As found over-expressed in human breast cancer cells and targeted by all forms of endocrine therapy, the zinc finger transcription factor ER is sensitive to either reversible or irreversible impairment after even brief exposures to various forms of oxidative stress [18
]. Although there have been a number of gene expression studies attempting to identify the suite of estrogen responsive genes expressed in ER-positive human breast cancers [29
], none have yet asked what subset of these estrogen responsive genes are also susceptible to modulation by oxidative stress. To address this question, we performed gene expression microarry studies on the model ER-positive breast cancer cell line MCF7, subjected to either estrogen deprivation or ER-α knockdown, in order to identify a comprehensive set of genes responsive to loss of ER function and sensitive to oxidant stress. Modulation of oxidative stress was accomplished by short-term (8 hours) exposure to one of three different chemical oxidants, each given at a predetermined titre associated with more than 50% loss of intracellular ER transcriptional activity (assayed by transient transfection of on ER element-luciferase reporter gene) without significant loss of ER content or cell viability. Despite the chemical differences between the thiol-specific reactant diamide, the redox-cycling quinone menadione (vitamin K3), and ROS-producing hydrogen peroxide, each of these oxidants has been shown to attack zinc-binding cysteine residues within the ER DNA-binding domain, preventing ER dimerization and direct DNA binding [51
]. By comparing the differentially expressed genes between control and treatment conditions, we identified 891 estrogen/ER-regulated probes containing a core set of 75 probes (62 unique genes) that were responsive to all three chemical oxidants; these 62 core genes constituted our newly defined Ox-E/ER gene signature. An alternative Ox' signature was experimentally determined by oxidant exposure of MCF7 cells following knockdown of ER-α, from which an alternate Ox'-E/ER signature was derived.
The 891 estrogen/ER-regulated probes exhibited only a 30% overlap with genes previously described as responsive to estrogen stimulation in the same MCF7 cell line model, using the same basic microarray assay platform [29
]. Although gene expression changes due to estrogen deprivation have been evaluated in the context of hormone therapy [53
], neither the effects of estrogen/ER withdrawal nor any comparison of estrogen/ER withdrawal with estrogen-stimulated genes has been reported. False discovery might account for a small fraction of this discrepancy, but the magnitude of difference between the estrogen/ER-responsive gene set identified here and the estrogen-inducible gene set identified previously [29
] suggests that estrogen withdrawal and ER loss of function are not entirely reciprocal conditions relative to estrogen stimulation.
Because oxidant-induced loss of ER DNA-binding might not impair all forms of gene regulation by ER (including co-activation of other promoter-bound transcription factor complexes like activator protein-1 and CREB1 [cAMP response element-binding protein 1]), it is notable that 24 out of 27 estrogen/ER-stimulated genes within the Ox-E/ER signature were downregulated by all three oxidants. Because little is known about estrogen/ER-induced gene suppression, in particular its ER DNA-binding requirement, it is not too surprising that only 29 of 48 genes originally suppressed by estrogen/ER were subsequently upregulated by the ER-inactivating oxidant treatments. These observations are consistent with recent genome surveys indicating that ER DNA-binding sites are significantly enriched in the promoters of early estrogen upregulated genes but not enriched in early estrogen suppressed genes [56
]. Regarding gene expression differences between the three forms of oxidative stress, the thiol-specific oxidant diamide affected a much smaller subset of estrogen/ER-responsive genes than the less specific oxidants hydrogen peroxide and menadione, both of which are also known to activate kinase pathways. Expression of PR and the estrogen-inducible growth regulator GREB1 were suppressed by hydrogen peroxide and menadione but not by diamide, explaining why these well known ER-associated genes were not part of the 62 Ox-E/ER gene signature and suggesting that their suppression was in part due to oxidant-activated kinase signaling in addition to structural changes in ER. This possibility is supported by previous evidence showing PR downregulation without alteration in ER content or function induced by activation of the phosphoinositide-3 kinase/AKT pathway [8
The relationships of the five gene signatures identified (E/ER, Ox, Ox', Ox-E/ER, and Ox'-E/ER) to clinical breast cancer cases were explored using data from 394 ER-positive primary human breast cancers pooled from three independently published microarray studies [33
]. Although PR expression was not commonly affected by all three forms of oxidant treatment, GSEA revealed that oxidant suppressed genes, in particular the oxidant suppressed E/ER regulated genes identified from ER over-expressing MCF7, were suppressed in ER-positive/PR-negative tumors relative to ER-positive/PR-positive tumors, suggesting that loss of PR expression might be a partial surrogate for increased oxidative stress. This suggestion is consistent with our earlier reported correlation between loss of ER DNA-binding in ER-positive/PR-negative breast cancers [20
], and with the present observation that numeric Ox-E/ER index values correlate inversely with PR transcript levels and tumor PR status. Recent evidence also indicates that ERBB2 over-expression is associated with loss of PR co-expression in ER-positive breast cancers [57
]. Although suppression of oxidant-repressed genes was not shown to be associated with ERBB2 over-expression, there was an enrichment of oxidant-induced genes among the ERBB2 over-expressing ER-positive breast tumors, suggesting that elevated oxidative stress is associated with ERBB2 overexpression and may contribute to the loss of PR co-expression seen in ERBB2 overexpressing ER-positive breast cancers.
GSEA comparisons performed using other established gene signatures demonstrated that ER-positive/PR-negative breast cancers express significantly higher levels of proliferation genes relative to ER-positive/PR-positive tumors. Proliferation genes were also expressed at higher levels in the ERBB2 overexpressing breast tumors relative to ERBB2-negative tumors. The observed associations between tumors bearing higher proliferation and oxidative stress signatures are consistent with numerous past observations that mitogenic signaling pathways generate and require increased ROS. Quite consistently, when the oxidant stress gene signatures were considered as numeric indices, these oxidant stress indices were significantly higher in those ER-positive tumors showing higher proliferation gene expression, including ER-positive/PR-negative tumors.
With regard to the putative relationships between aging, breast cancer incidence, and oxidative stress, our GSEA findings were somewhat unexpected in that they demonstrated a weak inverse relationship between oxidative stress and age at diagnosis, suggesting a decoupling of oxidative stress from cellular aging mechanisms that are thought to contribute to breast cancer development. Both GSEA and the Ox-E/ER numeric index also associated oxidative stress with breast tumors of higher grade, a pathologic score strongly influenced by mitotic index. The correlations between oxidative stress and tumor proliferation provide a potential explanation for the apparent decoupling of oxidative stress from aging and breast cancer development, because breast tumors arising earlier in life are known to be more proliferative [33
] and to have higher growth rates [59
]. Therefore, early age onset breast cancers are probably subject to greater oxidative stress because of their greater proliferative activity.
Focusing on the 62-gene Ox-E/ER signature, which best correlated with clinical PR status in the pooled set of 394 ER-positive breast cancer tumors, Ingenuity Pathway Systems analysis highlighted three top networks relating to cancer, cell development, and cell motility. In accordance with studies associating oxidative stress with kinase signaling [21
], the top scoring network contained 19 of the Ox-E/ER genes linked through various kinases and growth factors as well as two stress-activated transcription factors nuclear factor-κB and CREB1 (cAMP response element-binding protein 1). Of particular interest is this network's inclusion of TGF-β and platelet-derived growth factor-BB, two growth factors extensively evaluated for their involvement in breast cancer progression and metastasis [62
]. A closer look at the eight Ox-E/ER genes linked to these growth factors revealed that most (EGR1 [65
], PHLDA1 [66
], IGFBP5 [67
], TAGLN [68
], DAB2 [69
], and FHL2 [70
]) have been associated with breast cancer. In particular, downregulation of IGFBP5 and upregulation of TAGLN, as observed following oxidant treatment, have been implicated in tamoxifen resistance in a mouse mammary xenograft model [68
], suggesting that components of this Ox-E/ER signature may be clinically relevant to the variable endocrine responsiveness of ER-positive breast cancers.
Given that clinical studies have been unable to show why ER-positive/PR-negative breast cancers are significantly more resistant to tamoxifen and other hormonal therapies [4
], our observations linking the Ox-E/ER signature with both the ER-positive/PR-negative clinical phenotype and a preclinical model of tamoxifen resistance provides new insight into how oxidative stress may contribute to the development of clinically more aggressive forms of ER-positive breast cancers. Of the 16 Ox-E/ER genes involved in the second highest scoring network identified by Ingenuity, nine show a coordinated expression pattern signifying activated TNF signaling. Because oxidative stress is a known byproduct of TNF activation [71
], it may not be coincidental that several members of the Ox-E/ER gene signature are downstream targets of activated TNF signaling. Contained in the 11 Ox-E/ER genes that are involved in the third network involving cell motility pathways are two metastasis associated genes, namely MMP2 and CTNNB1 (β-catenin). Although the cell motility pathway was the smallest of the three Ox-E/ER gene networks identified by Ingenuity analysis, it appears to correspond to the only over-represented molecular function linked to the Ox-E/ER gene signature by Gene Ontology analysis in DAVID (Database for Annotation, Visualization, and Integrated Discovery; set size > 5, Expression Analysis Systematic Explorer score < 0.05) [72
], that of 'actin-binding' genes.
The numeric Ox-E/ER index allowed for its prognostic evaluation as either a continuous variable or as a categorical parameter, based on an optimized cut-point determination and dichotomization of the pooled ER-positive tumors into those with high versus low Ox-E/ER index values. Despite its correlation with proliferation genes, loss of PR expression, ERBB2 over-expression, and higher tumor grade, the Ox-E/ER index as a continuous variable did not achieve (although it exhibited a trend toward) significance with respect to DSS. However, when an optimized cut-point for the index value was determined (based on a maximized adjusted log-rank statistic) to achieve the greatest possible separation between the Kaplan-Meier DSS curves, the Ox-E/ER index proved capable of dichotomizing the pooled ER-positive breast cancers into two groups whose significant difference in survival exceeded that achieved by PR status alone. This suggests that oxidative stress, and its effects on E/ER signaling, contributes to the development of an aggressive subset of primary ER-positive breast cancers.