1 Division of Molecular Genetic Epidemiology C050, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, 2 Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden, 3 Department of Tumor Biology, Centre of Oncology, Maria Sklodowska-Curie Institue, Gliwice, Poland, 4 Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany and 5 Department of Genetic Toxicology, Institute of Occupational Medicine and Environmental Health, Sosnowiec, Poland
6 To whom correspondence should be addressed. Tel: +49 6221 421811; Fax: +49 6221 421810; Email: k.wagner{at}dkfz.de
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Abstract |
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Abbreviations: 95% CI, 95% confidence interval; GRB2, growth factor receptor bound protein 2; IGF-1, insulin-like growth factor-1; IGFBPs, insulin-like growth factor binding proteins; IRS, insulin receptor substrate; OR, odds ratios; PI, phosphatidylinositol; RFLP, restriction fragment length polymorphism; SH2, Scr homology 2; SHC1, Src homology 2 domain-containing transforming protein 1; SNPs, single nucleotide polymorphisms
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Introduction |
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The insulin-like growth factor 1 (IGF-1) pathway plays an important role in regulating cell proliferation, differentiation and apoptosis (68). The interaction between IGF-1 and its receptor is regulated by insulin-like growth factor binding proteins (IGFBPs). Clinical and epidemiological studies have suggested an association between the levels of circulating IGF-1 and IGFBP3 and the risk of breast cancer (68). A recent meta-analysis of 16 published studies showed that the increased risk associated with high IGF-1 and IGFBP3 levels is only seen in premenopausal women (9). Insulin receptor substrates (IRS) and Src homology 2 domain-containing transforming protein 1 (SHC1) are key mediators of the IGF-1 pathway (6,10,11). They act as docking proteins between the activated receptor and further downstream signalling proteins. IRS1 is a major substrate for the IGF-1 receptor while IRS2 and SHC1 are proteins that, beside the IGF-1 pathway, also are substrates for other tyrosine kinase growth factors. After IGF-1 binding and autophosphorylation of the IGF-I receptor, the IRS and SHC1 proteins are tyrosine phosphorylated and can therefore interact and activate especially Scr homology 2 (SH2) domain-containing proteins. Both IRS and SHC1 can bind to growth factor receptor bound protein 2 (GRB2) to activate the Ras/MAP kinase pathway that regulates cell proliferation. The IRS proteins can also bind to the p85 subunit of phosphatidylinositol (PI)-3 kinase and activate the serine kinase PKB/Akt pathway that regulates apoptosis. Changes in the activity of the IRS and SHC1 proteins may thus have an effect both on cell proliferation and apoptosis. It has been shown that IRS1 and SHC1 are constitutively activated in a high number of human tumours, including breast tumours, and may explain the overexpression of MAP kinase detected in many breast cancers (1214). Polymorphisms that are located in important regulatory regions, such as in the promoter region or functionally active domains, may affect the function of the protein (4). In IRS1, IRS2 and SHC1 polymorphisms that are located close to the tyrosine phosphorylation sites may cause a change in the tertiary structure of these proteins and therefore show an effect on p85 and GRB2 binding, resulting in impaired signalling. This has been shown for the IRS1 Gly972Arg polymorphism (15).
In this paper we have sequenced the coding region of the IRS1, IRS2 and SHC1 genes for polymorphisms. Of the confirmed polymorphisms, we chose three for further analysis because of their putative functional effects (IRS1 Gly972Arg, IRS2 Gly1057Asp and SHC1 Met300Val) and two polymorphisms because of their usefulness in haplotype analysis (IRS1 Ala804Ala and IRS2 Cys816Cys). To our knowledge, the role of these polymorphisms has never been tested in breast cancer. We carried out a casecontrol study on the effects of the polymorphisms on breast cancer risk among familiar breast cancer cases and controls.
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Materials and methods |
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PCR amplification
Amplification was performed with 5 ng genomic DNA in a 10 µl reaction volume using 1x PCR buffer, 1.5 mM MgCl2, 0.11 µM dNTP Mixture (Invitrogen, Paisley, UK), 0.15 µM each primer (MWG Biotech AG, Ebersberg, Germany) and 0.3 U Platinum Taq DNA polymerase (Invitrogen). The primer and probe sequences for detecting single nucleotide polymorphisms (SNPs) in each gene are available on request from the corresponding author. For the IRS2 Gly1057Asp polymorphism 5% DMSO was added to the reaction mixture. The PCR programme was: 94°C for 2 min; 3 cycles of 94°C for 1 min, the optimum annealing temperature for the gene (61, 65 and 59°C for IRS1, IRS2 and SHC1, respectively) for 1 min, 72°C for 1 min; 32 cycles of 94°C for 30 s, the optimum annealing temperature minus 1°C for 30 s, 72°C for 30 s; final extension at 72°C for 6 min. PCR was performed in GeneAmp PCR System 9700 thermocyclers (Applied Biosystems, Foster City, USA).
DNA sequencing
PCR was performed in 10 µl using the same primers and conditions as mentioned above. The PCR product was cleaned up using 0.75 µl ExoSapIT (USB Amersham, Uppsala, Sweden) for 40 min at 37°C followed by 15 min at 85°C. The sequencing reaction was carried out using a Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The sequencing reaction was performed using forward and reverse primers separately with the following PCR conditions: 96°C for 1 min; 27 cycles of 96°C for 16 s, 54°C for 5 s and 60°C for 4 min. The sequencing products were precipitated with isopropanol, resuspended in 25 µl of water and loaded onto an ABI PRISM 3100 Genetic analyser (Applied Biosystems). The original data were analysed using Sequencing Analysis 3.7 software (Applied Biosystems) for base calling. The obtained sequences were aligned using DNAStar Lasergene 5.0 software (DNAStar Inc., Madison, WI).
Genotyping using the Taq-Man assay
The polymorphisms Ala804Ala and Gly972Arg in IRS1 and Cys 816 Cys in IRS2 were investigated using the allelic discrimination method. Taq-Man primers and probes were ordered from Applied Biosystems. The reaction was performed in 10 µl using 225 nM each primer, 50 nM each probe and 5 µl Taq-Man Universal 2x PCR Master Mix (Applied Biosystems) per reaction. PCR was performed at 50°C for 2 min, 95°C for 10 min and for 3545 cycles at 92°C for 15 s and 60°C for 1 min. PCR was performed in GeneAmp PCR System 9700 thermocyclers and the number of cycles was dependent on the genotype clustering. The samples were read and analysed in an ABI Prism 7900HT sequence detection system using SDS 1.2 software (Applied Biosystems). We confirmed 10% of the results by sequencing.
Genotyping using RFLP assay
For the IRS2 Gly1057Asp and SHC1 Met300Val polymorphisms we were not able to design Taq-Man probes and therefore restriction fragment length polymorphism analysis (RFLP) was used. The IRS2 polymorphism was digested with 4 U SmaI directly after PCR. The PCR fragment had an additional restriction site for SmaI that we used as an internal control for digestion. The resulting fragments (GG, 124 + 115 + 54 bp; AA, 178 + 115 bp; GA, 178 + 124 + 115 + 54 bp) were analyzed on ethidium bromide stained 15% PAGE mini-gels (Gel Casting System; Bio-Rad, Hercules, CA). For the SHC1 polymorphism we used a long tail forward primer for the PCR which contained a second restriction site for BshNI (MBI Fermentas, St Leon-Rot, Germany) in addition to the polymorphic site, to be used as an internal digestion control. The resulting fragments (AA, 262 + 21 bp; GG, 146 + 108 + 21 bp; AG, 262 + 146 + 108 + 21 bp) were analyzed on ethidium bromide stained 8% PAGE mini-gels (Gel Casting System). The temperatures and digestion times used were as recommended by the manufacturers. About 10% of the RFLP assays were randomly repeated and the results checked for concordance. Additionally, we confirmed 5% of the RFLP results by DNA sequencing.
Haplotype analysis
Haplotypes for the IRS1 and IRS2 genes were determined using PHASE 2 software, created by Stephens et al. (19) (http://archimedes.well.ox.ac.uk/pise/PHASE-simple.html).
Statistical analysis
The observed genotype frequencies in the breast cancer cases and controls were tested for HardyWeinberg equilibrium and the difference between the observed and expected frequencies was tested for significance using the 2 test. Statistical significance for the differences in the genotype and haplotype frequencies was determined by the
2 test. The joint analysis was carried out using MantelHaenzsel adjustment. Whenever the expected number of cases was smaller than five, Fisher's exact test was used. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated for associations between genotypes and breast cancer. With the present sample size we had a power of 90% to detect an OR of 0.5 for polymorphisms with a 5% rare allele frequency in the control population. For the more common polymorphisms with a variant allele frequency of 35% we had a 90% power to detect an OR of 0.7. All calculations were carried out using the HardyWeinberg equilibrium test tool offered by the Institute of Human Genetics, Technische Universität Munich (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) and Epi Info 2000 software.
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Results |
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IRS1 variants are not associated with breast cancer risk
The Ala804Ala and Gly972Arg polymorphisms were used for a casecontrol study and haplotype analysis of the IRS1 gene. The allele frequencies were concordant with so far published Caucasian studies (2022). For the Ala804Ala polymorphism, the allele and genotype frequencies did not differ between the breast cancer cases and controls (Table II) and no homozygous individuals for the GG genotype could be detected. For the Gly972Arg polymorphism, the GA frequency in controls was slightly higher than in cases (10.5 and 8.5%, respectively), but the homozygous AA genotype was only detected in two breast cancer cases (Table II). The genotypes did not show any significant relationship to breast cancer risk, not even when stratified according to age of diagnosis, <50 (OR 0.69, 95% CI 0.391.22, P = 0.20) or 50 years (OR 1.09, 95% CI 0.562.13, P = 0.81). Mutual adjustment for the other SNP in the IRS1 gene did not change the OR (data not shown). There was no correlation between the haplotypes and breast cancer risk (Table II).
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SHC1 Val300 reduces breast cancer risk
We continued to investigate the Met300Val polymorphism in the SHC1 coding region because of its possible functional activity. Initially, only the Polish population was screened. The allele frequency was in concordance with the literature for Caucasian populations (25,26). The results showed a significant difference in the genotype frequency which led to a decreased risk of breast cancer with an OR of 0.54 (95% CI 0.320.90, P = 0.016) (Table III). The protective effect was observed in women diagnosed both below and over the age of 50, but the effect was significant only in women below the age of 50 (OR 0.51, 95% CI 0.280.93, P = 0.03). Heterozygotes were more common in controls than in cases. The only homozygote was found among the control population. Because of this finding we included 217 German familial breast cancer cases and 243 German controls in our study. In this population only a small difference in the frequency of the AG genotype between the case and control subjects was observed (7.4 and 9.1%, respectively), however, a trend for a lower breast cancer risk in heterozygotes could still be seen. No homozygous variants could be detected. Interestingly, the protective effect was observed only in women diagnosed below the age of 50 (OR 0.59, 95% CI 0.261.31, P = 0.19). A joint analysis of the two populations confirmed the protective effect of this polymorphism (OR 0.62, 95% CI 0.410.93, P = 0.02). Again, the protective effect of the Met300Val variant was stronger in women diagnosed below the age of 50 (OR 0.54, 95% CI 0.320.89, P = 0.01).
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Discussion |
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The IRS1 Gly972Arg polymorphism may change the function of the IRS1 protein. The arginine variant has been shown to have decreased binding to the p85 regulatory subunit of PI-3 kinase, resulting in decreased kinase activity and, finally, increased apotosis (15,30,31). This impaired interaction may be due to an altered tertiary structure of the variant IRS1. The variant is located close to Tyr939 and Tyr987, both included in a YMXM motif which, upon phosphorylation, binds to the SH2 domain of PI-3K. Compared with the wild-type protein, the IRS1 variant may also show decreased binding to GRB2 and thus lower MAP kinase activity and impaired proliferation (15). The Gly972Arg polymorphism also influences insulin secretion and insulin signalling. It therefore plays an important role in insulin resistance disorders (20,21,30). Transgenic mice lacking IRS1 show a 50% growth retardation and resistance to insulin and IGF-1; insulin action can be partly compensated by IRS2 (30,32). In the case of cancer the arginine allele would be expected to have a protective effect. However, in a recent population-based study the arginine allele was associated with an increased risk of colon cancer (22). In our study no effect on breast cancer risk was observed. The synonymous change Ala804Ala showed no difference in the allele or genotype frequencies between cases and controls. The haplotype frequencies were also similar among cases and controls.
The IRS2 Gly1057Asp polymorphism is located close to two putative tyrosine phosphorylation sites at positions 1042 and 1072 and may change the tertiary structure and function of the protein, similar to the IRS1 Gly972Arg polymorphism (23,33). However, in the only functional study of the IRS2 variant, no altered binding to the p85 regulatory subunit of PI-3 kinase could be detected (23). In mice lacking IRS2 insulin resistance has been shown to prevail because no other protein can compensate for the altered insulin action. However, only minimal growth retardation was observed (30,34). Thus, IRS2 seems to be more important for regulating carbohydrate metabolism while IRS1 promotes somatic growth (30,32,34,35). Polymorphisms affecting the function of IRS2 would be expected to have a bigger effect in insulin resistance disorders than in cancer. The effects of the IRS2 Gly1057Asp polymorphism on insulin resistance disorders have been intensively studied, with controversial results (21,23,24,33,36,37). In a recent study heterozygotes for the Gly1057Asp polymorphism were shown to be at decreased risk of colon cancer (22). However, no effect on homozygotes for the Asp allele was observed. In our study we could not observe any differences between breast cancer cases and controls in allele or genotype frequencies. The Cys816Cys polymorphism also did not show any differences in the allele or genotype frequencies between breast cancer cases and controls. None of the haplotypes were associated with breast cancer risk.
SHC1 binds to the IGF-1 receptor upon stimulation and becomes phosphorylated so that it can bind to GRB2 and activate the Ras/MAPK pathway that leads to cell proliferation (11). A MetVal variant at codon 300 (according to the p52 isoform) has been described (25,26). The only high affinity binding site for GRB2, a tyrosine phosphorylation site at codon 317, is close by, so we hypothesize that the amino acid exchange may alter the 3-dimensional structure of the protein, as in the case of the IRS1 Gly972Arg polymorphism (11,38). It may thus result in decreased binding to GRB2 and finally to impaired signalling through the Ras/MAPK pathway. However, no functional tests have been reported so far. We could observe a higher frequency of the G allele coding for valine in the Polish control group than in the breast cancer case group, which led to a significant (
50%) decreased risk for breast cancer (OR 0.53, 95% CI 0.320.90, P = 0.016). Because we used incident cases, it is unlikely that our results would have been biased due to differential survival of the variant allele carriers. We included 217 German cases and 243 controls in the study to confirm the results in an independent population. For this population we could detect the same protective trend, which, however, was not statistically significant. In each population the protective effect was stronger in women diagnosed for breast cancer below the age of 50. In the joint analysis the effect was significant in all women (OR 0.62, 95% CI 0.410.93, P = 0.02) and with a stronger effect in the younger women (OR 0.54, 95% CI 0.320.89, P = 0.01). This suggests that the protective effect is stronger in premenopausal women than postmenopausal. Because of the selection criteria in our study
75% of women were under 50 years of age at diagnosis. Earlier, a higher risk for breast cancer with increasing circulating IGF-1 and IGFBP3 levels was observed only in premenopausal women (9). For a better functional understanding this variant needs to be tested in vitro and in vivo, particularly with regard to its interaction with GRB2.
Because of the potential effects of the polymorphisms and partly overlapping functions of these proteins (30,32,34), we performed a genotype combination analysis in each individual according to the number of observed variant alleles. No trend for a decreased or increased risk of breast cancer could be seen.
In summary, in this first study on polymorphisms in the IRS1, IRS2 and SHC1 genes in relation to breast cancer risk we could detect a significantly decreased risk for breast cancer in carriers of the variant Val allele at codon 300 in the SHC1 gene. The IRS1 and IRS2 polymorphisms did not show any influence on breast cancer risk nor did the haplotype or genotype combinations.
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Acknowledgments |
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References |
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