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Cancer Susceptibility and Prevention |
Rokavec1,2,3
3Authors' Affiliations: 1 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany; 2 Department of Clinical Pharmacology University Tübingen, Tübingen, Germany; 3 Department of Molecular Genetics, Institute of Pathology, Medical Faculty, University of Ljubljana, Slovenia; 4 Institute of Pathology, Medical Faculty of the University of Bonn, Germany; 5 GSF-National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany; 6 Deutsches Krebsforschungszentrum, Heidelberg, Germany; and 7 Evangelische Kliniken Bonn gGmbH, Johanniter Krankenhaus Bonn, Germany
Requests for reprints: Hiltrud Brauch, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Auerbachstrasse 112, 70376 Stuttgart, Germany. Phone: 49-711-8101-3705; Fax: 49-711-85-9295; E-mail: hiltrud.brauch{at}ikp-stuttgart.de.
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Abstract |
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Experimental Design: We screened colorectal tumors from 92 patients for genetic variants at the ERBB4 ATG –1000 bp 5'-regulatory region by denaturing high-performance liquid chromatography and sequencing. Variants were subjected to DNA-protein interaction analyses (electrophoretic mobility shift assay), reporter gene assays in breast cancer cell lines MDA134 and MDA157, and immunohistochemical analyses of breast tumors. We established genotype frequencies within a breast cancer case-control collection (1,021 cases, 1,015 population-based controls) and a colorectal cancer case-control collection (459 cases, 569 blood donors) using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were assessed by multivariate logistic regression.
Results: We identified five new germ line variants –815 A>T, –782 G>T, –638 insTC, –267 C>G, and –219 del10bp. Two variants showed in vitro functional effects. The –782T allele showed lower protein binding affinity and lower promoter activity compared with the –782G allele, however, the –815T allele showed higher protein binding affinity and higher promoter activity. The –782T variant was identified as a risk allele for breast and colorectal cancer (OR, 1.59; 95% CI, 1.06-2.34 and OR, 2.21; 95% CI, 1.22-3.99, respectively).
Conclusion: The ERBB4 –782 G>T polymorphism, by virtue of its in vitro functional implication and incidence, is a risk factor for breast and colorectal cancer.
ERBB4 is expressed at low levels in the majority of adult tissues including breast and colon (5). Insight into its relevance during embryonic development comes from targeted inactivation in mice, which die by embryonic day 11 due to defective trabeculation in the heart (6). Although ERBB4 seems to be dispensable for ductal branching during mammary gland development of the virgin mouse and early lobulo-alveolar development at the onset of pregnancy, it is required at the penultimate stage of lobulo-alveolar expansion and epithelial functional differentiation (7). The mechanism of ERBB4 regulation is not fully understood, but there is increasing evidence that the estrogen receptor (ER) plays a role (8). In tumors, ERBB4 is overexpressed in 
20% of breast cancers with mRNA levels reaching 170-fold expression compared with those observed in normal mammary glands (9). Although the frequency and range of overexpression are reminiscent of ERBB2, the underlying mechanism for ERBB4 overexpression cannot be explained by gene amplification. Also, the role of ERBB4 expression in tumors is controversial (discussed in ref. 9). Although ERBB4 expression has been found to be associated with a well-differentiated phenotype (10) and favorable progosis in breast cancer (5, 11, 12), other studies favor an oncogenic role (13, 14). Recently, it has been shown that the prognostic value of ERBB4 depends on the subcellular localization of the ERBB4 protein, i.e., nuclear ERBB4 staining has been associated with worse clinical outcome compared with cell membrane–associated ERBB4 staining (9). The subcellular localization depends on the expressed ERBB4 isoform in that only protease-cleavable isoforms can translocate to the nucleus (9).
On the gene structure level, ERBB4 is subject to genetic variation; however, as of August 2007, limited data on variants in the 5'-regulatory region and their functional consequences are available. Because genetic variants in the 5'-regulatory region of EGFR have been shown to alter promoter activity and gene expression in vitro and in vivo (15), there is a possibility that variants of the ERBB4 5'-regulatory region may also exert such a role. We therefore screened the ERBB4 ATG –1000 bp including the 5'-untranslated region with the transcriptional starting site at –98 for the presence of novel genetic variants and studied their functional consequences as well as potential roles in breast and colorectal cancer.
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Materials and Methods |
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Plasmid constructs. For the construction of a recombinant plasmid, the ERBB4 5'-regulatory region (–1006 to –5) was amplified from human genomic DNA with forward primer 5'-AGAACGCGTCCGTAAGCGGCCCTCC-3' and reverse primer 5'-AGACTCGAGGGAAGTCTCAGATCCCG-3'. These oligonucleotides introduced a MluI and XhoI restriction site at the 5'- and 3'-ends of the amplified fragment, respectively. The PCR product was digested with XhoI and MluI (GeneCraft) and cloned into appropriately digested luciferase reporter gene vector pGL3-basic (Promega). The identity of the cloned ERBB4 5'-regulatory region was verified by sequencing. Variants of the 5'-regulatory region representing the genetic variants of interest were generated by introducing point mutations using Quickchange II site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations.
Transient transfections and reporter gene assays. Reporter gene assays were carried out for the investigation of differential promoter activities of variant ERBB4 alleles according to Burk et al. (16). Two breast cancer cell lines MDA134 (ER positive) and MDA157 (ER negative) were used for transfection. Statistical significance was determined using one-way ANOVA test with Dunett's post-test and P < 0.01 was considered statistically significant. The statistical tests were done by GraphPAD Prism version 3.03 software (GraphPad Software, Inc.).
Electrophoretic mobility shift assays. DNA-protein interaction was investigated by electrophoretic mobility shift assay (EMSA). The nuclear extract from the MDA134 BC cell line was prepared as described in Schreiber et al. (17). We used the transcription factor binding sites prediction software (TFSEARCH, version 1.3; ref. 18) to predict proteins that might bind to the ERBB4 promoter regions containing the variants under investigation and identified Sp1 as a potential candidate. Therefore, nuclear extract or recombinant Sp1 protein (Promega) were used as protein sources in the EMSA experiments. The sequences of the oligonucleotide probes used are listed in Supplementary Table S1. The sense and antisense oligonucleotides of the respective probes were annealed by incubation at 99°C for 5 min and slow cooling to 25°C. Double-stranded probes were labeled with Cy5.5-dCTP (Amersham Biosciences) by Klenow polymerase (Fermentas UAB) for 2 h at 37°C and cleaned by Sephadex G50 (Amersham Biosciences) column purification. Gel retardation assays were carried out as follows: nuclear extract or recombinant Sp1 protein was preincubated with 1 µg of pdIdC in Goodwin binding buffer containing 10 mmol/L of HEPES, 60 mmol/L of KCl, 0.2% NP40, 2.5% Ficoll, 2 mmol/L of DTT for 10 min at room temperature in the absence or presence of specific competitors (unlabeled oligonucleotide probes) or 2 µg of Sp1 antibody (Sp1 mouse monoclonal antibody sc-17824X; Santa Cruz Biotechnology). The labeled probe (1 ng) was added and incubated for 20 min at room temperature. Samples were loaded on a 30-min prerun native 6% polyacrylamide gel and electrophoresis was carried out at room temperature in 0.5x Tris-borate EDTA buffer at 80 V for 80 min. Gel imaging was carried out using Odyssey Infrared Imaging System (LI-COR) at a wavelength of 700 nm.
Immunohistochemical ERBB4 analysis. Immunohistochemical stainings were done on 4-µm tissue sections of formalin-fixed paraffin-embedded breast tumors using a 1:50 dilution of monoclonal anti-ERBB4 (HFR-1) mouse monoclonal antibody (Labvision). Immunostaining and scoring was done as described elsewhere (19). Stained tissues were evaluated by an expert pathologist who was unaware of the clinopathologic data and genotype. The differences in mean expression with respect to the genotype were calculated using a nonparametric t test. Statistical tests were done using GraphPAD Prism 3.03 software (GraphPad Software).
ERBB4 promoter polymorphisms and investigation of an associated breast and colorectal cancer risk. A colorectal case-control collection from Slovenia and the German population–based breast cancer case-control collection GENICA were investigated. The colorectal case-control collection consisted of 459 patients with histologically confirmed colorectal carcinoma collected at the Institute of Oncology, Ljubljana between 1996 and 2004, and 569 unrelated blood donors collected at the Blood Transfusion Center of Slovenia (20). Genomic DNA from patients were isolated from snap-frozen normal colonic mucosa using NucPrep chemistry (Applied Biosystems) on ABI 6100 (Applied Biosystems) and of controls from blood mononuclear cells using ethanol/NaCl precipitation. The German GENICA breast cancer case-control collection (21, 22) included 1,021 cases with histologically confirmed breast cancer and 1,015 population-based controls matched in 5-year age classes. Genomic DNA was isolated from blood mononuclear cells using Puregene kit (Gentra Systems, Inc.). Patient and control characteristics are listed in Table 1 . All study participants were of European descent. The GENICA study was approved by the Ethics Committee of the University of Bonn, Germany, and the colorectal cancer study was approved by the National Medical Ethics Committee of the Republic of Slovenia.
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2 test. All OR and P values were calculated from a 2 x 2 table based on the frequency of each genotype versus the frequency of the major genotype between cases and controls. Multivariate logistic regression analyses were used to obtain adjusted ORs and 95% CIs. Because there was a significant difference with respect to breast cancer case and control status for the variables family history of breast cancer and hormone replacement therapy usage (Table 1), we adjusted for these variables in the multivariate analysis. Likewise, there was a significant difference with respect to the colorectal cancer case and control status (Table 1) for the variables age and sex, the reason why we adjusted for these variables in multivariate analysis. Pairwise linkage disequilibrium (LD) and haplotypes were calculated and estimated using Arlequin 2.0 software. |
Results |
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Effect of ERBB4 variants on DNA-protein interaction. We performed EMSAs using nuclear proteins from MDA134 cells (Figs. 2 and 3 ). The DNA-protein complex with the major ERBB4 –782G allele probe showed a more intense band when compared with the complex obtained with the minor T allele probe (Fig. 2A, lanes 2 and 6). In competition assays with labeled G allele probes and an excess of unlabeled competitor oligonucleotides, we observed that the protein-DNA complex 1 required higher concentrations of T allele competitor than G allele competitor to diminish the band intensity (Fig. 2C; paired t test, P = 0.0274). These data show that the binding affinity of nuclear proteins to the minor T allele was significantly lower compared with the major G allele.
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In the case of the ERBB4 –815 A>T variant, we observed strong Sp1 binding (Fig. 3B). Incubation of the –815A allele probe with MDA134 nuclear extract or recombinant Sp1 protein led to the formation of a Sp1 complex (Fig. 3B, lanes 1 and 5), which was confirmed by supershift with Sp1 antibody in both cases (Fig. 3B, lanes 2 and 6). In addition, we observed a faster migrating complex (complex 2), which was also present when using the Sp1 consensus probe (Fig. 3B, lanes 1 and 3). However, we infer that this complex may not contain Sp1 protein due to a lack of supershift (Fig. 3B, lanes 2 and 4). The same binding pattern was observed with the –815T probe (Supplementary Fig. S1, lanes 5-8). Both protein-DNA complexes (Sp1 complex and complex 2) were more pronounced when using the minor T allele probe in comparison with the major A allele probe (Fig. 3A, lanes 6 and 2, respectively). Competition assays with labeled T allele probe and excess of unlabeled competitor oligonucleotide showed that complex 2 was competed out at lower amounts of T allele competitor (Fig. 3C) compared with the same amounts of A allele competitor, a difference that was close to significance (paired t test, P = 0.078). There was no difference in competition of Sp1 complex with respect to the two competitor oligonucleotides (Supplementary Fig. S2; paired t test, P = 0.14). These data indicate that the binding affinity of some nuclear proteins to the minor T allele might be higher when compared with the major A allele. No DNA-protein interactions were observed for the other analyzed ERBB4 promoter variants.
Expression of ERBB4 protein in breast tumors does not differ between carriers of the –782G/G and the G/T genotype. We analyzed the expression of ERBB4 protein in breast tumor tissues from 36 patients carrying the major –782G/G genotype and 21 patients carrying the heterozygous G/T genotype. There was no difference in expression levels between the two groups with respect to membranous, cytoplasmic, or nuclear staining (data not shown).
The ERBB4 –782T allele is associated with an increased risk for breast and colorectal cancer. To perform cancer risk association studies, we first needed to investigate whether the newly identified variants were single nucleotide polymorphisms or rare disease–associated germ line mutations. Therefore, we established allele and genotype frequencies in populations from Slovenia and Germany. The variants ERBB4 –638 insTC and –219 del10bp were rare, with allele frequencies of <0.5%, and were not further analyzed. The minor allele frequencies of –815 A>T, –782 G>T, and –267 C>G ranged between 0.8% and 3%. These variants, together with the known polymorphisms, –718 C>T (minor allele frequency 43% and 45%, respectively) and –609 G>A (minor allele frequency 27% and 29%, respectively) met Hardy-Weinberg equilibrium in cases and controls and were further analyzed in subsequent association studies (Table 2 ). Based on genotype frequencies in breast cancer and colorectal cancer case-control collections, we calculated ORs to obtain genotype-associated disease risk estimations (Table 2). The heterozygous genotype ERBB4–782G/T was significantly associated with an increased risk of developing breast cancer (OR, 1.59; 95% CI, 1.06-2.34) and colorectal cancer (OR, 2.21; 95% CI, 1.22-3.99). No other cancer risk associations were observed.
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Discussion |
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In search of ERBB4 promoter variants, we first analyzed DNA from colorectal tumors for the presence or absence of mutations in the ATG –1000 bp 5'-regulatory region. We chose these tumors because ERBB4 is similarly expressed in colorectal and breast tumors; however, the latter have not been available to us. We observed seven genetic variants which, upon analyses of the corresponding normal mucosa, were of constitutional origin. Among these, ERBB4 –815 A>T, ERBB4 –782 G>T, ERBB4 –638 insTC, ERBB4 –267 C>G, and ERBB4 –219 del10bp are novel variants, which to the best of our knowledge, are described for the first time herein. The other variants, ERBB4 –718 C>T and ERBB4 –609 G>A, refer to known polymorphisms rs6735626 and rs6735267, respectively. The highest LD was between ERBB4 –718 C>T and ERBB4 –609 G>A (r2 = 0.47), whereas other paired polymorphisms showed low levels of LD. The three novel variants ERBB4 –815 A>T, ERBB4 –782 G>T, and ERBB4 –267 C>G had heterozygous genotype frequencies ranging from 1.5% to 5.2%, and therefore, may play a possible role in colorectal and breast cancer risk.
To provide supportive in vitro functional evidence for a possible role in cancer, we set out to investigate the binding capacity of major and minor alleles with respect to nuclear proteins and their influence on promoter activity. The two most upstream located polymorphisms, ERBB4 –815 A>T and ERBB4 –782 G>T, showed significant effects in experiments addressing these biochemical aspects. In binding experiments with nuclear extracts from MDA134 breast cancer cells, both polymorphic sites formed specific DNA-protein complexes. Because in silico analyses pointed to the transcription factor Sp1 as a likely binding protein due to the presence of a Sp1 binding site within the ERBB4 –815 and –782 polymorphic region, we further tested whether this DNA-protein interaction involved Sp1. This is of particular interest because Sp1 is an important regulator of ERBB2 (25) and EGFR (15) expression. Sp1 interaction was unanimously shown for the –815 polymorphic site, however, the situation with respect to the –782 site was more complex. Although the latter was able to bind recombinant Sp1, it did not visibly bind Sp1 from the nuclear extract. Altogether, our experiments suggest that in addition to Sp1, there may be at least one other nuclear protein with even higher binding affinity. It is of note that in the case of the ERBB4 –782 polymorphic site, the major G allele showed higher protein binding affinity in competition experiments compared with the minor T allele. Accordingly, the minor T allele may be considered compromised in its capacity to bind nuclear protein, a notion that is well in line with the >2.5-fold observed in vitro reduction of promoter activity of this variant. Interestingly, the protein binding affinity of the minor ERBB4 –815T allele was apparently stronger than that of the major A allele, a circumstance that might help to explain the up to 1.5-fold observed increase of promoter activity. Altogether, our results underscore that both polymorphisms show effects on promoter activity. Moreover, they suggest a more versatile role for the ERBB4 –782 G>T polymorphism due to its putative multiple protein binding partners and capacity to obliterate transcriptional activity to a substantial degree.
These interpretations are in line with our patient-based results from breast and colorectal cancer risk association studies from Germany and Slovenia. From the five ERBB4 promoter polymorphisms included in the analyses, the –782 G>T polymorphism was significantly associated with higher cancer risks. In detail, the frequency of the heterozygote genotype G/T was 3.3% in the GENICA population-based controls and 3.4% in the Slovenian blood donor controls. This frequency was significantly increased in the corresponding cancer groups, which translates into a 1.6-fold increased risk for breast cancer and a 2.2-fold increased risk for colorectal cancer. The –782 G>T polymorphism is less frequent than the known promoter polymorphisms rs673626 and rs6735267. Yet, our findings draw substance from the large sizes of case-control comparisons (i.e., >2,000 study subjects from the GENICA breast cancer collection and >1,000 study subjects from the Slovenian colorectal cancer collection), appropriate study design, and high genotyping call rates of >97%. Despite the lower size of the colorectal cancer collection, we had an 85% power to detect a risk allele with a frequency of 2%, which confers a relative risk of 2.2 at the 0.05 level of significance. In the colorectal cancer study, cases and controls did not match with respect to age and sex; however, genotype frequencies matched those of the larger GENICA control group and were therefore regarded relevant. Haplotype analysis did not add more information about the influential single nucleotide polymorphism's accuracy. The only haplotype associated with an increased risk for breast and colorectal cancer was the haplotype that included the –782T variant, which has been identified as the at-risk allele in the single locus analysis. At this point, we are aware of the possibility that some of our observed associations may vanish upon stringent correction for multiple testing. Due to the explorative nature of our study, we purposely did not perform multiple testing corrections in order to not eliminate potentially important results from this first investigation. To understand the relevance of our data, however, it will be important to replicate these findings in independent data sets.
Our mutational, in vitro functional, and population-based cancer studies suggest a role for the ERBB4 –782 G>T polymorphism in breast and colorectal cancer risk. Whether this polymorphism will be useful to identify individuals at increased cancer risk should be further pursued in large international population-based studies. This is encouraged by recent developments in which CASP8 (rs1045485), originally identified as a putative breast cancer susceptibility gene in two British populations (26), was successfully confirmed within a comprehensive international effort of the Breast Cancer Association Consortium on the basis of >30,000 breast cancer cases and controls (27).
In addition, the question arises whether the effects of the polymorphisms may also influence tumor biology and behavior. We observed cytoplasmic, nuclear, and membranous ERBB4 protein expression in breast tumors; however, there was no difference with respect to the underlying major or minor –782 genotypes. This may not be surprising because breast cancer risk may rather be reflected by features of the normal mammary gland, yet ERBB4 expression in normal breast is too low to be quantitatively analyzed (9). Therefore, our ERBB4 expression findings do not contradict the observed genotype-associated risk associations, but rather point to other regulatory mechanisms of ERBB4 expression in tumors. We infer that the role of ERBB4 genotypes may be limited to normal tissue.
Recently, it has been shown that ERBB4 functions as a proapoptotic protein suppressing the growth of malignant cells, and when activated, induces cell killing of breast tumor cells and enhances apoptosis (28). From this, we may infer that high levels of ERBB4 may have a protective cellular effect, thus explaining why a low constitutional expression genotype may confer breast and colorectal cancer risk. This interpretation is in line with the reported improved breast cancer survival times of patients with high ERBB4 expression in their tumors (11, 12). Moreover, there has been mention that the nuclear localization of an ERBB4 isoform is associated with worse outcome compared with membrane localization (9). Whether the ERBB4 –782 G>T polymorphism plays a role in the aberrant expression of this or any other deleterious ERBB4 isoform remains to be determined. Finally, it will be important to understand the role of this polymorphism on the ERBB effector pathways mitogen-activated protein and phosphatidylinositol-3 kinase–activated AKT, which may eventually stimulate progress into the development of targeted drug treatment for breast and colorectal cancer.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Received 2/21/07; revised 9/13/07; accepted 9/20/07.
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, G competitor; 
, T competitor). Bars, SE. Complex 1 was competed out at significantly lower amounts of G allele competitor compared with the same amounts of T allele competitor (paired t test, P = 0.0274).