STAT1-deficient mice spontaneously develop estrogen receptor alpha-positive luminal mammary carcinomas

Introduction: Although breast cancers expressing estrogen receptor-a (ERa) and progesterone receptors (PR) are the most common form of mammary malignancy in humans, it has been difficult to develop a suitable mouse model showing similar steroid hormone responsiveness. STAT transcription factors play critical roles in mammary gland tumorigenesis, but the precise role of STAT1 remains unclear. Herein, we show that a subset of human breast cancers display reduced STAT1 expression and that mice lacking STAT1 surprisingly develop ERa+/PR+ mammary tumors. Methods: We used a combination of approaches, including histological examination, gene targeted mice, gene expression analysis, tumor transplantaion, and immunophenotyping, to pursue this study. Results: Forty-five percent (37/83) of human ERa+ and 22% (17/78) of ERabreast cancers display undetectable or low levels of STAT1 expression in neoplastic cells. In contrast, STAT1 expression is elevated in epithelial cells of normal breast tissues adjacent to the malignant lesions, suggesting that STAT1 is selectively downregulated in the tumor cells during tumor progression. Interestingly, the expression levels of STAT1 in the tumorinfiltrating stromal cells remain elevated, indicating that single-cell resolution analysis of STAT1 level in primary breast cancer biopsies is necessary for accurate assessment. Female mice lacking functional STAT1 spontaneously develop mammary adenocarcinomas that comprise > 90% ERa+/PR+ tumor cells, and depend on estrogen for tumor engraftment and progression. Phenotypic marker analyses demonstrate that STAT1 mammary tumors arise from luminal epithelial cells, but not myoepithelial cells. In addition, the molecular signature of the STAT1 mammary tumors overlaps closely to that of human luminal breast cancers. Finally, introduction of wildtype STAT1, but not a STAT1 mutant lacking the critical Tyr701 residue, into STAT1 mammary tumor cells results in apoptosis, demonstrating that the tumor suppressor function of STAT1 is cellautonomous and requires its transcriptional activity. Conclusions: Our findings demonstrate that STAT1 suppresses mammary tumor formation and its expression is frequently lost during breast cancer progression. Spontaneous mammary tumors that develop in STAT1 mice closely recapitulate the progression, ovarian hormone responsiveness, and molecular characteristics of human luminal breast cancer, the most common subtype of human breast neoplasms, and thus represent a valuable platform for testing novel treatments and detection modalities. * Correspondence: [email protected] Department of Pathology and Immunology, Washington University School of Medicine, 425 S. Euclid Avenue, St. Louis, MO 63110, USA Full list of author information is available at the end of the article Chan et al. Breast Cancer Research 2012, 14:R16 http://breast-cancer-research.com/content/14/1/R16 © 2012 Chan et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Estrogen receptor-alpha-positive (ERa) and progesterone receptor-positive (PR) breast cancer account for approximately 60% to 70% of the breast cancer cases diagnosed in humans [1,2]. The majority of these tumors exhibit a molecular signature that is characteristic of the luminal subtype [3]. The standard of care for luminal breast cancer is either to inhibit ERa signaling using selective ER modulators or to deprive the tumors of estradiol (E2) by ovarian ablation or aromatase inhibition [4]. Despite the advances in the treatment of luminal breast cancers, progress has been hampered by a significant deficit in murine models that fully reproduce the hormonal responsiveness and dependency of human ERa/PR breast cancers [5-8] and that can be used to develop better methods to follow the disease after treatment. STAT1 is a transcription factor that plays a critical role in interferon (IFN) signaling [9]. Cells lacking STAT1 respond aberrantly to IFNa/b and IFNg, and STAT1 mice display immune defects rendering them highly susceptible to infection [10,11] and tumor development [12,13]. The latter finding shows that STAT1 is important in manifesting the IFN-dependent, cell-extrinsic tumor suppressor actions of immunity (that is, the elimination phase of cancer immunoediting [14]). Other studies have also suggested that STAT1 can function as a cell-intrinsic tumor suppressor by maintaining basal expression levels of caspases [15], upregulating p27 expression [16,17], or interacting with p53 or BRCA1 [18-20]. However, these latter studies were conducted mostly with cell lines in vitro and have not been validated by in vivo approaches. Most recently, in vivo studies indicated that STAT1 could suppress tumor development in the ErbB2/Neu-driven mammary tumor models [21,22], although its action in other types of mammary tumors remains undefined. Paradoxically, others have proposed that STAT1 can facilitate tumor outgrowth since elevated levels of STAT1 in melanoma cell lines result in their acquisition of resistance to radiation or chemotherapy [23,24]. This apparent paradox has also been observed in biopsies of human breast cancers [25,26]. However, it remains unclear whether the altered STAT1 levels were present in the breast cancer cells themselves or in stromal cells. Thus, the physiological role of STAT1 during cancer development remains poorly understood and may be contextdependent. Here, we show that STAT1 expression is lost or significantly diminished in the neoplastic cells of a subset of human patients with ERa/PR breast cancer relative to normal breast epithelium, suggesting that downregulation of STAT1 is associated with tumor progression. To further investigate this observation, we followed female mice lacking STAT1 longitudinally and found that they spontaneously develop ERa/PR, hormoneresponsive mammary gland cancers of the luminal subtype, thus closely recapitulating the characteristics of human ERa/PR luminal breast cancers. Materials and methods Immunohistochemistry on human breast cancer samples Formalin-fixed paraffin-embedded tissue blocks were retrieved from the archive of the Department of Pathology in Spedali Civili di Brescia, Brescia, Italy. This retrospective study was conducted in compliance with the Declaration of Helsinki and with policies approved by the Ethics Board of Spedali Civili di Brescia. For this largescale retrospective and exclusively observational study on archival materials, patient consent was not needed, as established by Italian regulations (Delibera del garante n. 52 del 24/7/2008 and DL 193/2003). The cohort consisted of 161 primary breast carcinomas selected from a series of routinely examined cases collected between 2006 and 2008, adjacent normal breast tissues from 11 patients with cancer, and normal breast tissues from five healthy individuals (tissues kindly provided by Monica Guaragni, Clinica S. Anna, Brescia, Italy). Breast carcinoma samples were characterized on the basis of histology, ERa (clone SP1; Thermo Fisher Scientific, Waltham, MA, USA) and PR (clone PgR 636; Dako, Glostrup, Denmark) expression, and HER2 amplification by immunohistochemistry (Herceptest; Dako) and fluorescence in situ hybridization (PathVision HER-2 DNA probe kit; Abbott Laboratories, Abbott Park, IL, USA) (summarized in Table 1). STAT1 protein expression was evaluated on four-micron (4-μm) tissue sections by using a rabbit polyclonal antibody against STAT1 (sc-346, 1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Antigen retrieval was performed by microwaving in citrate buffer (pH 6.0). Positive signal was revealed by the Super Sensitive polymer-horseradish peroxidase immunohistochemistry detection system in accordance with the instructions of the manufacturer (BioGenex, San Ramon, CA, USA). Mouse spleens obtained from WT and STAT1 mice were used as positive and negative controls, respectively, to confirm the specificity of the STAT1 antibody (Figure 1A). STAT1 expression of the human normal breast tissues and breast tumor samples was assessed according to the percentage of STAT1 tumor or stromal cells (percentage score: 1 = fewer than 5% of positive cells; 2 = 5% to 25% of positive cells; 3 = 25% to 75% of positive cells; and 4 = greater than 75% of positive cells) and to the intensity of the staining (intensity score: 1 = low; 2 = intermediate, and 3 = high) (Table 1 and Figure 1B), similar to the determination of ERa Chan et al. Breast Cancer Research 2012, 14:R16 http://breast-cancer-research.com/content/14/1/R16 Page 2 of 21