Yu et al. Diagnostic Pathology (2018) 13:36 https://doi.org/10.1186/s13000-018-0715-y RESEARCH Open Access Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer Bao-Hua Yu1,2, Bai-Zhou Li3, Xiao-Yan Zhou1,2*, Da-Ren Shi1,2 and Wen-Tao Yang1,2 Abstract Background: Nuclear forkhead box protein P1 (N-FOXP1) expression in invasive breast cancer has been documented in the literature. However, the FOXP1 expression patterns at different stages of breast cancer progression are largely unknown, and the significance of cytoplasmic FOXP1 (C-FOXP1) expression in breast cancer has not been well illustrated. The aims of this study were to investigate FOXP1 expression patterns in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), atypical ductal hyperplasia (ADH) and usual ductal hyperplasia (UDH), and to analyze the clinicopathological relevance of C-FOXP1 and its prognostic value in IDC. Methods: N-FOXP1 and C-FOXP1 expression in cases of IDC, DCIS, ADH and UDH was determined using immunohistochemistry. The correlation between C-FOXP1 expression and clinicopathological parameters as well as the overall survival (OS) and disease-free survival (DFS) rates of patients with IDC were analyzed. Results: Exclusive N-FOXP1 expression was found in 85.0% (17/20), 40.0% (8/20), 12.2% (5/41) and 10.8% (9/83) of UDH, ADH, DCIS, and IDC cases, respectively, and exclusive C-FOXP1 expression was observed in 0% (0/20), 0% (0/20), 4.9% (2/41), and 31.3% (26/83) of the cases, respectively. Both N- and C-FOXP1 staining were observed in 15.0% (3/20), 60.0% (12/20), 82.9% (34/41) and 48.2% (40/83) of the above cases, respectively, while complete loss of FOXP1 expression was observed in only 9.6% (8/83) of IDC cases. Estrogen receptor (ER) expression in C-FOXP1-positive IDC cases (31/66, 47.0%) was significantly lower than that in C-FOXP1-negative cases (13/17, 76.5%) (p = 0.030). Calpain II expression was observed in 83.3% (55/66) of C-FOXP1-positive IDC cases, which was significantly higher than that in C-FOXP1-negative cases (9/17, 52.9%) (p = 0.007). Calpain II was significantly associated with pAKT (p = 0.029), pmTOR (p = 0.011), p4E-BP1 (p < 0.001) and p-p70S6K (p = 0.003) expression levels. The 10-year OS and DFS rates of the C-FOXP1-positive patients were 60.5% and 48.7%, respectively, both of which were lower than those of the C-FOXP1-negative patients (93.3, 75.3%). The OS curve showed a dramatic impact of C-FOXP1 status on OS (p = 0.045). Conclusions: Cytoplasmic relocalization of FOXP1 protein was a frequent event in breast IDC. Calpain II might play an important role in nucleocytoplasmic trafficking of FOXP1 and the AKT pathway might be involved in this process. C-FOXP1 expression was inversely associated with ER expression and might be a predictor of poor OS in patients with IDC. Keywords: Breast cancer, FOXP1, ER, Calpain II, AKT pathway, Immunohistochemistry, Survival * Correspondence: [email protected] 1Department of Pathology, Fudan University Shanghai Cancer Center, Dong-an Road 270, Xuhui District, Shanghai 200032, China 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yu et al. Diagnostic Pathology (2018) 13:36 Page 2 of 9 Background The underlying mechanisms of the nucleocytoplasmic Breast cancer is the most common female malignancy shuttling of FOXP1 in breast cancer are largely un- and also the second leading cause of cancer-related known. The calpains are a family of calcium-dependent death among women worldwide [1]. However, its mo- cysteine proteases that function in a wide range of lecular pathogenesis is largely unknown, and clinically important cellular activities [19]. The ubiquitously useful prognostic and predictive parameters, apart from expressed family members, μ-calpain (calpain I) and human epidermal growth factor receptor-2 (HER2), es- m-calpain (calpain II), are the most extensively studied trogen receptor (ER), progesterone receptor (PR) and calpains [20, 21]. Calpain II activity is subject to many lymph node status, are still insufficient, emphasizing the forms of posttranslational control in vivo, including need for further investigating additional prognostic bio- translocation from the cytosol to the membrane [22]. markers and potential targets for selective therapies. Calpains are implicated in the cleavage of several The forkhead box protein P1 (FOXP1) gene, locating apoptosis-associated proteins, notably Bax, Bcl2, JNK on 3p14.1, is a member of the forkhead/winged helix and JUN, amongst others [19, 23], and are involved in transcription factor family, and the FOXP1 protein is the regulation of some cell cycle progression-associated widely expressed in normal tissues [2–5]. FOXP1 protein proteins, such as p21, cyclin D1, and p27Kip1 [24, 25]. subcellular localization varies between different tissues. For example, calpains may cleave Bcl-2 and Bid and per- A predominant nuclear FOXP1 (N-FOXP1) distribution mit translocation of Bax and Bid to the mitochondria, has been identified in the kidney, thyroid, cerebellum, amplifying the apoptotic signaling pathway in cancer tonsil, blood, thymus, spleen, skin, pancreas and colon, cells [26, 27]. In addition, calpains can mediate p27Kip1 whereas cytoplasmic FOXP1 (C-FOXP1) labeling was degradation, and nuclear export might be necessary for observed in other epithelial tissues, such as the stomach this process [24]. The PI3K/AKT/mTOR signaling path- [3]. Altered FOXP1 expression is also associated with vari- way, including its downstream molecules p4E-BP1 and ous types of tumors [6]. For example, N-FOXP1 protein is p-p70S6K, plays a crucial role in initiation and progres- up-regulated in diffuse large B-cell lymphoma (DLBCL) sion of breast tumorigenesis and drug resistance [28, and extranodal marginal zone or mucosa-associated 29]. Calpain II might promote breast cancer cell prolifer- lymphoid tissue (MALT) lymphoma [7], while loss of ation through the PI3K/AKT signaling pathway [30]. N-FOXP1 expression characterizes malignancy in certain However, whether calpain II plays a role in FOXP1 regu- solid tumors, including endometrial and prostate tumors lation in breast cancer has not yet been documented. as well as familial and sporadic breast cancer [3, 8–10]. Herein, we investigated both the cytoplasmic and nu- The presence of N-FOXP1 expression is correlated with clear expression of FOXP1 protein in cases of invasive ERα and/or ERβ reactivity in invasive breast cancers ductal cancer (IDC) or ductal carcinoma in situ (DCIS), as [8, 11, 12]. A correlation between N-FOXP1 and ERα has well as in atypical ductal hyperplasia (ADH) and usual also been observed in endometrial adenocarcinoma [9]. ductal hyperplasia (UDH) of the breast, and further Loss of FOXP1 nuclear expression is the most striking ob- analyzed the association of C-FOXP1 expression with ER, servation, and cytoplasmic expression is noted more fre- calpain II and other clinicopathological parameters in quently in endometrial adenocarcinoma according to the IDC, and also evaluated the prognostic value of C-FOXP1. literature. However, to date, data regarding C-FOXP1 expression in breast cancer are limited, and its clinicopath- Methods ological relevance, including its correlation with ER expres- Patient selection and tissue microarray (TMA) construction sion, has not been well illustrated. Altogether, 83 cases of IDC, 41 of DCIS, 20 of ADH, and The oncogenic functions of FOXP1 in tumors, such 20 of UDH were retrieved from the archival files of the De- as DLBCL, MALT lymphoma, and hepatocellular and partment of Pathology, Fudan University Shanghai Cancer renal cell carcinoma, have been well documented Center (Shanghai, China). The study was approved by the [4, 13, 14]. On the other hand, FOXP1 might attenuate Institutional Review Board of Fudan University Shanghai tumorigenicity to exert a tumor-suppressive effect in Cancer Center (Shanghai Cancer Center Ethics Commit- other tumors, such as neuroblastoma and prostate can- tee). H&E-stained sections for each case were independ- cer [4, 15–17]. Therefore, FOXP1 is associated with entlyreviewedbytwooftheauthors(BHYandBZL) cancer patient prognosis in a context-dependent according to the criteria described in the World Health manner [4, 18]. Overall, FOXP1 positivity, with either Organization classification of tumors of the breast [31]. nuclear or an unspecified distribution, is associated Clinical data, including follow-up data, were available for with favorable survival in patients with breast cancer all of the 83 IDC cases. For TMA construction, H&E-stained [4, 8, 18]. Nevertheless, the prognostic value of sections from each formalin-fixed paraffin-embedded block C-FOXP1 expression in breast cancer patients has not were first observed to define representative tumor cell-rich been discussed in the literature. areas and then 2 representative 0.6 mm cores were Yu et al. Diagnostic Pathology (2018) 13:36 Page 3 of 9 obtained from each IDC case and inserted into a recipient The status of ER, PR and HER2 were evaluated using paraffin block in a grid pattern using a tissue arrayer the scoring criteria of the American Society of Clinical (Beecher Instruments, Silver Spring, MD, USA).