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Gadd45a Levels in Human Breast Cancer Are Hormone Receptor Dependent Jennifer S Tront1, Alliric Willis2, Yajue Huang3, Barbara Hoffman1,4 and Dan a Liebermann1,4*

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Gadd45a Levels in Human Breast Cancer Are Hormone Receptor Dependent Jennifer S Tront1, Alliric Willis2, Yajue Huang3, Barbara Hoffman1,4 and Dan a Liebermann1,4* Tront et al. Journal of Translational Medicine 2013, 11:131 http://www.translational-medicine.com/content/11/1/131 RESEARCH Open Access Gadd45a levels in human breast cancer are hormone receptor dependent Jennifer S Tront1, Alliric Willis2, Yajue Huang3, Barbara Hoffman1,4 and Dan A Liebermann1,4* Abstract Background: Gadd45a is a member of the Gadd45 family of genes that are known stress sensors. Gadd45a has been shown to serve as an effector in oncogenic stress in breast carcinogenesis in murine models. The present study was aimed at clarifying the expression of Gadd45a in human breast cancer and its correlation with clinicopathologic features. Methods: The expression levels of Gadd45a in breast tissue samples of female breast surgery cases were examined by immunohistochemistry (IHC) using a Gadd45a antibody. Percent staining was determined and statistical analyses were applied to determine prognostic correlations. Results: 56 female breast surgery cases were studied: Normal (11), Luminal A (9), Luminal B (11), HER2+ (10), Triple Negative (15). There was a highly significant difference in percent Gadd45a staining between groups [Mean]: Normal 16.3%; Luminal A 65.3%; Luminal B 80.7%; HER2+ 40.5%; TN 32%, P < 0.001, ANOVA. Gadd45a IHC levels for Normal cases found 82% negative/low. Luminal A breast cancer cases were found to be 67% high. Luminal B breast cancers were 100% high. Her2+ cases were 50% negative/low. Triple Negative cases were 67% negative/low. This difference in distribution of Gadd45a levels across breast cancer receptor subtypes was significant, P = 0.0009. Conclusions: Gadd45a levels are significantly associated with hormone receptor status in human breast cancer. Normal breast tissue displays low Gadd45a levels. High Gadd45a levels are associated with Luminal A and Luminal B subtypes. Absence of hormone receptors in Triple Negative subtype is associated with Negative/Low levels of Gadd45a. Further studies are indicated to elucidate the role of Gadd45a in breast cancer as a potential prognosticator or target for treatment. Keywords: Gadd45a, Breast cancer, Hormone receptor Background Gadd45a is a member of the Gadd45 family of genes In the United States, breast cancer is the most common that are known stress sensors, which modulates the cel- non-skin cancer among women, with an estimated lular response to a variety of stress conditions, including 230,000 cases diagnosed in 2012 [1,2]. It is also one of genotoxic and oncogenic stress [3-6]. The complex role the leading causes of cancer death among women of all of stress sensors in monitoring tumor development is races and Hispanic origin populations. However, a full not fully understood, the best example being the mul- understanding of the molecular mechanisms that con- tiple functions attributed to p53 in tumor development tribute to the malignant growth of breast cancer cells re- and suppression [7,8]. Gadd45a is a transcriptional target mains unclear. Therefore, understanding the complete for tumor suppressors p53 and BRCA1, whose loss of biology of breast cancer is an important aspect for iden- function play key roles in cancer development, including tifying therapeutic targets. breast tumorigenesis [7,8]. In murine models, Gadd45a functions to either pro- mote or suppress breast tumor development via engage- * Correspondence: [email protected] ment of different signaling pathways depending on the 1 Fels Institute for Cancer Research and Molecular Biology, Philadelphia, USA molecular nature of the activated oncogene [9,10]. Al- 4Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA though data suggests that Gadd45a may have a function Full list of author information is available at the end of the article © 2013 Tront 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. Tront et al. Journal of Translational Medicine 2013, 11:131 Page 2 of 6 http://www.translational-medicine.com/content/11/1/131 in modulating human breast cancer, thus far there have Table 1 Clinicopathological characteristics been no reports on whether the expression of Gadd45a Number Percentage is correlated with any clinicopathological characteristics. Cases The present study was aimed at clarifying the expres- Normal 11 sion of Gadd45a in human breast cancer and correlation Cancer 45 of its expression with clinicopathologic features, such as hormone receptor status. In this study, we report for the Grade first time the expression pattern of Gadd45a in breast I 11 24% cancer. We found that the expression levels of Gadd45a II 14 31% are hormone receptor dependent. Our results suggest III 20 45% that Gadd45a may play a role in breast cancer pathogen- Estrogen receptor esis and could be used as a biomarker for breast cancer Positive 20 44% prognosis. Negative 25 56% Methods Progesterone receptor Tissue samples and patient clinical information Positive 19 42% A total of 56 formalin-fixed paraffin embedded tissue Negative 26 58% samples were analyzed in this study, 45 primary breast HEr2/Neu carcinomas and 11 normal breast tissue. Normal sam- Positive 21 47% ples were obtained from patients who had undergone re- duction mammaplasty surgery at Temple University Negative 24 53% Hospital. Breast carcinoma samples were obtained from Age patients who had undergone surgery and were histo- ≤45 9 20% pathologically and clinically diagnosed from 2005 to >45 36 80% 2011. Clinical diagnosis and staging were determined T Classification according to the WHO tumor classification (Inter- T1 16 36% national Classification of Diseases in Oncology) [11] and the AJCC Cancer Staging Manual (7th Edition) [12]. The T2 19 42% histopatholgical grading was done according to the T3 8 18% modified Bloom and Richardson system [11]. All work T4 2 4% was performed under a Temple University Institutional N Classification Review Board approved protocol (Protocol Number N0 21 47% 12485). Patient consent was obtain according to guide- N1 16 36% lines. Table 1 summarizes clinical and pathological infor- mation for all patients. N2 5 11% N3 3 6% Immunohistochemistry Immunohistochemical analysis was performed on 56 hu- man breast tissues. To summarize, paraffin-embedded mounted in Permount (Fisher Scientific). Slides were tissue slides were deparaffinized, rehydrated and subjec- prepared in triplicate. The degree of immunostaining ted to antigen unmasking by sodium citrate (pH 6.0) for was reviewed independently by three observers, includ- 30 minutes at a sub-boiling temperature. Endogenous ing an attending pathologist. Percent positive stainings peroxidase activity was blocked by incubation in 3% for each specimen were averaged and categorized as: hydrogen peroxide for 10 minutes. Sections were Negative/Low (0- < 30%, Neg), Medium (30–60%, Med), blocked with 5% serum for one hour at room tempera- or High (>60%). ture, followed by incubation with primary antibody overnight at 4°C. (Gadd45A - Abcam (ab7664)). For a Statistical analysis negative control, the anti–Gadd45a antibody was re- All statistical analyses were carried out using GraphPad placed with normal goat serum. Sections were incubated Prism Statistical Software. ANOVA was used to analyze with a peroxidase-conjugated secondary antibody for the correlation between the mean percent Gadd45a 30 min at room temperature, followed by treatment with staining and the clinicopathological characteristics. Chi ABC reagent (Vector Laboratories) for 30 min. Sections Square Analysis was performed on percent staining cat- were stained with 3,3′-diaminobenzidine substrate egories. P < 0.05 in all cases was considered statistically and counterstained with hematoxylin, dehydrated and significant. Tront et al. Journal of Translational Medicine 2013, 11:131 Page 3 of 6 http://www.translational-medicine.com/content/11/1/131 Results Gadd45a Expression is elevated in luminal A and luminal B Elevated expression of Gadd45a in primary breast tumors breast cancers To investigate Gadd45a expression in human breast can- In order to assess if Gadd45a expression levels corre- cer, 45 previously characterized human female mammary lated with any of breast cancer subtypes, the 56 carcinoma samples and 11 normal mammary samples breast surgery cases were grouped as follows: Normal were evaluated for Gadd45a expression by immunohisto- (11, benign mammoplasty), Luminal A (9, ER+, PR+, chemistry analysis with an antibody against Gadd45a. We HER2-; LumA), Luminal B (11, ER+, PR+, HER2+), found that Gadd45a was significantly overexpressed in HER2+ (10, ER-, PR-, HER2+), and Triple Negative breast cancer tissues compared to normal tissues obtained (15, ER-, PR-, HER2-; TN ). There was a highly sig- from patients who underwent reduction mammaplasty. nificant difference in percent Gadd45a staining be- (P < 0.001, Figure 1A). High levels of strong cytoplasmic tween groups as measured by mean percent staining: staining of Gadd45a were present in areas containing can- Normal 16.3%; LumA 65.3%; LumB 80.7%; HER2+ cer cells of the primary breast tumors. In addition, strong 40.5%; TN 32% (Figure 2A). Figure 2B illustrates high cytoplasmic staining of Gadd45a was also detected in adja- levels of Gadd45a in Luminal A and Luminal B breast cent noncancerous epithelial cells (Figure 1B). Taken to- cancers. However, in Her2+/Neu
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