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FOXA1 Expression in Breast Cancer—Correlation with Luminal Subtype A and Survival

Authors' Affiliations: Departments of ¹ Pathology and Laboratory Medicine, ² Internal Medicine, ³ Surgery, and ⁴ Biochemistry and Molecular Biology; ⁵ Walther Oncology Center, Indiana University School of Medicine; ⁶ Walther Cancer Institute, Indianapolis, Indiana; ⁷ Departments of Genetics and the Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; ⁸ Genetic Pathology Evaluation Centre and ⁹ Child and Family Research Institute, University of British Columbia, British Columbia, Vancouver, Canada

Requests for reprints: Harikrishna Nakshatri, R4-202, Indiana Cancer Research Institute, 1044 West Walnut Street, Indianapolis, IN 46202 and Sunil Badve, Department of Pathology, Indiana University School of Medicine, 635 Barnhill Drive, MS-A128, Indianapolis, IN 46202. Phone: 317-274-2053; Fax: 1-317-278-2018; E-mail: sbadve{at}iupui.edu.

	Abstract

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Purpose: FOXA1, a forkhead family transcription factor, is essential for optimum expression of 50% of estrogen receptor (ER):estrogen responsive genes. FOXA1 is expressed in breast cancer cells. It segregates with genes that characterize the luminal subtypes in DNA microarray analyses. The utility of FOXA1 as a possible independent prognostic factor has not been determined in breast cancers.

Materials and Methods: A tissue microarray comprising tumors from 438 patients with 15.4 years median follow-up was analyzed for FOXA1 expression by immunohistochemistry. Interpretable FOXA1 expression obtained in 404 patients was analyzed along with other prognostic factors like tumor grade, size, nodal status, ER, progesterone receptor (PR), and HER2/neu.

Results: FOXA1 expression (score >3) was seen in 300 of 404 breast cancers and it correlated with ER (P = 0.000001), PR (P = 0.00001), and luminal A subtype (P = 0.000001). Loss of expression was noted with worsening tumor grade (P = 0.001). Univariate analysis showed nodal status (P = 0.0000012), tumor size (P = 0.00001), FOXA1 (P = 0.0004), and ER (P = 0.012) to be predictors of breast cancer–specific survival. Multivariate analysis showed only nodal status (P = 0.001) and tumor size (P = 0.039) to be significant prognostic factors, whereas FOXA1 (P = 0.060) and ER (P = 0.131) were not significant. In luminal subtype A patient subgroup, FOXA1 expression was associated with better cancer-specific survival (P = 0.024) and in ER-positive subgroup, it was better predictor of cancer-specific survival (P = 0.009) than PR (P = 0.213).

Conclusion: FOXA1 expression correlates with luminal subtype A breast cancer and it is significant predictor of cancer-specific survival in patients with ER-positive tumors. Prognostic ability of FOXA1 in these low-risk breast cancers may prove to be useful in clinical treatment decisions.

Estrogen plays an important role in the growth, proliferation, and differentiation of mammary epithelium. ER and ERß mediate the biological action of estrogen by functioning as estrogen-activated transcription factors (1, 2). ER is expressed in 10% to 15% of luminal epithelial cells of normal breast and these cells are generally considered slowly proliferating and well-differentiated cell types (3). However, >50% of breast cancers express ER at the time of initial diagnosis (1). These findings suggest a distinct role for ER in the growth of normal, immortalized, and transformed mammary epithelial cells. Identifying signaling pathways that control these distinct functions of ER may provide a unique opportunity to develop agents that prevent transformation of ER-positive luminal epithelial cells from a slowly proliferating and well-differentiated phenotype to a rapidly proliferating aggressive phenotype.

Recent gene expression profiling studies have classified breast cancer into five distinct subtypes with unique molecular characteristics and prognostic significance (4, 5). These include luminal subtypes A and B, HER2+/ER–, basal-like, and normal-like subtypes. Luminal subtypes A and B are ER-positive breast cancers with subtype A expressing higher levels of ER and having a better prognosis than subtype B (5). Why patients with luminal subtype A tumors have better prognosis than patients with luminal subtype B tumors is not known. One possible explanation is that ER functions differently in luminal A versus luminal B cancers, which may be due to the influence of additional factors, including transcription factors, coactivators, and corepressors that modulate ER activity.

Transcription factors that are coexpressed with ER in luminal subtype A include GATA3 and XBP-1 (4, 6). FOXA1/HNF3, a forkhead family transcription factor, is receiving considerable attention with respect to ER function because it interacts with cis-regulatory regions in heterochromatin and enhances the interaction of ER with chromatin (7). Recent studies have shown the requirement of FOXA1 for optimum expression of 50% of ER-regulated genes and estrogen-induced proliferation (7–9). Thus, estrogen:ER dependency of breast cancers for survival or proliferation may be related to the expression levels of FOXA1. Although previous studies have shown the coexpression of FOXA1, GATA3, XBP-1, and ER in cell lines and breast cancer samples at the mRNA level (6, 10, 11), a detailed protein analysis relating FOXA1 to ER and other disease markers such as progesterone receptor (PR), HER2, nodal status, and histologic grade and prognostic significance of such association have not been reported. We did immunohistochemistry for FOXA1 expression in breast carcinoma tissues of 438 patients with median follow-up of 15.4 years and compared FOXA1 expression with various established disease markers and breast cancer–specific survival. We show that FOXA1 expression is associated with ER positivity, the luminal subtype A, and better breast cancer–specific survival.

	Materials and Methods

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Patient information and tissue microarray. A tissue microarray comprising duplicate cores of tumors from 438 patients with a median follow-up of 15.4 years was analyzed for FOXA1 expression. These samples were obtained from archival cases at Vancouver General Hospital between 1974 and 1995. Patient information, tumor pathology, tissue microarray preparation, and the expression of a number of biomarkers have been reported previously (Supplementary Information; ref. 12) and summarized in Table 1 . Complete clinical treatment details are not available for all patients, which is a limitation of this cohort. In addition to the tissue microarrays, analysis of FOXA1 expression was done on normal breast tissue obtained from reduction mammoplasty specimens. As all personal identifiers had been removed from these samples, information about age or coexisting disease was not available for the normal samples. The study was approved by institutional review boards at Vancouver General Hospital and Indiana University School of Medicine.

Immunohistochemical staining for ER, PR, and HER-2/neu. Immunohistochemical staining was done on serial sections for ER and PR (Clones SP1, NeoMarker and PR636, DAKO, respectively,) and Hercept Test using protocols recommended by the manufacturer (Dako). ER and PR status was assessed from immunohistochemistry stained sections using a 10% cutoff. HER-2/neu expression was scored as 0, 1+, 2+, and 3+ as recommended by the manufacturer (DAKO). In case of discrepant scores from two cores, the higher score of two was taken for the statistical analysis.

Statistical methods. FOXA1 expression was compared with tumor grade, nodal status, and expression of ER, PR, HER2, and the luminal subtype A (ref. 13; defined as ER and/or PR+, and HER2-neg) in Spearman's two-tailed correlation tests. Information regarding each of these variables was not available for all patients, a known limitation of tissue microarray based studies and old cohort studies. Number of patients for whom information was available is given variable-wise in Table 2 . Kaplan-Meier analysis was done using log-rank test for comparison of linear trends with cancer-specific survival as primary end point. Cox proportional hazard ratio (HR) model was used for the multivariate survival analysis. We used univariate Cox regression to calculate HRs for the subgroups. All tests were two-sided. We used 5% level to determine significance. SPSS 14.0 (SPSS, Inc.) statistical package was used to perform the analysis.

	Results

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FOXA1 expression in normal breast and breast cancer
Representative FOXA1 immunostaining of normal breast and breast cancers is shown in Fig. 1 . FOXA1 expression was observed in a few luminal epithelial cells of the normal breast. The expression was restricted to the nucleus with little or no cytoplasmic staining. Invasive cancers showed variable expression; none, weak, moderate, and strong. Staining was nuclear in both normal and cancerous tissues. Thus, it seems that FOXA1 is expressed in a specific subpopulation of normal luminal epithelial cells. High level of FOXA1 expression (FOXA1_high; score >3) was seen in 300 of 404 interpretable breast cancers.

View larger version (162K): [in this window] [in a new window]   Fig. 1. Immunohistochemical analysis of FOXA1 in breast cancer. Normal breast contains rare FOXA1-positive cells (A), whereas invasive carcinomas show three distinct patterns of expression: no expression (B), weak expression (C and D), and strong expression (E and F). Numerical scores: A = 3, B = 0, C = 6, D and E = 16, and F = 30.

Survival analysis
Univariate analysis. Univariate analysis showed (Table 3 ; Fig. 2 ) nodal status, tumor size, FOXA1, and ER as significant predictors of cancer-specific survival. HER-2/neu and tumor grade did not significantly affect cancer-specific survival.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Kaplan-Meier analysis of cancer-specific survival (all patients).

Subgroup analysis. We did two post hoc subgroup analyses; luminal subtype A subgroup and ER-positive lymph node–negative subgroup. Luminal subtype A patients expressing high FOXA1 had significantly better survival with relative risk of death due to breast cancer being 0.60 (P = 0.024; Fig. 3 ). ER-positive lymph node–negative patients expressing high FOXA1 also showed a trend toward better survival with relative risk of death due to breast cancer being 0.63 (P = 0.215 NS). Five and 10-year cancer-specific survival in ER-positive lymph node–negative patients expressing high FOXA1 was 92.6 ± 2.5% and 85.4 ± 3.5%, respectively, compared with 80.0 ± 10.3% and 72.7 ± 11.7% in FOXA1_low patients.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Kaplan-Meier analysis of cancer-specific survival (luminal subtype A patients).

Prognostic significance of FOXA1 in combination with ER and PR

View larger version (23K): [in this window] [in a new window]   Fig. 4. Kaplan-Meier analysis of cancer-specific survival (ER+ patients).

	Discussion

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FOXA1 is a "winged helix" transcription factor, which has recently been dubbed as a "pioneer factor" responsible for the recruitment of ER to the genome (7). Depletion of FOXA1 protein in MCF-7 breast cancer cells leads to reduced estrogen-dependent gene expression and proliferation, which is consistent with its role in mediating the effects of estrogen (8, 9). The COOH-terminal region of FOXA1 interacts with histones H3 and H4 and this interaction is responsible for opening compacted chromatin (16). By opening chromatin, FOXA1 may permit efficient interaction of ER with its response elements and subsequent interaction of ER-associated histone modifying enzymes with histones. Consistent with this possibility, about half of estrogen-regulated genes contain binding sites for FOXA1 (8). Optimum expression of these estrogen-regulated genes may occur only in cells that coexpress ER and FOXA1 and only these cells may be addicted to estrogen-dependent survival and proliferation signaling pathways.

PR negativity is often considered as a marker for the gain of hormone-independent growth properties by ER-positive breast cancers through increased cross-talk between ER and growth factor signaling pathways (15). We observed that patients who are ER+/FOXA1–/PR– have worse prognoses than patients who are ER+/FOXA1+/PR+. It is possible that ER+/FOXA1–/PR– tumors have acquired hormone-independent growth properties because of the shift in ER function from traditional ER:FOXA1:estrogen regulated pathway to a ER:growth factor–activated signaling pathways. Thus, simultaneous analysis of ER, FOXA1, and PR may provide the earliest indication for hormone independence and/or ER:growth factor signaling pathway cross-talk in ER-positive breast cancers.

We also did an exploratory subgroup analysis in ER-positive lymph node–negative patients. We aimed at testing possible utility of FOXA1 as a classifier in this so-called low-risk patient group, which is toughest to deal with when it comes to taking adjuvant treatment decisions, especially decisions like whether to give chemotherapy. A prognostic marker that can further identify patients with very low risk of recurrence will be very useful to avoid toxic therapies in patients who are not likely to derive any additional benefits. Although FOXA1 expression was not a statistically significant predictor of survival in this subgroup, a 10-year cancer-specific survival of 85.4 ± 3.5% in FOXA1_high patients (in this cohort with >50% T₂ or larger tumors from early systemic therapy days) seems promising. It is certainly worth further exploration in larger studies to see if FOXA1 can be used as a prognostic marker in small ER-positive lymph node–negative tumors to avoid toxic therapies in patients with high FOXA1 expression.

We did not have complete clinical treatment details available for all patients in this old cohort (1974-1995). However, because this is a patient cohort spreading over many years, during which breast cancer treatment changed significantly, treatment information in any case cannot be analyzed meaningfully. Because this is a tissue microarray–based study, information regarding all variables is not available for all 404 patients with interpretable FOXA1 staining. This resulted in individual analyses having lower number of patients than 404, decreasing statistical power of the study.

Expression patterns of FOXA1 in normal breast and tumors are strikingly similar to that of ER. In normal breast, ER expression is observed only in 10% to 15% of luminal epithelial cells (3), which is similar to the FOXA1 expression that we observed in the normal breast (Fig. 1). Most of the ER-positive normal cells are nondividing and do not express Ki67, a proliferation marker (3). To determine whether FOXA1 is coexpressed with ER or is excluded from Ki67-positive cells, we attempted dual immunohistochemistry, which was not successful. The intensity of FOXA1 staining in luminal epithelial cells of normal breast and tumors that are highly positive for FOXA1 is similar; an identical finding has also been observed for ER and GATA3 (17). Hence, FOXA1 expression in breast tumors seems not due to cancer-specific overexpression but rather reflects initiation of cancers from luminal cells that express FOXA1.

The functional role of FOXA1 in normal breast is yet to be determined. FOXA1–/– mice are viable for 2 weeks and these animals have been analyzed extensively for prostate development (18). FOXA1–/– animals show a severely altered ductal pattern lacking differentiated or mature luminal epithelial cells. It has been suggested that FOXA1 in concert with the androgen receptor is involved in differentiation of prostate epithelium by regulating the expression of genes such as Nkx3.1, Shh, and FOXA2 (18). By analogy, FOXA1 in concert with ER may be involved in mammary ductal morphogenesis and differentiation. ER-expressing cells have previously been shown to control proliferation and gene expression pattern in stromal and ER-negative luminal cells through paracrine mechanisms (19–21). This function of ER may be dependent on FOXA1. Interestingly, FOXA1 expression is regulated by estrogen (9). Thus, proliferation and differentiation of normal breast epithelium may be under the control of the FOXA1:ER axis. Because of their mutual dependency for normal function, signaling events that alter the expression levels or function of either of them may be sufficient for initiating transformation of FOXA1+/ER+ cells or acquisition of estrogen-independent growth properties.

Emerging data based on molecular signatures clearly shows the existence of subclasses within ER-positive tumors, which have different clinical courses and different likelihoods of response to therapy (5, 22, 23). The differences between these classes involve multiple pathways and are not limited to differences in proliferation rates. This feature, thus far, has been difficult to duplicate using simple protein/immunohistochemistry markers. In this light, it is of interest to note that FOXA1 is an ER-regulated transcription factor that additionally controls downstream transcription of estrogen:ER–regulated genes as well as exerts direct control on cell proliferation possibly via regulation of p27^Kip1 (8, 9, 24).

FOXA1 is a prognostic factor in ER-positive breast cancers and is associated with luminal subtype A. Loss of FOXA1 expression may mean shifting of ER function from traditional ER:FOXA1:estrogen–regulated pathway to a ER:growth factor activated signaling pathway resulting in resistance to hormonal therapy. It will be worthwhile to study FOXA1 in a more recent and larger cohort with smaller tumors to validate the utility of FOXA1 expression analysis in identifying and stratifying ER positive patients and to define its exact clinical utility as a prognostic marker.

	Acknowledgments

 
We thank P. Bhat-Nakshatri for technical assistance.

	Footnotes

 
Grant support: NIH grants RO1 CA89153 (H. Nakshatri), RO1 CA101227-01 (C.M. Perou), and National Cancer Institute/Eastern Cooperative Oncology Group grant CA37403 (S. Badve). The Genetic Pathology Evaluation Centre is supported by an unrestricted educational grant from Sanofi-Aventis. T.O. Nielsen and D.G. Huntsman are scholars of the Michael Smith Foundation for Health Research.

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 1/16/07; revised 4/ 4/07; accepted 4/17/07.

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