MEK1 and AKT2 Mutations in Japanese Lung Cancer

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Hidefumi Sasaki, MD, PhD, Yu Hikosaka, MD, Osamu Kawano, MD, Satoru Moriyama, MD, PhD, Motoki Yano, MD, PhD, and Yoshitaka Fujii, MD, PhD

lung cancer.5 In addition, expression of the activated form of the Background: Recently, to identify potential somatic mutations in cell survival protein Akt (phospho Akt) in primary non-small genes of epidermal growth factor receptor (EGFR) signaling path- cell lung cancer is an independent indicator of poor prognosis.6 way, MEK1 gene mutation at exon 2 and mutation of the AKT2 Previous report indicated that there were AKT2 mutations (v-akt murine thymoma viral oncogene homologue 2) gene at kinase (2.5%) at kinase domain in lung cancers; however, no AKT 1 domain have been reported in non-small cell lung cancer. and 3 mutations at kinase domain.7 Methods: We investigated the MEK1 mutation (n ϭ 280) and AKT2 ϭ Because we have previously found the other kinase mutation (n 273) in surgically treated non-small cell lung cancer mutation, EGFR mutation was predominantly found in Jap- cases. The presence or absence of MEK1 mutation (exon 2) and anese population,8–11 we investigated MEK1 and AKT2 gene AKT2 mutation at kinase domain was analyzed by direct sequences. status, mainly focused on adenocarcinomas by direct se- Results: MEK1 mutation (K57K) was found from 1 of 280 patients quences. After retrospective pathologic review of lung can- with lung cancer (0.4%) and detected only one case (0.4%) of AKT2 cers, some of the sampled defined as other pathologic sub- mutation (R371H) in our cohort. MEK1 mutation was exclusive with types. The findings were compared with the clinicopathologic EGFR, K-ras, and B-raf mutations at kinase domain. However, features of lung cancer. AKT2 mutation was coexisted with EGFR and PIK3CA mutations. Conclusion: This study demonstrated that mutation in the kinase PATIENTS AND METHODS domain of AKT2 and MEK1 exon 2 mutation occurred in a small fraction of Japanese lung cancers. Patients Key Words: MEK1, Lung cancer, AKT2, Mutation. The study group included patients with lung cancer who had undergone surgery at the Department of Surgery, (J Thorac Oncol. 2010;5: 597–600) Nagoya City University Hospital. The study was approved by ethics committee of Nagoya City University Graduate School of Medical Sciences. The lung tumors were classified accord- he mitogen-activated protein kinase (MAPK) pathway ing to the general rule for clinical and pathologic record of Tplays a major role in the epidermal growth factor receptor lung cancer in Japan.12 The clinical and pathologic charac- (EGFR) signaling cascade. After activation of EGFR signal- teristics of the 280 patients with lung cancer for MEK1 gene ing, key downstream steps involve phosphorylation by RAF1 analyses were as follows: 164 (58.6%) were men, and 116 kinase of two distinct serine residues on both MEK1 and were women. Four were diagnosed as squamous cell carci- MEK2, upstream of the ERK1/ERK2 MAPK module.1 nomas, 273 were adenocarcinomas, and three were adeno- MEK1 is the main activator of both ERK1 and ERK2 and the squamous cell were carcinoma. One hundred fifty-three (54.6%) critical isoform regulating tumor cell proliferation in vitro were smokers, and 127 were nonsmokers. The clinical and and in vivo.2 More recently, MEK1 mutation at exon 2 was pathologic characteristics of the 273 patients with lung cancer identified by mutational analysis EGFR signaling pathway genes for AKT2 gene analyses were as follows: 149 (54.6%) were in lung adenocarcinoma.3 Activation of the intracellular prosur- men and 124 were women. Fifteen were diagnosed as squa- vival signal transduction protein AKT(v-akt murine thymoma mous cell carcinomas, 250 were adenocarcinomas, and eight viral oncogene) has been proposed as a central signaling event in were adenosquamous cell were carcinoma. One hundred carcinogenesis,4 key downstream steps of EGFR, and has been thirty-five (49.5%) were smokers, and 138 were nonsmokers shown in experimental models to confer chemoresistance in (Table 1). Polymerase Chain Reaction Assays for MEK1 Department of Oncology, Immunology and Surgery II, Nagoya City Univer- and AKT2 sity Graduate School of Medical Sciences, Nagoya, Japan. Disclosure: The authors declare no conflicts of interest. All tumor samples were immediately frozen and stored Address for correspondence: Hidefumi Sasaki, MD, PhD, Department of at Ϫ80°C until assayed. Total RNA was extracted from lung Oncology, Immunology and Surgery II, Nagoya City University Gradu- cancer tissues using Isogen kit (Nippon gene, Tokyo, Japan) ate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, according to the manufacturer’s instructions. RNA concen- Nagoya 467-8601, Japan. E-mail: hisasaki @med.nagoya-cu.ac.jp Copyright © 2010 by the International Association for the Study of Lung tration was determined by spectrophotometer and adjusted to Cancer a concentration of 200 ng/ml. About 10 cases were excluded ISSN: 1556-0864/10/0505-0597 for each assay because tumor cells were too few to suffi-

Journal of Thoracic Oncology • Volume 5, Number 5, May 2010 597 Sasaki et al. Journal of Thoracic Oncology • Volume 5, Number 5, May 2010

TABLE 1. Clinicopathologic Data of Patients with Lung Cancer Status Factors MEK1 Study AKT2 Study Mean age (yr) 65.6 Ϯ 9.1 64.6 Ϯ 9.1 Stage I 175 (62.5%) 170 (62.3%) II–IV 105 (37.5%) 103 (37.7%) Lymph node metastasis N0 203 (72.5%) 192 (70.3%) FIGURE 1. MEK1 gene mutation. One hundred seventy-one Nϩ 77 (27.5%) 81 (29.7%) nucleotide change from G to T at exon 2, introduces a sub- Smoking stitution of lysine (K) to asparagine (N) at amino acid posi- tion 57 (K57N) of the MEK1 protein. Never smoker 127 (45.3%) 135 (49.5%) Smoker 153 (54.6%) 138 (50.5%) EGFR mutation Wild type 160 (57.1%) 162 (55.7%) Statistical Analyses Mutation 120 (42.9%) 121 (44.3%) Statistical analyses were done using the Mann-Whitney Pathologic subtypes U test for unpaired samples and Wilcoxon singed rank test for Adeno 273 (97.5%) 250 (91.6%) paired samples. Linear relationships between variables were Nonadeno 7 (2.5%) 23 (8.4%) determined by means of simple linear regression. Correlation Age coefficients were determined by rank correlation using Spear- Յ65 138 (49.3%) 131 (48.0%) man test and ␹2 test. The overall survival of patients with lung Ͼ65 142 (50.7%) 142 (52.0%) cancer was examined by the Kaplan-Meier methods, and Gender differences were examined by the log-rank test. All analysis Male 164 (58.6%) 149 (54.6%) was done using the Stat-View software package (Abacus Female 116 (41.4%) 124 (45.4%) Concepts Inc. Berkeley, CA) and was considered significant Nϩ, lymph node metastasis positive; adeno, adenocarcinoma. when the p value was less than 0.05. ciently extract tumor RNA. RNA (1 ␮g) was reverse tran- RESULTS scribed by Superscript II enzyme (Gibco BRL, Gaithersburg, Using the primers sets for MEK1 exon 2, when we ␮ visualized the PCR products with 1% agarose gel, these MD) with 0.5 g oligo (dT)12–16 (Amersham Pharmacia Biotech Inc. Piscataway, NJ). The reaction mixture was samples were further studied. In our cohort, 1 of 280 patients incubated at 42°C for 50 minutes and then at 72°C for 15 had MEK1 mutation, K57N (Figure 1). The patient was male minutes. We then used 1 ␮L of each DNA for polymerase and smoker. The MEK1 mutation was exclusive with B-raf, chain reaction (PCR) analyses. The PCR reactions were K-ras, and EGFR mutation at kinase domain. performed using LA-Taq kit (Takara Bio Inc, Shiga, Japan) in Retrospective pathologic review demonstrated that the a 25-␮L reaction volume. The primer sequences for MEK1 MEK1 mutant sample was adenocarcinoma (poorly differen- gene for exon 2 were as follows: the forward primer, tiated) with mixed subtype, 60% of solid and 40% of acinar 5-CCGACGGCTCTGCAGTTA-3, and the reverse primer, components. The patient was dead 5 months after surgery for 5-TGTTCAGGAATTCTTCCAGCTT-3 (448bp). The cy- his recurrence of cancer. Because of the rapid growth, the cling conditions were as follows: initial denaturation at 94°C patients did not receive chemotherapy. for 5 minutes, followed by 40 cycles at 94°C for 45 seconds, We have also sequenced for kinase domain of AKT2 gene 60°C for 45 seconds, and 72°C for 45 seconds. The primer from 273 non-small cell lung cancer samples. Of 273 patients, sequences for AKT2 gene for kinase domain were as follows: from direct sequencing using cDNA samples, we found only one the forward primer, 5-GACAAAGATGGCCACATCAA-3, mutation at exon 11 (G1112A, R371H) (Figure 2). Adjacent and the reverse primer, 5-CTGGGCGGTAAATTCATCAT-3 normal lung tissue sample showed wild-type sequence that (489bp, exons 9–12). The cycling conditions were as follows: suggested that the mutation was somatic. This patient was initial denaturation at 94°C for 5 minutes, followed by 40 female, nonsmoker with well differentiated adenocarcinoma. cycles at 94°C for 45 seconds, 58°C for 45 seconds, and 72°C Pathologic stage was T1N2 (stage IIIa). The patient also had the for 45 seconds. The products were purified by Qiagen PCR L861Q type mutation in EGFR exon 21 and PIK3CA mutation purification kit (Qiagen, Valencia, CA). These samples were (E542Q). The patients received EGFR TKI-based therapy for sequenced by ABI prism 3100 analyzer (Applied Biosystems her recurrence, however, less effective than would be expected Japan Ltd., Tokyo, Japan) and analyzed by BLAST and in the broader population of patients with an activating EGFR chromatograms by manual review. The results of EGFR mutation. The patient was dead 33 months after surgery for her mutation statuses at kinase domain were already reported.8–11 recurrence of cancer.

FIGURE 2. AKT2 mutation at kinase domain, R371H, at exon 11. Left: forward sequence from lung cancer samples. Right: reverse sequence.

DISCUSSION pancreatic adenocarcinoma cell lines inhibits its tumorigen- We obtained findings that MEK1 K57N mutation status esity.20 AKT2 expression correlated with prognosis of human was rare in Japanese cohort (0.4%) and exclusive with B-raf, hepatocellular carcinoma.21 Previous report investigated that K-ras, and EGFR mutation of adenocarcinoma patients. We one of 79 lung cancer case had missense AKT2 mutation. have also found somatic AKT2 mutation at kinase domain in Proteins along the AKT signaling pathway, including 0.4% of Japanese lung cancers. Although the incidence of K-ras, EGFR, erbB2, and Braf, are frequently mutated in lung AKT2 mutations was low, the location of the mutation at an cancers8,21–23 and act as oncogenic proteins. Of these genes, important functional site (the kinase domain) suggested that we found a coincidence of an AKT2 mutation in a lung AKT2 mutation might possibly contribute to the pathogenesis adenocarcinoma, suggesting that alterations in the AKT sig- of lung cancer. Because we used direct sequencing for naling by both AKT2 and EGFR mutation can occur and screening purpose, the frequency of these mutations might be could possibly contribute together to the pathogenesis of lung underestimated, probably not enough for detection of all adenocarcinomas. mutated cases. In summary, MEK1 and AKT2 mutations in Japanese lung The previous report showed the somatic mutation in cancers seemed to be extremely rare; however, these mutations MEK1 in human lung tumors, but not in colon, breast, might be a role in function. Because we used surgical resected prostate, and pancreas carcinomas, identified by mutational samples, we would like to test for these mutations in a cohort of profiling of genes encoding EGFR signaling pathway proteins patients with more advanced lung cancer. in a large cohort of lung adenocarcinomas.1,13 In the previous report, the same heterozygous mutation, ACKNOWLEDGMENTS K57N, was found in two of 207 lung adenocarcinomas, Supported by AstraZeneca Research Grant 2004, neither of which harbored mutations in other genes encoding Grand-in-Aid for Research in Nagoya City University (2006), components of the EGFR signaling pathway, such as EGFR, and Grants-in-Aid for Scientific Research, Japan Society for K-ras, B-raf, and PIK3CA.3 Functional characterization of the Promotion of Science (JSPS) (Nos., 19390367, 21591820, mutant in vitro indicated that its expression in 293T cells lead and 21390367). to constitutive activation of downstream signaling compo- The authors thank Mrs. Tomomi Shibata for her excel- nents.14 Thus, MEK1 K57N mutant display gain-of-function lent technical assistances. properties.14 K57 is highly conserved among various species3 and located in a region between the nuclear export signal REFERENCES (amino acids 33–44) and catalytic domain (amino acids 1. Kyriakis JM, App H, Zhang XF, et al. Raf-1 activates MAP kinase- 68–271) of MEK1. Consistent with the G to T mutation, a kinase. Nature 1992;358:417–421. type of transversion known to be smoking related, the sample 2. Shama J, Garcia-Medina R, Pouyssegur J, et al. Major contribution of MEK1 to the activation of ERK1/ERK2 and to the growth of LS174T colon was from smokers. The K57N substitution leads to constitu- carcinoma cells. Biochem Biophys Res Commun 2008;372:845–849. 3 tive activation of the MAPK pathway in vitro (Ba/F3 cell). 3. Marks JI, Gong Y, Chitale D, et al. Novel MEK1 mutation identified Akt has been found to play a role in the survival of by mutational analysis of epidermal growth factor receptor signaling cancer cells.15–18 The importance of AKT in human cancer is pathway genes in lung adenocarcinoma. Cancer Res 2008;68:5524– largely inferred from common recurring mutations in en- 5528. 4. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT path- zymes that produce or degrade short-lived second messenger way in human cancer. Nat Rev Cancer 2002;2:489–501. phospholipids and consequently indirectly activate AKT 5. Brognard J, Clark AS, Ni Y, et al. Akt/protein B is constitutively active through membrane recruitment. in non-small cell lung cancer cells and promotes cellular survival and Among the members of the AKT family, AKT2 is resistance to chemotherapy and radiation. Cancer Res 2001;61:3986– 3997. associated with the development of human cancers. Overex- 6. David O, Jett J, LeBean H, et al. Phospho-Akt overexpression in pression of the AKT2 is observed in 20% of pancreatic non-small cell lung cancer confers significant stage-independent survival adenocarcinoma.19 Transfection of antisense AKT2 RNA into disadvantage. Clin Cancer Res 2004;10:6865–6871.

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