The estrogen receptor-α A908G (K303R) mutation occurs at a low frequency in invasive breast tumors: results from a population-based study

The principal risk factors for breast cancer are hormonal or reproductive factors that increase exposure to estrogen [1]. The importance of estrogen in breast cancer development is further supported by studies demonstrating the occurrence of marked changes in estrogen signaling and expression of the two estrogen receptors (ERs) ER-α and ER-β during breast tumorigenesis and progression [2‐8]. Although mutations in the ER-α gene are relatively rare in primary breast cancers [2, 3], Fuqua and colleagues recently described a point mutation in ER-α in one-third of typical breast hyperplasias [9], and also observed this mutation in a high percentage of breast tumors [10]. This A→G base substitution at nucleotide 908 in codon 303, referred to as ER-α A908G or K303R, results in an amino acid change of lysine to arginine. The mutation affects the border of the hinge and the hormone-binding domains of ER-α and has been reported to confer hypersensitivity to estrogen compared with wild-type ER-α, leading to increased cellular proliferation at sub-physiologic levels of estrogen [9]. No difference in estradiol affinity was detected between the mutant and wild-type ER-α; however, the mutant exhibited enhanced binding to the TIF-2 coactivator at low hormone levels [9]. Recent studies also indicate that the ER-α A908G or K303R mutation renders the receptor hypersensitive to phosphorylation at Ser305 through the phosphatidylinositol 3-kinase/Akt signaling cascade [11], protein kinase A [12] and p21-activated kinase [10].

The enhanced function of the hypersensitive ER-α A908G mutant and its discovery in early hyperplastic breast lesions makes it a potentially important marker for studies of breast cancer etiology and progression. In the present study, we screened a series of newly diagnosed invasive breast tumors from patients enrolled in the Carolina Breast Cancer Study (CBCS), a population-based case-control study of breast cancer in African American and white women in North Carolina, for the A908G point mutation in ER-α by using a combination of single-strand conformational polymorphism (SSCP) analysis and ³³P-cycle DNA sequencing. Our results extend the initial observations of Fuqua and coworkers [9, 10] by confirming the presence of this mutation in some invasive breast carcinomas.

Our results confirm the presence of the ER-α A908G mutation in invasive breast cancer, although the overall mutation frequency is low. This finding is consistent with the literature, which indicates that mutations of the ER-α gene occur at low frequency in primary breast tumors [2, 3]. The A908G base change was not detected in the germline of any case; this is consistent with the study of Fuqua and colleagues [9], which failed to find the mutation in normal breast epithelium adjacent to A908G mutation-positive breast hyperplasias. Schubert and colleagues [16] also evaluated the ER-α A908G base change as a potential polymorphic variant in the CBCS but failed to detect it in germline DNA.

Despite the initial finding of this mutation in hyperplastic breast lesions [9] and subsequently its detection in a high proportion of breast cancers [10], four recent studies by other researchers have failed to find the A908G mutation in either benign or malignant breast tissues [17‐20]. It seems likely that multiple factors have contributed to the variable results across studies. These include laboratory screening methods, different tumor or patient characteristics of the populations evaluated, relatively small numbers of breast tumors evaluated previously, the apparent low prevalence of the mutation, its presence in a minority of cells within some tumor tissues, and a histologic preference for mixed lobular/ductal carcinomas that constituted fewer than 10% of breast tumors in the CBCS.

The previous negative studies evaluated primarily ductal carcinomas and included few, if any, mixed lobular/ductal carcinomas [17‐20]. Two of the negative studies screened Japanese breast cancer patients who may have experienced different hormonal exposures from cases in our study [17, 18]. One recent negative study assessed a highly selected group of ER expression-positive breast tumors from postmenopausal cancer patients treated with anti-estrogen therapy [20]. Our study, in contrast, assessed a large population-based series of more than 650 breast tumors of various histologic types from both premenopausal and postmenopausal women.

Laboratory screening techniques might have significantly contributed to the negative findings of several previous studies. One negative study relied on restriction digestion with the MboII restriction enzyme to detect the point mutation [17]. Detection of restriction products on agarose gels is relatively insensitive in comparison with radiolabeling techniques, because small amounts of undigested template, which could correspond to mutant, might not be visible, and incomplete digestion of wild-type template could be mistaken for mutant. In our hands, MboII was an inefficient cutter that never digested wild-type ER sequence to completion. This enzyme can also exhibit star activity, or inappropriate cutting, when digestion is allowed to proceed for more than the recommended time. Incomplete or inappropriate digestion by MboII may have contributed to background bands that we observed in sequencing and the false positive findings we obtained with the combination of MboII pre-digestion followed by SNaPshot. Similar anomalies associated with use of MboII were also reported recently by Davies and colleagues [20].

Three negative studies used automated fluorescent sequencing to screen for the A908G mutation [18‐20]. Fluorescent sequencing has been reported to be less sensitive than radiolabeled sequencing for detecting somatic mutations, failing to detect one-third of mutations in the p53 gene [21]. This inferior sensitivity has been attributed to unevenness in peak heights and suppression of peaks, particularly G when it follows A, resulting in inaccuracies in base calling [22, 23]. Although the use of Big Dye terminators has at least partly corrected the differences in peak heights [22, 23], we still observed a fourfold to fivefold variation in peak sizes within the ER exon 4 sequence and diminished G bases following A bases within the codon 302 to 303 sequence (AAG-AAG) using this method. The sensitivity of detection of the mutant G base in codon 303 (AAG to AGG) might be particularly low when it is present in only a small proportion of tumor cells.

Since the initial report by Fuqua and colleagues [9], we are the only researchers to have used radiolabeled techniques to detect the ER-α A908G mutation in breast tissues. The combination of SSCP and ³³P-cycle sequencing proved to be a sensitive and reliable approach because SSCP demonstrated a consistent band shift pattern when the mutation was present, and even faint bands at the correct position on SSCP gels were usually indicative of the mutation. ³³P-cycle sequencing, which permits more uniform labeling of all bases in a sequence, clearly detected the mutation, whereas fluorescent sequencing did not. It should be noted that ³⁵S sequencing and SNaPshot dideoxy primer extension supported our findings with ³³P-cycle sequencing.

The increased responsiveness of the mutant receptor to sub-physiologic levels of estrogen, and its detection in breast hyperplasias and now invasive carcinomas, raise the question of its role in breast cancer etiology and prognosis. The mechanism of mutant hypersensitivity is not an altered affinity for estradiol, because the mutant receptor binds hormone with affinity similar to that of wild-type ER-α [9]. Rather, it seems to be related primarily to an enhancement of phosphorylation at the downstream serine 305 residue via multiple kinase signaling pathways [10‐12]. Thus, the mutation could function in the early stages of neoplastic development by stimulating cellular proliferation, increasing the formation of genetic changes associated with breast tumorigenesis. Our finding of higher tumor grade among the A908G mutation-positive tumors suggests that upregulated and/or aberrant estrogen signaling might be associated with the mutation and could lead to more aggressive tumor growth. It will be of interest to determine whether oral contraceptives, hormone replacement therapy or other endogenous hormonal factors interact with the ER-α A908G mutant to augment the growth of preneoplastic cells or established tumors. From the standpoint of therapy, it will also be important to determine whether the mutation influences response or the development of resistance to tamoxifen or other anti-estrogen therapies. A recent report by Michalides and colleagues [24] found that phosphorylation of serine 305 by PKA induces resistance to tamoxifen and may even convert tamoxifen from an antagonist to an agonist, although Fuqua and colleagues [9] did not find evidence for this in studies in vitro.

Our data from the CBCS indicate that the ER-α A908G mutation is present at a low frequency in invasive breast tumors and may occur more frequently in higher-grade cancers. The mutation may be associated with the mixed lobular/ductal tumor type, a less-characterized histologic entity, although this result was not statistically significant. Although the relationship of the hypersensitive ER-α A908G mutation to clinical and tumor growth characteristics is of significant interest, our results are based on a relatively small number of mutation-positive tumors even though the CBCS is the largest series of breast cancers yet screened for this mutation. Clearly, additional larger studies are needed to clarify the role of this mutant in breast cancer development.

Our results confirm the presence of the ER-α A908G mutation, originally described in hyperplastic benign breast tissues, in some invasive breast cancers. Although the frequency of this mutation was low, it was more prevalent in subgroups of higher-grade breast tumors and those with mixed lobular/ductal histology. The presence of the hypersensitive ER-α A908G point mutation in invasive breast tumors may have important implications for breast cancer etiology and prognosis.