- Email: [email protected]
Mutation analysis of the FRK gene in non-small cell lung cancers
Letters to the Editor / Lung Cancer 71 (2011) 113–117 [7] Amatori S, Bagaloni I, Macedi E, Formica M, Giorgi L, Fusi V, et al. Malten, a new synthetic molecule showing in vitro antiproliferative activity against tumor cells and induction of complex DNA structural alterations. Br J Cancer 2010;103:239–48. [8] Gewirtz DA, Holt SE, Elmore LW. Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation. Biochem Pharmacol 2008;76:947–57. [9] Tront JS, Hoffman B, Liebermann DA. Gadd45a suppresses Ras-driven mammary tumorigenesis by activation of c-Jun NH2-terminal kinase and p38 stress signaling resulting in apoptosis and senescence. Cancer Res 2006;66:8448–54. [10] Narita M, Nunez S, Heard E, Narita M, Lin AW, Hearn SA, et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 2003;113:703–13. [11] Soriani A, Zingoni A, Cerboni C, Iannitto ML, Ricciardi MR, Di Gialleonardo V, et al. ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype. Blood 2009;113(15):3503–11.
S. Amatori I. Bagaloni D. Viti M. Fanelli ∗ Molecular Pathology and Oncology Lab. “Paola”, Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Via Campanella 1, 61032 Fano (PU), Italy ∗ Corresponding
author. Tel.: +39 0722 304951; fax: +39 0721 862834. E-mail address: [email protected] (M. Fanelli) 13 July 2010 doi: 10.1016/j.lungcan.2010.10.016
Mutation analysis of the FRK gene in non-small cell lung cancers Keywords: FRK Lung cancer Mutation
To the Editor, The epidermal growth factor receptor (EGFR) signaling pathway is one of the important oncogenic signaling cascades in lung carcinogenesis. The EGFR is a transmembrane tyrosine kinase receptor that plays an important role in many tumorigenic processes, including cell growth, proliferation, invasion, and metastasis. These effects are mediated by activation of downstream signaling pathways, including RAS-RAF-mitogen-activated protein kinase and phosphatidylinositol-3 kinase (PI3K)-AKT pathways [1,2]. Mutations in the tyrosine kinase (TK) domain of the EGFR gene are present in a subset of non-small cell lung cancers (NSCLCs), and those tumors with EGFR mutations have been reported to be highly sensitive to EGFR TK inhibitors [3]. In addition, alterations of downstream molecules of the EGFR have been reported to be associated with a clinical response to the EGFR TK inhibitors [4,5]. Therefore, a comprehensive evaluation of mutations in the EGFR signaling pathway genes may lead to optimized therapeutic approaches to lung cancer. Fyn-related kinase (FRK, also known as RAK) is a 54 kDa TK that belongs to the SRC non-receptor kinase family [6,7]. FRK was originally identified in human breast cancer cells and is expressed predominantly in epithelial tissues, including the kidney, liver, lung, and mammary gland [6]. Several lines of evidence suggest that FRK functions as a tumor suppressor; FRK inhibits cell proliferation and invasion, and tumorigenesis [8–10]. Interestingly, a recent
115
study has demonstrated that FRK positively regulates PTEN protein stability by phosphorylating PTEN on Try336, which in turn prevents PTEN degradation [11]. In addition, the FRK gene is located in 6q21-23, which is frequently deleted in NSCLCs [12]. These findings suggest that FRK may be involved in lung carcinogenesis. Therefore, we searched for mutations of the FRK gene in a cohort of NSCLCs that was previously investigated for mutations of the EGFR signaling pathway genes [13]. The study population has been described in our previous study [13]. Briefly, primary lung tumors and matching non-malignant lung tissues were obtained from 173 Korean patients with NSCLC who underwent curative resection at Kyungpook National University Hospital, in Daegu, Korea between January 2003 and July 2007. Patients who underwent chemotherapy or radiotherapy prior to surgery were excluded to avoid the effects on DNA. This study included 56 patients with squamous cell carcinoma and 117 patients with adenocarcinoma (AC). There were 113 males and 60 females in the study cohort. The patients consisted of 56 neversmokers and 117 smokers. Of the 117 patients with AC, 53 were never-smokers. All of the tumor and macroscopically normal lung tissue samples were obtained at the time of surgery, rapidly frozen in liquid nitrogen, and stored at −80 ◦ C. Only tumors with ≥80% of the tumor component were sent for analysis. This study was approved by the Institutional Review Board of Kyungpook National University Hospital, and written informed consent was obtained from each patient. FRK mutation analysis of the entire coding exons (exons 1–8) and exon–intron junctions was performed using polymerase chain reaction (PCR) and direct sequencing. Intron-based PCR primers were designated based on the GenBank reference sequence (accession no. NT 025741.15). The forward and reverse primers were as follows: exon 1 (5 -GCATACTCTCCGAAGTATGGTG-3 and 5 -GCATACTCTCCGAAGTATGGTG-3 ); exon 2 (5 -CTATCTTGCCAGGCTTTTTCCTC-3 and 5 -CGTGTCCATGTTCAC ACAGAAAG-3 ); exon 3 (5 -CCCAGTTCTCACACTCTCTCTTTTG-3 and 5 -CTCA TAGGCCATGGTTTGCCTATAC-3 ); exon 4 (5 -CAAATGCAGATTCTCCCACCAT AC-3 and 5 -GTATTAGCCACCCTATGACCTCTT-3 ); exon 5 (5 -GCAGGAGGCAA TCTTGATGATTC-3 and 5 -CCTTTCGGTTACAGAAGGCTTG-3 ); exon 6 (5 -GTGC CATTGTACTTAATCTCACTG-3 and 5 -GTAGTTTGTAAGGCTTTGGACTAC-3 ); exon 7 (5 -GCTATTCTTGGCACATAGTAGGC-3 and 5 -CTCATAACTCTGC CCTCTAAGTC-3 ); and exon 8 (5 -GGCTATAAGTTTAAGACTCTGCC3 and 5 - CATGGCCAACTTTATCCTATCAC-3 ). The PCR reactions were performed in a total volume of 20 L containing 50 ng of genomic DNA, 0.2 mM of each primer, 0.2 mM dNTPs, 1 unit of Taq polymerase (Takara, Shuzo Company, Otus, Shiga, Japan), and 1× reaction buffer (10 mM Tris–HCl [pH 8.3], 50 mM KCl, and 1.5 mM MgCl2 ). The PCR cycle conditions consisted of an initial denaturation step at 95 ◦ C for 5 min, followed by 35 cycles of 30 s at 95 ◦ C; 30 s at 53 ◦ C to 58 ◦ C; 30 s at 72 ◦ C; and a final elongation at 72 ◦ C for 10 min. The PCR products were purified using a GENECLEAN Turbo kit (Q-Biogene, Carlsbad, CA, USA). Sequencing was done using an ABI Prism 3100 Genetic Analyzer (PE Biosystems, Foster City, CA, USA). Both the forward and reverse sequences obtained were analyzed by manual review. The detected sequence variant (c.551C>G [pS184X]) was confirmed by sequencing the product of independent PCR amplification in both directions. To examine the potential effect of the p.S184X mutant on the expression of FRK protein, we constructed wild-type and p.S184X mutant plasmids. The entire open reading frame of FRK was amplified using 5 -AAGCTTGCGGCCGCGAGCAACATCTGTCAGAGGCTCT-3 (forward) and 5 -GATCTGATATCCCAGTGTTCATCTTATGAAGTTATT TGC-3 (reverse) primers. The PCR products were restricted by NotI and EcoRV, and then the restricted fragments were inserted into the p3×FLAG pCMV10 vector. The S184X mutant construct was created from p3×FLAG-FRK using the
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Letters to the Editor / Lung Cancer 71 (2011) 113–117
Quick-change II Site-Directed Mutagenesis kit (Stratagene, Cedar Creek, TX, USA). Both constructs were verified by sequencing. In addition, to determine whether or not the p.S184X mutation found in the present study affects the expression of PTEN protein, we examined the level of expression of PTEN in 293T cells after cotransfection of wild-type PTEN plasmid with FRK constructs. The 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum. The wild-type and mutant plasmids were transfected to cells using Effectene (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Cells were harvested with 200 l of RIPA lysis buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS), supplemented with protease inhibitor (Sigma, St. Louis, MO, USA). Western blots were prepared by standard procedures using anti-FLAG or anti-actin antibody (Sigma). Immunoreactivity was detected by an Immunobilon Western chemiluminescence reaction (Millipore, Billerica, MA, USA). Five genetic alterations (c.203A>G [p.K68R], c.364A>G [p.G122R], c.375T>G [p.D125E], c.551C>G [p.S184X], and c.1017C>T [A339A]) were found in the study population. The c.551C>G (p.S184X) was detected in one of the AC cases (a female neversmoker, Fig. 1A). This nonsense mutation was not detected in DNA from the matched non-malignant lung tissues and blood lymphocytes, indicating that the c.551C>G was a somatic mutation. The patient with the c.551C>G did not have mutations of the EGFR, ERBB2, KRAS, PTEN, LKB1, or TP53 genes. To determine whether or not the novel FRK mutation identified in the present study affected the expression on FRK protein, we overexpressed wild-type FRK and p.S184X mutant FRK in 293T cells. The p.S184X mutant created a low molecular weight variant of FRK (Fig. 1B), suggesting that the p.S184X mutation results in premature termination of the open reading frame and produces a truncated FRK protein. In addition, we examined the level of expression of PTEN in 293T cells expressing the wild-type FRK and p.S184X mutant FRK. The level of expression of PTEN was lower in the p.S184X mutant FRK-expressing cells than in the wild-type FRK-expressing cells (Fig. 1C). This finding suggests that a truncated FRK protein produced by the p.S184X mutation leads to a reduction of PTEN protein stability and expression. The remaining four variants (c.203A>G, c.364A>G, c.375T>G, and c.1017C>T) were also detected in the matched nonmalignant lung tissues and blood lymphocytes, suggesting that these variants are polymorphisms. The c.364A>G and c.1017C>T of the five polymorphisms has been previously reported in the public database (rs3756772 and rs56094152, respectively, http://www.ncbi.nlm.nih.gov/snp), and the c.203A>G and c.375T>G were novel polymorphisms. The minor allele frequency of the c.203A>G, c.364A>G, c.375T>G, and c.1017C>T polymorphisms was 0.014, 0.234, 0.020, and 0.023, respectively. The FRK gene contains SRC-homology (SH) domains SH3 (codons 42–110) and SH2 (codons 116–208) at the amino terminus, and a TK domain (codons 234–491) at the carboxy terminus [6]. There have been no published reports investigating FRK mutations in human cancers, but the results of FRK mutational analysis in human cancer cell lines have been deposited in a database (http://www.sanger.ac.uk), in which a nonsense mutation (c.579C>A [p.Y193X]) was found in one AC cell line (overall, 3.0%; and 7.7% [1/13] of the AC cell lines) of the 31 human lung cancer cell lines evaluated. The frequency of somatic FRK mutations in our series of NSCLCs was 0.6% (0.9% [1/117] in ACs and 2.0% [1/49] in female never-smokers with AC). Like the FRK mutation deposited in the database, the FRK mutation found in the present study was also a nonsense mutation (p.S184X) in the SH2 domain. The nonsense mutation p.S184X in the SH2 domain produced a truncated FRK protein lacking a part of the SH2 domain and a whole kinase domain, which may lead to loss of PTEN phosphorylation activity,
Fig. 1. A somatic missense mutation, c.551C>G (arrow), at the exon 2 of the FRK gene (A). Western blot analysis of wild-type FRK and the p.S184X mutant FRK (B). 293T cells were transfected with vector, and FLAG-tagged FRK and p.S184X mutant FRK. Proteins were resolved by SDS-PAGE and analyzed by Western blotting with antiFLAG antibodies recognizing FLAG-tagged and mutant FRK, or anti-actin antibody. Arrow indicates the wild-type FRK and mutant FRK. PTEN protein expression in 293T cells expressing the wild-type FRK and p.S184X mutant FRK (C).
and thereby a reduction of PTEN protein stability and expression. The FRK mutation identified in the present study was a heterozygous mutation. Considering the Knudson’s ‘two hit’ model in which both alleles of a tumor suppressor gene must be inactivated to contribute to tumor formation, the mechanism for inactivation of the remaining allele in the tumor with the heterozygous mutation remains to be determined. Given that tumor suppressor genes could be inactivated by allele loss, loss-of-function mutation, and aberrant promoter hypermethylation and that loss of heterozygosity occurs in NSCLC at a relatively high frequency [12], it is possible that the remaining allele is inactivated by allele loss. Otherwise, the truncated FRK protein that possesses the SH3 domain and a part of the SH2 domain but not the TK domain, may dominantnegatively inhibit wild-type FRK protein, and thereby contribute to tumorigenesis. In addition, recent data suggest that the function
Letters to the Editor / Lung Cancer 71 (2011) 113–117
of some tumor suppressor genes can be disrupted solely by haploinsufficiency; thus, there is alteration of one allele of a gene with the other allele remaining normal [14]. Therefore, whether or not haploinsufficiency of FRK provides a selective growth advantage in the tumor lacking a second hit in the remaining FRK allele should be also investigated. This study searched for mutations in the FRK gene, a recently identified tumor suppressor gene, for the first time in NSCLC. Somatic FRK mutations were found at a very low frequency in NSCLCs. However, considering that PTEN is required for a response to EGFR TK inhibitors, our findings indicate that an evaluation of loss-of-function mutations in the FRK gene is required for an optimal therapeutic approach for patients with NSCLC. Because the prevalence of genetic alterations often varies depending on ethnicity, additional studies are needed to determine the frequency of FRK mutations and the association with other EGFR pathway gene mutations in diverse ethnic populations. Conflict of interest statement None declared. References [1] Tang X, Varella-Garcia M, Xavier AC, et al. Epidermal growth factor receptor abnormalities in the pathogenesis and progression of lung adenocarcinomas. Cancer Prev Res 2008;1:192–200. [2] Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007;7:169–81. [3] Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39. [4] Han SW, Hwang PG, Chung DH, et al. Epidermal growth factor receptor (EGFR) downstream molecules as response predictive markers for gefitinib (Irresa, ZD1839) in chemotherapy-resistant non-small cell lung cancer. Int J Cancer 2005;113:109–15. [5] Shigematsu H, Gazdar AF. Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int J Cancer 2006;118:257–62. [6] Cance WG, Craven RJ, Bergman M, et al. Rak, a novel nuclear tyrosine kinase expressed in epithelial cells. Cell Growth Diff 1994;5:1347–55. [7] Brown MT, Cooper JA. Regulation, substrates and functions of src. Biochim Biophys Acta 1996;1287:121–49. [8] Chandrasekharan S, Qiu TH, Alkharouf N, et al. Characterization of mice deficient in the Src family nonreceptor tyrosine kinase Frk/rak. Mol Cell Biol 2002;22:5235–47. [9] Meyer T, Xu L, Chang J, et al. Breast cancer cell line proliferation blocked by the SRC-related RAK tyrosine kinase. Int J Cancer 2003;104:139–46. [10] Yim EK, Siwko S, Lim SY. Exploring Rak tyrosine kinase function in breast cancer. Cell Cycle 2009;8:2360–4. [11] Yim EK, Peng G, Dai H, et al. Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell 2009;15:304–14. [12] Girard L, Zochbauer-Muller S, Virmani AK, et al. Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering. Cancer Res 2000;60:4894–906. [13] Lee SY, Kim MJ, Jin G, et al. Somatic mutations in epidermal growth factor receptor signaling pathway genes in non-small cell lung cancers. J Thorac Oncol 2010;5:1734–40.
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[14] Chalhoub N, Baker SJ. PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol Mech Dis 2009;4:127–50.
Guang Jin a,b,1 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea b Department of Pharmacology, Yanbian University School of Basic Medicine, Yanji 133000, Jilin Province, China
a
Hyo-Sung Jeon a,c,1 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea c Cancer Research Institute, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea
a
Enyue Yang a,b Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea b Department of Pharmacology, Yanbian University School of Basic Medicine, Yanji 133000, Jilin Province, China
a
Jae Yong Park a,c,d,∗ Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea c Cancer Research Institute, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea d Department of Internal Medicine, Kyungpook National University Hospital, Samduk 2Ga 50, Daegu 700-412, South Korea
a
∗ Corresponding
author at: Department of Internal Medicine, Kyungpook National University Hospital, Samduk 2Ga 50, Daegu 700-412, South Korea. Tel.: +82 53 420 5536; fax: +82 53 426 2046. E-mail address: [email protected] (J.Y. Park) 1
These two authors contributed equally to this paper. 26 July 2010
doi: 10.1016/j.lungcan.2010.10.002
S. Amatori I. Bagaloni D. Viti M. Fanelli ∗ Molecular Pathology and Oncology Lab. “Paola”, Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, Via Campanella 1, 61032 Fano (PU), Italy ∗ Corresponding
author. Tel.: +39 0722 304951; fax: +39 0721 862834. E-mail address: [email protected] (M. Fanelli) 13 July 2010 doi: 10.1016/j.lungcan.2010.10.016
Mutation analysis of the FRK gene in non-small cell lung cancers Keywords: FRK Lung cancer Mutation
To the Editor, The epidermal growth factor receptor (EGFR) signaling pathway is one of the important oncogenic signaling cascades in lung carcinogenesis. The EGFR is a transmembrane tyrosine kinase receptor that plays an important role in many tumorigenic processes, including cell growth, proliferation, invasion, and metastasis. These effects are mediated by activation of downstream signaling pathways, including RAS-RAF-mitogen-activated protein kinase and phosphatidylinositol-3 kinase (PI3K)-AKT pathways [1,2]. Mutations in the tyrosine kinase (TK) domain of the EGFR gene are present in a subset of non-small cell lung cancers (NSCLCs), and those tumors with EGFR mutations have been reported to be highly sensitive to EGFR TK inhibitors [3]. In addition, alterations of downstream molecules of the EGFR have been reported to be associated with a clinical response to the EGFR TK inhibitors [4,5]. Therefore, a comprehensive evaluation of mutations in the EGFR signaling pathway genes may lead to optimized therapeutic approaches to lung cancer. Fyn-related kinase (FRK, also known as RAK) is a 54 kDa TK that belongs to the SRC non-receptor kinase family [6,7]. FRK was originally identified in human breast cancer cells and is expressed predominantly in epithelial tissues, including the kidney, liver, lung, and mammary gland [6]. Several lines of evidence suggest that FRK functions as a tumor suppressor; FRK inhibits cell proliferation and invasion, and tumorigenesis [8–10]. Interestingly, a recent
115
study has demonstrated that FRK positively regulates PTEN protein stability by phosphorylating PTEN on Try336, which in turn prevents PTEN degradation [11]. In addition, the FRK gene is located in 6q21-23, which is frequently deleted in NSCLCs [12]. These findings suggest that FRK may be involved in lung carcinogenesis. Therefore, we searched for mutations of the FRK gene in a cohort of NSCLCs that was previously investigated for mutations of the EGFR signaling pathway genes [13]. The study population has been described in our previous study [13]. Briefly, primary lung tumors and matching non-malignant lung tissues were obtained from 173 Korean patients with NSCLC who underwent curative resection at Kyungpook National University Hospital, in Daegu, Korea between January 2003 and July 2007. Patients who underwent chemotherapy or radiotherapy prior to surgery were excluded to avoid the effects on DNA. This study included 56 patients with squamous cell carcinoma and 117 patients with adenocarcinoma (AC). There were 113 males and 60 females in the study cohort. The patients consisted of 56 neversmokers and 117 smokers. Of the 117 patients with AC, 53 were never-smokers. All of the tumor and macroscopically normal lung tissue samples were obtained at the time of surgery, rapidly frozen in liquid nitrogen, and stored at −80 ◦ C. Only tumors with ≥80% of the tumor component were sent for analysis. This study was approved by the Institutional Review Board of Kyungpook National University Hospital, and written informed consent was obtained from each patient. FRK mutation analysis of the entire coding exons (exons 1–8) and exon–intron junctions was performed using polymerase chain reaction (PCR) and direct sequencing. Intron-based PCR primers were designated based on the GenBank reference sequence (accession no. NT 025741.15). The forward and reverse primers were as follows: exon 1 (5 -GCATACTCTCCGAAGTATGGTG-3 and 5 -GCATACTCTCCGAAGTATGGTG-3 ); exon 2 (5 -CTATCTTGCCAGGCTTTTTCCTC-3 and 5 -CGTGTCCATGTTCAC ACAGAAAG-3 ); exon 3 (5 -CCCAGTTCTCACACTCTCTCTTTTG-3 and 5 -CTCA TAGGCCATGGTTTGCCTATAC-3 ); exon 4 (5 -CAAATGCAGATTCTCCCACCAT AC-3 and 5 -GTATTAGCCACCCTATGACCTCTT-3 ); exon 5 (5 -GCAGGAGGCAA TCTTGATGATTC-3 and 5 -CCTTTCGGTTACAGAAGGCTTG-3 ); exon 6 (5 -GTGC CATTGTACTTAATCTCACTG-3 and 5 -GTAGTTTGTAAGGCTTTGGACTAC-3 ); exon 7 (5 -GCTATTCTTGGCACATAGTAGGC-3 and 5 -CTCATAACTCTGC CCTCTAAGTC-3 ); and exon 8 (5 -GGCTATAAGTTTAAGACTCTGCC3 and 5 - CATGGCCAACTTTATCCTATCAC-3 ). The PCR reactions were performed in a total volume of 20 L containing 50 ng of genomic DNA, 0.2 mM of each primer, 0.2 mM dNTPs, 1 unit of Taq polymerase (Takara, Shuzo Company, Otus, Shiga, Japan), and 1× reaction buffer (10 mM Tris–HCl [pH 8.3], 50 mM KCl, and 1.5 mM MgCl2 ). The PCR cycle conditions consisted of an initial denaturation step at 95 ◦ C for 5 min, followed by 35 cycles of 30 s at 95 ◦ C; 30 s at 53 ◦ C to 58 ◦ C; 30 s at 72 ◦ C; and a final elongation at 72 ◦ C for 10 min. The PCR products were purified using a GENECLEAN Turbo kit (Q-Biogene, Carlsbad, CA, USA). Sequencing was done using an ABI Prism 3100 Genetic Analyzer (PE Biosystems, Foster City, CA, USA). Both the forward and reverse sequences obtained were analyzed by manual review. The detected sequence variant (c.551C>G [pS184X]) was confirmed by sequencing the product of independent PCR amplification in both directions. To examine the potential effect of the p.S184X mutant on the expression of FRK protein, we constructed wild-type and p.S184X mutant plasmids. The entire open reading frame of FRK was amplified using 5 -AAGCTTGCGGCCGCGAGCAACATCTGTCAGAGGCTCT-3 (forward) and 5 -GATCTGATATCCCAGTGTTCATCTTATGAAGTTATT TGC-3 (reverse) primers. The PCR products were restricted by NotI and EcoRV, and then the restricted fragments were inserted into the p3×FLAG pCMV10 vector. The S184X mutant construct was created from p3×FLAG-FRK using the
116
Letters to the Editor / Lung Cancer 71 (2011) 113–117
Quick-change II Site-Directed Mutagenesis kit (Stratagene, Cedar Creek, TX, USA). Both constructs were verified by sequencing. In addition, to determine whether or not the p.S184X mutation found in the present study affects the expression of PTEN protein, we examined the level of expression of PTEN in 293T cells after cotransfection of wild-type PTEN plasmid with FRK constructs. The 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum. The wild-type and mutant plasmids were transfected to cells using Effectene (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Cells were harvested with 200 l of RIPA lysis buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS), supplemented with protease inhibitor (Sigma, St. Louis, MO, USA). Western blots were prepared by standard procedures using anti-FLAG or anti-actin antibody (Sigma). Immunoreactivity was detected by an Immunobilon Western chemiluminescence reaction (Millipore, Billerica, MA, USA). Five genetic alterations (c.203A>G [p.K68R], c.364A>G [p.G122R], c.375T>G [p.D125E], c.551C>G [p.S184X], and c.1017C>T [A339A]) were found in the study population. The c.551C>G (p.S184X) was detected in one of the AC cases (a female neversmoker, Fig. 1A). This nonsense mutation was not detected in DNA from the matched non-malignant lung tissues and blood lymphocytes, indicating that the c.551C>G was a somatic mutation. The patient with the c.551C>G did not have mutations of the EGFR, ERBB2, KRAS, PTEN, LKB1, or TP53 genes. To determine whether or not the novel FRK mutation identified in the present study affected the expression on FRK protein, we overexpressed wild-type FRK and p.S184X mutant FRK in 293T cells. The p.S184X mutant created a low molecular weight variant of FRK (Fig. 1B), suggesting that the p.S184X mutation results in premature termination of the open reading frame and produces a truncated FRK protein. In addition, we examined the level of expression of PTEN in 293T cells expressing the wild-type FRK and p.S184X mutant FRK. The level of expression of PTEN was lower in the p.S184X mutant FRK-expressing cells than in the wild-type FRK-expressing cells (Fig. 1C). This finding suggests that a truncated FRK protein produced by the p.S184X mutation leads to a reduction of PTEN protein stability and expression. The remaining four variants (c.203A>G, c.364A>G, c.375T>G, and c.1017C>T) were also detected in the matched nonmalignant lung tissues and blood lymphocytes, suggesting that these variants are polymorphisms. The c.364A>G and c.1017C>T of the five polymorphisms has been previously reported in the public database (rs3756772 and rs56094152, respectively, http://www.ncbi.nlm.nih.gov/snp), and the c.203A>G and c.375T>G were novel polymorphisms. The minor allele frequency of the c.203A>G, c.364A>G, c.375T>G, and c.1017C>T polymorphisms was 0.014, 0.234, 0.020, and 0.023, respectively. The FRK gene contains SRC-homology (SH) domains SH3 (codons 42–110) and SH2 (codons 116–208) at the amino terminus, and a TK domain (codons 234–491) at the carboxy terminus [6]. There have been no published reports investigating FRK mutations in human cancers, but the results of FRK mutational analysis in human cancer cell lines have been deposited in a database (http://www.sanger.ac.uk), in which a nonsense mutation (c.579C>A [p.Y193X]) was found in one AC cell line (overall, 3.0%; and 7.7% [1/13] of the AC cell lines) of the 31 human lung cancer cell lines evaluated. The frequency of somatic FRK mutations in our series of NSCLCs was 0.6% (0.9% [1/117] in ACs and 2.0% [1/49] in female never-smokers with AC). Like the FRK mutation deposited in the database, the FRK mutation found in the present study was also a nonsense mutation (p.S184X) in the SH2 domain. The nonsense mutation p.S184X in the SH2 domain produced a truncated FRK protein lacking a part of the SH2 domain and a whole kinase domain, which may lead to loss of PTEN phosphorylation activity,
Fig. 1. A somatic missense mutation, c.551C>G (arrow), at the exon 2 of the FRK gene (A). Western blot analysis of wild-type FRK and the p.S184X mutant FRK (B). 293T cells were transfected with vector, and FLAG-tagged FRK and p.S184X mutant FRK. Proteins were resolved by SDS-PAGE and analyzed by Western blotting with antiFLAG antibodies recognizing FLAG-tagged and mutant FRK, or anti-actin antibody. Arrow indicates the wild-type FRK and mutant FRK. PTEN protein expression in 293T cells expressing the wild-type FRK and p.S184X mutant FRK (C).
and thereby a reduction of PTEN protein stability and expression. The FRK mutation identified in the present study was a heterozygous mutation. Considering the Knudson’s ‘two hit’ model in which both alleles of a tumor suppressor gene must be inactivated to contribute to tumor formation, the mechanism for inactivation of the remaining allele in the tumor with the heterozygous mutation remains to be determined. Given that tumor suppressor genes could be inactivated by allele loss, loss-of-function mutation, and aberrant promoter hypermethylation and that loss of heterozygosity occurs in NSCLC at a relatively high frequency [12], it is possible that the remaining allele is inactivated by allele loss. Otherwise, the truncated FRK protein that possesses the SH3 domain and a part of the SH2 domain but not the TK domain, may dominantnegatively inhibit wild-type FRK protein, and thereby contribute to tumorigenesis. In addition, recent data suggest that the function
Letters to the Editor / Lung Cancer 71 (2011) 113–117
of some tumor suppressor genes can be disrupted solely by haploinsufficiency; thus, there is alteration of one allele of a gene with the other allele remaining normal [14]. Therefore, whether or not haploinsufficiency of FRK provides a selective growth advantage in the tumor lacking a second hit in the remaining FRK allele should be also investigated. This study searched for mutations in the FRK gene, a recently identified tumor suppressor gene, for the first time in NSCLC. Somatic FRK mutations were found at a very low frequency in NSCLCs. However, considering that PTEN is required for a response to EGFR TK inhibitors, our findings indicate that an evaluation of loss-of-function mutations in the FRK gene is required for an optimal therapeutic approach for patients with NSCLC. Because the prevalence of genetic alterations often varies depending on ethnicity, additional studies are needed to determine the frequency of FRK mutations and the association with other EGFR pathway gene mutations in diverse ethnic populations. Conflict of interest statement None declared. References [1] Tang X, Varella-Garcia M, Xavier AC, et al. Epidermal growth factor receptor abnormalities in the pathogenesis and progression of lung adenocarcinomas. Cancer Prev Res 2008;1:192–200. [2] Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007;7:169–81. [3] Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39. [4] Han SW, Hwang PG, Chung DH, et al. Epidermal growth factor receptor (EGFR) downstream molecules as response predictive markers for gefitinib (Irresa, ZD1839) in chemotherapy-resistant non-small cell lung cancer. Int J Cancer 2005;113:109–15. [5] Shigematsu H, Gazdar AF. Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int J Cancer 2006;118:257–62. [6] Cance WG, Craven RJ, Bergman M, et al. Rak, a novel nuclear tyrosine kinase expressed in epithelial cells. Cell Growth Diff 1994;5:1347–55. [7] Brown MT, Cooper JA. Regulation, substrates and functions of src. Biochim Biophys Acta 1996;1287:121–49. [8] Chandrasekharan S, Qiu TH, Alkharouf N, et al. Characterization of mice deficient in the Src family nonreceptor tyrosine kinase Frk/rak. Mol Cell Biol 2002;22:5235–47. [9] Meyer T, Xu L, Chang J, et al. Breast cancer cell line proliferation blocked by the SRC-related RAK tyrosine kinase. Int J Cancer 2003;104:139–46. [10] Yim EK, Siwko S, Lim SY. Exploring Rak tyrosine kinase function in breast cancer. Cell Cycle 2009;8:2360–4. [11] Yim EK, Peng G, Dai H, et al. Rak functions as a tumor suppressor by regulating PTEN protein stability and function. Cancer Cell 2009;15:304–14. [12] Girard L, Zochbauer-Muller S, Virmani AK, et al. Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering. Cancer Res 2000;60:4894–906. [13] Lee SY, Kim MJ, Jin G, et al. Somatic mutations in epidermal growth factor receptor signaling pathway genes in non-small cell lung cancers. J Thorac Oncol 2010;5:1734–40.
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[14] Chalhoub N, Baker SJ. PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol Mech Dis 2009;4:127–50.
Guang Jin a,b,1 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea b Department of Pharmacology, Yanbian University School of Basic Medicine, Yanji 133000, Jilin Province, China
a
Hyo-Sung Jeon a,c,1 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea c Cancer Research Institute, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea
a
Enyue Yang a,b Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea b Department of Pharmacology, Yanbian University School of Basic Medicine, Yanji 133000, Jilin Province, China
a
Jae Yong Park a,c,d,∗ Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea c Cancer Research Institute, School of Medicine, Kyungpook National University, Dong In 2Ga 101, Daegu 700-422, South Korea d Department of Internal Medicine, Kyungpook National University Hospital, Samduk 2Ga 50, Daegu 700-412, South Korea
a
∗ Corresponding
author at: Department of Internal Medicine, Kyungpook National University Hospital, Samduk 2Ga 50, Daegu 700-412, South Korea. Tel.: +82 53 420 5536; fax: +82 53 426 2046. E-mail address: [email protected] (J.Y. Park) 1
These two authors contributed equally to this paper. 26 July 2010
doi: 10.1016/j.lungcan.2010.10.002