Clinical Cancer Research Vol. 12, 7252-7260, December 15, 2006
© 2006 American Association for Cancer Research
Mechanisms of egfr Gene Transcription Modulation: Relationship to Cancer Risk and Therapy Response
Burkhard Brandt1,
Sönke Meyer-Staeckling1,
Hartmut Schmidt2,
Konstantin Agelopoulos2 and
Horst Buerger2
Authors' Affiliations: 1 Institute for Tumor Biology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany and 2 Gerhard-Domagk Institute of Pathology, University Medical Centre Münster, Münster, Germany
Requests for reprints: Burkhard Brandt, Institut für Tumorbiologie, University Medical Centre, Centre for Experimental Medicine, Martinistr. 52, 20246 Hamburg, Germany. Phone: 49-251-8347226; Fax: 49-251-8347226; E-mail: bu.brandt{at}uke.uni-hamburg.de.
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Abstract
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The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation, and motility of normal as well as cancer cells. For predictive cancer diagnostics and therapeutic targeting of EGFR, it is important to know how the expression level of EGFR is controlled and related to receptor signaling. A novel transcriptional regulation mechanism has been described that depends on the length of a CA repeat in intron 1 [CA simple sequence repeat 1 (CA SSR I)] of the EGFR gene. Thereby, the number of CA repeats is inversely correlated to pre-mRNA synthesis. Indirect evidence for the importance of this mechanism includes the preferential occurrence of amplifications in cancer tissue harboring short CA repeats in this sequence and the discovery of distinct alleles in young breast cancer patients with a family history of the disease and in Japanese breast cancer patients. It can be postulated that the length of the CA repeat influences DNA bendability and, in consequence, the binding of repressor proteins. In summary, it seems that the CA SSR I represents an inherited variable for response to anti-EGFR therapies that could be determined before therapy. Moreover, the potential for synergistic effects with other polymorphism [e.g., EGFR R497K (HER-1 497K) and CCND1 A870G] leading to a simultaneous increase of EGFR signaling activity and expression should be investigated. From a practical perspective, assessment of the CA SSR I number of CA dinucleotide repeats as a predictor for clinical outcome is very attractive because it is a constant feature that does not change over time and can be easily measured in normal and cancer tissues (blood cells, skin, and tumor biopsies) in an assay that is technically simple, objective, and even quantitative.
The erbB family of receptor tyrosine kinases consists of four members: epidermal growth factor receptor (EGFR; erbB-1), erbB-2 (Her2 or neu), erbB-3, and erbB-4 (1–4). The cell surface allosteric enzymes EGFR, erbB-3, and erbB-4 contain an extracellular ligand-binding domain, a single transmembrane domain, and, for EGFR, erbB-2, and erbB-4, an intracellular tyrosine kinase domain (5). For EGFR, six direct binding ligands are known, including EGF, transforming growth factor, amphiregulin, betacellulin, epiregulin, and heparin-binding EGF (5, 6). Ligand binding to the extracellular domain of EGFR leads to receptor dimerization, causing the activation of signaling pathways by activation of the cytosolic kinase domain of the receptor tyrosine kinase and cross-autophosphorylation of the receptors (6, 7). It has also been shown that erbB-2 is the preferential heterodimerization partner of EGFR compared with the rest of the erbB family members (8). At least three main pathways of EGFR signaling supporting cancer development and progression can be identified: first, a pathway via phosphatidylinositol 3-kinase leading to Akt activation and suppression of apoptosis; second, a pathway via Grb2 and Sos leading to the activation of p21ras pushing the cell cycle; and third, via phospholipase C-
1 phosphorylation leading to PIP2-related actin reorganization.
In most cell types, EGFR is found in amounts varying from 2 x 104 to 2 x 105 receptors per cell. Overexpression of EGFR up to >106 receptors per cell has been described for many cancer types and is mostly associated with poor prognosis (9–13). Several studies reported a positive correlation of increased amounts of the receptor with shortened survival of cancer patients, poor response to chemotherapy, and, especially, even failure of endocrine therapy in breast cancer (14, 15). Furthermore, the increasing knowledge about EGFR-related pathways has led to the development of EGFR-targeted therapies (16). Two predominant classes of EGFR inhibitors have been developed, including monoclonal antibodies that target the extracellular domain of EGFR, such as cetuximab, panitumumab, and matuzumab, and small-molecule tyrosine kinase inhibitors that target the receptor catalytic domain of EGFR, such as gefitinib (Iressa) and erlotinib (Tarceva; ref. 17). Mutations within the EGFR tyrosine kinase domain have been associated with sensitivity to EGFR tyrosine kinase inhibitors in lung cancer (18–20). Therefore, understanding the transcriptional regulatory mechanisms that control EGFR proto-oncogene expression in the absence of activating mutations within the catalytic kinase domain of egfr is important. The identification of polymorphic simple repetitive sequences, such as CA repeats [CA simple sequence repeat (CA SSR)], in relation to negative or positive enhancers provides new insights into individually different gene expression and the linkage of inherited polymorphisms to cancer. Whether the CA repeat numbers alter response to the EGFR tyrosine kinase inhibitors or to other EGFR-targeted therapy is a subject for speculation and for study.
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EGFR Gene Transcription and Its Regulation
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The 5'-regulatory sequence of the egfr gene contains a GC-rich promoter without any consensus sequences, such as TATA or CAAT boxes. Therefore, transcription starts at multiple initiation sites within the promoter region (21). One enhancer element is located in direct proximity to the promoter (Fig. 1
; ref. 22). Two others show a cooperative function: a downstream enhancer, located in intron 1 (+1,788 to 2,318) close to a polymorphic CA dinucleotide repeat, which only functions in the presence of an upstream element, located upstream of the promoter (–1,409 to –1,109; Fig. 1; ref. 23). Therefore, a complex regulation of the egfr gene could be assumed, which is further supported by studies showing transcription factor binding within the 5'-region down to the enhancer in intron 1. Basal transcription of the egfr gene is regulated by the transcription factor Sp1 (24, 25). Additionally, a functional common single-nucleotide polymorphism in the Sp1 recognition site (G216T) was discovered. The replacement of G by T at position –216 increased the promoter activity by 30% (26). The egfr gene was also identified as a target for transcription factor c-Jun with DNA topoisomerase I as a cofactor. Overexpression of topoisomerase I increased the c-Jun-mediated gene activation of egfr (27). Topoisomerase I inhibitors (e.g., camptothecins) have a long history in the treatment of lung cancers, which frequently express high EGFR levels (28). Therefore, novel camptothecins displaying superior preclinical activity might improve the outcome of these patients.
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Fig. 1. Structure of the 5'-region of the egfr gene. The sequence shown comprises  4,000 bp and contains the upstream enhancers 1 (position not determined) and 3 (–1,409 to –1,109) and the downstream enhancer 2 (+1,788 to +2,318) in intron 1 (solid boxes). For simplicity, the start of exon 1 (striped box) is shown as +1 at the ATG, start of translation. In reality, the EGFR gene has multiple start sites for transcription 5' to the start of translation. Intron 1, which contains the polymorphic CA SSR I sequence, extends 150 kb of the 177 kb of EGFR gene. Inset, potential transcription factor binding sites upstream the transcription start.
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A Polymorphic CA Simple Sequence Repeat 1 in the egfr Gene Modulates Transcription
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For several genes, including egfr, it has been shown that the first intron has an important regulatory function (29). A highly polymorphic simple sequence repeat with 14 to 21 CA dinucleotides is located close to the downstream enhancer element of the egfr gene (30). Variation in the number of CA dinucleotides in intron 1 causes different levels of pre-RNA transcription. In vitro, EGFR transcription activity declines with an increasing number of CA repeats in intron 1 (31). Taking the most frequent allele containing 16 CA dinucleotide pairs as the "normal" level of egfr transcription (32), modulation by an increasing number of CA pairs, physiologically up to 21, represses transcription in vitro by a factor of 5 and a decreasing number of CA pairs, down to 12, enhances transcription up to 5-fold. The in vitro observed effect translates in vivo to protein expression level. Allele-dependent modulation of egfr transcription level can be observed in carcinoma cell lines in vivo; heterozygous breast tumors with short CA dinucleotide repeats (<17 CA) showed elevated EGFR expression in contrast to tumors with longer repeats (31, 33). The measurement of EGFR protein expression in head and neck tumors confirmed that, in tumors homozygous for <17 CA alleles, a higher EGFR protein concentration was observed compared with tumors with heterozygous alleles (Fig. 2
; ref. 33).
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Fig. 2. Detection of the minimal amplified region of EGFR by PCR-based microsatellite analysis, capillary electrophoresis for evaluation of fluorescent dye-labeled PCR fragments, and real-time PCR showing amplification of the CA SSR I. A series of microsatellite markers along the p arm of chromosome 7 were evaluated by PCR. The markers between and including CA SSR I and CA SSR VI are located in intron 1 of EGFR. The remaining markers are outside the EGFR gene in chromosome 7p. Microsatellite analysis showed LOH at CA SSR I by PCR. Right, Top histogram, derived from lymphocyte DNA, shows two major peaks corresponding to the two alleles of the CA repeat. Approximately 80% of individuals are heterozygous for the number of CA dinucleotides in CA SSR I. The longer allele of the two shows up as a shorter peak in PCR quantification by capillary electrophoresis due to less efficient PCR amplification and less PCR product. Bottom histogram, PCR products from amplification of tumor DNA. Allele 2 now gives a much smaller PCR product or apparent LOH. However, this is in reality only compared with allele 1, which is amplified, as confirmed in the real-time PCR on the right. Amplification of two to four copies can be distinguished by this method. Less than 5% of tumors show amplification of the entire EGFR gene, whereas 30% to 35% show amplification of the CA SSR I. Although the amplicon may extend to other microsatellite markers, the CA SSR I comprises the minimal region of amplification. The microsatellite assay cannot be used to detect "LOH" or amplification in patients who are homozygous for the CA repeat length, which could only be detected by quantitation by real-time PCR comparing tumor with lymphocyte DNA. DNA from MDA-MB-468 cells harboring an egfr amplification of 60 to 80 copies as a positive control for the real-time PCR assay. Adapted with permission from Tidow et al. (36).
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The influence of a repressor protein inhibiting transcription of DNA molecules by binding to the higher numbers of CA repeats might be one way to explain the modulating effect of the CA SSR I. Alternatively, one can speculate that the CA SSR is a highly bendable hinge-like sequence located between the promoter region and a downstream enhancer element bearing several binding sites. A loop structure bringing these regulatory elements together by the cooperation of one or more DNA-binding proteins might be favored the more the polymorphic stretch is elongated. In this way, a transcriptional repressor protein binding downstream of the CA SSR I could serve as a mediator of allele-dependent inhibition in egfr transcription. If confirmed, this would represent a novel transcriptional regulation mechanism involving the action of a presumed repressor depending on the length of a CA SSR and its influence on DNA bendability (36).
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Amplifications of Regulatory Sequences within the First Intron of egfr Are Common and Are Associated with EGFR Expression in Breast Tumors
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Amplifications of oncogenes are common mechanisms in the initiation and progression of malignant tumors and can circumvent basic transcription mechanisms. Using microsatellite analysis, data from our studies showed that amplifications in the egfr gene were restricted to region of the regulatory sequence in the 5'-end of intron 1 and associated with EGFR expression in epithelial breast tumors irrespective of the detection method (immunohistochemistry and ELISA). Figure 3
shows that the detection of a loss of heterozygosity (LOH) in microsatellite analysis of the EGFR gene can be due to gene amplification. Furthermore, retrospective studies revealed an association with tumor dedifferentiation and prognosis (33, 37).
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Fig. 3. Length of CA SSR I and EGFR expression. A, comparison of EGFR expression for Germans and Japanese. Mean values of intratumoral EGFR protein concentrations in fmol/mg membrane protein for breast (37) and head and neck cancer (right; ref. 36). B, EGFR expression of colon cancer tissue normalized for the EGFR concentration of A431 cells in relation to the sum of the number of CA repeats of both alleles (54). C, mean values of intratumoral EGFR protein concentrations in fmol/mg membrane protein for head and neck cancer (36). Differences in EGFR expression reached statistical significance (P < 0.001) only for the interethnic comparison between German and Japanese breast cancer patients.
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Interestingly, amplifications involving the above-described sequences of intron 1 of egfr belong to the first initial hits described in breast carcinogenesis because they have been noted in normal-appearing epithelial and stromal breast tissue next to the respective benign and malignant tumors (36, 37). At the present state, it is only possible to speculate about the underlying mechanisms. However, the presence of a fragile site, predisposing for chromosomal strand break, next to the egfr locus points to a genetic locus with a generally increased genetic instability. As shown below, the length of the CA repeat within intron 1 of egfr might influence this feature.
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Allelic Length of a CA Dinucleotide Repeat in the egfr Gene Correlates with the Frequency of Amplifications of This Sequence
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Further indirect evidence that the length of a CA SSR I repeat in intron 1 of the egfr gene might indicate a potential clinical response for these anti-EGFR-based therapies comes from an interethnic study comprising German and Japanese breast cancer cases. Applying microsatellite DNA analysis, Japanese breast cancer patients displayed significantly longer alleles for the CA SSR I repeat (67% carried >18 CA alleles), associated with significantly lower EGFR expression (Fig. 3; ref. 38). Allelic imbalance, determined by the same method, was observed in 55% of the informative Japanese breast cancers compared with only 34% of the German breast cancer reference group. Using a quantitative five-nuclease PCR assay for egfr CA SSR, a significantly higher percentage of Japanese breast cancer patients revealed amplifications of the CA SSR I repeat. Japanese patients with these amplifications were characterized by a significantly higher EGFR content compared with the German breast cancer patients (Fig. 3; refs. 32, 38). The data show clearly an interaction between the length of a polymorphism in intron 1 of egfr as an inherited genetic factor and the frequency of egfr amplifications as an acquired genetic factor, both factors contributing to EGFR overexpression in breast cancer. This mechanism might seem to overcome the transcriptional limitations imposed by long alleles, resulting in tumors with high content of EGFR that could be more sensitive to the anti-EGFR agents. But it is not yet known whether Japanese patients with other tumor types will derive benefit from EGFR tyrosine kinase inhibitors as the lung cancer patients did (39). Nevertheless, this new knowledge about mechanisms of regulation of EGFR expression might serve as an additional basis for the evaluation of EGFR tyrosine kinase inhibitor therapies.
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A Polymorphic Sequence in the egfr Gene Is Suspected to Modify Breast Cancer Risk in Young Women Related to Dietary Factors
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In a population-based case-control study in a German population, an association of the allelic length of the egfr CA SSR polymorphism in intron 1 with breast cancer risk was not found (40). However, the presence of two long alleles, particularly when defined as 19 CA repeats, of the egfr CA SSR was associated with a highly significantly elevated odds ratio among women with a first-degree family history of breast cancer but not among women without such a family history. Furthermore, the risk increase was associated with high red meat consumption and the protective effect of high vegetable intake among carriers of two longer alleles, again particularly for >18 CA repeats, compared with the total population (41). From these data, it might be concluded that EGFR expression underlies further complex regulation mechanisms influenced by extrinsic factors. These factors seem to concern predominantly lifestyle, especially dietary factors, which are likely to be shared in a family. Therefore, longer alleles of the egfr CA SSR may contribute to a higher risk of breast cancer caused by environmental factors that might be considered in cancer prevention concepts (42).
These data might be further supported by the observation that LOHs in the egfr CA SSR I are frequently coincident with LOHs in CA repeat markers of the BRCA1 gene as shown in Table 1
. It might be speculated that aberrations of the egfr gene occur as a second hit in familial breast cancer as a consequence of genomic instability caused by BRCA1 inactivation. Preliminary data in 17 patients as shown in Table 1 suggest that a simple explanation of a generalized defect in the mismatch repair system as in hereditary nonpolyposis colon cancer is not the explanation. As the table shows, although there seems to be a concordance of BRCA1 and EGFR allelic imbalance, this breast cancer might not be linked to defect mismatch repair system (mutator phenotype) like in hereditary nonpolyposis colon cancer as shown by the low number of coincident alterations in BRCA1 and egfr microsatellites and a panel of polymorphic markers, which are primary targets for replication errors (43).
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Table 1. Comprehensive analysis of microsatellite instability markers, BRCA1, and egfr CA SSR I in tumors from breast cancer patients
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Allelic Length of the CA SSR I Is Associated with Response to EGFR-Targeted Therapy
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The application of antibodies inhibiting EGFR and small-molecule inhibitors impairing EGFR tyrosine kinase activity in clinical therapy regimens led to the requirement for predictive variables. Disappointingly, first results from studies have noted that the level of expression of EGFR cannot predict the sensitivity of cells to the inhibitors in vivo (16, 43–45). Moreover, in clinical phase II trials, the widely expected efficacy of EGFR inhibitors in the treatment of a wide range of solid tumors with high levels of EGFR, including breast, ovarian, lung, renal, head and neck, pancreatic, and colorectal malignancies, was not obtained (16, 43–50). The identification of mutations in the ATP-binding domain of the EGFR that predict for dramatic responses introduces the possibility of truly individualized therapy, but it seems to apply only in a subset of advanced non–small cell lung cancer patients (18–20) These mutations are more prevalent in certain patient populations, including patients of Asian ethnicity, female gender, and never-smoker status. Conceivably, egfr polymorphisms may be a relevant factor in unraveling the mechanism of the benefit of EGFR tyrosine kinase inhibitors in patients who do not harbor an EGFR ATP-binding site mutation.
The clinical observation that acneiform rash predicts for superior outcome and the question of how this rash may relate to polymorphisms in the egfr gene is emerging. In a clinical study with colorectal cancer, subjects carrying a lower number of the CA SSR I more frequently developed skin toxicity (51). Eighty-four percent of patients with a sum of alleles of <35 developed an acneiform rash, whereas only 33% of those with a sum of alleles of 
35 did (P = 0.04; ref. 51). Interestingly, multiple investigators have noted that the occurrence of rash associates with response to anti-EGFR therapy. For example, Soulieres et al. (52) found, in a subgroup analysis of their erlotinib study with metastatic head and neck squamous cell carcinoma, a significant difference in overall survival favoring patients who developed at least grade 2 skin rashes versus those who did not.
From a practical perspective, assessment of intron 1 number of CA dinucleotide repeats as a predictor for clinical outcome is very attractive because it can be easily measured in normal tissues (blood cells and skin), is a constant feature that does not change over time, and is technically simple, objective, and quantitative in fresh-frozen tissue and also in formalin-fixed, paraffin-embedded tissue (52, 53). It is important to note, however, that this measurement is unlikely to be the only predictor because, as noted above, tumors may have other somatic genetic alterations, such as the CA SSR I amplification, which can compensate the deficient transcription activity of long alleles or suppress those of short alleles as shown for Japanese patients (38).
Besides polymorphism in the egfr gene, it could be hypothesized that differences in genes downstream of EGFR activation, such as cyclins, cyclin-dependent kinases, and the cell cycle inhibitor p27KIP1, might be of clinical relevance for cetuximab (C225) therapy. First evidence for these assumptions might be given by Zhang et al. (54) showing that cyclin D1 (CCND1) A870G polymorphism is a potential prognostic marker for the clinical outcome of metastatic colorectal cancer patients receiving third-line C225 therapy.
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Allelic Length of the CA SSR I Dinucleotide Repeat Along with Other egfr Polymorphisms May Predict Poor Response to Conventional Therapy
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Clinical treatment regimens for cancer patients with chemotherapeutics do not account for genetic interindividual variability in the expression of genes that are targets or modulators of therapy response. In certain cases, this variability might lead to unpredictable tumor responses or host toxicity. Thus, the allelic length of the CA SSR I of the egfr gene has also been evaluated as a predictor of response to conventional chemotherapy in various cancers. Table 2
presents several examples of studies linking the EGFR intron 1 polymorphism to clinical outcome.
Preliminary data for an association between the length of the CA SSR I and pelvic recurrence of rectal cancer following chemoradiation were obtained retrospectively in a study of genomic polymorphisms of 20 genes involved in drug metabolism, DNA repair, angiogenesis, and cell cycle regulation (55). There was a trend toward patients with EGFR genotypes associated with high expression being at a higher risk for pelvic recurrence (P = 0.10). Later, the authors reported that the response of locally advanced rectal cancer treated with adjuvant or neoadjuvant chemoradiation therapy was found to be related to the additive effect of the EGFR R497K polymorphism and the length of the CA SSR I (56). An association between EGFR signaling pathways and response of cancer cells to ionizing radiation has been reported already. The EGFR R497K polymorphic variant in the egfr gene leads to an arginine-to-lysine substitution in the extracellular domain at codon 497 within subdomain IV of EGFR. The lysine variant EGFR seems to have attenuated ligand binding, growth stimulation, tyrosine kinase activation, and proto-oncogene induction. Patients with EGFR 497 Arg/Arg phenotype or lower number of CA repeats (both alleles <20) only tended to have a higher risk of local recurrence. A combined analysis showed significantly the highest risk for local recurrence in patients who possessed both an EGFR 497 Arg allele and <20 CA repeats (Fig. 4
; Table 2). The data support the assumption that the obvious synergistic effect is based on the simultaneous effect of higher EGFR signaling activity and expression level, which are both encoded by the two polymorphisms.
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Fig. 4. Recurrence-free survival of patients with rectal cancer by EGFR polymorphisms. Vertical hash marks, time of last follow-up for those patients who were still recurrence-free at the time of the analysis ofd ata. All censored patients and thosewho were locally recurrent are accounted for. Adapted with permission from Zhang et al. (56).
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The authors extended their molecular analysis to samples from 26 patients treated at an outside center. Surprisingly, a much higher proportion of the "outside" patients (22 of 26, 85%) had local recurrence compared with patients treated at their center (12 of 30, 40%) within 5 years of completion of chemoradiotherapy. The explanation for these differences in patient outcome might be explained by the results of EGFR polymorphism analysis: only 1 of 24 (4%) patients treated at outside facilities carried the favorable prognosis genotype (EGFR R497K Lys/Lys, regardless of CA SSR I genotype) compared with 8 of 46 (17%) patients treated in their center (Fig. 4; ref. 56).
Finally, PCR assays for the CA SSR I length were done to establish whether the polymorphism could predict clinical outcome to 5-fluorouracil/oxaliplatin chemotherapy in patients with metastatic colorectal cancer. Among all patients assessed, those possessing <20 EGFR CA repeats were more likely to show disease progression than were patients with 20 CA repeats (P < 0.05; Table 2; ref. 57). Taken together, these results suggest that the short CA SSR I intron 1 polymorphism and the resulting higher EGFR expression may predict for worse outcome following conventional therapy. These results complement earlier studies showing that increased EGFR expression confers a poorer overall prognosis (9–15).
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Conclusions
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From mechanistic considerations and also from a practical perspective, the assessment of the CA SSR I number of CA dinucleotide repeats and other egfr polymorphisms as predictors for therapy response and clinical outcome is very attractive. The measurement of CA repeat length is very easy, reproducible, and quantitative and does not change over time. It can be measured in normal as well as cancer tissues. Microsatellite PCR also sensitively detects allelic imbalances and provides an additional variable for EGFR activation in cancer tissues. In our opinion, the data from studies evaluating anti-EGFR drugs should be recalculated after determination of the CA SSR I length of the CA dinucleotide stretch. Furthermore, more experimental data on the dependency of the activation of EGFR-induced pathways (e.g., Akt phosphorylation) to the CA SSR I number of CA dinucleotides and whole sequence copies need to be obtained. Finally, a comprehensive screen needs to be done, seeking further polymorphisms that activate EGFR signaling and/or modulate transcription.
Received 3/15/06;
revised 9/27/06;
accepted 10/26/06.
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Abstract
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