慕容鲜卑DOI:10.1093/jnci/djt338
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Novel Germline Mutation in the
Transmembrane Domain of HER2 in Familial Lung Adenocarcinomas
Hiromasa Yamamoto, Koichiro Higasa, Masakiyo Sakaguchi,
Kazuhiko Shien, Junichi Soh, Koichi Ichimura, Masashi Furukawa,
Shinsuke Hashida, Kazunori Tsukuda, Nagio Takigawa, Keitaro Matsuo,
Katsuyuki Kiura, Shinichiro Miyoshi, Fumihiko Matsuda, Shinichi Toyooka
Manuscript received July 7, 2013; revised October 14, 2013; accepted October 16, 2013.Correspondence to: Shinichi Toyooka, MD, PhD, O kayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Clinical Genomic Medicine/Thoracic, Breast and Endocrinological Surgery, 2-5-1 Shikata-cho, Kita-ku, O kayama, O kayama 700–8558, Japan (e-mail: toyooka@md.okayama-u.ac.jp ).We encountered a family of Japanese descent in which multiple members developed
lung cancer. Using whole-exome sequencing, we identified a novel germline muta-tion in the transmembrane domain of the human epidermal growth factor recep-tor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in
the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas.
Because the transmembrane domain of HER2 is considered to be responsible for the
dimerization and subsequent activation of the HER family and downstream signaling
特区报
pathways, we performed functional analyses of these HER2 mutants. Mutant HER2
G660D and V659E proteins were more stable than wild-type protein. Both the G660D
and V659E mutants activated Akt. In addition, they activated p38, which is thought
to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest
that mutations in the transmembrane domain of HER2 may be oncogenic, causing
hereditary and sporadic lung adenocarcinomas.
J Natl Cancer Inst;2014;106:1–4
Familial lung cancers are rare among human malignancies. Recent studies have reported that germline mutations in the epidermal growth factor receptor (EGFR ) gene predispose the development of lung cancer. Reported familial lung adenocar-cinomas with a germline EGFR mutation, such as T790M, carry secondary somatic EGFR mutations, including exon 19 dele-tion and exon 21 L858R mutation (1–4). We
encountered a family of Japanese descent in which multiple members developed lung cancer (Figure 1). The proband (III-4) was a 53-year-old woman with multiple lung adenocarcinomas in bilateral lungs. She was
巴塞杜氏病a light smoker with a 1.2-pack-year history
of smoking. She had undergone a left lower
lobectomy for multiple lung adenocarci-nomas at the age of 44 years. Her mother
(II-4), a never smoker, also had multiple
lung adenocarcinomas. Partial pulmonary
resections of two tumors were performed
for II-4 for the purpose of diagnosis after
pleural dissemination was found during sur-gery, and multiple lesions were removed in
a lobectomy or partial resections in III-4.
A histological examination of the resected
tumors in II-4 revealed nonmucinous ade-nocarcinoma in situ and nonmucinous min-imally invasive adenocarcinoma, whereas BrieF CoMMuNiCATioN
Figure 1. Pedigree chart of a Japanese family in which multiple mem-bers developed lung cancer. T
he boxes and circles indicate men and
women, respectively. The numbers at the bottom of each member indi-cate the age at the time of death or the time of the analysis. An oblique line shows deceased family members. The proband (III-4) had multiple lung adenocarcinomas (arrow ). T umor tissue, nonmalignant lung tissue, and peripheral blood samples were obtained from III-4. The proband’s mother (II-4) also had multiple lung adenocarcinomas, and tumor and nonmalignant lung tissue samples were available. The proband’s father (II-5) and sister (III-5) were both unaffected, and peripheral blood sam-ples were obtained from these individuals. Some family members who were not considered as critical for this study were excluded from the pedigree chart to preserve confidentiality. Whole-exome sequencing was performed for individuals II-4, II-5, III-4, and III-5.
the histological findings of pleural dissemi-nation indicated mucus-containing adeno-carcinoma. Those of III-4 contained various subtypes of adenocarcinoma, including non-mucinous and mucinous adenocarcinoma in situ and invasive mucinous adenocarci-noma. In addition, normal-appearing lung parenchyma obtained from a lobectomy in III-4 revealed innumerable small pre-invasive lesions, implying the presence of precancerous changes throughout the lung (Supplementary Figure 1, available online). Sequencing analyses of EGFR exons 18 to 21 and KRA
S as well as an immunohisto-chemical staining for AL K protein in the resected tumors indicated no genetic altera-tions in these genes. The pedigree chart
suggested that lung cancer was inherited in an autosomal dominant manner.
After obtaining permission from the Institutional Review Board at Okayama University Hospital and informed consent from the patients and other family members, we performed a whole-exome sequencing study. T umor DNA samples from II-4, tumor and peripheral blood DNA samples from III-4, and peripheral blood DNA samples from two unaffected family members (II-5 and III-5) were used for the analysis. The candi-date germline alterations were restricted to 29 variants by comparing the whole-exome sequencing results between the patients and the unaffected family members. Among them, we focused on a point mutation in the human epidermal growth factor receptor 2 (HER2/neu ) gene (NM_004448, G660D, GGC to GAC), which was located in exon 17 encoding the transmembrane domain of HER2 (Supplementary T ables 1–3). This alteration was confirmed by direct sequenc-ing (Figure 2A ). We also confirmed that there was no copy number gain of HER2 in the examined tumors based on the degree of read-depth in the whole-exome sequenc-ing results. Of note, no mutations in genes known to cause lung cancers were detected for tumors from III-4 and II-4.
We considered that somatic mutations in the HER2 transmembrane domain might act as driver mutations in lung cancer. Hence, we sequenced exon 17 of the HER2
A
B
C
Figure 2. DNA and amino acid sequences in the transmembrane domain of HER2. A ) Direct Sanger sequencing of the proband (III-4), her affected mother (II-4), and her unaffected sister (III-5). The results indicated that G660D was a germline mutation. B ) Direct sequencing of a sporadic lung adenocarci-noma with a HER2 V659E mutation. V659E was found to be of somatic ori-gin based on the sequencing results of the peritumoral lung tissue from the same specimen. All the sequence variants were confirmed by independent polymerase chain reaction amplifications and were sequenced in both direc-tions. C ) Interspecies conservation of the transmembrane domain of HER2
(UCSC Genome Browser, genome.ucsc.edu , accessed September 12, 2013). The yellow highlight indicates the N-terminal glycine zipper motif Thr 652-X 3-Ser 656-X 3-Gly 660, a tandem variant of a GG4-like motif of human HER2. Codons 659 and 660 in human HER2 are highly conserved among the listed vertebrate species (shown in red ). X. tropicalis = Xenopus tropicalis .
in the tumor samples of 315 sporadic non–small cell lung cancer patients, of which 253 were adenocarcinomas. Although the HER2 G660D mutation was not detected, a novel nonsynonymous mutation, V659E (GTT to GAA), next to codon 660 was identified in one of these patients. This patient was histologically diagnosed as nonmucinous adenocarcinoma in situ, and the patient had neither smoking history nor apparent family history of lung can-cer. This V659E mutation was certainly a somatic mutation because it was not iden-tified in the peritumoral lung tissue of the same patient (Figure 2B ). The alignment of HER2 amino acid sequences showed high conservation of valine 659 and glycine 660 among vertebrates (Figure 2C ).
HER2 somatic mutations have been
reported in 2% to 4% of lung adenocarci-nomas (5–7). However, all reported muta-membrane domain (V664E) of the rat neu gene, which corresponds to V659E in its human homolog HER2, induced oncogenic transformation (8). In addition, in vivo experiments showed that the HER2 V659E
mutation contributed to the stability of HER2 dimers, resulting in the dysregu-lated receptor activation and subsequent cell transformation (9,10). Furthermore, the novel mutations were located within the glycine zipper motif Thr 652-X 3-Ser 656-X 3-Gly 660, a tandem variant of the GG4-like motif, at the N-terminal portion of the transmembrane domain, which was critically related to the dimerization of HER2 (Figure 2C ) (9,11). Accordingly, we performed a functional analysis of the mutant HER2 proteins. We found that the degradation of HER2 protein after the administration of cycloheximide was slower in G660D and V659E mutants as compared with wild-type (Supplementary Figure 2A ), indicating the higher stabil-ity of the mutant proteins than wild-type protein. In addition, results of a phospho-mitogen–activated protein kinase array indicated the activation of Akt and p38α (data not shown). Indeed, Akt is known
to be activated by HER2 by phosphati-dylinositol 3-kinase and leads to increased cell growth and survival (12,13). Also, the activation of p38 was shown to contribute to the viability of lung adenocarcinoma cells derived from never or light smokers (14,15). A western blot analysis for Akt and p38 successfully confirmed the upregula-tion of both phospho-Akt and phospho-p38 expression in the mutant HER2 transfect-ants (Supplementary Figure 2B ).
Because the G660D alteration in HER2 might have been the cause of the lung can-cer in the pedigr
ee studied, we investigated whether familial aggregation of cancer in other organs could be seen in this pedi-gree. We found that II-1 and II-6 devel-oped renal and gastric cancers, respectively; however, both of them also had lung cancer. The reason why other types of clinically apparent malignances were rarely found in this pedigree is unclear. The G660D ger-mline mutation may be tolerated in organs other than the lung.
太中银铁路
This study had some limitations. First, the carcinogenic potential of the HER2 mutation at the transmembrane domain should be confirmed in other models such as transgenic mice. Second, the rarity of these mutations in sporadic lung cancers may be the limitation for generalizability to other cases even if targeting therapies for similar types of HER2 mutation were developed.
In conclusion, we identified a novel germline mutation in the transmembrane domain of the HER2 in familial lung adeno-carcinomas. Somatic mutation in the HER2 transmembrane domain may be a possible cause of sporadic lung adenocarcinomas.
references
1. Bell DW, Gore I, Okimoto RA, et al. Inherited
susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet . 2005;37(12):1315–1316. 2. Ikeda K, Nomori H, Mori T , Sasaki J,
Kobayashi T . Novel germline mutation: EGFR V843I in patient with multiple lung adenocar-cinomas and family members with lung cancer. Ann Thorac Surg . 2008;85(4):1430–1432.
3. Ohtsuka K, Ohnishi H, Kurai D, et al. Familial
lung adenocarcinoma caused by the EGFR V843I germ-line mutation. J Clin Onc o l . 2011;29(8):e191–e192.
4. van Noesel J, van der Ven WH, van Os TA,
et al. Activating germline R776H mutation in the epidermal growth factor receptor associ-ated with lung cancer with squamous differen-tiation. J Clin Oncol . 2013;31(10):e161–e164.
5. Pao W, Girard N. New driver mutations
in non-small-cell lung cancer. Lancet Onco l . 2011;12(2):175–180. 6. Shigematsu H, T akahashi T , Nomura M,
et al. Somatic mutations of the HER2 kinase domain in lung adenocarcinomas. Cancer Res . 2005;65(5):1642–1646.尘世佛心 7. Stephens P , Hunter C, Bignell G, et al. Lung
cancer: intragenic ERBB2 kinase mutations in tumours. Nature . 2004;431(7008):525–526. 8. Bargmann CI, Hung MC, Weinberg RA.
Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell . 1986;45(5):649–657. 9. Bocharov EV , Mineev KS, Volynsky PE,
et al. Spatial structure of the dimeric trans-membrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state. J Bio l Chem . 2008;283(11):6950–6956.
10. Fleishman SJ, Schlessinger J, Ben-T al N. A
斯特林冲锋putative molecular-activation switch in the transmembrane domain of erbB2. Pro c Natl Acad Sci U S A . 2002;99(25):15937–15940. 11. Mineev KS, Bocharov EV , Pustovalova
YE, Bocharova OV , Chupin VV , Arseniev AS. Spatial structure of the transmem-brane domain heterodimer of ErbB1 and ErbB2 receptor tyrosine kinases. J Mo l Bio l . 2010;400(2):231–243.
12. Baselga J, Swain SM. Novel anticancer targets:
revisiting ERBB2 and discovering ERBB3. Nat Rev Cancer . 2009;9(7):463–475.
13. Engelman JA. T argeting PI3K signalling in
cancer: opportunities, challenges and limita-tions. Nat Rev Cancer . 2009;9(8):550–562.
14. Mountzios G, Planchard D, Besse B, et al.
Mitogen-activated protein kinase activation in lung adenocarcinoma: a comparative study between ever smokers and never smokers. Clin Cancer Res . 2008;14(13):4096–4102.
15. Planchard D, Camara-Clayette V , Dorvault
N, Soria JC, Fouret P . p38 Mitogen-activated protein kinase signaling, ERCC1 expres-sion, and viability of lung cancer cells from never or light smoker patients. Cancer . 2012;118(20):5015–5025.
Funding
This study was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and T echnology of Japan (25293302 to ST).
Note
H. Y amamoto, J. Soh, S. Miyoshi, and S. T oyooka conceived the project. K. Higasa, M. Sakaguchi, K. Shien, and K. Ichimura performed the experi-ments. H. Y amamoto, J. Soh, M. Furukawa, S. Hashida, N. T akigawa, K. Kiura, K. T sukuda, and S. T oyooka collected the samples and assisted with the experi-ments. H. Y amamoto, K. Higasa, K. Shien, and K. Matsuo analyzed the data. H. Y amamoto, K. Higasa, M. Sakaguchi, F . Matsuda, and S. T oyooka prepared the manuscript with input from the other authors. S. Miyoshi, F . Matsuda, and S. T oyooka supervised the project. The authors declared no conflicts of interest.
Affiliations of authors: Department of Thoracic, Breast and Endocrinological Surgery (HY, KS, JS, MF, SH, KT, SM, ST), Department of Clinical Genomic Medicine (KS, ST), Department of Cell Biology (MS), Department of Pathology (KI), and Department of Hematology, O ncology and
Respiratory Medicine (KK), O kayama University
Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences, Okayama, Japan; Center
for Genomic Medicine, Kyoto University School of
Medicine, Kyoto, Japan (KH, FM); Department of
General Internal Medicine 4, Kawasaki Medical
School, O kayama, Japan (NT); Department of
Preventive Medicine, Kyushu University Faculty of
Medical Sciences, Fukuoka, Japan (KM).
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