (31) values of FAK were downloaded from the TCGA SpliceSeq PSI download page. The positivity of Box 6/7-containing splicing variants should fulfill two criteria: PSI greater than .05 and junction reads of 2 or greater (31). Reads per kilobase per million mapped reads (RPKM) were used to measure the expression of the specific exon, and the Log2 (RPKM + 1) values were calculated. To compare the expression of Box 6/7-containing FAK transcripts between smokers and reformed/nonsmokers, the RPKM values of each spliced exon were normalized by dividing the sum RPKM of all exons from the FAK gene (32). Cell Culture The normal human lung epithelial 16HBE (Clonetics, Walkersville, MD), embryonic lung fibroblasts (HLF), embryonic kidney HEK293, NSCLC lines A549, Calu-6, EPLC32M1, H460, H1975 (the American Type Culture Collection [ATCC], Manassas, VA), and L78 (the Cell Resource Center, Chinese Academy of Medical Sciences, Beijing) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco/BRL, Grand Island, NY). To knockdown FAK in A549 cells, CRISPR-Cas9 system and a specific sgRNA (5’-TCCAGTCTACAGATTTGATA-3’) targeting FAK were used. Cas9 and sgRNA plasmids were cotransfected into A549 cells, and FAK knockdown (designated A549-FAK-Cas9) cells were selected by puromycin (5 µg/mL). The cells were transfected with indicated plasmids using Lipofectamine 3000 (Invitrogen, Frederick, MD), and FAK–stably expressing clones were selected with G418 (Amresco Inc., Cleveland, OH). The cells were treated with PF-562271 and PF-573228, and cell viability was estimated by trypan blue exclusion assay and MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide; Amresco, Solon, OH) assay. For transwell assay, the cells were seeded into the inserts (1 × 105), and complete medium was added into the bottom chamber. Twenty-four hours later, the cells in the upper chamber were removed, and cells in the lower surface were fixed with methanol, stained with crystal violet, and counted with a microscope. Immunoblot and Kinase Activity Assays Proteins were extracted from frozen tissues or cells and subjected to immunoblot using indicated antibodies (see the Supplementary Materials, available online) as described (33). For kinase activity, proteins were harvested from EGFP-FAK-expressing HLF cells and purified by protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GFP antibody-mediated immunoprecipitation and analyzed with a Universal Tyrosine Kinase Assay Kit (Clontech, Palo Alto, CA). Statistical Analysis All the statistical analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). Statistically significant differences were determined by Fisher’s exact test or Mann-Whitney test. The survival curve for each group was estimated by the Kaplan-Meier method and log-rank test. P values of less than .05 were considered statistically significant. All statistical tests were two-sided. Results FAK abnormalities By analyzing the sequences, four types of FAK somatic mutations/splicing variants were identified: an in-frame ITD, one mutation that led to A1004S amino acid substitution, one deletion variant lacking exons 5–27 (ΔE5-27), and four splicing variants (FAK6,7) (Table 1). Interestingly, smokers had more (6/39, 15.4%) FAK abnormalities than nonsmokers (1/50, 2.0%; P = .04) (Table 1). Table 1. The demographic characteristics of the 91 lung cancer patients Characteristics  Total (n = 91)  FAK mutations  Splicing variant  Ratio, %  P  Sex             Male  52  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  11.5  .23   Female  37    1 FAK6,7  2.7     n.d.  2          Age, y             <65  66  1 ITD, 1 point mutation, 1 truncation variant  1 FAK6,7  6.1  .37   ≥65  23    3 FAK6,7  13.0     n.d.  2          Smoking             Smoker  39  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  15.4  .04   Nonsmoker  50    1 FAK6,7  2     n.d.  2          Histology             LUAD  60  1 point mutation, 1 truncation variant    3.3  .07   LUSC  27  1 ITD  3 FAK6,7  14.8     Large cell  carcinoma  1           SCLC  1    1 FAK6,7       n.d.  2          TNM stage             IA–IIB  49  1 point mutation  3 FAK6,7  8.2  1.00   IIIA–IV  35  1 ITD, 1 truncation variant    5.7     n.d.  7    1 FAK6,7      Characteristics  Total (n = 91)  FAK mutations  Splicing variant  Ratio, %  P  Sex             Male  52  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  11.5  .23   Female  37    1 FAK6,7  2.7     n.d.  2          Age, y             <65  66  1 ITD, 1 point mutation, 1 truncation variant  1 FAK6,7  6.1  .37   ≥65  23    3 FAK6,7  13.0     n.d.  2          Smoking             Smoker  39  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  15.4  .04   Nonsmoker  50    1 FAK6,7  2     n.d.  2          Histology             LUAD  60  1 point mutation, 1 truncation variant    3.3  .07   LUSC  27  1 ITD  3 FAK6,7  14.8     Large cell  carcinoma  1           SCLC  1    1 FAK6,7       n.d.  2          TNM stage             IA–IIB  49  1 point mutation  3 FAK6,7  8.2  1.00   IIIA–IV  35  1 ITD, 1 truncation variant    5.7     n.d.  7    1 FAK6,7      * P values were calculated using a two-sided Fisher’s exact test. LCLC = large cell lung cancer; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; n.d. = not determined. FAK-ITD Mutation A FAK-ITD mutation was identified in a male smoker patient, age 38 years, with stage IIIB LUSC. In this mutant, exons 6–27 were duplicated and inserted into the FAK gene body right after exon 27 (Figure 1A). To confirm this mutation, the break point cluster region in genomic DNA was amplified by nested PCR and sequenced. The results showed the junction of break points 141,865,885 and 141,690,508, giving rise to the fusion of FAK intron 27 and intron 5 in the same orientation (Figure 1B). Reverse transcription PCR (RT-PCR) was performed using F1 and R1 primers spanning exons 3 through 7, and wild-type (WT) and ITD FAK were detected in the tumor sample (Figure 1C, left). Sequencing results revealed that an additional copy of exons 6–27 was inserted between exon 5 and exon 6 of the WT transcript, confirming the tandem duplication (Figure 1C, right). By using primers F2 and R2 mapping to exons 22 and 7, respectively, a fusion transcript between exon 27 and exon 6 was detected (Figure 1D). FISH assay showed the existence of FAK-ITD in tumor cells (Figure 1E). FAK-ITD encodes an elongated FAK with an additional 704 amino acids (duplicated 151–854 residues), which is comprised of the autophosphorylation site Y397, the whole tyrosine kinase domain, and the truncated FERM domain (Figure 1F). Immunoblot showed the presence of FAK-ITD in tumor but not paired normal lung tissues of the patient (Figure 1G). Figure 1. View largeDownload slide Identification of a focal adhesion kinase–internal tandem duplication (FAK-ITD) in lung cancer. A) Chromosomal localization and structure of wild-type (WT) FAK and FAK-ITD. The loci 141,690,508 and 141,865,885 were genomic break points, and the duplicated region encompassed exons 6 through 27 of WT FAK transcript. Primers used for detection of FAK-ITD are shown. B-D) Validation of FAK-ITD using the genomic (g) DNA (B) and cDNA (C, D) of tumor and paired normal lung tissues of the patient, by nested polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) followed by sequencing. Samples from a FAK-ITD (-) patient were used as controls. Sanger sequencing results showed the junction region of FAK-ITD. E) Detection of FAK-ITD mRNA by RNA fluorescence in situ hybridization using FITC-labeled probe mapping to the exon junction of duplicated regions. Signals from FAK-ITD are shown as green, and nuclei stained with DAPI are shown as blue. Scale bar = 5 μm. F) Schematic representation of the protein encoded by FAK-ITD. G) Immunoblot analysis of FAK in tumor and paired normal lung tissues of the patient. FAK = focal adhesion kinase; ITD = internal tandem duplication; N = normal; pt # = patient number; T = tumor; WT = wild-type. Figure 1. View largeDownload slide Identification of a focal adhesion kinase–internal tandem duplication (FAK-ITD) in lung cancer. A) Chromosomal localization and structure of wild-type (WT) FAK and FAK-ITD. The loci 141,690,508 and 141,865,885 were genomic break points, and the duplicated region encompassed exons 6 through 27 of WT FAK transcript. Primers used for detection of FAK-ITD are shown. B-D) Validation of FAK-ITD using the genomic (g) DNA (B) and cDNA (C, D) of tumor and paired normal lung tissues of the patient, by nested polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) followed by sequencing. Samples from a FAK-ITD (-) patient were used as controls. Sanger sequencing results showed the junction region of FAK-ITD. E) Detection of FAK-ITD mRNA by RNA fluorescence in situ hybridization using FITC-labeled probe mapping to the exon junction of duplicated regions. Signals from FAK-ITD are shown as green, and nuclei stained with DAPI are shown as blue. Scale bar = 5 μm. F) Schematic representation of the protein encoded by FAK-ITD. G) Immunoblot analysis of FAK in tumor and paired normal lung tissues of the patient. FAK = focal adhesion kinase; ITD = internal tandem duplication; N = normal; pt # = patient number; T = tumor; WT = wild-type. FAK-ITD’s Functions and Sensitivity to FAK Inhibitors FAK-ITD was transfected into HEK293 cells (Figure 2A, left), and we found that this mutant exhibited elevated level of phosphorylated Y397 (p-Y397) and increased overall tyrosine phosphorylation (Figure 2A, middle and right panels) than WT FAK. FAK-ITD showed higher kinase activity than WT FAK (Figure 2B). In WT FAK- and FAK-ITD-expressing 293 cells adhering to the culture plate dish, p-Y397 was detected; in cells held in suspension for 60 to 90 minutes, p-Y397 of WT and ITD FAK was reduced (Figure 2C). In 293 cells transfected with FAK-ITD, the expression of p-AKT and p-ERK was increased, as compared with cells expressing WT FAK (Figure 2D). Ectopic expression of FAK-ITD statistically significantly promoted proliferation of HLF cells (P = .02) (Figure 2E). Figure 2. View largeDownload slide Increased autophosphorylation of Y397 and the responsiveness of focal adhesion kinase–internal tandem duplication (FAK-ITD) to FAK inhibitors. A) Wild-type (WT) FAK and FAK-ITD were transfected into HEK293 cells, and 48 hours later the cells were harvested and lysed. The cell lysates were subjected to immunoprecipitation and immunoblot using indicated antibodies. B) Embryonic lung fibroblasts (HLF) cells stably expressing EGFP-FAK were lysed, and the lysates were immunoprecipitated with an anti-GFP antibody and assayed for tyrosine kinase activity. The amount of FAK proteins in immunoprecipitated samples was determined by immunoblots. C) HEK293 cells expressing Flag-FAK were cultured and attached onto dish plates or kept suspended for 60 minutes or 90 minutes, they were lysed, and the lysates were subjected to immunoblot. D) Immunoblot assays of p-AKT and p-ERK using lysates of HEK293 cells stably expressing Flag-FAK. E) The growth curves of HLF cells expressing EGFP-FAK. Trypan blue exclusion analyses were conducted. *WT-FAK-expressing cells vs FAK-ITD-expressing cells (P = .02, two-sided Student’s t test). F and G) HLF cells stably expressing Flag-FAK were treated with PF-562271 (F) or PF-573228 (G) for 15 minutes, they were lysed, and lysates were analyzed by immunoblot. Error bars on the bar charts represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; WT = wild-type. Figure 2. View largeDownload slide Increased autophosphorylation of Y397 and the responsiveness of focal adhesion kinase–internal tandem duplication (FAK-ITD) to FAK inhibitors. A) Wild-type (WT) FAK and FAK-ITD were transfected into HEK293 cells, and 48 hours later the cells were harvested and lysed. The cell lysates were subjected to immunoprecipitation and immunoblot using indicated antibodies. B) Embryonic lung fibroblasts (HLF) cells stably expressing EGFP-FAK were lysed, and the lysates were immunoprecipitated with an anti-GFP antibody and assayed for tyrosine kinase activity. The amount of FAK proteins in immunoprecipitated samples was determined by immunoblots. C) HEK293 cells expressing Flag-FAK were cultured and attached onto dish plates or kept suspended for 60 minutes or 90 minutes, they were lysed, and the lysates were subjected to immunoblot. D) Immunoblot assays of p-AKT and p-ERK using lysates of HEK293 cells stably expressing Flag-FAK. E) The growth curves of HLF cells expressing EGFP-FAK. Trypan blue exclusion analyses were conducted. *WT-FAK-expressing cells vs FAK-ITD-expressing cells (P = .02, two-sided Student’s t test). F and G) HLF cells stably expressing Flag-FAK were treated with PF-562271 (F) or PF-573228 (G) for 15 minutes, they were lysed, and lysates were analyzed by immunoblot. Error bars on the bar charts represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; WT = wild-type. The FAK-expressing HLF cells were treated with PF-562271 or PF-573228 for 15 minutes, and the expression of p-Y397 was detected. We found that PF562271 at 0.01 µM drastically suppressed p-Y397 of WT FAK; interestingly, PF562271 repressed p-Y397 of FAK-ITD at an even lower concentration, 0.003 µM (Figure 2F). Densitometry analyses of the immunoblot bands confirmed that FAK-ITD responded to a lower concentration of PF-562271 compared with WT FAK (Supplementary Figure 1A, available online). Similarly, PF-573228 inhibited p-Y397 of both WT FAK and FAK-ITD, while FAK-ITD was more sensitive to this inhibitor (Figure 2G;Supplementary Figure 1A, available online). Other FAK Mutations We performed RT-PCR and subsequent sequencing to identify aberrant FAK transcripts using cDNA samples of the other 90 patients and primers spanning the whole coding region of FAK (F3-R3) (Supplementary Table 1, available online). One G to T mutation that led to A1004S amino acid substitution was found in a male smoker patient, age 57 years, with stage IB LUAD (Figure 3, A and B). In a male smoker patient, age 62 years, with stage IV LUAD, a PCR band of about 1200 bp was observed (Figure 3C). Exons 5–27 were deleted in this mutant (Figure 3D), yielding a truncated FAK ΔE5-27 that encodes a protein of 194 amino acids (Figure 3B). Figure 3. View largeDownload slide Identification of focal adhesion kinase (FAK) mutations and splicing variants in lung cancer. A) A somatic nucleotide substitution, c.3010G>T, was detected in FAK in one tumor. This mutation encodes an A to S amino acid change at residue 1004 (B). C and D) Identification of a ΔE5-27 FAK transcript in one tumor by reverse transcription polymerase chain reaction (RT-PCR) (C) and sequencing (D). This transcript encodes a truncated FAK (B). E–G) Identification of Boxes 6 and 7–containing FAK6,7 in four tumors. E) Primers used for RT-PCR for validation of this variant. F) Bands of RT-PCR assay using cDNA of samples isolated from the four patients. G) Sequencing results of bands in (F). This transcript encodes a protein inclusion with Box 6 and Box 7 on either side of Y397 (B). FAK = focal adhesion kinase; pt # = patient number; WT = wild-type. Figure 3. View largeDownload slide Identification of focal adhesion kinase (FAK) mutations and splicing variants in lung cancer. A) A somatic nucleotide substitution, c.3010G>T, was detected in FAK in one tumor. This mutation encodes an A to S amino acid change at residue 1004 (B). C and D) Identification of a ΔE5-27 FAK transcript in one tumor by reverse transcription polymerase chain reaction (RT-PCR) (C) and sequencing (D). This transcript encodes a truncated FAK (B). E–G) Identification of Boxes 6 and 7–containing FAK6,7 in four tumors. E) Primers used for RT-PCR for validation of this variant. F) Bands of RT-PCR assay using cDNA of samples isolated from the four patients. G) Sequencing results of bands in (F). This transcript encodes a protein inclusion with Box 6 and Box 7 on either side of Y397 (B). FAK = focal adhesion kinase; pt # = patient number; WT = wild-type. Alternative Splicing Variant FAK6,7 in Lung Cancer By RT-PCR assay using primers spanning the whole coding region, one FAK splicing variant was identified in the tumor but not paired normal lung tissues of four (4.4%) patients (Table 1). RT-PCR with primers spanning exons 14 through 16 (Figure 3E) confirmed the existence of this splicing variant (Figure 3F). Sequencing results (Figure 3G) showed that this variant contains additional alternatively spliced exons of 18 bp (Box 6) and 21 bp (Box 7), respectively (Figure 3E), coding for two short peptides of six and seven amino acids on either side of Y397 (Figure 3B). RNA FISH assays confirmed the existence of FAK6,7 in tumor cells (Supplementary Figure 2A, available online). To identify the potential intron abnormalities that lead to splicing variants, exons 14–16 (chr8: 141771264–141799625; a region of 28.3 kb) of genomic FAK were sequenced, and 13 single nucleotide polymorphisms (SNPs) were detected in four patients (Supplementary Table 2 and Supplementary Figure 2B, available online). Two SNPs (ID rs7840381 and rs10875458) were seen in NSCLC line Calu-6. By ACESCAN2 method (34), we found that three SNPs, for example, G to T in rs4246123, T to G in rs7840381, and A to G in rs4579574, located in regions of intronic splicing enhancers. SNPs were able to enhance alternative splicing (35); therefore, the above SNPs may pave the way to FAK6,7 alternative splicing. Box 6 and/or 7–Containing Variants FAK6/7 in Lung Cancer FAK alternative splicing events were explored in TCGA RNA-seq data. In 508 LUADs, the PSI values of Box 6 alternative splicing ranged from 0 to 0.47, and those of Box 7 ranged from 0 to 0.72 (Figure 4A). By screening with the number of corresponding splice junctions and the PSI values (Supplementary Table 3, available online), 42 (8.3%) LUAD patients were identified as “Box 6 and/or Box 7 (Box 6/7) positive,” and 12 (2.4%) patients harbored FAK6,7. Moreover, 29 (5.7%) patients showed high expression of exon coding for Box 6/7 (RPKM > 2) (Figure 4B). In 501 LUSCs, the PSI values of Box 6/7 variants were up to 0.69 and 0.90, respectively (Figure 4C). Thirty-seven (7.4%) patients were positive for Box 6 or 7, and 5 (1.0%) patients had FAK6,7, while 20 (4.0%) patients showed high expression of Box 6/7 exons (RPKM > 2) (Figure 4D). Figure 4. View largeDownload slide Box 6 and/or Box 7 (Box 6/7)–containing focal adhesion kinase (FAK) splicing variants in lung cancer. A) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 508 The Cancer Genome Atlas (TCGA) lung adenocarcinomas (LUADs). B) Box 6/7–containing FAK variants were found in 42 (8.3%) LUADs. The values of Log2 (reads per kilobase per million mapped reads [RPKM] + 1) were calculated, and the junction reads of the corresponding exons were indicated. C) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 501 TCGA lung squamous cell carcinoma (LUSCs). D) Box 6/7–containing FAK variants were found in 37 (7.4%) LUSCs. The values of Log2 (RPKM+1) were calculated, and the junction reads of the corresponding exons were indicated. E and F) The expression of Box 6 or Box 7–containing FAK splicing variants in current smoker, reformed smoker, and nonsmoker LUADs (E) and LUSCs (F). The expression level is shown as normalized RPKM ×1000. The Mann-Whitney test was used for calculating statistical significance. G) Overall survival (OS) of LUADs and LUSCs with or without Box 6/7–containing FAK splicing variants. H) Overall survival of LUADs with or without Box 6/7–containing FAK splicing variants. I) Overall survival of LUSCs with or without Box 6/7–containing FAK splicing variants. The log-rank test was used for analyzing survival differences. All statistical tests were two-sided. LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; OS = overall survival; PSI = percent spliced-in; RPKM = reads per kilobase per million mapped reads. Figure 4. View largeDownload slide Box 6 and/or Box 7 (Box 6/7)–containing focal adhesion kinase (FAK) splicing variants in lung cancer. A) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 508 The Cancer Genome Atlas (TCGA) lung adenocarcinomas (LUADs). B) Box 6/7–containing FAK variants were found in 42 (8.3%) LUADs. The values of Log2 (reads per kilobase per million mapped reads [RPKM] + 1) were calculated, and the junction reads of the corresponding exons were indicated. C) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 501 TCGA lung squamous cell carcinoma (LUSCs). D) Box 6/7–containing FAK variants were found in 37 (7.4%) LUSCs. The values of Log2 (RPKM+1) were calculated, and the junction reads of the corresponding exons were indicated. E and F) The expression of Box 6 or Box 7–containing FAK splicing variants in current smoker, reformed smoker, and nonsmoker LUADs (E) and LUSCs (F). The expression level is shown as normalized RPKM ×1000. The Mann-Whitney test was used for calculating statistical significance. G) Overall survival (OS) of LUADs and LUSCs with or without Box 6/7–containing FAK splicing variants. H) Overall survival of LUADs with or without Box 6/7–containing FAK splicing variants. I) Overall survival of LUSCs with or without Box 6/7–containing FAK splicing variants. The log-rank test was used for analyzing survival differences. All statistical tests were two-sided. LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; OS = overall survival; PSI = percent spliced-in; RPKM = reads per kilobase per million mapped reads. We analyzed the association between FAK6/7 expression and patients’ smoking status by analyzing the normalized RPKM value (×1000) and found that in LUADs the current smokers had higher FAK6/7 than reformed and nonsmokers (Figure 4E); in patients with LUSCs, the current smokers had similar FAK6/7 to nonsmokers (Figure 4F). Among the 990 patients whose survival information was available, the median overall survival (OS) for Box 6/7 (+) patients was 1293 days, shorter than Box 6/7 (-) patients (1622 days, P = .40) (Figure 4G). In LUADs (Figure 4H) and LUSCs (Figure 4I), patients with Box 6/7 (+) FAK had shorter survival than patients with Box 6/7 (-) FAK. Kinase Activity and Sensitivity to Targeted Therapy of FAK6,7 By immunohistochemical assays, we showed that the four FAK6,7 (+) patients had more elevated p-FAK (Y397) than WT FAK patients (P = .04) (Figure 5, A and B). Immunoblot assays confirmed the increased p-Y397 in tumor samples of the FAK6,7 (+) patients compared with patients with WT FAK (Figure 5C). Immunoblot analysis using proteins from EGFP-FAK-transfected HLF cells confirmed that FAK6,7 had more elevated p-Y397 than WT FAK (Figure 5D). In H460, A549-FAK-Cas9 (Supplementary Figure 3, available online), and 16HBE cells transfected with FAK, FAK6,7 showed elevated phosphorylation level compared with WT FAK and the kinase dead (KD) (Figure 5E;Supplementary Figure 1B, available online). FAK6,7 promoted proliferation (Figure 5F;Supplementary Figure 1C, available online) and migration (Figure 5G) of the cells. Figure 5. View largeDownload slide Autophosphorylation of focal adhesion kinase (FAK)6,7 and its sensitivity to FAK inhibitors. A–C) p-FAK (Y397) in tumors of FAK6,7 or wild-type (WT) FAK patients detected by immunohistochemistry (A) scoring (B) and immunoblots (C). Scale bar = 2000 µm. D) p-Y397 of HLF cells stably expressing WT FAK and FAK6,7. E-G) Flag-FAK vectors were transfected into H460 and A549-FAK-Cas9 cells (E). Cells were counted by trypan blue exclusion analysis at 48 and 72 hours after transfection (F), and cell migration was assessed by transwell assay 48 hours after transfection (G). Scale bar = 200 µm. H) HLF cells expressing WT FAK or FAK6,7 were treated with PF562271 and PF573228 for 15 minutes, and the expression of p-Y397 was tested. I) H460 and A549-FAK-Cas9 cells were transfected with Flag-FAK and then treated with or without 2 μM PF562271 for 48 hours. Inhibition rates of cell proliferation were determined by trypan blue exclusion assay. J) Non–small cell lung cancer lines seeded in 96-well plates were treated with PF562271, and cell viability was measured by MTT assays. K) The cells were treated with PF562271 at indicated concentrations for 15 minutes, and p-Y397 was tested. Error bars represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; pt # = patient number; WT = wild-type. Figure 5. View largeDownload slide Autophosphorylation of focal adhesion kinase (FAK)6,7 and its sensitivity to FAK inhibitors. A–C) p-FAK (Y397) in tumors of FAK6,7 or wild-type (WT) FAK patients detected by immunohistochemistry (A) scoring (B) and immunoblots (C). Scale bar = 2000 µm. D) p-Y397 of HLF cells stably expressing WT FAK and FAK6,7. E-G) Flag-FAK vectors were transfected into H460 and A549-FAK-Cas9 cells (E). Cells were counted by trypan blue exclusion analysis at 48 and 72 hours after transfection (F), and cell migration was assessed by transwell assay 48 hours after transfection (G). Scale bar = 200 µm. H) HLF cells expressing WT FAK or FAK6,7 were treated with PF562271 and PF573228 for 15 minutes, and the expression of p-Y397 was tested. I) H460 and A549-FAK-Cas9 cells were transfected with Flag-FAK and then treated with or without 2 μM PF562271 for 48 hours. Inhibition rates of cell proliferation were determined by trypan blue exclusion assay. J) Non–small cell lung cancer lines seeded in 96-well plates were treated with PF562271, and cell viability was measured by MTT assays. K) The cells were treated with PF562271 at indicated concentrations for 15 minutes, and p-Y397 was tested. Error bars represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; pt # = patient number; WT = wild-type. In EGFP-FAK-expressing HLF cells, p-Y397 of FAK6,7 responded to PF562271 and PF573228 at a lower concentration and an earlier time point compared with WT FAK (Figure 5H;Supplementary Figure 1D, available online). In FAK-expressing H460 and A549-FAK-Cas9 cells, p-Y397 of FAK6,7 was more sensitive to PF562271 compared with WT FAK (Figure 5I, upper), and inhibition rates of FAK6,7-expressing cells were higher than those of WT FAK cells (Figure 5I, lower). To identify NSCLC lines harboring FAK6,7, RT-PCR and subsequent sequencing were conducted, and we found that Calu-6 had FAK6,7 (Supplementary Figure 4, A and B, available online) and elevated p-Y397 (Supplementary Figure 4C, available online). PF562271 suppressed the viability of Calu-6 cells in a dose- and time-dependent manner, and this line was the most sensitive one among the lines treated with PF562271 (Figure 5J). PF562271 suppressed FAK p-Y397 in Calu-6 cells at a lower concentration than in H460 and A549 cells (Figure 5K). Discussion FAK serves as an essential intracellular mediator of extracellular signals such as growth factors, extracellular matrix remodeling, and nutrient availability. FAK is activated in a variety of cancers, but only a few FAK missense mutations (R65S, etc.) and a PTDSS1_FAK fusion transcript in one tumor were reported (36,37). Here, by detailed analysis of genomic and cDNA sequence of FAK, we reported four types of somatic variants of FAK in seven (7.7%) of 91 lung cancer patients, indicating that FAK has several types of structural variations, and scrutinizing its sequence is helpful for lung cancer genotyping. FAK activation involves integrin receptor clustering upon cell binding to extracellular matrix proteins, and Y397 is a major autophosphorylation site (5) that can be blocked by FERM domain (38,39). In the duplicated FERM domain of FAK-ITD, the N-terminal 116 amino acids (R35–V151) including K38, which is important for auto-inhibitory activity of FERM (40), were deleted, suggesting that this mutant might have elevated kinase activity. This hypothesis was confirmed by our cellular experiments. On the other hand, the truncated protein encoded by the ΔE5-27 allele may lack its kinase activity; however, this protein may probably form heterodimer with the FERM domain of the full-length FAK encoded by the WT allele, thus releasing the auto-inhibitory effect of the FERM domain and leading to elevation of FAK activity. This possibility warrants further investigation. Alternative splicing, the process by which splice sites are differentially utilized to produce different mRNA isoforms, contributes to oncogenic activation in several types of cancers (41,42). Alternative splicing in genes such as VEGFA had been reported in lung cancer (43). FAK6, FAK7, and FAK6,7 have been identified in brain (44–46). Here we showed for the first time that FAK6,7 was expressed in tumor but not paired normal lung tissues in four (4.4%) of the 91 patients. A previous study showed that FAK6,7 has elevated kinase activity (44). We found that compared with WT FAK, FAK6,7 exhibited increased p-Y397 in patient samples and lung epithelial cells. FAK6,7 promoted cell proliferation and migration, suggesting a role for FAK6,7 in lung carcinogenesis. By analyzing the RNA-seq data of TCGA, we found that 8.3% of LUADs and 7.4% LUSCs had Box 6– or Box 7–containing FAK splicing events. These results suggest that alternative splicing represents an important mechanism of FAK activation in lung cancer. Smoker NSCLCs bear more genomic mutations than nonsmokers (47,48). Here we showed that smoker patients had more FAK abnormalities than nonsmokers. In TCGA patients, current smokers had more Box 6/7–containing FAK variants than reformed and nonsmokers. The association between FAK splicing variants and tobacco smoke suggested that tobacco carcinogens might affect molecules involved in splicing. This speculation was confirmed by previous studies showing that smokers had a higher frequency of MDM2 variant splicing than nonsmokers, owing to the effects of the carcinogen benzo[a]pyrene and its metabolite benzo[a]pyrene diolepoxide on the PI3K or MAPK pathway (49). CD44 splicing variants were also induced by benzo[a]pyrene treatment (50). These results suggest that lung carcinogenesis driven by tobacco smoke is very complicated, and how carcinogens induce FAK alternative splicing in patients remains to be elucidated. Our study has some limitations. First, the role of FAK-ITD and FAK6,7 in malignant transformation and tumor growth is partially demonstrated at the cellular level, which should be further tested in animal models (xenograft or transgenic/knockin mice). Second, the greater sensitivity of FAK-ITD/FAK6,7 to FAK inhibitors is shown in vitro. However, in vivo experiments are warranted for further evaluating the translational potentials of this study. Third, whether FAK-ITD is a recurrent somatic mutation needs to be determined. Several FAK tyrosine kinase inhibitors, for example, PF-562271, VS-4718, and VS-6063, are currently undergoing clinical investigation and show therapeutic potentials in selected solid cancers (5). We found that FAK-ITD and FAK6,7 were more sensitive to FAK inhibitors than WT FAK. Therefore, smokers with FAK mutations and splicing variants may represent biomarkers for FAK-targeted therapies, and this possibility should be tested in future clinical trials. Funding This work was supported by the National Natural Science Funds for Distinguished Young Scholars (81425025), the National Key Research and Development Program of China (2016YFC0905500), the “Personalized Medicines–Molecular Signature-based Drug Discovery and Development” Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12010307), the National Natural Science Foundation of China (81672765), and grants from the State Key Laboratory of Membrane Biology. Notes The study sponsor had no role in the design of the study; the data collection, analysis, or interpretation; the writing of the article; or the decision to submit for publication. References 1 Fiedorek FTJr, Kay ES. Mapping of the focal adhesion kinase (Fak) gene to mouse chromosome 15 and human chromosome 8. Mamm Genome.  1995; 6( 2): 123– 126. Google Scholar CrossRef Search ADS PubMed  2 Schaller MD, Hildebrand JD, Shannon JD, et al.   Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2- dependent binding of pp60src. Mol Cell Biol.  1994; 14( 3): 1680– 1688. Google Scholar CrossRef Search ADS PubMed  3 Owens LV, Xu L, Craven RJ, et al.   Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res.  1995; 55( 13): 2752– 2755. Google Scholar PubMed  4 Agochiya M, Brunton VG, Owens DW, et al.   Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells. Oncogene.  1999; 18( 41): 5646– 5653. Google Scholar CrossRef Search ADS PubMed  5 Lee BY, Timpson P, Horvath LG, et al.   FAK signaling in human cancer as a target for therapeutics. Pharmacol Ther.  2015; 146: 132– 149. Google Scholar CrossRef Search ADS PubMed  6 Yoon H, Dehart JP, Murphy JM, et al.   Understanding the roles of FAK in cancer: Inhibitors, genetic models, and new insights. J Histochem Cytochem.  2015; 63( 2): 114– 128. Google Scholar CrossRef Search ADS PubMed  7 Despeaux M, Chicanne G, Rouer E, et al.   Focal adhesion kinase splice variants maintain primitive acute myeloid leukemia cells through altered Wnt signaling. Stem Cells.  2012; 30( 8): 1597– 1610. Google Scholar CrossRef Search ADS PubMed  8 Yao L, Li K, Peng W, et al.   An aberrant spliced transcript of focal adhesion kinase is exclusively expressed in human breast cancer. J Transl Med.  2014; 12( 1): 1– 9. Google Scholar CrossRef Search ADS PubMed  9 Jiang H, Hegde S, Knolhoff BL, et al.   Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nat Med.  2016; 22( 8): 851– 860. Google Scholar CrossRef Search ADS PubMed  10 Golubovskaya VM. Targeting FAK in human cancer: From finding to first clinical trials. Front Biosci.  2014; 19: 687– 706. Google Scholar CrossRef Search ADS   11 Infante JR, Camidge DR, Mileshkin LR, et al.   Safety, pharmacokinetic, and pharmacodynamic phase I dose-escalation trial of PF-00562271, an inhibitor of focal adhesion kinase, in advanced solid tumors. J Clin Oncol.  2012; 30( 13): 1527– 1533. Google Scholar CrossRef Search ADS PubMed  12 Roy-Luzarraga M, Hodivala-Dilke K. Molecular pathways: Endothelial cell FAK—a target for cancer treatment. Clin Cancer Res.  2016; 22( 15): 3718– 3724. Google Scholar CrossRef Search ADS PubMed  13 Shapiro IM, Kolev VN, Vidal CM, et al.   Merlin deficiency predicts FAK inhibitor sensitivity: A synthetic lethal relationship. Sci Transl Med.  2014; 6( 237): 237ra68– 237ra68. Google Scholar CrossRef Search ADS PubMed  14 Torre LA, Bray F, Siegel RL, et al.   Global cancer statistics, 2012. CA Cancer J Clin.  2015; 65( 2): 87– 108. Google Scholar CrossRef Search ADS PubMed  15 Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med.  2008; 359( 13): 1367– 1380. Google Scholar CrossRef Search ADS PubMed  16 Hecht SS. Lung carcinogenesis by tobacco smoke. Int J Cancer.  2012; 131( 12): 2724– 2732. Google Scholar CrossRef Search ADS PubMed  17 Shen J, Xu L, Owonikoko TK, et al.   NNK promotes migration and invasion of lung cancer cells through activation of c-Src/PKCι/FAK loop. Cancer Lett.  2012; 318( 1): 106– 113. Google Scholar CrossRef Search ADS PubMed  18 Wang B, Qi X, Li D, et al.   Expression of pY397 FAK promotes the development of non-small cell lung cancer. Oncol Lett.  2016; 11( 2): 979– 983. Google Scholar CrossRef Search ADS PubMed  19 Kang Y, Hu W, Ivan C, et al.   Role of focal adhesion kinase in regulating YB–1–mediated paclitaxel resistance in ovarian cancer. J Natl Cancer Inst.  2013; 105( 19): 1485– 1495. Google Scholar CrossRef Search ADS PubMed  20 Lu H, Wang L, Gao W, et al.   IGFBP2/FAK pathway is causally associated with dasatinib resistance in non–small cell lung cancer cells. Mol Cancer Ther.  2013; 12( 12): 2864– 2873. Google Scholar CrossRef Search ADS PubMed  21 Konstantinidou G, Ramadori G, Torti F, et al.   RHOA-FAK is a required signaling axis for the maintenance of KRAS-driven lung adenocarcinomas. Cancer Disc.  2013; 3( 4): 444– 457. Google Scholar CrossRef Search ADS   22 Carelli S, Zadra G, Vaira V, et al.   Up-regulation of focal adhesion kinase in non-small cell lung cancer. Lung Cancer.  2006; 53( 3): 263– 271. Google Scholar CrossRef Search ADS PubMed  23 Ji HF, Pang D, Fu SB, et al.   Overexpression of focal adhesion kinase correlates with increased lymph node metastasis and poor prognosis in non-small-cell lung cancer. J Cancer Res Clin Oncol.  2013; 139( 3): 429– 435. Google Scholar CrossRef Search ADS PubMed  24 Nishimura M, Machida K, Imaizumi M, et al.   Tyrosine phosphorylation of 100-130 kDa proteins in lung cancer correlates with poor prognosis. Br J Cancer.  1996; 74( 5): 780– 787. Google Scholar CrossRef Search ADS PubMed  25 Rikova K, Guo A, Zeng Q, et al.   Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell.  2007; 131( 6): 1190– 1203. Google Scholar CrossRef Search ADS PubMed  26 Meng XN, Jin Y, Yu Y, et al.   Characterisation of fibronectin-mediated FAK signalling pathways in lung cancer cell migration and invasion. Br J Cancer.  2009; 101( 2): 327– 334. Google Scholar CrossRef Search ADS PubMed  27 Howe GA, Xiao B, Zhao H, et al.   Focal adhesion kinase inhibitors in combination with erlotinib demonstrate enhanced anti-tumor activity in non-small cell lung cancer. PLoS One.  2016; 11( 3): e0150567. Google Scholar CrossRef Search ADS PubMed  28 Roberts WG, Ung E, Whalen P, et al.   Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res.  2008; 68( 6): 1935– 1944. Google Scholar CrossRef Search ADS PubMed  29 Slack-Davis JK, Martin KH, Tilghman RW, et al.   Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem.  2007; 282( 20): 14845– 14852. Google Scholar CrossRef Search ADS PubMed  30 Kurobe M, Kojima T, Nishimura K, et al.   Development of RNA-FISH assay for detection of oncogenic FGFR3-TACC3 fusion genes in FFPE samples. PLoS One.  2016; 11( 12): e0165109. Google Scholar CrossRef Search ADS PubMed  31 Ryan M, Wong WC, Brown R, et al.   TCGASpliceSeq a compendium of alternative mRNA splicing in cancer. Nucl Acids Res.  2016; 44( D1): D1018– D1022. Google Scholar CrossRef Search ADS   32 Zhao Q, Caballero OL, Davis ID, et al.   Tumor-specific isoform switch of the fibroblast growth factor receptor 2 underlies the mesenchymal and malignant phenotypes of clear cell renal cell carcinomas. Clin Cancer Res.  2013; 19( 9): 2460– 2472. Google Scholar CrossRef Search ADS PubMed  33 Wang GZ, Cheng X, Zhou B, et al.   The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution. eLife.  2015; 4: e09419. Google Scholar PubMed  34 Yeo GW, Van Nostrand E, Holste D, et al.   Identification and analysis of alternative splicing events conserved in human and mouse. Proc Natl Acad Sci U S A.  2005; 102( 8): 2850– 2855. Google Scholar CrossRef Search ADS PubMed  35 Narla G, DiFeo A, Reeves HL, et al.   A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk. Cancer Res.  2005; 65( 4): 1213– 1222. Google Scholar CrossRef Search ADS PubMed  36 Sulzmaier FJ, Jean C, Schlaepfer DD. FAK in cancer: Mechanistic findings and clinical applications. Nat Rev Cancer.  2014; 14( 9): 598– 610. Google Scholar CrossRef Search ADS PubMed  37 Yoshihara K, Wang Q, Torres-Garcia W, et al.   The landscape and therapeutic relevance of cancer-associated transcript fusions. Oncogene.  2015; 34( 37): 4845– 4854. Google Scholar CrossRef Search ADS PubMed  38 Lietha D, Cai X, Ceccarelli DFJ, et al.   Structural basis for the autoinhibition of focal adhesion kinase. Cell.  2007; 129( 6): 1177– 1187. Google Scholar CrossRef Search ADS PubMed  39 Cooper LA, Shen TL, Guan JL. Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction. Mol Cell Biol.  2003; 23( 22): 8030– 8041. Google Scholar CrossRef Search ADS PubMed  40 Cohen LA, Guan JL. Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation. J Biol Chem.  2005; 280( 9): 8197– 8207. Google Scholar CrossRef Search ADS PubMed  41 Danan-Gotthold M, Golan-Gerstl R, Eisenberg E, et al.   Identification of recurrent regulated alternative splicing events across human solid tumors. Nucl Acid Res.  2015; 43( 10): 5130– 5144. Google Scholar CrossRef Search ADS   42 Wiesner T, Lee W, Obenauf AC, et al.   Alternative transcription initiation leads to expression of a novel ALK isoform in cancer. Nature.  2015; 526( 7573): 453– 457. Google Scholar CrossRef Search ADS PubMed  43 Misquitta-Ali CM, Cheng E, O'Hanlon D, et al.   Global profiling and molecular characterization of alternative splicing events misregulated in lung cancer. Mol Cell Biol.  2011; 31( 1): 138– 150. Google Scholar CrossRef Search ADS PubMed  44 Burgaya F, Toutant M, Studler JM, et al.   Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity. J Biol Chem.  1997; 272: 28720– 28725. Google Scholar CrossRef Search ADS PubMed  45 Corsi JM, Rouer E, Girault JA, et al.   Organization and post-transcriptional processing of focal adhesion kinase gene. BMC Genomics.  2006; 7( 1): 1– 22. Google Scholar CrossRef Search ADS PubMed  46 Toutant M, Costa A, Studler JM, et al.   Alternative splicing controls the mechanisms of FAK autophosphorylation. Mol Cell Biol.  2002; 22: 7731– 7743. Google Scholar CrossRef Search ADS PubMed  47 Govindan R, Ding L, Griffith M, et al.   Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell.  2012; 150( 6): 1121– 1134. Google Scholar CrossRef Search ADS PubMed  48 Yu XJ, Yang MJ, Zhou B, et al.   Characterization of somatic mutations in air pollution-related lung cancer. EBioMedicine.  2015; 2( 6): 583– 590. Google Scholar CrossRef Search ADS PubMed  49 Weng MW, Lai JC, Hsu CP, et al.   Alternative splicing of MDM2 mRNA in lung carcinomas and lung cell lines. Environ Mol Mutagen.  2005; 46( 1): 1– 11. Google Scholar CrossRef Search ADS PubMed  50 Yan C, Wu W, Li H, et al.   Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing. Mutat Res.  2010; 686( 1–2): 47– 56. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JNCI: Journal of the National Cancer Institute Oxford University Press

Somatic Mutations and Splicing Variants of Focal Adhesion Kinase in Non–Small Cell Lung Cancer

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Abstract

Abstract Background Overexpression of focal adhesion kinase (FAK) has been reported in lung cancer, but the somatic mutations and alternative splicing variants of this nonreceptor tyrosine kinase remain to be investigated. Methods FAK in 91 lung cancer patients was sequenced using genomic DNA and cDNA samples of tumor and paired normal lung tissues as templates, and the RNA-seq data of The Cancer Genome Atlas (TCGA) data set were assessed. The biological functions of abnormal FAK transcripts and their response to FAK inhibitors were analyzed in eight cell lines using tyrosine kinase activity assay, trypan blue exclusion assay, MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide) assay, and transwell assay. Results We identified an internal tandem duplication (ITD), an A1004S point mutation, an exon 5–27 deletion (ΔE5-27) truncation variant, and four FAK6,7 splicing variants (containing exons for Boxes 6 and 7) in seven (7.7%) patients. Smokers had more FAK abnormalities than nonsmokers. In FAK-ITD, the sequence encoding the C-terminal of the FERM domain and kinase domain was duplicated in-frame and produced a protein product with elevated autophosphorylation and sensitivity to FAK inhibitors. FAK6,7 was detected in the tumor but not counterpart normal lung tissues of four (4.4%) patients. In TCGA RNA-seq data, Box 6 and/or Box 7 (Box 6/7)–containing FAK variants were positive in 42 (8.3%) of 508 lung adenocarcinomas (LUADs) and 37 (7.4%) of 501 lung squamous cell carcinomas, and smokers had higher expression of Box 6/7 (+) FAK than reformed or nonsmokers with LUAD. FAK6,7 promoted cell proliferation and migration, exhibited increased autophosphorylation, and was more sensitive to FAK inhibitor compared with wild-type FAK. Conclusions Somatic mutations and splicing variants of FAK may have a role in lung carcinogenesis and represent potential biomarkers for FAK-targeted therapies. Focal adhesion kinase (FAK), a cytoplasmic nonreceptor tyrosine kinase encoded by PTK2 (or FAK) (1,2), is overexpressed in a variety of cancers and associated with poor clinical outcome (3–6). While mutations are rarely reported in cancer genome sequencing projects, FAK splice variants are detected in leukemia (7) and breast cancer (8). FAK promotes cancer cell proliferation, survival, invasion, and stem cell activities (5). Inhibition of FAK renders pancreatic cancer responsiveness to checkpoint immunotherapy (9) and exerts beneficial effects in selected solid tumors (5,10–12). However, the underlying mechanism of FAK hyperactivation in most cancers remains obscure, and biomarkers (13) for FAK-targeting therapies are desired. Lung cancer, the most common cause of cancer-related mortality worldwide (14), is comprised of small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC); the latter type consists of lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large cell carcinoma (15). About 90% of lung cancer deaths are caused by cigarette smoke (16), which activates the c-Src/FAK loop to promote cancer cell migration and invasion (17). FAK promotes cancer development (18) and induces drug resistance (19,20) in lung cancer and is required for the maintenance of KRAS-driven LUAD (21). FAK is overexpressed and inversely associates with prognosis of patients (22–25). Inhibition of FAK suppresses cancer cells (21,26) and enhances the antitumor activity of erlotinib (27). Notably, beneficial effects were seen in one of the five patients receiving FAK inhibitor PF-562271 (11, 28). However, whether lung cancer cells harbor FAK mutations or structural variations and how to precisely select patients for FAK inhibitor treatment warrant further investigation. To uncover FAK somatic variations in lung cancer, here we analyzed the sequence of the whole FAK gene in tumor and paired normal lung tissues of 91 patients. The biological functions and sensitivity to FAK inhibitors PF-562271 and PF-573228 (29) of the identified mutants were investigated. Methods Patient Samples The study was approved by the research ethics committees of all participating sites; all samples were collected with written informed consent from the patient’s family. Tumor and adjacent normal lung tissues were immediately frozen in liquid nitrogen after surgical resection. Genomic DNA and total RNAs were extracted with an AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), and RNAs were reverse-transcribed (RT) into complementary DNA (cDNA) using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Basingstoke, UK) and random primers (Takara Biotechnology, Dalian, China). Polymerase chain reaction (PCR) was performed using DNA or cDNA templets and the primers listed in Supplementary Table 1 (available online), and the products were sequenced. FAK transcripts were cloned for functional studies. RNA Fluorescence In Situ Hybridization and Immunohistochemical Staining Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned at 5 µm intervals. Fluorescently labeled probes (Supplementary Table 1, available online) were designed at the exon junction of the duplicated region for FAK- internal tandem duplication (ITD) and at the Box 6/7 locus for FAK6,7 (refers to the FAK splicing variant containing exons for Boxes 6 and 7, respectively). Fluorescence in situ hybridization (FISH) was conducted as described (30). Immunohistochemical assay was conducted and scored (22) using FFPE tissue specimens and anti-FAK (Cell Signaling, Beverly, MA) and anti-p-FAK (Y397; Thermo Fisher Scientific, Basingstoke, UK) antibodies. The Cancer Genome Atlas RNA-Seq Data The Cancer Genome Atlas (TCGA) level 3 IlluminaHiseq RNAseqV2 data were downloaded from the Broad GDAC Firehose 2015_11_01 run. The percent spliced-in (PSI) (31) values of FAK were downloaded from the TCGA SpliceSeq PSI download page. The positivity of Box 6/7-containing splicing variants should fulfill two criteria: PSI greater than .05 and junction reads of 2 or greater (31). Reads per kilobase per million mapped reads (RPKM) were used to measure the expression of the specific exon, and the Log2 (RPKM + 1) values were calculated. To compare the expression of Box 6/7-containing FAK transcripts between smokers and reformed/nonsmokers, the RPKM values of each spliced exon were normalized by dividing the sum RPKM of all exons from the FAK gene (32). Cell Culture The normal human lung epithelial 16HBE (Clonetics, Walkersville, MD), embryonic lung fibroblasts (HLF), embryonic kidney HEK293, NSCLC lines A549, Calu-6, EPLC32M1, H460, H1975 (the American Type Culture Collection [ATCC], Manassas, VA), and L78 (the Cell Resource Center, Chinese Academy of Medical Sciences, Beijing) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco/BRL, Grand Island, NY). To knockdown FAK in A549 cells, CRISPR-Cas9 system and a specific sgRNA (5’-TCCAGTCTACAGATTTGATA-3’) targeting FAK were used. Cas9 and sgRNA plasmids were cotransfected into A549 cells, and FAK knockdown (designated A549-FAK-Cas9) cells were selected by puromycin (5 µg/mL). The cells were transfected with indicated plasmids using Lipofectamine 3000 (Invitrogen, Frederick, MD), and FAK–stably expressing clones were selected with G418 (Amresco Inc., Cleveland, OH). The cells were treated with PF-562271 and PF-573228, and cell viability was estimated by trypan blue exclusion assay and MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide; Amresco, Solon, OH) assay. For transwell assay, the cells were seeded into the inserts (1 × 105), and complete medium was added into the bottom chamber. Twenty-four hours later, the cells in the upper chamber were removed, and cells in the lower surface were fixed with methanol, stained with crystal violet, and counted with a microscope. Immunoblot and Kinase Activity Assays Proteins were extracted from frozen tissues or cells and subjected to immunoblot using indicated antibodies (see the Supplementary Materials, available online) as described (33). For kinase activity, proteins were harvested from EGFP-FAK-expressing HLF cells and purified by protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GFP antibody-mediated immunoprecipitation and analyzed with a Universal Tyrosine Kinase Assay Kit (Clontech, Palo Alto, CA). Statistical Analysis All the statistical analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). Statistically significant differences were determined by Fisher’s exact test or Mann-Whitney test. The survival curve for each group was estimated by the Kaplan-Meier method and log-rank test. P values of less than .05 were considered statistically significant. All statistical tests were two-sided. Results FAK abnormalities By analyzing the sequences, four types of FAK somatic mutations/splicing variants were identified: an in-frame ITD, one mutation that led to A1004S amino acid substitution, one deletion variant lacking exons 5–27 (ΔE5-27), and four splicing variants (FAK6,7) (Table 1). Interestingly, smokers had more (6/39, 15.4%) FAK abnormalities than nonsmokers (1/50, 2.0%; P = .04) (Table 1). Table 1. The demographic characteristics of the 91 lung cancer patients Characteristics  Total (n = 91)  FAK mutations  Splicing variant  Ratio, %  P  Sex             Male  52  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  11.5  .23   Female  37    1 FAK6,7  2.7     n.d.  2          Age, y             <65  66  1 ITD, 1 point mutation, 1 truncation variant  1 FAK6,7  6.1  .37   ≥65  23    3 FAK6,7  13.0     n.d.  2          Smoking             Smoker  39  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  15.4  .04   Nonsmoker  50    1 FAK6,7  2     n.d.  2          Histology             LUAD  60  1 point mutation, 1 truncation variant    3.3  .07   LUSC  27  1 ITD  3 FAK6,7  14.8     Large cell  carcinoma  1           SCLC  1    1 FAK6,7       n.d.  2          TNM stage             IA–IIB  49  1 point mutation  3 FAK6,7  8.2  1.00   IIIA–IV  35  1 ITD, 1 truncation variant    5.7     n.d.  7    1 FAK6,7      Characteristics  Total (n = 91)  FAK mutations  Splicing variant  Ratio, %  P  Sex             Male  52  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  11.5  .23   Female  37    1 FAK6,7  2.7     n.d.  2          Age, y             <65  66  1 ITD, 1 point mutation, 1 truncation variant  1 FAK6,7  6.1  .37   ≥65  23    3 FAK6,7  13.0     n.d.  2          Smoking             Smoker  39  1 ITD, 1 point mutation, 1 truncation variant  3 FAK6,7  15.4  .04   Nonsmoker  50    1 FAK6,7  2     n.d.  2          Histology             LUAD  60  1 point mutation, 1 truncation variant    3.3  .07   LUSC  27  1 ITD  3 FAK6,7  14.8     Large cell  carcinoma  1           SCLC  1    1 FAK6,7       n.d.  2          TNM stage             IA–IIB  49  1 point mutation  3 FAK6,7  8.2  1.00   IIIA–IV  35  1 ITD, 1 truncation variant    5.7     n.d.  7    1 FAK6,7      * P values were calculated using a two-sided Fisher’s exact test. LCLC = large cell lung cancer; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; n.d. = not determined. FAK-ITD Mutation A FAK-ITD mutation was identified in a male smoker patient, age 38 years, with stage IIIB LUSC. In this mutant, exons 6–27 were duplicated and inserted into the FAK gene body right after exon 27 (Figure 1A). To confirm this mutation, the break point cluster region in genomic DNA was amplified by nested PCR and sequenced. The results showed the junction of break points 141,865,885 and 141,690,508, giving rise to the fusion of FAK intron 27 and intron 5 in the same orientation (Figure 1B). Reverse transcription PCR (RT-PCR) was performed using F1 and R1 primers spanning exons 3 through 7, and wild-type (WT) and ITD FAK were detected in the tumor sample (Figure 1C, left). Sequencing results revealed that an additional copy of exons 6–27 was inserted between exon 5 and exon 6 of the WT transcript, confirming the tandem duplication (Figure 1C, right). By using primers F2 and R2 mapping to exons 22 and 7, respectively, a fusion transcript between exon 27 and exon 6 was detected (Figure 1D). FISH assay showed the existence of FAK-ITD in tumor cells (Figure 1E). FAK-ITD encodes an elongated FAK with an additional 704 amino acids (duplicated 151–854 residues), which is comprised of the autophosphorylation site Y397, the whole tyrosine kinase domain, and the truncated FERM domain (Figure 1F). Immunoblot showed the presence of FAK-ITD in tumor but not paired normal lung tissues of the patient (Figure 1G). Figure 1. View largeDownload slide Identification of a focal adhesion kinase–internal tandem duplication (FAK-ITD) in lung cancer. A) Chromosomal localization and structure of wild-type (WT) FAK and FAK-ITD. The loci 141,690,508 and 141,865,885 were genomic break points, and the duplicated region encompassed exons 6 through 27 of WT FAK transcript. Primers used for detection of FAK-ITD are shown. B-D) Validation of FAK-ITD using the genomic (g) DNA (B) and cDNA (C, D) of tumor and paired normal lung tissues of the patient, by nested polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) followed by sequencing. Samples from a FAK-ITD (-) patient were used as controls. Sanger sequencing results showed the junction region of FAK-ITD. E) Detection of FAK-ITD mRNA by RNA fluorescence in situ hybridization using FITC-labeled probe mapping to the exon junction of duplicated regions. Signals from FAK-ITD are shown as green, and nuclei stained with DAPI are shown as blue. Scale bar = 5 μm. F) Schematic representation of the protein encoded by FAK-ITD. G) Immunoblot analysis of FAK in tumor and paired normal lung tissues of the patient. FAK = focal adhesion kinase; ITD = internal tandem duplication; N = normal; pt # = patient number; T = tumor; WT = wild-type. Figure 1. View largeDownload slide Identification of a focal adhesion kinase–internal tandem duplication (FAK-ITD) in lung cancer. A) Chromosomal localization and structure of wild-type (WT) FAK and FAK-ITD. The loci 141,690,508 and 141,865,885 were genomic break points, and the duplicated region encompassed exons 6 through 27 of WT FAK transcript. Primers used for detection of FAK-ITD are shown. B-D) Validation of FAK-ITD using the genomic (g) DNA (B) and cDNA (C, D) of tumor and paired normal lung tissues of the patient, by nested polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) followed by sequencing. Samples from a FAK-ITD (-) patient were used as controls. Sanger sequencing results showed the junction region of FAK-ITD. E) Detection of FAK-ITD mRNA by RNA fluorescence in situ hybridization using FITC-labeled probe mapping to the exon junction of duplicated regions. Signals from FAK-ITD are shown as green, and nuclei stained with DAPI are shown as blue. Scale bar = 5 μm. F) Schematic representation of the protein encoded by FAK-ITD. G) Immunoblot analysis of FAK in tumor and paired normal lung tissues of the patient. FAK = focal adhesion kinase; ITD = internal tandem duplication; N = normal; pt # = patient number; T = tumor; WT = wild-type. FAK-ITD’s Functions and Sensitivity to FAK Inhibitors FAK-ITD was transfected into HEK293 cells (Figure 2A, left), and we found that this mutant exhibited elevated level of phosphorylated Y397 (p-Y397) and increased overall tyrosine phosphorylation (Figure 2A, middle and right panels) than WT FAK. FAK-ITD showed higher kinase activity than WT FAK (Figure 2B). In WT FAK- and FAK-ITD-expressing 293 cells adhering to the culture plate dish, p-Y397 was detected; in cells held in suspension for 60 to 90 minutes, p-Y397 of WT and ITD FAK was reduced (Figure 2C). In 293 cells transfected with FAK-ITD, the expression of p-AKT and p-ERK was increased, as compared with cells expressing WT FAK (Figure 2D). Ectopic expression of FAK-ITD statistically significantly promoted proliferation of HLF cells (P = .02) (Figure 2E). Figure 2. View largeDownload slide Increased autophosphorylation of Y397 and the responsiveness of focal adhesion kinase–internal tandem duplication (FAK-ITD) to FAK inhibitors. A) Wild-type (WT) FAK and FAK-ITD were transfected into HEK293 cells, and 48 hours later the cells were harvested and lysed. The cell lysates were subjected to immunoprecipitation and immunoblot using indicated antibodies. B) Embryonic lung fibroblasts (HLF) cells stably expressing EGFP-FAK were lysed, and the lysates were immunoprecipitated with an anti-GFP antibody and assayed for tyrosine kinase activity. The amount of FAK proteins in immunoprecipitated samples was determined by immunoblots. C) HEK293 cells expressing Flag-FAK were cultured and attached onto dish plates or kept suspended for 60 minutes or 90 minutes, they were lysed, and the lysates were subjected to immunoblot. D) Immunoblot assays of p-AKT and p-ERK using lysates of HEK293 cells stably expressing Flag-FAK. E) The growth curves of HLF cells expressing EGFP-FAK. Trypan blue exclusion analyses were conducted. *WT-FAK-expressing cells vs FAK-ITD-expressing cells (P = .02, two-sided Student’s t test). F and G) HLF cells stably expressing Flag-FAK were treated with PF-562271 (F) or PF-573228 (G) for 15 minutes, they were lysed, and lysates were analyzed by immunoblot. Error bars on the bar charts represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; WT = wild-type. Figure 2. View largeDownload slide Increased autophosphorylation of Y397 and the responsiveness of focal adhesion kinase–internal tandem duplication (FAK-ITD) to FAK inhibitors. A) Wild-type (WT) FAK and FAK-ITD were transfected into HEK293 cells, and 48 hours later the cells were harvested and lysed. The cell lysates were subjected to immunoprecipitation and immunoblot using indicated antibodies. B) Embryonic lung fibroblasts (HLF) cells stably expressing EGFP-FAK were lysed, and the lysates were immunoprecipitated with an anti-GFP antibody and assayed for tyrosine kinase activity. The amount of FAK proteins in immunoprecipitated samples was determined by immunoblots. C) HEK293 cells expressing Flag-FAK were cultured and attached onto dish plates or kept suspended for 60 minutes or 90 minutes, they were lysed, and the lysates were subjected to immunoblot. D) Immunoblot assays of p-AKT and p-ERK using lysates of HEK293 cells stably expressing Flag-FAK. E) The growth curves of HLF cells expressing EGFP-FAK. Trypan blue exclusion analyses were conducted. *WT-FAK-expressing cells vs FAK-ITD-expressing cells (P = .02, two-sided Student’s t test). F and G) HLF cells stably expressing Flag-FAK were treated with PF-562271 (F) or PF-573228 (G) for 15 minutes, they were lysed, and lysates were analyzed by immunoblot. Error bars on the bar charts represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; WT = wild-type. The FAK-expressing HLF cells were treated with PF-562271 or PF-573228 for 15 minutes, and the expression of p-Y397 was detected. We found that PF562271 at 0.01 µM drastically suppressed p-Y397 of WT FAK; interestingly, PF562271 repressed p-Y397 of FAK-ITD at an even lower concentration, 0.003 µM (Figure 2F). Densitometry analyses of the immunoblot bands confirmed that FAK-ITD responded to a lower concentration of PF-562271 compared with WT FAK (Supplementary Figure 1A, available online). Similarly, PF-573228 inhibited p-Y397 of both WT FAK and FAK-ITD, while FAK-ITD was more sensitive to this inhibitor (Figure 2G;Supplementary Figure 1A, available online). Other FAK Mutations We performed RT-PCR and subsequent sequencing to identify aberrant FAK transcripts using cDNA samples of the other 90 patients and primers spanning the whole coding region of FAK (F3-R3) (Supplementary Table 1, available online). One G to T mutation that led to A1004S amino acid substitution was found in a male smoker patient, age 57 years, with stage IB LUAD (Figure 3, A and B). In a male smoker patient, age 62 years, with stage IV LUAD, a PCR band of about 1200 bp was observed (Figure 3C). Exons 5–27 were deleted in this mutant (Figure 3D), yielding a truncated FAK ΔE5-27 that encodes a protein of 194 amino acids (Figure 3B). Figure 3. View largeDownload slide Identification of focal adhesion kinase (FAK) mutations and splicing variants in lung cancer. A) A somatic nucleotide substitution, c.3010G>T, was detected in FAK in one tumor. This mutation encodes an A to S amino acid change at residue 1004 (B). C and D) Identification of a ΔE5-27 FAK transcript in one tumor by reverse transcription polymerase chain reaction (RT-PCR) (C) and sequencing (D). This transcript encodes a truncated FAK (B). E–G) Identification of Boxes 6 and 7–containing FAK6,7 in four tumors. E) Primers used for RT-PCR for validation of this variant. F) Bands of RT-PCR assay using cDNA of samples isolated from the four patients. G) Sequencing results of bands in (F). This transcript encodes a protein inclusion with Box 6 and Box 7 on either side of Y397 (B). FAK = focal adhesion kinase; pt # = patient number; WT = wild-type. Figure 3. View largeDownload slide Identification of focal adhesion kinase (FAK) mutations and splicing variants in lung cancer. A) A somatic nucleotide substitution, c.3010G>T, was detected in FAK in one tumor. This mutation encodes an A to S amino acid change at residue 1004 (B). C and D) Identification of a ΔE5-27 FAK transcript in one tumor by reverse transcription polymerase chain reaction (RT-PCR) (C) and sequencing (D). This transcript encodes a truncated FAK (B). E–G) Identification of Boxes 6 and 7–containing FAK6,7 in four tumors. E) Primers used for RT-PCR for validation of this variant. F) Bands of RT-PCR assay using cDNA of samples isolated from the four patients. G) Sequencing results of bands in (F). This transcript encodes a protein inclusion with Box 6 and Box 7 on either side of Y397 (B). FAK = focal adhesion kinase; pt # = patient number; WT = wild-type. Alternative Splicing Variant FAK6,7 in Lung Cancer By RT-PCR assay using primers spanning the whole coding region, one FAK splicing variant was identified in the tumor but not paired normal lung tissues of four (4.4%) patients (Table 1). RT-PCR with primers spanning exons 14 through 16 (Figure 3E) confirmed the existence of this splicing variant (Figure 3F). Sequencing results (Figure 3G) showed that this variant contains additional alternatively spliced exons of 18 bp (Box 6) and 21 bp (Box 7), respectively (Figure 3E), coding for two short peptides of six and seven amino acids on either side of Y397 (Figure 3B). RNA FISH assays confirmed the existence of FAK6,7 in tumor cells (Supplementary Figure 2A, available online). To identify the potential intron abnormalities that lead to splicing variants, exons 14–16 (chr8: 141771264–141799625; a region of 28.3 kb) of genomic FAK were sequenced, and 13 single nucleotide polymorphisms (SNPs) were detected in four patients (Supplementary Table 2 and Supplementary Figure 2B, available online). Two SNPs (ID rs7840381 and rs10875458) were seen in NSCLC line Calu-6. By ACESCAN2 method (34), we found that three SNPs, for example, G to T in rs4246123, T to G in rs7840381, and A to G in rs4579574, located in regions of intronic splicing enhancers. SNPs were able to enhance alternative splicing (35); therefore, the above SNPs may pave the way to FAK6,7 alternative splicing. Box 6 and/or 7–Containing Variants FAK6/7 in Lung Cancer FAK alternative splicing events were explored in TCGA RNA-seq data. In 508 LUADs, the PSI values of Box 6 alternative splicing ranged from 0 to 0.47, and those of Box 7 ranged from 0 to 0.72 (Figure 4A). By screening with the number of corresponding splice junctions and the PSI values (Supplementary Table 3, available online), 42 (8.3%) LUAD patients were identified as “Box 6 and/or Box 7 (Box 6/7) positive,” and 12 (2.4%) patients harbored FAK6,7. Moreover, 29 (5.7%) patients showed high expression of exon coding for Box 6/7 (RPKM > 2) (Figure 4B). In 501 LUSCs, the PSI values of Box 6/7 variants were up to 0.69 and 0.90, respectively (Figure 4C). Thirty-seven (7.4%) patients were positive for Box 6 or 7, and 5 (1.0%) patients had FAK6,7, while 20 (4.0%) patients showed high expression of Box 6/7 exons (RPKM > 2) (Figure 4D). Figure 4. View largeDownload slide Box 6 and/or Box 7 (Box 6/7)–containing focal adhesion kinase (FAK) splicing variants in lung cancer. A) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 508 The Cancer Genome Atlas (TCGA) lung adenocarcinomas (LUADs). B) Box 6/7–containing FAK variants were found in 42 (8.3%) LUADs. The values of Log2 (reads per kilobase per million mapped reads [RPKM] + 1) were calculated, and the junction reads of the corresponding exons were indicated. C) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 501 TCGA lung squamous cell carcinoma (LUSCs). D) Box 6/7–containing FAK variants were found in 37 (7.4%) LUSCs. The values of Log2 (RPKM+1) were calculated, and the junction reads of the corresponding exons were indicated. E and F) The expression of Box 6 or Box 7–containing FAK splicing variants in current smoker, reformed smoker, and nonsmoker LUADs (E) and LUSCs (F). The expression level is shown as normalized RPKM ×1000. The Mann-Whitney test was used for calculating statistical significance. G) Overall survival (OS) of LUADs and LUSCs with or without Box 6/7–containing FAK splicing variants. H) Overall survival of LUADs with or without Box 6/7–containing FAK splicing variants. I) Overall survival of LUSCs with or without Box 6/7–containing FAK splicing variants. The log-rank test was used for analyzing survival differences. All statistical tests were two-sided. LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; OS = overall survival; PSI = percent spliced-in; RPKM = reads per kilobase per million mapped reads. Figure 4. View largeDownload slide Box 6 and/or Box 7 (Box 6/7)–containing focal adhesion kinase (FAK) splicing variants in lung cancer. A) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 508 The Cancer Genome Atlas (TCGA) lung adenocarcinomas (LUADs). B) Box 6/7–containing FAK variants were found in 42 (8.3%) LUADs. The values of Log2 (reads per kilobase per million mapped reads [RPKM] + 1) were calculated, and the junction reads of the corresponding exons were indicated. C) The percent spliced-in (PSI) values of FAK transcripts containing Box 6/7 in 501 TCGA lung squamous cell carcinoma (LUSCs). D) Box 6/7–containing FAK variants were found in 37 (7.4%) LUSCs. The values of Log2 (RPKM+1) were calculated, and the junction reads of the corresponding exons were indicated. E and F) The expression of Box 6 or Box 7–containing FAK splicing variants in current smoker, reformed smoker, and nonsmoker LUADs (E) and LUSCs (F). The expression level is shown as normalized RPKM ×1000. The Mann-Whitney test was used for calculating statistical significance. G) Overall survival (OS) of LUADs and LUSCs with or without Box 6/7–containing FAK splicing variants. H) Overall survival of LUADs with or without Box 6/7–containing FAK splicing variants. I) Overall survival of LUSCs with or without Box 6/7–containing FAK splicing variants. The log-rank test was used for analyzing survival differences. All statistical tests were two-sided. LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; OS = overall survival; PSI = percent spliced-in; RPKM = reads per kilobase per million mapped reads. We analyzed the association between FAK6/7 expression and patients’ smoking status by analyzing the normalized RPKM value (×1000) and found that in LUADs the current smokers had higher FAK6/7 than reformed and nonsmokers (Figure 4E); in patients with LUSCs, the current smokers had similar FAK6/7 to nonsmokers (Figure 4F). Among the 990 patients whose survival information was available, the median overall survival (OS) for Box 6/7 (+) patients was 1293 days, shorter than Box 6/7 (-) patients (1622 days, P = .40) (Figure 4G). In LUADs (Figure 4H) and LUSCs (Figure 4I), patients with Box 6/7 (+) FAK had shorter survival than patients with Box 6/7 (-) FAK. Kinase Activity and Sensitivity to Targeted Therapy of FAK6,7 By immunohistochemical assays, we showed that the four FAK6,7 (+) patients had more elevated p-FAK (Y397) than WT FAK patients (P = .04) (Figure 5, A and B). Immunoblot assays confirmed the increased p-Y397 in tumor samples of the FAK6,7 (+) patients compared with patients with WT FAK (Figure 5C). Immunoblot analysis using proteins from EGFP-FAK-transfected HLF cells confirmed that FAK6,7 had more elevated p-Y397 than WT FAK (Figure 5D). In H460, A549-FAK-Cas9 (Supplementary Figure 3, available online), and 16HBE cells transfected with FAK, FAK6,7 showed elevated phosphorylation level compared with WT FAK and the kinase dead (KD) (Figure 5E;Supplementary Figure 1B, available online). FAK6,7 promoted proliferation (Figure 5F;Supplementary Figure 1C, available online) and migration (Figure 5G) of the cells. Figure 5. View largeDownload slide Autophosphorylation of focal adhesion kinase (FAK)6,7 and its sensitivity to FAK inhibitors. A–C) p-FAK (Y397) in tumors of FAK6,7 or wild-type (WT) FAK patients detected by immunohistochemistry (A) scoring (B) and immunoblots (C). Scale bar = 2000 µm. D) p-Y397 of HLF cells stably expressing WT FAK and FAK6,7. E-G) Flag-FAK vectors were transfected into H460 and A549-FAK-Cas9 cells (E). Cells were counted by trypan blue exclusion analysis at 48 and 72 hours after transfection (F), and cell migration was assessed by transwell assay 48 hours after transfection (G). Scale bar = 200 µm. H) HLF cells expressing WT FAK or FAK6,7 were treated with PF562271 and PF573228 for 15 minutes, and the expression of p-Y397 was tested. I) H460 and A549-FAK-Cas9 cells were transfected with Flag-FAK and then treated with or without 2 μM PF562271 for 48 hours. Inhibition rates of cell proliferation were determined by trypan blue exclusion assay. J) Non–small cell lung cancer lines seeded in 96-well plates were treated with PF562271, and cell viability was measured by MTT assays. K) The cells were treated with PF562271 at indicated concentrations for 15 minutes, and p-Y397 was tested. Error bars represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; pt # = patient number; WT = wild-type. Figure 5. View largeDownload slide Autophosphorylation of focal adhesion kinase (FAK)6,7 and its sensitivity to FAK inhibitors. A–C) p-FAK (Y397) in tumors of FAK6,7 or wild-type (WT) FAK patients detected by immunohistochemistry (A) scoring (B) and immunoblots (C). Scale bar = 2000 µm. D) p-Y397 of HLF cells stably expressing WT FAK and FAK6,7. E-G) Flag-FAK vectors were transfected into H460 and A549-FAK-Cas9 cells (E). Cells were counted by trypan blue exclusion analysis at 48 and 72 hours after transfection (F), and cell migration was assessed by transwell assay 48 hours after transfection (G). Scale bar = 200 µm. H) HLF cells expressing WT FAK or FAK6,7 were treated with PF562271 and PF573228 for 15 minutes, and the expression of p-Y397 was tested. I) H460 and A549-FAK-Cas9 cells were transfected with Flag-FAK and then treated with or without 2 μM PF562271 for 48 hours. Inhibition rates of cell proliferation were determined by trypan blue exclusion assay. J) Non–small cell lung cancer lines seeded in 96-well plates were treated with PF562271, and cell viability was measured by MTT assays. K) The cells were treated with PF562271 at indicated concentrations for 15 minutes, and p-Y397 was tested. Error bars represent standard deviation. FAK = focal adhesion kinase; HLF = embryonic lung fibroblasts; ITD = internal tandem duplication; pt # = patient number; WT = wild-type. In EGFP-FAK-expressing HLF cells, p-Y397 of FAK6,7 responded to PF562271 and PF573228 at a lower concentration and an earlier time point compared with WT FAK (Figure 5H;Supplementary Figure 1D, available online). In FAK-expressing H460 and A549-FAK-Cas9 cells, p-Y397 of FAK6,7 was more sensitive to PF562271 compared with WT FAK (Figure 5I, upper), and inhibition rates of FAK6,7-expressing cells were higher than those of WT FAK cells (Figure 5I, lower). To identify NSCLC lines harboring FAK6,7, RT-PCR and subsequent sequencing were conducted, and we found that Calu-6 had FAK6,7 (Supplementary Figure 4, A and B, available online) and elevated p-Y397 (Supplementary Figure 4C, available online). PF562271 suppressed the viability of Calu-6 cells in a dose- and time-dependent manner, and this line was the most sensitive one among the lines treated with PF562271 (Figure 5J). PF562271 suppressed FAK p-Y397 in Calu-6 cells at a lower concentration than in H460 and A549 cells (Figure 5K). Discussion FAK serves as an essential intracellular mediator of extracellular signals such as growth factors, extracellular matrix remodeling, and nutrient availability. FAK is activated in a variety of cancers, but only a few FAK missense mutations (R65S, etc.) and a PTDSS1_FAK fusion transcript in one tumor were reported (36,37). Here, by detailed analysis of genomic and cDNA sequence of FAK, we reported four types of somatic variants of FAK in seven (7.7%) of 91 lung cancer patients, indicating that FAK has several types of structural variations, and scrutinizing its sequence is helpful for lung cancer genotyping. FAK activation involves integrin receptor clustering upon cell binding to extracellular matrix proteins, and Y397 is a major autophosphorylation site (5) that can be blocked by FERM domain (38,39). In the duplicated FERM domain of FAK-ITD, the N-terminal 116 amino acids (R35–V151) including K38, which is important for auto-inhibitory activity of FERM (40), were deleted, suggesting that this mutant might have elevated kinase activity. This hypothesis was confirmed by our cellular experiments. On the other hand, the truncated protein encoded by the ΔE5-27 allele may lack its kinase activity; however, this protein may probably form heterodimer with the FERM domain of the full-length FAK encoded by the WT allele, thus releasing the auto-inhibitory effect of the FERM domain and leading to elevation of FAK activity. This possibility warrants further investigation. Alternative splicing, the process by which splice sites are differentially utilized to produce different mRNA isoforms, contributes to oncogenic activation in several types of cancers (41,42). Alternative splicing in genes such as VEGFA had been reported in lung cancer (43). FAK6, FAK7, and FAK6,7 have been identified in brain (44–46). Here we showed for the first time that FAK6,7 was expressed in tumor but not paired normal lung tissues in four (4.4%) of the 91 patients. A previous study showed that FAK6,7 has elevated kinase activity (44). We found that compared with WT FAK, FAK6,7 exhibited increased p-Y397 in patient samples and lung epithelial cells. FAK6,7 promoted cell proliferation and migration, suggesting a role for FAK6,7 in lung carcinogenesis. By analyzing the RNA-seq data of TCGA, we found that 8.3% of LUADs and 7.4% LUSCs had Box 6– or Box 7–containing FAK splicing events. These results suggest that alternative splicing represents an important mechanism of FAK activation in lung cancer. Smoker NSCLCs bear more genomic mutations than nonsmokers (47,48). Here we showed that smoker patients had more FAK abnormalities than nonsmokers. In TCGA patients, current smokers had more Box 6/7–containing FAK variants than reformed and nonsmokers. The association between FAK splicing variants and tobacco smoke suggested that tobacco carcinogens might affect molecules involved in splicing. This speculation was confirmed by previous studies showing that smokers had a higher frequency of MDM2 variant splicing than nonsmokers, owing to the effects of the carcinogen benzo[a]pyrene and its metabolite benzo[a]pyrene diolepoxide on the PI3K or MAPK pathway (49). CD44 splicing variants were also induced by benzo[a]pyrene treatment (50). These results suggest that lung carcinogenesis driven by tobacco smoke is very complicated, and how carcinogens induce FAK alternative splicing in patients remains to be elucidated. Our study has some limitations. First, the role of FAK-ITD and FAK6,7 in malignant transformation and tumor growth is partially demonstrated at the cellular level, which should be further tested in animal models (xenograft or transgenic/knockin mice). Second, the greater sensitivity of FAK-ITD/FAK6,7 to FAK inhibitors is shown in vitro. However, in vivo experiments are warranted for further evaluating the translational potentials of this study. Third, whether FAK-ITD is a recurrent somatic mutation needs to be determined. Several FAK tyrosine kinase inhibitors, for example, PF-562271, VS-4718, and VS-6063, are currently undergoing clinical investigation and show therapeutic potentials in selected solid cancers (5). We found that FAK-ITD and FAK6,7 were more sensitive to FAK inhibitors than WT FAK. Therefore, smokers with FAK mutations and splicing variants may represent biomarkers for FAK-targeted therapies, and this possibility should be tested in future clinical trials. Funding This work was supported by the National Natural Science Funds for Distinguished Young Scholars (81425025), the National Key Research and Development Program of China (2016YFC0905500), the “Personalized Medicines–Molecular Signature-based Drug Discovery and Development” Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12010307), the National Natural Science Foundation of China (81672765), and grants from the State Key Laboratory of Membrane Biology. Notes The study sponsor had no role in the design of the study; the data collection, analysis, or interpretation; the writing of the article; or the decision to submit for publication. References 1 Fiedorek FTJr, Kay ES. Mapping of the focal adhesion kinase (Fak) gene to mouse chromosome 15 and human chromosome 8. Mamm Genome.  1995; 6( 2): 123– 126. Google Scholar CrossRef Search ADS PubMed  2 Schaller MD, Hildebrand JD, Shannon JD, et al.   Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2- dependent binding of pp60src. Mol Cell Biol.  1994; 14( 3): 1680– 1688. Google Scholar CrossRef Search ADS PubMed  3 Owens LV, Xu L, Craven RJ, et al.   Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res.  1995; 55( 13): 2752– 2755. Google Scholar PubMed  4 Agochiya M, Brunton VG, Owens DW, et al.   Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells. Oncogene.  1999; 18( 41): 5646– 5653. Google Scholar CrossRef Search ADS PubMed  5 Lee BY, Timpson P, Horvath LG, et al.   FAK signaling in human cancer as a target for therapeutics. Pharmacol Ther.  2015; 146: 132– 149. Google Scholar CrossRef Search ADS PubMed  6 Yoon H, Dehart JP, Murphy JM, et al.   Understanding the roles of FAK in cancer: Inhibitors, genetic models, and new insights. J Histochem Cytochem.  2015; 63( 2): 114– 128. Google Scholar CrossRef Search ADS PubMed  7 Despeaux M, Chicanne G, Rouer E, et al.   Focal adhesion kinase splice variants maintain primitive acute myeloid leukemia cells through altered Wnt signaling. Stem Cells.  2012; 30( 8): 1597– 1610. Google Scholar CrossRef Search ADS PubMed  8 Yao L, Li K, Peng W, et al.   An aberrant spliced transcript of focal adhesion kinase is exclusively expressed in human breast cancer. J Transl Med.  2014; 12( 1): 1– 9. Google Scholar CrossRef Search ADS PubMed  9 Jiang H, Hegde S, Knolhoff BL, et al.   Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nat Med.  2016; 22( 8): 851– 860. Google Scholar CrossRef Search ADS PubMed  10 Golubovskaya VM. Targeting FAK in human cancer: From finding to first clinical trials. Front Biosci.  2014; 19: 687– 706. Google Scholar CrossRef Search ADS   11 Infante JR, Camidge DR, Mileshkin LR, et al.   Safety, pharmacokinetic, and pharmacodynamic phase I dose-escalation trial of PF-00562271, an inhibitor of focal adhesion kinase, in advanced solid tumors. J Clin Oncol.  2012; 30( 13): 1527– 1533. Google Scholar CrossRef Search ADS PubMed  12 Roy-Luzarraga M, Hodivala-Dilke K. Molecular pathways: Endothelial cell FAK—a target for cancer treatment. Clin Cancer Res.  2016; 22( 15): 3718– 3724. Google Scholar CrossRef Search ADS PubMed  13 Shapiro IM, Kolev VN, Vidal CM, et al.   Merlin deficiency predicts FAK inhibitor sensitivity: A synthetic lethal relationship. Sci Transl Med.  2014; 6( 237): 237ra68– 237ra68. Google Scholar CrossRef Search ADS PubMed  14 Torre LA, Bray F, Siegel RL, et al.   Global cancer statistics, 2012. CA Cancer J Clin.  2015; 65( 2): 87– 108. Google Scholar CrossRef Search ADS PubMed  15 Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med.  2008; 359( 13): 1367– 1380. Google Scholar CrossRef Search ADS PubMed  16 Hecht SS. Lung carcinogenesis by tobacco smoke. Int J Cancer.  2012; 131( 12): 2724– 2732. Google Scholar CrossRef Search ADS PubMed  17 Shen J, Xu L, Owonikoko TK, et al.   NNK promotes migration and invasion of lung cancer cells through activation of c-Src/PKCι/FAK loop. Cancer Lett.  2012; 318( 1): 106– 113. Google Scholar CrossRef Search ADS PubMed  18 Wang B, Qi X, Li D, et al.   Expression of pY397 FAK promotes the development of non-small cell lung cancer. Oncol Lett.  2016; 11( 2): 979– 983. Google Scholar CrossRef Search ADS PubMed  19 Kang Y, Hu W, Ivan C, et al.   Role of focal adhesion kinase in regulating YB–1–mediated paclitaxel resistance in ovarian cancer. J Natl Cancer Inst.  2013; 105( 19): 1485– 1495. Google Scholar CrossRef Search ADS PubMed  20 Lu H, Wang L, Gao W, et al.   IGFBP2/FAK pathway is causally associated with dasatinib resistance in non–small cell lung cancer cells. Mol Cancer Ther.  2013; 12( 12): 2864– 2873. Google Scholar CrossRef Search ADS PubMed  21 Konstantinidou G, Ramadori G, Torti F, et al.   RHOA-FAK is a required signaling axis for the maintenance of KRAS-driven lung adenocarcinomas. Cancer Disc.  2013; 3( 4): 444– 457. Google Scholar CrossRef Search ADS   22 Carelli S, Zadra G, Vaira V, et al.   Up-regulation of focal adhesion kinase in non-small cell lung cancer. Lung Cancer.  2006; 53( 3): 263– 271. Google Scholar CrossRef Search ADS PubMed  23 Ji HF, Pang D, Fu SB, et al.   Overexpression of focal adhesion kinase correlates with increased lymph node metastasis and poor prognosis in non-small-cell lung cancer. J Cancer Res Clin Oncol.  2013; 139( 3): 429– 435. Google Scholar CrossRef Search ADS PubMed  24 Nishimura M, Machida K, Imaizumi M, et al.   Tyrosine phosphorylation of 100-130 kDa proteins in lung cancer correlates with poor prognosis. Br J Cancer.  1996; 74( 5): 780– 787. Google Scholar CrossRef Search ADS PubMed  25 Rikova K, Guo A, Zeng Q, et al.   Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell.  2007; 131( 6): 1190– 1203. Google Scholar CrossRef Search ADS PubMed  26 Meng XN, Jin Y, Yu Y, et al.   Characterisation of fibronectin-mediated FAK signalling pathways in lung cancer cell migration and invasion. Br J Cancer.  2009; 101( 2): 327– 334. Google Scholar CrossRef Search ADS PubMed  27 Howe GA, Xiao B, Zhao H, et al.   Focal adhesion kinase inhibitors in combination with erlotinib demonstrate enhanced anti-tumor activity in non-small cell lung cancer. PLoS One.  2016; 11( 3): e0150567. Google Scholar CrossRef Search ADS PubMed  28 Roberts WG, Ung E, Whalen P, et al.   Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res.  2008; 68( 6): 1935– 1944. Google Scholar CrossRef Search ADS PubMed  29 Slack-Davis JK, Martin KH, Tilghman RW, et al.   Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem.  2007; 282( 20): 14845– 14852. Google Scholar CrossRef Search ADS PubMed  30 Kurobe M, Kojima T, Nishimura K, et al.   Development of RNA-FISH assay for detection of oncogenic FGFR3-TACC3 fusion genes in FFPE samples. PLoS One.  2016; 11( 12): e0165109. Google Scholar CrossRef Search ADS PubMed  31 Ryan M, Wong WC, Brown R, et al.   TCGASpliceSeq a compendium of alternative mRNA splicing in cancer. Nucl Acids Res.  2016; 44( D1): D1018– D1022. Google Scholar CrossRef Search ADS   32 Zhao Q, Caballero OL, Davis ID, et al.   Tumor-specific isoform switch of the fibroblast growth factor receptor 2 underlies the mesenchymal and malignant phenotypes of clear cell renal cell carcinomas. Clin Cancer Res.  2013; 19( 9): 2460– 2472. Google Scholar CrossRef Search ADS PubMed  33 Wang GZ, Cheng X, Zhou B, et al.   The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution. eLife.  2015; 4: e09419. Google Scholar PubMed  34 Yeo GW, Van Nostrand E, Holste D, et al.   Identification and analysis of alternative splicing events conserved in human and mouse. Proc Natl Acad Sci U S A.  2005; 102( 8): 2850– 2855. Google Scholar CrossRef Search ADS PubMed  35 Narla G, DiFeo A, Reeves HL, et al.   A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk. Cancer Res.  2005; 65( 4): 1213– 1222. Google Scholar CrossRef Search ADS PubMed  36 Sulzmaier FJ, Jean C, Schlaepfer DD. FAK in cancer: Mechanistic findings and clinical applications. Nat Rev Cancer.  2014; 14( 9): 598– 610. Google Scholar CrossRef Search ADS PubMed  37 Yoshihara K, Wang Q, Torres-Garcia W, et al.   The landscape and therapeutic relevance of cancer-associated transcript fusions. Oncogene.  2015; 34( 37): 4845– 4854. Google Scholar CrossRef Search ADS PubMed  38 Lietha D, Cai X, Ceccarelli DFJ, et al.   Structural basis for the autoinhibition of focal adhesion kinase. Cell.  2007; 129( 6): 1177– 1187. Google Scholar CrossRef Search ADS PubMed  39 Cooper LA, Shen TL, Guan JL. Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction. Mol Cell Biol.  2003; 23( 22): 8030– 8041. Google Scholar CrossRef Search ADS PubMed  40 Cohen LA, Guan JL. Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation. J Biol Chem.  2005; 280( 9): 8197– 8207. Google Scholar CrossRef Search ADS PubMed  41 Danan-Gotthold M, Golan-Gerstl R, Eisenberg E, et al.   Identification of recurrent regulated alternative splicing events across human solid tumors. Nucl Acid Res.  2015; 43( 10): 5130– 5144. Google Scholar CrossRef Search ADS   42 Wiesner T, Lee W, Obenauf AC, et al.   Alternative transcription initiation leads to expression of a novel ALK isoform in cancer. Nature.  2015; 526( 7573): 453– 457. Google Scholar CrossRef Search ADS PubMed  43 Misquitta-Ali CM, Cheng E, O'Hanlon D, et al.   Global profiling and molecular characterization of alternative splicing events misregulated in lung cancer. Mol Cell Biol.  2011; 31( 1): 138– 150. Google Scholar CrossRef Search ADS PubMed  44 Burgaya F, Toutant M, Studler JM, et al.   Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity. J Biol Chem.  1997; 272: 28720– 28725. Google Scholar CrossRef Search ADS PubMed  45 Corsi JM, Rouer E, Girault JA, et al.   Organization and post-transcriptional processing of focal adhesion kinase gene. BMC Genomics.  2006; 7( 1): 1– 22. Google Scholar CrossRef Search ADS PubMed  46 Toutant M, Costa A, Studler JM, et al.   Alternative splicing controls the mechanisms of FAK autophosphorylation. Mol Cell Biol.  2002; 22: 7731– 7743. Google Scholar CrossRef Search ADS PubMed  47 Govindan R, Ding L, Griffith M, et al.   Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell.  2012; 150( 6): 1121– 1134. Google Scholar CrossRef Search ADS PubMed  48 Yu XJ, Yang MJ, Zhou B, et al.   Characterization of somatic mutations in air pollution-related lung cancer. EBioMedicine.  2015; 2( 6): 583– 590. Google Scholar CrossRef Search ADS PubMed  49 Weng MW, Lai JC, Hsu CP, et al.   Alternative splicing of MDM2 mRNA in lung carcinomas and lung cell lines. Environ Mol Mutagen.  2005; 46( 1): 1– 11. Google Scholar CrossRef Search ADS PubMed  50 Yan C, Wu W, Li H, et al.   Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing. Mutat Res.  2010; 686( 1–2): 47– 56. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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JNCI: Journal of the National Cancer InstituteOxford University Press

Published: Feb 1, 2018

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