- Email: [email protected]
A Single-Nucleotide Polymorphism of SMARCB1 in Human Breast Cancers
Article
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
A Single-Nucleotide Polymorphism of SMARCB1 in Human Breast Cancers Koshi Mimori,1 Hiroshi Inoue,1 Takeshi Shiraishi,1 Hiroaki Ueo,2 Ken-ichi Mafune,3 Yoichi Tanaka,3 and Masaki Mori1,* 1
Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan 2 Department of Surgery, Oita Prefectural Hospital, Oita, Japan 3 Department of Surgery, Saitama Cancer Center, Saitama, Japan
*To whom correspondence and reprint requests should be addressed. Fax: +81-977-27-1651. E-mail: [email protected].
The gene SMARCB1 has been considered a candidate for a tumor-suppressor gene. Nucleotide alterations in SMARCB1 have been reported, primarily in association with malignant rhabdoid tumor cases. We carried out a search for mutations in SMARCB1 in 60 human gastro-intestinal tract carcinoma cases, 122 breast cancer cases, and 36 human cancer cell lines. A single-nucleotide polymorphism (SNP) at codon 152 with an amino acid change (Asn to Asp) was found in 2 of 122 (1.6%) breast cancer cases, and another SNP at codon 299 without an amino acid change was found in tumor and normal tissues from 7 (5.7%) cases. Codons 152 and 299 of SMARCB1 are localized near or within the binding site for the cMYC protein. The amount of immunoprecipitated cMYC protein was reduced in two different cell lines expressing the codon 152 polymorphic SMARCB1 clone compared with those expressing wild-type SMARCB1, regardless of the identical expression of SMARCB1 protein in both cell lines. Therefore, the SNP at codon 152 is considered to be one of the coding SNPs that alters the SMARCB1–cMYC complex, which regulates various tumor-suppressor related genes against cancer. In addition, we identified three types of splicing isoforms, a 27-bp deleted gene, a 51-bp inserted gene, and a consensus gene, in both carcinoma tissues and in normal tissues; however, no clinical significance was observed for those isoforms. We found a nucleotide change at codon 152 of SMARCB1 that may alter the amount of immunoprecipitated cMYC protein, but we finally determined that SMARCB1 is highly conserved in human solid carcinomas. Key Words: SMARCB1, cMYC, polymorphism, breast cancer, gastro-intestinal tract cancer, splicing isoform
INTRODUCTION SMARCB1 (also known as hSNF5 and INI1) is located at chromosomal position 22q.11.2, and several previous studies have reported alterations within the 22q.11 region [1–3]. A gene in this region has intrigued investigators for years as a possible tumor-suppressor gene candidate. Versteege et al. reported that somatic alterations, such as nucleotide deletions, insertions, and a nonsense mutation in SMARCB1, are associated with malignant rhabdoid tumor (MRT) cases [4–8]. Their report was fully consistent with the paradigm of the two-hit theory of oncogenesis, and supports the hypothesis that SMARCB1 is a tumor-suppressor gene.
254
Considering the important role of SMARCB1 in chromatin remodeling during cell growth [9,10], abnormalities in this gene would be expected to occur frequently in carcinomas. Alterations of SMARCB1 have been reported mostly in association with rhabdoid tumors in brain and kidney, and in leukemia. Furthermore, there has been no definitive search for genetic alterations of SMARCB1 in gastro-intestinal (G-I) tract or breast carcinoma cases. Here, sequences of the entire SMARCB1 gene (Fig. 1) in 182 human solid cancers and in 36 other human carcinoma cell lines were examined to explore the possibility that SMARCB1 may be a universal tumor-suppressor gene for a wide range of human carcinomas.
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved. 0888-7543/02 $35.00
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
Article FIG. 1. cDNA and genomic DNA of SMARCB1. (Top) Schematic of SMARCB1 showing nine exons and four amplified segments. (Bottom) Genomic structure of SMARCB1. Each exon was digested with the SmaI enzyme. The primer for each exon was located within the flanking regions. There were two splicing isoforms: one was a short form in exon 2 (EX2S), and the other was a long isoform in exon 4 (EX4L).
RESULTS Nucleotide Changes in SMARCB1 from Both cDNA and Genomic DNA A search was carried out for mutations in 218 clinical and cell line samples. A single-nucleotide change was found at the first nucleotide of codon 152 with an amino acid change in 2 of 122 breast cancer cases. On the other hand, a change in the third nucleotide of codon 299, which would not result in an amino acid change, was found in seven samples from breast carcinoma cases (Fig. 2). The identical region in genomic DNA, from both the carcinoma and the corresponding normal sample, was sequenced directly, and the results confirmed that those nucleotide changes were observed in each carcinoma tissue as well as in the corresponding normal tissue. According to the LOH study, 4 cases of all 122 breast cancer cases showed LOH at the marker located near SMARCB1. In those four cases with LOH, one case showed the polymorphic change at codon 299 in genomic DNA extracted from tumor tissue (data not shown); however, the corresponding normal tissue from the case also showed the identical alteration at codon 299. On the other hand, two cases of codon 152 alteration showed no LOH in the genetic region of SMARCB1. Therefore, those alterations are considered to be polymorphic changes, not mutations. In addition, the coding SNP (cSNP) [11] at codon 152 observed in two breast cancer cases and the silent SNP (sSNP) at codon 299 in seven breast cancer cases showed no significant correlations with any clinicopathologic features and prognosis. DNA samples from esophageal carcinoma cell lines showed several point mutations in introns of SMARCB1 (data not shown), whereas there were no nucleotide changes in DNA from G-I tract carcinoma samples. We found two sequence errors in the original paper by Kalpana et al. [12]. Nucleotides 416 and 1145 were read as TCC (Ser) and GCC (Gly), respectively, in the original paper,
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
whereas we found them to be CCC (Pro) and GCC (Ala) in these positions. The Amount of Immunoprecipitated cMYC Protein We confirmed that identical amounts of SMARCB1 protein were expressed in the transfectant clone with the SMARCB1 polymorphic change at codon 152 and in the clone with wildtype SMARCB1. In MCF7 cells, we found immunoprecipitated cMYC protein at a reduced level in cells with the polymorphic clone compared with cells with the wild-type clone (Fig. 3A). This result indicated that the coding nucleotide change at codon 152 may in fact have altered the binding affinity of SMARCB1 for cMYC protein. Reduced expression of the immunoprecipitated cMYC protein in cells with the polymorphic SMARCB1 clone was also confirmed in KYSE150 cells (Fig. 3B). Cloning of Two Splicing Isoforms of SMARCB1 SMARCB1 consists of nine exons containing 1857 bp (1158 bp coding). Transcripts of SMARCB1, including a short form of the aberrant gene isolated from an esophageal carcinoma sample, were obtained. We isolated 49 colonies corresponding to transformants of the PCR product amplified from fetal brain cDNA, and 20 colonies were obtained in a similar manner from testis cDNA. In 49 colonies of fetal brain cDNA, the DNA sequences of 40 (81.6%) colonies were in complete agreement with the previous study [12]. Seven (14.3%) of the colonies had a short form of the consensus sequence deleted for 27 bp (at exon 2S), which was reported by Kalpana et al. [12] (Figs. 4 and 5). A 51-bp insertion (in exon 4L) was added to the 3⬘ end of exon 4 of the SMARCB1 cDNA in the remaining two colonies (4.1%). Expression of the corresponding long sequence of SMARCB1 cDNA was observed in fetal brain, fetal liver, human adult testis, and human adult prostate.
255
Article
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
FIG. 2. A silent mutation at codon 152 in a representative breast cancer case is indicated by a red arrow. This nucleotide change was found in 2 of 122 (1.6%) breast carcinoma cases. The corresponding sequence in samples derived from normal tissue was also positive for the change.
We directly sequenced the RT-PCR products with primer sets A and B to determine the incidence and clinical significance of both isoforms in the representative 145 (20 esophageal, 20 gastric, 20 colon, and 85 breast) cancer cases; however, we found that they carried none of the isoforms. The consensus sequences are highly conserved in all human cancers.
First, we have to determine the magnitude of the SNP at codon 152 of SMARCB1 in breast cancer. The nucleotide (amino acid 152) located near the repeat 1 fragment (amino acids 186–245) is the actual binding site for cMYC, and SMARCB1 protein is necessary for transactivation mediated by cMYC protein [13]. A reduction of cMYC transcription was seen when amino acids 146–181 of SMARCB1 were eliminated [13]. In our present study, the clone polymorphic for codon 152 reduced the amount of immunoprecipitated cMYC protein. As codon 152 is located within amino acids 146–181, the polymorphic change should alter the binding affinity for cMYC and affect the activity of cMYC transcription. Once the cMYC–SMARCB1 interaction or the cMYC-mediated transactivation is damaged, the chromatin remodeling systems would be disrupted, which may promote the malignant process because the SWI/SNF complex contributes to the regulation of gene expression by altering the chromatin structure, and this complex has been considered to inhibit several oncogenes and activate tumor-suppressor genes and/or control their transcriptional activity [14]. Therefore, the complex between cMYC and SMARCB1 regulates transactivations of genes that may have critical roles in the oncogenesis or progression of carcinoma, especially in breast cancer. We expect
A
DISCUSSION In 122 breast cancer cases, we observed nucleotide changes at codon 152 in 2 cases, and at codon 299 in 7 cases in tumor samples as well as corresponding normal samples. There was no correlation between the presence of LOH and the nucleotide change in those cases (except one case with a codon 299 alteration), but it is interesting that among various human solid cancers, nine cases with polymorphism were observed only in breast cancer. Therefore, we have focused on the role of the polymorphism at codon 152 in oncogenesis or progression of human breast cancer because the change accompanies an amino acid change.
FIG. 3. Reduced immunoprecipitated cMYC protein with polymorphic SMARCB1. (A) Immunoprecipitation of cMYC protein in the SMARCB1 mutant clone and the wild-type clone. The NIH Image program calculated intensities of signals. The amount of the immunoprecipitated cMYC protein was normalized by the expression of SMARCB1 protein by western blot. MCF7 showed the reduced amount of the immunoprecipitated amount of cMYC protein in the mutant SMARCB1 clone compared with the wild-type clone. (B) KYSE150 also showed similar result. (C) Comparison of the amount of immunoprecipitated cMYC protein between SMARCB1 wild-type and SMARCB1 mutant clones at codon 152. The amount of the immunoprecipitated cMYC protein in each parent cell was defined as the control (1.0). The wildtype SMARCB1 clone showed a more than 2.5 times higher amount of the cMYC protein in both cell lines, whereas the mutant clone showed less than half of the amount of cMYC protein of both parental cell lines.
256
B
C
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
Article
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
FIG. 4. This schematic shows three isoforms of SMARCB1. (Top) The sequences of 40 of 49 (81.6%) colonies of fetal brain cDNA library showed complete agreement with a previous study [12]. (Middle) Seven of 49 (14.3%) colonies had a 27 bp deleted (in exon 2S) short form of the consensus sequence. (Bottom) A 51-bp long form had an addition to the 3⬘ end of exon 4 (in exon 4L) in the SMARCB1 cDNA. This change was noted in two of the remaining colonies (4.1%).
that the cSNP at codon 152 will have a key role in the cure of breast cancer patients. In the library of fetal brain cDNA, the proportion of the DNA corresponding to the long isoform, which had a 51-bp insertion (4.1%), was lower than that of the short form, which had a deletion of 27 bp (14.3%). A BLAST search detected no “hits” corresponding to proteins with sequences that aligned well with either the long or short isoforms. The location of both isoforms was not correlated with cMYC binding sites described above. In addition, both the short isoform and the long isoform were not observed in any of the representative 145 G-I tract carcinoma samples including breast cancers as examined by RT-PCR. We found a reduction of the amount of immunoprecipitated cMYC in the polymorphic clone in breast cancer. Further study will be required to clarify the actual role of the polymorphism in the progression of breast cancer. However, the sequence of SMARCB1 is highly conserved in human G-I tract carcinoma, and we conclude that SMARCB1 alterations occur primarily in association with the rhabdoid carcinomas, as reported in previous papers.
MATERIALS AND METHODS cDNA preparation for mutation search. cDNAs were generated from the following primary carcinoma cases: 20 esophageal carcinoma cases, 20 gastric carcinoma cases, 20 colorectal carcinomas, and 122 breast carcinoma cases. cDNAs were also prepared from 36 carcinoma cell lines: TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE12, KYSE140, KYSE170, KYSE180, KYSE300, KYSE450, and KYSE700 (14 esophageal carcinomas); KATOIII, MKN1, MKN28, MKN45, MKN74, NVGC3, NS8, SCH, SW1, SW5, SW16, and RF (11 gastric carcinomas); LNCaP, DU145, and PC-3 (3 prostate carcinomas); NCI-H82 and NCI-H446 (2 lung carcinomas); PA-1 and SK-OV (2 ovarian carcinomas); 769P and Caki-1 (2 kidney carcinomas); HeLa (cervical carcinoma); and RD (rhabdomyosarcoma). Sequencing of SMARCB1 and the SMARCB1 genomic region. Total RNA was purified from tumor lines, and cDNA was generated from RNA described elsewhere [15]. PCR amplification was performed in a 30 l reaction mixture containing 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3 (20⬚C), 0.2 M each of dATP, dGTP, dCTP, and dTTP, 100 ng of each primer, and 2 units of Taq DNA polymerase (Boehringer). Figure 1 showed the four primer sets for SMARCB1 amplification. PCR reactions with primer set A (F, 5⬘-GCCGCAATGATGATGATGGC-3⬘; R, 5⬘-GTTCCTCTTGGCCTTCTGTTC-3⬘) for exons 1, 2, and 3, and with set B (F, 5⬘-GATCACGGATACACGACTCTAG-3⬘; R, 5⬘-CATTCATGTTCCAGGTGAAGG-3⬘) for exons 3, 4, and 5 were carried out following an initial denaturation at 95⬚C for 5 minutes, 40 cycles of 95⬚C for 15 seconds, 57⬚C for 1 minute. PCR reactions with primer set C (F, 5⬘-CTTTGATGACCATGACCCAGC-3⬘; R, 5⬘-TGCGATGGTGGTGA-
FIG. 5. DNA sequences of the altered regions. (Top) The sequence of the 27-bp-deleted short form was determined from nucleotides 206 to 232. (Bottom) The sequences of the 51-bp long form that corresponded to an insertion between codons 500 and 501.
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
257
Article
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
CAAACTC-3⬘) for exons 5, 6, and 7 were performed by an initial denaturation at 95⬚C for 5 minutes followed by 40 cycles of 95⬚C for 15 seconds, 60⬚C for 30 seconds, and 72⬚C for 1 minute. PCR reactions with primer set D (F, 5⬘-CATCAGACAGCAGATCGAGTC-3⬘; R, 5⬘-CAACAAATGGAATGTGTACCGG-3⬘) for exons 7, 8, and 9 were performed by an initial denaturation at 95⬚C for 5 minutes followed by 40 cycles of 95⬚C for 15 seconds, 54⬚C for 30 seconds, and 72⬚C for 1 minute on a Perkin-Elmer/Cetus DNA Thermal Cycler 9600 (Perkin Elmer, CA). To confirm alterations of bases on cDNA, we also examined the sequence of the identical position on genomic DNA, which was detected by both directions of primers on cDNA. PCR reactions and sequencing were performed using di-deoxy-terminator reaction chemistry on a Perkin-Elmer/Cetus DNA Thermal Cycler 9600 and the Applied Biosystems Model 377 DNA sequencing systems. The faint amplified products were subcloned by TOPO TA Cloning kit (Invitrogen, CA), and then sequencing was carried out. The expression of SMARCB1 was observed in all four segments, and sequencing of all amplified PCR products was carried out, then nucleotide sequences of each region were aligned by the Sequencher 3.0 (Gene Codes Corporation). Furthermore, we examined whether both or one allele was lost or retained at the locus near SMARCB1 in 122 breast cancer cases as follows. The Gene Scan program was applied according to our previous studies [16,17]. We amplified the D22S345 marker for LOH study between primers 5⬘-CACTTCTGAGTTGTTTGAATCTC-3⬘ and 5⬘-AGAATCTGCTGCTTGCTTTT-3⬘, and the amplified products were electrophoresed and analyzed by an ABI377 sequencer (Applied Biosystems). The latter primer was fluorolabeled by 6-FAM fluoroamidide. Mutagenesis study. For evaluation of the significance of mutation of SMARCB1 at codon 152 from AAC (Asn) to GAC (Asp) without an amino acid change, we obtained the mutant clone by the Site-Directed Mutagenesis system (Gibco BRL). The primer for the target the codon 152 was 5⬘-GCTCCACAACCATCGACAGGAACCGCATGG-3⬘ instead of the normal consensus sequence, 5⬘-TTAGATGCCGTGCCATGCTCCACAACCATCAAC (codon152) AGGAACCGCATGGGCCGAGACAAGAAG-3⬘. The primer for clone selection was 5⬘-CTGAGAGTGCACCATGGGCGGTGTGAAATACC-3⬘, with a NcoI site at nucleotide 12. We obtained 10 clones of mutant plasmids, then selected two mutant plasmids with SMARCB1 expression to be transfected into MCF7, a breast cancer cell line, and KYSE150, an esophageal cancer cell line. Immunoprecipitation with SMARCB1 and cMYC protein. The wild-type SMARCB1 clone with consensus sequence and a SMARCB1 mutant clone at the codon 152 polymorphism were obtained and ligated into the expression vector pcDNA 3.1 (Invitrogen). Both clones, wild-type SMARCB1 and a mutant clone, were transfected into MCF7 as well as KYSE150. The expression of SMARCB1 protein was confirmed by western blot analysis with SMARCB1/Snf5 polyclonal antibody (SMARCB1 sc-9749; Santa Cruz). Four clones were immunoprecipitated with cMYC antibody (Santa Cruz) to compare the amounts of immunoprecipitated cMYC protein. Cloning of the SMARCB1 transcript. We prepared the full length of the SMARCB1 cDNA, whose sequence was confirmed by the GenBank accession number U04847 (1857 bp). Initially, the wild-type SMARCB1 cDNA was amplified by the PCR primer upper primer, 5⬘-GCCGCAATGATGATGGCGCTGAGCAAGAC-3⬘, and lower primer, 5⬘-ATCTTCTGAGATGCTC-
258
CGTGGGAGCCGTGT-3⬘. We purified and cloned the amplified products using the TOPO cloning kit (Invitrogen) using the manufacturer’s protocol. We have performed subcloning of the SMARCB1 PCR products and obtained 52 isolated mono-clones. Then, those clones were sequenced. [12]. We examined the expression of SMARCB1 in 14 representative carcinoma cell lines: TE1, TE3, TE4, TE6, KYSE110, KYSE170, KYSE180, KYSE270, KYSE700, KATOIII, MKN7, MKN45, and COLO201. Sixty cases of tumor tissue and the corresponding normal tissue were also examined by RT-PCR with the above primers.
ACKNOWLEDGMENTS We thank T. Shimo-oka and J. Miyake for technical support. RECEIVED FOR PUBLICATION FEBRUARY 20; ACCEPTED JUNE 2, 2002.
REFERENCES 1. Rey, J. A., et al. (1993). Abnormalities of chromosome 22 in human brain tumors determined by combined cytogenetic and molecular genetic approaches. Cancer Genet. Cytogenet. 66: 1–10. 2. Schofield, D. E., Beckwith, J. B., and Sklar, J. (1996). Loss of heterozygosity at chromosome regions 22q11-12 and 11p15.5 in renal rhabdoid tumors. Genes Chromosomes Cancer 15: 10–17. 3. Shin, E., et al. (1993). Deletion mapping of chromosome 1p and 22q in pheochromocytoma. Jpn. J. Cancer Res. 84: 402–408. 4. Biegel, J. A., et al. (1999). Germ-line and acquired mutations of INI1 in atypical teratoid and rhabdoid tumors. Cancer Res. 59: 74–79. 5. Savla, J., et al. (2000). Mutations of the hSNF5/INI1 gene in renal rhabdoid tumors with second primary brain tumors. J. Natl. Cancer Inst. 92: 648–650. 6. Sevenet, N., et al. (1999). Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotype-phenotype correlations. Hum. Mol. Genet. 8: 2359–2368. 7. Sevenet, N., et al. (1999). Constitutional mutations of the hSNF5/INI1 gene predispose to a variety of cancers. Am. J. Hum. Genet. 65: 1342–1348. 8. Versteege, I., et al. (1998). Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 394: 203–206. 9. Wang, W., et al. (1996). Purification and biochemical heterogeneity of the mammalian SWI-SNF complex. EMBO J. 15: 5370–5382. 10. Wang, W., et al. (1996). Diversity and specialization of mammalian SWI/SNF complexes. Genes Dev. 10: 2117–2130. 11. Ponomarenko, J. V., et al. (2001). rSNP_Guide, a database system for analysis of transcription factor binding to target sequences: application to SNPs and site-directed mutations. Nucleic Acids Res. 29: 312–316. 12. Kalpana, G. V., Marmon, S., Wang, W., Crabtree, G. R., and Goff, S. P. (1994). Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5. Science 266: 2002–2006. 13. Cheng, S. W., et al. (1999). c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function. Nat. Genet. 22: 102–105. 14. Klochendler-Yeivin, A., Muchardt, C., and Yaniv, M. (2002). SWI/SNF chromatin remodeling and cancer. Curr. Opin. Genet. Dev. 12: 73–79. 15. Mori, M., et al. (1998). Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription-polymerase chain reaction in patients with gastrointestinal or breast carcinomas. J. Clin. Oncol. 16: 128–132. 16. Mori, M., et al. (2001). Absence of Msh2 protein expression is associated with alteration in the FHIT locus and Fhit protein expression in colorectal carcinoma. Cancer Res. 61: 7379–382. 17. Mori, M., et al. (2000). Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus. Cancer Res. 60: 1177–1182.
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
A Single-Nucleotide Polymorphism of SMARCB1 in Human Breast Cancers Koshi Mimori,1 Hiroshi Inoue,1 Takeshi Shiraishi,1 Hiroaki Ueo,2 Ken-ichi Mafune,3 Yoichi Tanaka,3 and Masaki Mori1,* 1
Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan 2 Department of Surgery, Oita Prefectural Hospital, Oita, Japan 3 Department of Surgery, Saitama Cancer Center, Saitama, Japan
*To whom correspondence and reprint requests should be addressed. Fax: +81-977-27-1651. E-mail: [email protected].
The gene SMARCB1 has been considered a candidate for a tumor-suppressor gene. Nucleotide alterations in SMARCB1 have been reported, primarily in association with malignant rhabdoid tumor cases. We carried out a search for mutations in SMARCB1 in 60 human gastro-intestinal tract carcinoma cases, 122 breast cancer cases, and 36 human cancer cell lines. A single-nucleotide polymorphism (SNP) at codon 152 with an amino acid change (Asn to Asp) was found in 2 of 122 (1.6%) breast cancer cases, and another SNP at codon 299 without an amino acid change was found in tumor and normal tissues from 7 (5.7%) cases. Codons 152 and 299 of SMARCB1 are localized near or within the binding site for the cMYC protein. The amount of immunoprecipitated cMYC protein was reduced in two different cell lines expressing the codon 152 polymorphic SMARCB1 clone compared with those expressing wild-type SMARCB1, regardless of the identical expression of SMARCB1 protein in both cell lines. Therefore, the SNP at codon 152 is considered to be one of the coding SNPs that alters the SMARCB1–cMYC complex, which regulates various tumor-suppressor related genes against cancer. In addition, we identified three types of splicing isoforms, a 27-bp deleted gene, a 51-bp inserted gene, and a consensus gene, in both carcinoma tissues and in normal tissues; however, no clinical significance was observed for those isoforms. We found a nucleotide change at codon 152 of SMARCB1 that may alter the amount of immunoprecipitated cMYC protein, but we finally determined that SMARCB1 is highly conserved in human solid carcinomas. Key Words: SMARCB1, cMYC, polymorphism, breast cancer, gastro-intestinal tract cancer, splicing isoform
INTRODUCTION SMARCB1 (also known as hSNF5 and INI1) is located at chromosomal position 22q.11.2, and several previous studies have reported alterations within the 22q.11 region [1–3]. A gene in this region has intrigued investigators for years as a possible tumor-suppressor gene candidate. Versteege et al. reported that somatic alterations, such as nucleotide deletions, insertions, and a nonsense mutation in SMARCB1, are associated with malignant rhabdoid tumor (MRT) cases [4–8]. Their report was fully consistent with the paradigm of the two-hit theory of oncogenesis, and supports the hypothesis that SMARCB1 is a tumor-suppressor gene.
254
Considering the important role of SMARCB1 in chromatin remodeling during cell growth [9,10], abnormalities in this gene would be expected to occur frequently in carcinomas. Alterations of SMARCB1 have been reported mostly in association with rhabdoid tumors in brain and kidney, and in leukemia. Furthermore, there has been no definitive search for genetic alterations of SMARCB1 in gastro-intestinal (G-I) tract or breast carcinoma cases. Here, sequences of the entire SMARCB1 gene (Fig. 1) in 182 human solid cancers and in 36 other human carcinoma cell lines were examined to explore the possibility that SMARCB1 may be a universal tumor-suppressor gene for a wide range of human carcinomas.
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved. 0888-7543/02 $35.00
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
Article FIG. 1. cDNA and genomic DNA of SMARCB1. (Top) Schematic of SMARCB1 showing nine exons and four amplified segments. (Bottom) Genomic structure of SMARCB1. Each exon was digested with the SmaI enzyme. The primer for each exon was located within the flanking regions. There were two splicing isoforms: one was a short form in exon 2 (EX2S), and the other was a long isoform in exon 4 (EX4L).
RESULTS Nucleotide Changes in SMARCB1 from Both cDNA and Genomic DNA A search was carried out for mutations in 218 clinical and cell line samples. A single-nucleotide change was found at the first nucleotide of codon 152 with an amino acid change in 2 of 122 breast cancer cases. On the other hand, a change in the third nucleotide of codon 299, which would not result in an amino acid change, was found in seven samples from breast carcinoma cases (Fig. 2). The identical region in genomic DNA, from both the carcinoma and the corresponding normal sample, was sequenced directly, and the results confirmed that those nucleotide changes were observed in each carcinoma tissue as well as in the corresponding normal tissue. According to the LOH study, 4 cases of all 122 breast cancer cases showed LOH at the marker located near SMARCB1. In those four cases with LOH, one case showed the polymorphic change at codon 299 in genomic DNA extracted from tumor tissue (data not shown); however, the corresponding normal tissue from the case also showed the identical alteration at codon 299. On the other hand, two cases of codon 152 alteration showed no LOH in the genetic region of SMARCB1. Therefore, those alterations are considered to be polymorphic changes, not mutations. In addition, the coding SNP (cSNP) [11] at codon 152 observed in two breast cancer cases and the silent SNP (sSNP) at codon 299 in seven breast cancer cases showed no significant correlations with any clinicopathologic features and prognosis. DNA samples from esophageal carcinoma cell lines showed several point mutations in introns of SMARCB1 (data not shown), whereas there were no nucleotide changes in DNA from G-I tract carcinoma samples. We found two sequence errors in the original paper by Kalpana et al. [12]. Nucleotides 416 and 1145 were read as TCC (Ser) and GCC (Gly), respectively, in the original paper,
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
whereas we found them to be CCC (Pro) and GCC (Ala) in these positions. The Amount of Immunoprecipitated cMYC Protein We confirmed that identical amounts of SMARCB1 protein were expressed in the transfectant clone with the SMARCB1 polymorphic change at codon 152 and in the clone with wildtype SMARCB1. In MCF7 cells, we found immunoprecipitated cMYC protein at a reduced level in cells with the polymorphic clone compared with cells with the wild-type clone (Fig. 3A). This result indicated that the coding nucleotide change at codon 152 may in fact have altered the binding affinity of SMARCB1 for cMYC protein. Reduced expression of the immunoprecipitated cMYC protein in cells with the polymorphic SMARCB1 clone was also confirmed in KYSE150 cells (Fig. 3B). Cloning of Two Splicing Isoforms of SMARCB1 SMARCB1 consists of nine exons containing 1857 bp (1158 bp coding). Transcripts of SMARCB1, including a short form of the aberrant gene isolated from an esophageal carcinoma sample, were obtained. We isolated 49 colonies corresponding to transformants of the PCR product amplified from fetal brain cDNA, and 20 colonies were obtained in a similar manner from testis cDNA. In 49 colonies of fetal brain cDNA, the DNA sequences of 40 (81.6%) colonies were in complete agreement with the previous study [12]. Seven (14.3%) of the colonies had a short form of the consensus sequence deleted for 27 bp (at exon 2S), which was reported by Kalpana et al. [12] (Figs. 4 and 5). A 51-bp insertion (in exon 4L) was added to the 3⬘ end of exon 4 of the SMARCB1 cDNA in the remaining two colonies (4.1%). Expression of the corresponding long sequence of SMARCB1 cDNA was observed in fetal brain, fetal liver, human adult testis, and human adult prostate.
255
Article
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
FIG. 2. A silent mutation at codon 152 in a representative breast cancer case is indicated by a red arrow. This nucleotide change was found in 2 of 122 (1.6%) breast carcinoma cases. The corresponding sequence in samples derived from normal tissue was also positive for the change.
We directly sequenced the RT-PCR products with primer sets A and B to determine the incidence and clinical significance of both isoforms in the representative 145 (20 esophageal, 20 gastric, 20 colon, and 85 breast) cancer cases; however, we found that they carried none of the isoforms. The consensus sequences are highly conserved in all human cancers.
First, we have to determine the magnitude of the SNP at codon 152 of SMARCB1 in breast cancer. The nucleotide (amino acid 152) located near the repeat 1 fragment (amino acids 186–245) is the actual binding site for cMYC, and SMARCB1 protein is necessary for transactivation mediated by cMYC protein [13]. A reduction of cMYC transcription was seen when amino acids 146–181 of SMARCB1 were eliminated [13]. In our present study, the clone polymorphic for codon 152 reduced the amount of immunoprecipitated cMYC protein. As codon 152 is located within amino acids 146–181, the polymorphic change should alter the binding affinity for cMYC and affect the activity of cMYC transcription. Once the cMYC–SMARCB1 interaction or the cMYC-mediated transactivation is damaged, the chromatin remodeling systems would be disrupted, which may promote the malignant process because the SWI/SNF complex contributes to the regulation of gene expression by altering the chromatin structure, and this complex has been considered to inhibit several oncogenes and activate tumor-suppressor genes and/or control their transcriptional activity [14]. Therefore, the complex between cMYC and SMARCB1 regulates transactivations of genes that may have critical roles in the oncogenesis or progression of carcinoma, especially in breast cancer. We expect
A
DISCUSSION In 122 breast cancer cases, we observed nucleotide changes at codon 152 in 2 cases, and at codon 299 in 7 cases in tumor samples as well as corresponding normal samples. There was no correlation between the presence of LOH and the nucleotide change in those cases (except one case with a codon 299 alteration), but it is interesting that among various human solid cancers, nine cases with polymorphism were observed only in breast cancer. Therefore, we have focused on the role of the polymorphism at codon 152 in oncogenesis or progression of human breast cancer because the change accompanies an amino acid change.
FIG. 3. Reduced immunoprecipitated cMYC protein with polymorphic SMARCB1. (A) Immunoprecipitation of cMYC protein in the SMARCB1 mutant clone and the wild-type clone. The NIH Image program calculated intensities of signals. The amount of the immunoprecipitated cMYC protein was normalized by the expression of SMARCB1 protein by western blot. MCF7 showed the reduced amount of the immunoprecipitated amount of cMYC protein in the mutant SMARCB1 clone compared with the wild-type clone. (B) KYSE150 also showed similar result. (C) Comparison of the amount of immunoprecipitated cMYC protein between SMARCB1 wild-type and SMARCB1 mutant clones at codon 152. The amount of the immunoprecipitated cMYC protein in each parent cell was defined as the control (1.0). The wildtype SMARCB1 clone showed a more than 2.5 times higher amount of the cMYC protein in both cell lines, whereas the mutant clone showed less than half of the amount of cMYC protein of both parental cell lines.
256
B
C
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
Article
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
FIG. 4. This schematic shows three isoforms of SMARCB1. (Top) The sequences of 40 of 49 (81.6%) colonies of fetal brain cDNA library showed complete agreement with a previous study [12]. (Middle) Seven of 49 (14.3%) colonies had a 27 bp deleted (in exon 2S) short form of the consensus sequence. (Bottom) A 51-bp long form had an addition to the 3⬘ end of exon 4 (in exon 4L) in the SMARCB1 cDNA. This change was noted in two of the remaining colonies (4.1%).
that the cSNP at codon 152 will have a key role in the cure of breast cancer patients. In the library of fetal brain cDNA, the proportion of the DNA corresponding to the long isoform, which had a 51-bp insertion (4.1%), was lower than that of the short form, which had a deletion of 27 bp (14.3%). A BLAST search detected no “hits” corresponding to proteins with sequences that aligned well with either the long or short isoforms. The location of both isoforms was not correlated with cMYC binding sites described above. In addition, both the short isoform and the long isoform were not observed in any of the representative 145 G-I tract carcinoma samples including breast cancers as examined by RT-PCR. We found a reduction of the amount of immunoprecipitated cMYC in the polymorphic clone in breast cancer. Further study will be required to clarify the actual role of the polymorphism in the progression of breast cancer. However, the sequence of SMARCB1 is highly conserved in human G-I tract carcinoma, and we conclude that SMARCB1 alterations occur primarily in association with the rhabdoid carcinomas, as reported in previous papers.
MATERIALS AND METHODS cDNA preparation for mutation search. cDNAs were generated from the following primary carcinoma cases: 20 esophageal carcinoma cases, 20 gastric carcinoma cases, 20 colorectal carcinomas, and 122 breast carcinoma cases. cDNAs were also prepared from 36 carcinoma cell lines: TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE12, KYSE140, KYSE170, KYSE180, KYSE300, KYSE450, and KYSE700 (14 esophageal carcinomas); KATOIII, MKN1, MKN28, MKN45, MKN74, NVGC3, NS8, SCH, SW1, SW5, SW16, and RF (11 gastric carcinomas); LNCaP, DU145, and PC-3 (3 prostate carcinomas); NCI-H82 and NCI-H446 (2 lung carcinomas); PA-1 and SK-OV (2 ovarian carcinomas); 769P and Caki-1 (2 kidney carcinomas); HeLa (cervical carcinoma); and RD (rhabdomyosarcoma). Sequencing of SMARCB1 and the SMARCB1 genomic region. Total RNA was purified from tumor lines, and cDNA was generated from RNA described elsewhere [15]. PCR amplification was performed in a 30 l reaction mixture containing 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3 (20⬚C), 0.2 M each of dATP, dGTP, dCTP, and dTTP, 100 ng of each primer, and 2 units of Taq DNA polymerase (Boehringer). Figure 1 showed the four primer sets for SMARCB1 amplification. PCR reactions with primer set A (F, 5⬘-GCCGCAATGATGATGATGGC-3⬘; R, 5⬘-GTTCCTCTTGGCCTTCTGTTC-3⬘) for exons 1, 2, and 3, and with set B (F, 5⬘-GATCACGGATACACGACTCTAG-3⬘; R, 5⬘-CATTCATGTTCCAGGTGAAGG-3⬘) for exons 3, 4, and 5 were carried out following an initial denaturation at 95⬚C for 5 minutes, 40 cycles of 95⬚C for 15 seconds, 57⬚C for 1 minute. PCR reactions with primer set C (F, 5⬘-CTTTGATGACCATGACCCAGC-3⬘; R, 5⬘-TGCGATGGTGGTGA-
FIG. 5. DNA sequences of the altered regions. (Top) The sequence of the 27-bp-deleted short form was determined from nucleotides 206 to 232. (Bottom) The sequences of the 51-bp long form that corresponded to an insertion between codons 500 and 501.
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
257
Article
doi:10.1006/geno.2002.6829, available online at http://www.idealibrary.com on IDEAL
CAAACTC-3⬘) for exons 5, 6, and 7 were performed by an initial denaturation at 95⬚C for 5 minutes followed by 40 cycles of 95⬚C for 15 seconds, 60⬚C for 30 seconds, and 72⬚C for 1 minute. PCR reactions with primer set D (F, 5⬘-CATCAGACAGCAGATCGAGTC-3⬘; R, 5⬘-CAACAAATGGAATGTGTACCGG-3⬘) for exons 7, 8, and 9 were performed by an initial denaturation at 95⬚C for 5 minutes followed by 40 cycles of 95⬚C for 15 seconds, 54⬚C for 30 seconds, and 72⬚C for 1 minute on a Perkin-Elmer/Cetus DNA Thermal Cycler 9600 (Perkin Elmer, CA). To confirm alterations of bases on cDNA, we also examined the sequence of the identical position on genomic DNA, which was detected by both directions of primers on cDNA. PCR reactions and sequencing were performed using di-deoxy-terminator reaction chemistry on a Perkin-Elmer/Cetus DNA Thermal Cycler 9600 and the Applied Biosystems Model 377 DNA sequencing systems. The faint amplified products were subcloned by TOPO TA Cloning kit (Invitrogen, CA), and then sequencing was carried out. The expression of SMARCB1 was observed in all four segments, and sequencing of all amplified PCR products was carried out, then nucleotide sequences of each region were aligned by the Sequencher 3.0 (Gene Codes Corporation). Furthermore, we examined whether both or one allele was lost or retained at the locus near SMARCB1 in 122 breast cancer cases as follows. The Gene Scan program was applied according to our previous studies [16,17]. We amplified the D22S345 marker for LOH study between primers 5⬘-CACTTCTGAGTTGTTTGAATCTC-3⬘ and 5⬘-AGAATCTGCTGCTTGCTTTT-3⬘, and the amplified products were electrophoresed and analyzed by an ABI377 sequencer (Applied Biosystems). The latter primer was fluorolabeled by 6-FAM fluoroamidide. Mutagenesis study. For evaluation of the significance of mutation of SMARCB1 at codon 152 from AAC (Asn) to GAC (Asp) without an amino acid change, we obtained the mutant clone by the Site-Directed Mutagenesis system (Gibco BRL). The primer for the target the codon 152 was 5⬘-GCTCCACAACCATCGACAGGAACCGCATGG-3⬘ instead of the normal consensus sequence, 5⬘-TTAGATGCCGTGCCATGCTCCACAACCATCAAC (codon152) AGGAACCGCATGGGCCGAGACAAGAAG-3⬘. The primer for clone selection was 5⬘-CTGAGAGTGCACCATGGGCGGTGTGAAATACC-3⬘, with a NcoI site at nucleotide 12. We obtained 10 clones of mutant plasmids, then selected two mutant plasmids with SMARCB1 expression to be transfected into MCF7, a breast cancer cell line, and KYSE150, an esophageal cancer cell line. Immunoprecipitation with SMARCB1 and cMYC protein. The wild-type SMARCB1 clone with consensus sequence and a SMARCB1 mutant clone at the codon 152 polymorphism were obtained and ligated into the expression vector pcDNA 3.1 (Invitrogen). Both clones, wild-type SMARCB1 and a mutant clone, were transfected into MCF7 as well as KYSE150. The expression of SMARCB1 protein was confirmed by western blot analysis with SMARCB1/Snf5 polyclonal antibody (SMARCB1 sc-9749; Santa Cruz). Four clones were immunoprecipitated with cMYC antibody (Santa Cruz) to compare the amounts of immunoprecipitated cMYC protein. Cloning of the SMARCB1 transcript. We prepared the full length of the SMARCB1 cDNA, whose sequence was confirmed by the GenBank accession number U04847 (1857 bp). Initially, the wild-type SMARCB1 cDNA was amplified by the PCR primer upper primer, 5⬘-GCCGCAATGATGATGGCGCTGAGCAAGAC-3⬘, and lower primer, 5⬘-ATCTTCTGAGATGCTC-
258
CGTGGGAGCCGTGT-3⬘. We purified and cloned the amplified products using the TOPO cloning kit (Invitrogen) using the manufacturer’s protocol. We have performed subcloning of the SMARCB1 PCR products and obtained 52 isolated mono-clones. Then, those clones were sequenced. [12]. We examined the expression of SMARCB1 in 14 representative carcinoma cell lines: TE1, TE3, TE4, TE6, KYSE110, KYSE170, KYSE180, KYSE270, KYSE700, KATOIII, MKN7, MKN45, and COLO201. Sixty cases of tumor tissue and the corresponding normal tissue were also examined by RT-PCR with the above primers.
ACKNOWLEDGMENTS We thank T. Shimo-oka and J. Miyake for technical support. RECEIVED FOR PUBLICATION FEBRUARY 20; ACCEPTED JUNE 2, 2002.
REFERENCES 1. Rey, J. A., et al. (1993). Abnormalities of chromosome 22 in human brain tumors determined by combined cytogenetic and molecular genetic approaches. Cancer Genet. Cytogenet. 66: 1–10. 2. Schofield, D. E., Beckwith, J. B., and Sklar, J. (1996). Loss of heterozygosity at chromosome regions 22q11-12 and 11p15.5 in renal rhabdoid tumors. Genes Chromosomes Cancer 15: 10–17. 3. Shin, E., et al. (1993). Deletion mapping of chromosome 1p and 22q in pheochromocytoma. Jpn. J. Cancer Res. 84: 402–408. 4. Biegel, J. A., et al. (1999). Germ-line and acquired mutations of INI1 in atypical teratoid and rhabdoid tumors. Cancer Res. 59: 74–79. 5. Savla, J., et al. (2000). Mutations of the hSNF5/INI1 gene in renal rhabdoid tumors with second primary brain tumors. J. Natl. Cancer Inst. 92: 648–650. 6. Sevenet, N., et al. (1999). Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotype-phenotype correlations. Hum. Mol. Genet. 8: 2359–2368. 7. Sevenet, N., et al. (1999). Constitutional mutations of the hSNF5/INI1 gene predispose to a variety of cancers. Am. J. Hum. Genet. 65: 1342–1348. 8. Versteege, I., et al. (1998). Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 394: 203–206. 9. Wang, W., et al. (1996). Purification and biochemical heterogeneity of the mammalian SWI-SNF complex. EMBO J. 15: 5370–5382. 10. Wang, W., et al. (1996). Diversity and specialization of mammalian SWI/SNF complexes. Genes Dev. 10: 2117–2130. 11. Ponomarenko, J. V., et al. (2001). rSNP_Guide, a database system for analysis of transcription factor binding to target sequences: application to SNPs and site-directed mutations. Nucleic Acids Res. 29: 312–316. 12. Kalpana, G. V., Marmon, S., Wang, W., Crabtree, G. R., and Goff, S. P. (1994). Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5. Science 266: 2002–2006. 13. Cheng, S. W., et al. (1999). c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function. Nat. Genet. 22: 102–105. 14. Klochendler-Yeivin, A., Muchardt, C., and Yaniv, M. (2002). SWI/SNF chromatin remodeling and cancer. Curr. Opin. Genet. Dev. 12: 73–79. 15. Mori, M., et al. (1998). Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription-polymerase chain reaction in patients with gastrointestinal or breast carcinomas. J. Clin. Oncol. 16: 128–132. 16. Mori, M., et al. (2001). Absence of Msh2 protein expression is associated with alteration in the FHIT locus and Fhit protein expression in colorectal carcinoma. Cancer Res. 61: 7379–382. 17. Mori, M., et al. (2000). Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus. Cancer Res. 60: 1177–1182.
GENOMICS Vol. 80, Number 3, September 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.