Frameshift mutations in TGFßRII, IGFIIR, BAX, hMSH3 and hMSH6 are absent in lung cancers
Kunihiko Gotoh1,5,
Yasushi Yatabe2,
Takahiko Sugiura1,
Kenzo Takagi5,
Makoto Ogawa1,
Takashi Takahashi4 and
Tetsuya Mitsudomi3,6
1 Department of Internal Medicine and Pulmonary Medicine,
2 Pathology and Clinical Laboratories and
3 Thoracic Surgery, Aichi Cancer Center Hospital and
4 Laboratory of Ultrastructure Research, Aichi Cancer Center Research Institute, Kanokoden, Chikusa-ku, Nagoya 464-8681 and
5 Department of Internal Medicine II, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan
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Abstract
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A genome-wide instability at simple repeat sequences characterizes gastrointestinal and endometrial cancers of the microsatellite mutator phenotype (MMP). The genes encoding transforming growth factor-ß receptor type II (TGFßRII), insulin-like growth factor II receptor (IGFIIR), Bcl-2 associated X protein (BAX), hMSH3 and hMSH6 have simple repeat sequences in their coding regions. Consequently, mutations in the single repeat sequences in these genes provide one major route for carcinogenesis in these cancers. We examined 43 non-small cell lung carcinomas and 16 small cell carcinomas for frameshift mutations in simple repeat sequences of TGFßRII, IGFIIR, BAX, hMSH3 and hMSH6. In addition, MMP was assessed using a primer set for BAT-26. None of 59 lung cancers exhibited frameshift mutations or MMP. It is concluded that somatic frameshift mutations in these genes and MMP do not constitute important mechanisms in lung carcinogenesis. The possibility of some sort of genetic instability undetectable as a form of MMP cannot be precluded.
Abbreviations: BAX, Bcl-2-associated X protein; HNPCC, hereditary non-polyposis colorectal cancer; IGFIIR, insulin-like growth factor II receptor gene; MMP, microsatellite mutator phenotype; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancers; TGFßRII, transforming growth factor-ß receptor type II gene.
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Introduction
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Cancers with the microsatellite mutator phenotype (MMP) show exaggerated genomic instability at simple repeat sequences (1). This behavior is thought to be caused by mutator mutations occurring in mismatch repair genes (2) and an MMP leading to intrinsic genomic instability and mutations of genes important for growth regulation has been proposed as an early step in colonic carcinogenesis (1). However, the target gene(s) for mutations remained unidentified until the first report of frameshift mutations in the transforming growth factor-ß receptor type II gene (TGFßRII) in colon cancer cells (3) TGFßRII has a 10 bp poly(A) tract at codons 125128 of its open reading frame and this locus is thought to be prone to slippage-related frameshift mutations. Subsequently, other genes involved in cell growth, such as that for the insulin-like growth factor II receptor (IGFIIR) (4), or apoptosis, such as the Bcl-2 associated X protein (BAX) (5,6), were found to be mutational targets for the MMP in cancers of the stomach and colorectum. In addition, some members of the DNA mismatch repair gene family, such as hMSH3 and hMSH6, have been reported to display simple repeat sequences in their coding regions and slippage-related frameshift mutations in colorectal tumors (7). Frameshift mutations of secondary mutators are presumably induced by primary mutators such as hMLH1 and hMSH2 (6).
Frameshift mutations in TGFßRII, IGFIIR, BAX, hMSH3 and hMSH6 in gastric and colorectal tumors are relatively common. On the other hand, the frequency of MMP in lung cancer has been a matter of controversy (8,9) and frameshift mutations in such genes have not been reported except in TGFßRII (10,11). To assess the significance of this type of change to lung cancers, a total of 59 tumors were analyzed in the present study for the frequency of frameshift mutations in TGFßRII, IGFIIR, BAX, hMSH3 and hMSH6.
We examined 43 tumor specimens from patients with non-small cell lung cancer (NSCLC) who underwent pulmonary resection from 1991 to 1994 in the Department of Thoracic Surgery, Aichi Cancer Center Hospital, and three tumor samples and 13 tumor cell lines from small cell lung cancers (SCLC). (Ten of the SCLC cell lines were established at the Aichi Cancer Center Research Institute and three were obtained from the NCINavy Medical Oncology Branch, Bethesda, MD.) All tissue samples were immediately frozen at surgery in liquid nitrogen and maintained at 70°C until use. Total RNA was isolated using the acid guanidium thiocyanate/cesium chloride procedure (12). First strand cDNA was synthesized using 5 µg total cellular RNA with random hexanucleotide primers (Boehringer Mannheim, Yamanouchi, Tokyo, Japan). Genomic DNA was extracted from tumor samples and cell lines using standard methods (12).
A 100 bp region of the TGFßRII gene containing a 10 bp poly(A) tract (nucleotides 671770) was amplified from the synthesized cDNA. The primer sequences were 5'-TCTGGAAGATGCTGCTTCTC-3' and 5'-GTCATTGCACTCATCAGAGC-3'. Amplification of TGFßRII was performed using a 0.5 µl aliquot of cDNA and 10 µl of amplification reaction mixture containing 200 µM each of four dNTPs, 50 mM Kcl, 1.5 mM MgCl2, 10 mM TrisHCl (pH 8.3), 5 ng/ml each of two appropriate primers, 0.25 µl of [
-32P]dCTP (3000 Ci/mmol, 10 mCi/ml; Amersham, Arlington Heights, IL) and 0.25 U TaKaRa Taq DNA polymerase (Takara Biomedicals, Shiga, Japan). The annealing temperature was 55°C. The colon cancer cell line LS174T DNA with a known TGFßRII frameshift mutation was used as a positive control.
We also amplified 110, 94, 152 and 93 bp regions encompassing the IGFIIR G8, BAX G8, hMSH3 A8 and hMSH6 C8 tracts by PCR in a similar way. The primer sequences used were as follows: IGFIIR, 5'-CAGGTCTCCTGACTCAGAAG-3' and 5'-GAAGAAGATGGCTGTGGAGC-3'; BAX, 5'-ATCCAGGATCGAGCAGGGCG-3' and 5'-ACTCGCTCAGCTTCTTGGTG-3'; hMSH3, 5'-AGATGTGAATCCCCTAATCAAGC-3' and 5'-ACTCCCACAATGCCAATAAAAAT-3'; hMSH6, 5'-GGGTGATGGTCCTATGTGTC-3' and 5'-CGTAATGCAAGGATGGCGT-3'. The annealing temperatures were: IGFIIR, 58°C; BAX, 55°C; hMSH3, 58°C; hMSH6, 55°C.
PCR products were subjected to electrophoresis in denaturing 6% polyacrylamide gels containing 89 mmol/l Trisborate, 2 mmol/l EDTA, pH 8.3 (1x TBE) and 8 M urea at 60 W for 2 h. After electrophoresis, the gels were dried on filter paper and subjected to autoradiography.
Since it has been reported that a single poly(A) tract, BAT-26, is sufficient to confirm the MMP status of 159 of 160 tumors and cell lines (13) and to be the best marker for MMP assessment in colorectal cancers (14), we assessed the MMP status by examining this locus.
To detect frameshift mutations, we performed a PCR-based assay for mutational hot-spots comprising small repeat sequences in the TGFßRII, IGFIIR, BAX, hMSH3 and hMSH6 genes. Representative gels are shown in Figure 1
. In the 43 NSCLC and 16 SCLC studied, we did not find any frameshift mutations.

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Fig. 1. Evaluation of frameshift mutations in lung cancers occurring in the (a) TGFßRII, (b) IGFIIR, (c) BAX, (d) hMSH3 and (e) hMSH6 genes. MMP was also examined using the BAT-26 locus (f). LS174T, E90T, LOVO, LS411, DU145, LS180, AN3CA and HCT116 are positive controls for the corresponding loci. LS174T, LOVO, LS411, LS180 and HCT116 are colon cancer cell lines, DU145 is a prostate cancer cell line and AN3CA and E90T are an endometrial cancer cell line and a resected tumor sample, respectively. Lanes 17 and 1627 are NSCLC samples and lanes 815 are SCLC samples.
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Determination of MMP in lung cancers was also performed using a primer set for BAT-26, which amplifies an adenine mononucleotide repeat and reportedly has the highest sensitivity for detecting MMP (13). BAT-26 is a repeat of 26 deoxyadenosines localized in an intron of hMSH2. PCR amplification of BAT-26 was performed with primers 5'-TGACTACTTTTGACTTCAGCC-3' and 5'-AACCATTCAACATTTTTAACCC-3'. The annealing temperature was 45°C and PCR products were analyzed as detailed above. In each case, tumor DNA and, if available, matching normal DNA were analyzed. Figure 1f
shows representative results. No positive cases were found in 41 NSCLC and 11 SCLC analyzed (Figure 1
).
In the present study, we could not detect a single case of MMP or frameshift mutations in a series of NSCLC and SCLC. The literature concerning the incidence of MMP in lung cancer is conflicting. Peltomäki et al. (15) reported alteration in only one microsatellite locus in two (2%) of 87 NSCLC cases, unlike tumors of hereditary non-polyposis colorectal cancer (HNPCC), where instability involves multiple genetic loci. Adachi et al. (8) also could not find any MMP (0/37, 0%) in SCLC, while Fong et al. (16) and Mao et al. (17) reported MMP frequencies in NSCLC of 7 and 9%, respectively. However, other investigators reported higher incidences in lung cancers in the range 3466% for NSCLC (9,18) and 4550% for SCLC (17,19). The reasons for the wide variation are unclear. However, the following are candidates as possible explanations: (i) the definition of MMP is not universal and the numbers and loci of MMP markers are not standardized, with sensitivity dependent on the repeat unit length of MMP markers; (ii) gel patterns are sometimes difficult to interpret or even unreproducible; (iii) racial differences may exist; and (iv) other factors such as age (20) or nature of sample (frozen or paraffin-embedded material) may affect the incidence. The frequency of MMP in paraffin-embedded samples has tended to be higher in the literature.
HNPCC or HNPCC-associated tumors of the endometrium, stomach, pancreaticobiliary system, ovary, small intestine or upper urological tract (renal pelvis and ureter) are characterized by a high frequency of MMP (21). However, in HNPCC families there appears to be no increase or even a lower risk of lung cancer (22). In this context, the high incidence of MMP in lung cancer reported by some investigators is difficult to interpret.
The target gene(s) for MMP associated with a neoplastic phenotype remained unidentified until the reports of frequent frameshift mutations in genes such as TGFßRII, IGFIIR, BAX, hMSH3 and hMSH6 in tumors with MMP. The reported frequencies of mutations are summarized in Table I
. Recently, Takenoshita et al. (10) reported no mutations in TGFßRII, including an A10 nucleotide repeat at codons 125128 in lung cancers with MMP. Tani et al. (11) reported a mutation in only one of 15 SCLC, in good agreement with the present study. To our knowledge, this is the first systematic survey of frameshift mutations in lung cancer. The fact that we did not detect any frameshift mutations is in line with the absence of MMP as examined by BAT-26.
Multiple genetic alterations in such genes as p53, Rb, ras, myc or putative tumor suppressor genes on 3p are thought to be involved in lung carcinogenesis (23). The multiplicity of genetic abnormalities found in lung cancers thus appears at odds with the absence of MMP at first sight. Alternatively, one might assume that lung cancer cells have some sort of genetic instability that cannot be detected as a form of MMP. Lengauer et al. (24) have recently reported that colorectal tumors without MMP exhibit a striking defect in chromosome segregation, resulting in gains or losses in excess of 102 per chromosome per generation. They also concluded that this form of chromosomal instability reflects a continuing cellular defect that persists throughout the lifetime of the tumor cell and that it is not simply related to chromosome number (24). In colorectal carcinomas, loss of heterozygosity of the p53 gene is strongly associated with DNA aneuploidy (25), whereas tumors with MMP are usually diploid and have a low frequency of p53 mutations (2628). The prevalence of p53 mutations and aneuploidy and absence of MMP in lung cancer thus allows the tempting hypothesis that most lung cancer cells may have chromosomal instability. Recently, Cahill et al. (29) have reported that chromosomal instability is consistently associated with loss of function of a mitotic checkpoint. Thus, loss of function of a mitotic checkpoint might be a common characteristic of lung cancer cells.
In conclusion, we could not find any frameshift mutations in the single repeat sequences of TGFßRII, IGFIIR, BAX, hMSH3 and hMSH6 or MMP in a series of lung cancers. The mechanism(s) causing the multiple genetic alterations found in neoplasia in the lung remains to be elucidated.
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Acknowledgments
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We would like to thank Dr A.Horii (Tohoku University School of Medicine) for providing E90T and E90N tumor DNA and Drs M.Perucho and H.Yamamoto (La Jolla Cancer Research Center, CA) for providing DU145, LS411, AN3CA and LS180. K.G. would like to express his gratitude to Prof. T.Hayakawa (Nagoya University School of Medicine) for his encouragement throughout the study. This work was partly supported by the Bristol-Meyers Squibb Biomedical Research Grant Program and a Grant-in-Aid (09671403) from the Ministry of Education, Science, Sports and Culture of Japan.
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Notes
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6 To whom correspondence should be addressed Email: mitsudom{at}leo.bekkoame.or.jp 
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References
|
---|
-
Ionov,Y., Peinado,M.A., Malkhosyan,S., Shibata,D. and Perucho,M. (1993) Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature, 363, 558561.[ISI][Medline]
-
Marra,G. and Boland,C.R. (1995) Hereditary nonpolyposis colorectal cancer: the syndrome, the genes and historical perspectives. J. Natl Cancer Inst., 87, 11141125.[Abstract]
-
Markowitz,S., Wang,J., Myeroff,L. et al. (1995) Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability [see comments]. Science, 268, 13361338.[ISI][Medline]
-
Ouyang,H., Shiwaku,H.O., Hagiwara,H. et al. (1997) The insulin-like growth factor II receptor gene is mutated in genetically unstable cancers of the endometrium, stomach and colorectum. Cancer Res., 57, 18511854.[Abstract]
-
Rampino,N., Yamamoto,H., Ionov,Y., Li,Y., Sawai,H., Reed,J.C. and Perucho,M. (1997) Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science, 275, 967969.[Abstract/Free Full Text]
-
Yamamoto,H., Sawai,H. and Perucho,M. (1997) Frameshift somatic mutations in gastrointestinal cancer of the microsatellite mutator phenotype. Cancer Res., 57, 44204426.[Abstract]
-
Malkhosyan,S., Rampino,N., Yamamoto,H. and Perucho,M. (1996) Frameshift mutator mutations [letter]. Nature, 382, 499500.[ISI][Medline]
-
Adachi,J., Shiseki,M., Okazaki,T., Ishimaru,G., Noguchi,M., Hirohashi,S. and Yokota,J. (1995) Microsatellite instability in primary and metastatic lung carcinomas. Genes Chromosomes Cancer, 14, 301306.[ISI][Medline]
-
Pifarre,A., Rosell,R., Monzo,M., De Anta,J.M., Moreno,I., Sanchez,J.J., Ariza,A., Mate,J.L., Martinez,E. and Sanchez,M. (1997) Prognostic value of replication errors on chromosomes 2p and 3p in non-small-cell lung cancer. Br. J. Cancer, 75, 184189.[ISI][Medline]
-
Takenoshita,S., Hagiwara,K., Gemma,A., Nagashima,M., Ryberg,D., Lindstedt,B.A., Bennett,W.P., Haugen,A. and Harris,C.C. (1997) Absence of mutations in the transforming growth factor-beta type II receptor in sporadic lung cancers with microsatellite instability and rare H-ras1 alleles. Carcinogenesis, 18, 14271429.[Abstract]
-
Tani,M., Takenoshita,S., Kohno,T., Hagiwara,K., Nagamachi,Y., Harris,C.C. and Yokota,J. (1997) Infrequent mutations of the transforming growth factor beta-type II receptor gene at chromosome 3p22 in human lung cancers with chromosome 3p deletions. Carcinogenesis, 18, 11191121.[Abstract]
-
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
-
Hoang,J.M., Cottu,P.H., Thuille,B., Salmon,R.J., Thomas,G. and Hamelin,R. (1997) BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines. Cancer Res., 57, 300303.[Abstract]
-
Dietmaier,W., Wallinger,S., Bocker,T., Kullmann,F., Fishel,R. and Ruschoff,J. (1997) Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res., 57, 47494756.[Abstract]
-
Peltomäki,P., Lothe,R.A., Aaltonen,L.A. et al. (1993) Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome. Cancer Res., 53, 58535855.[Abstract]
-
Fong,K.M., Zimmerman,P.V. and Smith,P.J. (1995) Microsatellite instability and other molecular abnormalities in non-small cell lung cancer. Cancer Res., 55, 2830.[Abstract]
-
Mao,L., Lee,D.J., Tockman,M.S., Erozan,Y.S., Askin,F. and Sidransky,D. (1994) Microsatellite alterations as clonal markers for the detection of human cancer. Proc. Natl Acad. Sci. USA, 91, 98719875.[Abstract/Free Full Text]
-
Shridhar,V., Siegfried,J., Hunt,J., del Mar Alonso,M. and Smith,D.I. (1994) Genetic instability of microsatellite sequences in many non-small cell lung carcinomas. Cancer Res., 54, 20842087.[Abstract]
-
Merlo,A., Mabry,M., Gabrielson,E., Vollmer,R., Baylin,S.B. and Sidransky,D. (1994) Frequent microsatellite instability in primary small cell lung cancer. Cancer Res., 54, 20982101.[Abstract]
-
Sekine,I., Yokose,T., Ogura,T., Suzuki,K., Nagai,K., Kodama,T., Mukai,K., Nishiwaki,Y. and Esumi,H. (1997) Microsatellite instability in lung cancer patients 40 years of age or younger. Jpn. J. Cancer Res., 88, 559563.[ISI][Medline]
-
Lynch,H.T. and Smyrk,T. (1996) Hereditary nonpolyposis colorectal cancer (Lynch syndrome). An updated review. Cancer, 78, 11491167.[ISI][Medline]
-
Watson,P. and Lynch,H.T. (1993) Extracolonic cancer in hereditary nonpolyposis colorectal cancer. Cancer, 71, 677685.[ISI][Medline]
-
Minna,J.D., Sekido,Y., Fong,K.M. and Gazdar,A.F. (1997) Cancer of the lung. In DeVita,V.T.Jr, Hellman,S. and Rosenberg,S.A. (eds) Cancer: Principles and Practice of Oncology, 5th Edn. Lippincott-Raven, Philadelphia, PA, Vol. 1, pp. 849857.
-
Lengauer,C., Kinzler,K.W. and Vogelstein,B. (1997) Genetic instability in colorectal cancers. Nature, 386, 623627.[ISI][Medline]
-
Meling,G.I., Lothe,R.A., Borresen,A.L., Graue,C., Hauge,S., Clausen,O.P. and Rognum,T.O. (1993) The TP53 tumour suppressor gene in colorectal carcinomas. II. Relation to DNA ploidy pattern and clinicopathological variables. Br. J. Cancer, 67, 9398.[ISI][Medline]
-
Schlegel,J., Stumm,G., Scherthan,H., Bocker,T., Zirngibl,H., Ruschoff,J. and Hofstadter,F. (1995) Comparative genomic in situ hybridization of colon carcinomas with replication error. Cancer Res., 55, 60026005.[Abstract]
-
Kim,H., Jen,J., Vogelstein,B. and Hamilton,S.R. (1994) Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences. Am. J. Pathol., 145, 148156.[Abstract]
-
Konishi,M., Kikuchi-Yanoshita,R., Tanaka,K. et al. (1996) Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis and sporadic colon cancer [see comments]. Gastroenterology, 111, 307317.[ISI][Medline]
-
Cahill,D.P., Lengauer,C., Yu,J., Riggins,G.J., Willson,J.K., Markowitz,S.D., Kinzler,K.W. and Vogelstein,B. (1998) Mutations of mitotic checkpoint genes in human cancers [see comments]. Nature, 392, 300303.[ISI][Medline]
-
Parsons,R., Myeroff,L.L., Liu,B., Willson,J.K., Markowitz,S.D., Kinzler,K.W. and Vogelstein,B. (1995) Microsatellite instability and mutations of the transforming growth factor beta type II receptor gene in colorectal cancer. Cancer Res., 55, 55485550.[Abstract]
-
Myeroff,L.L., Parsons,R., Kim,S.J. et al. (1995) A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability. Cancer Res., 55, 55455547.[Abstract]
Received July 27, 1998;
revised October 14, 1998;
accepted October 29, 1998.