The recent characterisation of molecular phenotypes of breast cancer defines biological subgroups, independent of histological type, which provides a further insight into the disease at a functional level. Luminal A cancers defined by the presence of ER and/or PR positivity and HER-2 negativity form a favourable prognostic group. However, further defining this group may provide new insights into the underlying biology of oestrogen sensitivity/resistance and provide clinically useful markers for routine clinical practice. This study demonstrates that a high BAG-1 expression identifies a good prognosis group of cancers with a luminal A phenotype, which may have enhanced therapeutic sensitivity to tamoxifen.
We first addressed the question of identifying an association between BAG-1 mRNA expression levels and patient outcome in two independent cohorts, which are broadly equivalent to our validation cohort in terms of clinicopathological characteristics. Using serially determined cut points, we identified two populations of patients with high and low BAG-1 expressions, which correlated with patient outcome. Thus, the high BAG-1 mRNA expression, found within the top 80% of patients, is associated with a favourable outcome. Correspondingly, the frequency distribution of immunohistochemically detected BAG-1 protein expression in our clinical cohort identifies two distinct subgroups of patients of similar proportions to those identified in the gene expression profiling analyses. High BAG-1 protein expression, defined as greater than 40% positive nuclear staining of any intensity, identified 78% of patients with a good prognosis. The predictive value of high BAG-1 expression was greatest in ER+ cancer in which a high nuclear expression was an independent predictor of prognosis for local recurrence, distant metastases and death. Furthermore, for breast cancer-specific death, BAG-1 expression was of superior predictive power to tumour grade, tumour size and lymph node status. This group of patients has a strong positive correlation with a luminal A phenotype and low histological grade, which suggests that BAG-1 may be a useful surrogate marker of intact ER signalling and identifies those tumours maintaining a luminal A-differentiated phenotype. Therefore, BAG-1 is a marker with potentially useful prognostic applications in ER+ disease.
Outcome studies, published earlier, of BAG-1 expression using immunohistochemistry have shown inconsistent results but with a trend towards improved prognosis with high expression levels. However, its role as a predictive biomarker has not yet been fully defined or adequately validated. The first published study (Tang et al, 1999
) of 140 patients included both early and metastatic disease, ER status was unknown in 38% of patients and only 35% of patients were ER+. Consequently, ER and PR were excluded from multivariate analysis, which showed that an elevated nuclear BAG-1 expression was associated with shorter disease-free and overall survival, although BAG-1 was not significant in univariate analysis. These findings were not replicated in a subsequent study by the same group of investigators (Tang et al, 2004
). The second study of 122 patients (Turner et al, 2001
) consisted predominantly of pre-menopausal patients (mean age, 54 years), only 41% of whom had ER+ cancers, and lymph node status was unknown in 48% of cases. In addition, well-documented prognostic indicators, such as tumour size, grade, ER, PR and HER-2, were not significant in univariate analysis. In this study, elevated cytoplasmic BAG-1 expression was associated with improved prognosis in a multivariate model that included ER, BCL-2 and stage. In a more homogeneous and representative cohort of early breast cancer, Cutress et al (2003)
described improved prognosis in univariate analysis with high nuclear, but not high cytoplasmic, BAG-1 expression in a cohort of 138 patients, 60% of whom were ER+. All patients were treated with surgery and endocrine therapy without chemotherapy. The largest and the most recent study of 517 patients (Nadler et al, 2008
) used image analysis-based assessment of immunofluorescent staining and found that both high nuclear and cytoplasmic expression of BAG-1 was associated with improved prognosis in the whole cohort and in lymph node-positive patients only in univariate analysis, with a strong correlation with ER, PR and Bcl-2. However, again in this study, only 52% of patients were ER+, with a predominance of large tumours (59%>2
cm), and details on histological grade and treatment were not available. Two other studies were unable to identify any association with outcome in patients treated with hormonal therapy (Townsend et al, 2002
) or in a cohort with advanced breast cancer treated with chemotherapy (Sjostrom et al, 2002
). There are many possible explanations for these discordant findings: differences in the composition of the clinical cohorts, incomplete clinical information, different antigen retrieval methods, differing monoclonal antibodies used in the detection of BAG-1 and divergent cut points used to determine a high or low expression. This study, therefore, confirms the findings described earlier of improved prognosis with high nuclear BAG-1 expression described by Cutress et al (2003)
and represents a detailed analysis of BAG-1 expression and its potential relationship with therapeutic responsiveness in a large cohort of uniform histological type with well-documented clinical outcome.
The finding of improved responsiveness to tamoxifen and better patient outcome associated with a high expression of BAG-1, a pro-survival antiapoptotic protein, is somewhat counter-intuitive but is mirrored by several studies identifying the overexpression of BCL-2, a major target of BAG-1, also being consistently associated with improved prognosis in low-grade ER+ tumours (Callagy et al, 2006
) and also in patients treated with tamoxifen (Linke et al, 2006
). Furthermore, the strong relationship between high nuclear BAG-1 expression and improved patient outcome reported by Cutress et al (2003)
emanated from a cohort of tamoxifen-treated patients. BAG-1
feature among the 16 cancer-related genes of the Oncotype Dx assay (Paik et al, 2004
), which predicts distant failure in ER+ lymph node-negative patients treated with tamoxifen. The derived recurrence-score algorithm, which is largely weighted towards proliferation-related genes, assigns a negative value to the BAG-1 mRNA expression level, in turn supporting our observation of improved prognosis with high expression level. Several other gene expression profiling studies have identified signatures predictive of outcome in ER+ disease treated with tamoxifen (Ma et al, 2004
; Jansen et al, 2005
; Loi et al, 2008
did not feature among genes within these signatures, although there is often a limited overlap in signatures between studies (Miller, 2007
). Interestingly, an expression profiling study (Cleator et al, 2006
) of pre-treatment biopsies from 40 patients treated with AC chemotherapy identified BAG-1
among a diverse group of 178 genes overexpressed in sensitive tumours. Thus, the potential role of BAG-1 as a predictive marker of therapeutic responsiveness to both endocrine and chemotherapy requires further investigation. This is best performed within the context of randomised clinical trials and these studies are ongoing.
gene is located on chromosome 9p12 and is expressed as three protein isoforms generated through alternative initiation sites from a single mRNA (Packham et al, 1997
). The overexpression of BAG-1 has been described in several breast cancer cell lines (Takayama et al, 1998
; Brimmell et al, 1999
), with the three isoforms demonstrating differing intracellular localisations: BAG-1L is predominantly nuclear, BAG-1S is predominantly cytoplasmic and BAG-1M is present in both the cellular compartments (Brimmell et al, 1999
). This differential subcellular localisation of BAG-1 isoforms is altered under different experimental conditions, possibly representing a regulatory mechanism for protein activity: BAG-1M relocalises from the cytoplasm to the nucleus following heat shock (Zeiner et al, 1999
) and when bound to the glucocorticoid receptor, possibly downregulating the receptor (Schneikert et al, 1999
). Nuclear-to-cytoplasmic relocalisation of BAG-1M has also been observed during epidermal and neuronal differentiation and during breast epithelial involution, both in vitro
and in vivo
(Takayama et al, 1998
; Schorr et al, 1999
; Kermer et al, 2002
). BAG-1 possesses a range of pro-survival properties through its ability to interact with diverse downstream target molecules, originally described by its ability to bind to and enhance the activity of the antiapoptotic protein BCL-2 mediated by its binding to the heat-shock proteins HSP70 and HSC70 (Takayama et al, 1995
). Differential staining and subcellular localisation of the isoforms have not been investigated in the normal breast or breast cancer and are impaired by the absence of isoform-specific antibodies, all current studies in breast detecting ‘total' BAG-1 expression. The mechanism of overexpression of BAG-1 in breast cancer is also not known although in prostate cancer it is amplified in 7.4% of hormone-refractory cancers (Maki et al, 2007
). Our data with normal and malignant breast epithelial cell lines confirm overexpression in ER+ carcinoma cell lines compared with normal epithelial cells. The BAG-1S isoform appeared to be preferentially overexpressed, but there was evidence for elevated expression of all three isoforms in a cell line-specific manner. The relative contribution of the respective isoforms to the relationships reported here must await further studies with isoform-specific antibodies.
A key target of BAG-1 is ERα
, which when bound by the BAG-1L isoform increases its transcriptional activity by up to five-fold in MCF-7 cells (Cutress et al, 2003
). The ability of high nuclear BAG-1 expression to predict improved outcome in ER+ cancer and also in those treated with tamoxifen is of potential mechanistic importance as it suggests that it may have a role in responsiveness to adjuvant endocrine therapy. In our subgroup of ER+ patients treated with tamoxifen (n
=107), high BAG-1 expression predicted improved prognosis, which may indicate sensitivity to therapy or possibly a better definition of a good prognostic luminal A group of patients. To address the question of whether BAG-1 expression could confer enhanced sensitivity to antioestrogen treatment in vitro
, we analysed cell cycle arrest in BAG-1-overexpressing MCF-7 cells. The data presented here demonstrate a significant increase in sensitivity to the induction of cell cycle arrest by both tamoxifen and the pure steroidal antioestrogen ICI 182780.
As antioestrogen therapy, targeted at oestrogen synthesis (aromatase inhibitors), or the ER (tamoxifen), is the single most-effective treatment for women with hormone receptor-positive disease, the ability to predict likely success or failure of these therapies would enable potential alternative therapeutic strategies to be targeted to a group of patients most likely to fail on tamoxifen or aromatase therapy up-front or at an earlier stage in treatment, which may result in improved outcome. The ability of BAG-1 to predict responsiveness to antioestrogen therapy now merits further investigation by examining the relationship between expression and response in large randomised clinical trials of endocrine therapy in ER+ patients.
In summary, we have demonstrated that the high BAG-1 expression is associated with the luminal A phenotype, is an independent predictor of outcome and may indicate enhanced responsiveness to tamoxifen in ER+ invasive ductal carcinoma. These effects may be related to the ability of BAG-1 overexpression to confer increased sensitivity to antioestrogens in vitro. These findings suggest that BAG-1 immunohistochemistry may have a role in a routine pathology setting as a marker for better defining luminal A breast cancers and as a therapeutic response marker for ER-targeted therapy with tamoxifen or aromatase inhibitors.