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Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor... 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: xyzhou100@163.com Department of Pathology, Fudan University Shanghai Cancer Center, Dong-an Road 270, Xuhui District, Shanghai 200032, China Department 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 entlyreviewedbytwo of theauthors (BHY andBZL) 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). Oncology (ASCO)/College of American Pathologists Four-micrometer-thick sections were then cut from the (CAP) guideline [34, 35]. Staining was considered TMA blocks for routine hematoxylin and eosin (H&E) positive for ER when nuclear staining was present in staining and immunohistochemical procedures. The more than 1% of the tumor cells. Immunohistochemistry H&E-stained sections were used to verify the adequate for HER2 as 3+ was defined as positive. For cases of representation of the diagnostic biopsies. HER2 IHC 2+, Abbott-Vysis HER2 FISH assay was employed to further confirm the status of HER2 gene amplification. Immunohistochemical staining Following deparaffinization and heat-mediated antigen re- Statistical analysis trieval, immunohistochemical staining was carried out All statistical analyses were performed using the SPSS soft- using an Envision system (DAKO, Glostrup, Denmark) ware package (SPSS version 19.0, SPSS Inc., Chicago, IL, with primary antibodies against FOXP1 (JC12, AbD Sero- USA). Categorical variables were compared with a χ test, tec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), cal- and measurement data were analyzed using Pearson correl- pain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, ation analysis. Overall survival (OS) was defined as the Roche Tucson), pAKT (736E11, Cell signaling, Danvers, interval from the initial diagnosis to the time of death or MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 the last contact. Surviving patients were censored at the (53H11, Cell signaling) and p-p70S6K (49D7, Cell signal- last known date of contact. Disease-free survival (DFS) ing). The stained sections were then counterstained with was determined according to the time from diagnosis to hematoxylin. Appropriate positive and negative controls the time of recurrence or the last contact. Patient survival were carried out simultaneously for all stains. was estimated using the Kaplan-Meier method and was The immunostaining results were reviewed by 2 inde- compared by means of a log-rank test. All p-values were pendent qualified pathologists. Nuclear and cytoplasmic two sided, and a p-value < 0.05 was considered statistically tumor cell staining for FOXP1 protein was analyzed separ- significant. ately. FOXP1 nuclear expression was scored using the following system: negative = 0; weak/focal staining = 1; Results strong focal/wide spread moderate staining = 2; or strong/ FOXP1 protein expression patterns in UDH, ADH, DCIS, widespread staining = 3. Tumors that scored 2 or 3 were and IDC of the breast considered positive for N-FOXP1 [8]. Scoring of C-FOXP1, Most UDH cases (17/20, 85%) showed uniform strong calpain II, pAKT, pmTOR, p4E-BP1, p-p70S6K were per- N-FOXP1 staining and the other 3 cases (15.0%) showed formed in terms of the staining intensity (intensity score: 0, both N- and C-FOXP1 staining (Fig. 1). As for ADH none; 1, weak; 2, moderate; and 3, strong) and the propor- group, 40.0% (8/20) of cases demonstrated nuclear tion of positive tumor cells (proportion score: less than 5% positivity and 60.0% (12/20) showed both nuclear and positive cells were scored as 0; 5 to 25% as 1; 26 to 50% as cytoplasmic positivity. Nevertheless, solely cytoplasmic 2; 51 to 75% as 3; greater than 75% as 4) according to previ- staining was not found in these two groups. In compari- ously described scoring methods with a slight modification son, exclusive N-FOXP1 expression was present only in [9, 32, 33]. These two scores were then multiplied to yield 12.2% (5/41) of DCIS cases, while both N- and C-FOXP1 the final score. A final score of ≥3was definedas positive. expression was observed in the majority of this group Fig. 1 Representative cases of FOXP1 expression in UDH and ADH. FOXP1 positive staining was located in the nuclei of ductal cells in UDH (a ×400). In ADH, FOXP1 positivity was observed in the nuclei of tumor cells (b ×400) or in both the nuclei and the cytoplasm (c ×400) Yu et al. Diagnostic Pathology (2018) 13:36 Page 4 of 9 (34/41, 82.9%), and the remaining 2 cases (4.9%) revealed with 52.9% (9/17) of C-FOXP1-negative ones, and the exclusive cytoplasmic labeling (Fig. 2). The FOXP1 ex- difference was statistically significant (p = 0.007, Fig. 4). pression pattern in IDC samples varied. In this group, ex- Nevertheless, there was no significant relevance between clusive cytoplasmic staining (26/83, 31.3%) was more C-FOXP1 expression and patient age, tumor size, grade, frequently observed than solely nuclear staining (9/83, tumor stage, nodal status, distant metastasis or HER2 10.8%), both nuclear and cytoplasmic staining accounted expression (all p > 0.05). pAKT, pmTOR, p4E-BP1 and for 48.2% (40/83) of cases and complete loss of expression p-p70S6K, as key members in the AKT pathway, was was observed in 8 cases (9.6%) (Fig. 3). Moreover, exclu- expressed in 72.3% (60/83), 74.7% (62/83), 69.9% (58/83) sive cytoplasmic FOXP1 expression was more common in and 73.5% (61/83) of IDC cases in the current series, IDC than that in DCIS, ADH and UDH (31.3% vs 4.9, 0 respectively. Interestingly, calpain II expression was and 0%). The FOXP1 expression patterns were signifi- statistically associated with the expression of pAKT cantly different in UDH, ADH, DCIS and IDC (p <0.001, (p = 0.029), pmTOR (p = 0.011), p4E-BP1 (p < 0.001) Table 1). Even within a single case, different lesions and p-p70S6K (p = 0.003, Table 3). showed diverse FOXP1 staining patterns. For example, clear nuclear and cytoplasmic staining was observed in Correlation between C-FOXP1 expression and the survival the DCIS region, while solely nuclear staining was seen in of patients with breast IDC the epithelium of adjacent benign ducts. Among the 83 patients with breast IDC, the follow-up period ranged from 2 to 146 months (median, 67 months), Correlation between C-FOXP1 expression and and there were 32 relapses and 21 deaths. Twenty out of clinicopathological variables in breast IDC 66 C-FOXP1-positive patients died of disease, and 28 had The identified associations between C-FOXP1 expres- relapses, whereas 1 and 4 out of 17 C-FOXP1-negative sion and histopathological and clinical variables in IDC patients died or relapsed, respectively. The 10-year OS are shown in Table 2. ER staining was observed in 47.0% and DFS rates of the C-FOXP1-positive patients were 60.5 (31/66) of C-FOXP1-positive staining cases, which was and 48.7%, respectively, both of which were lower than lower than that in C-FOXP1-negative cases (13/17, that of the C-FOXP1-negative patients (93.3, 75.3%). The 76.5%) (p = 0.030). Calpain II expression was found in OS curve showed that C-FOXP1 status had an impact on 83.3% (55/66) of C-FOXP1 positive cases, compared outcome (p = 0.045). The DFS curve suggested that Fig. 2 FOXP1 expression patterns in DCIS. FOXP1 immunostaining was observed in the nuclei of tumor cells (a ×200, b ×400) or both the nuclei and cytoplasm (c ×200, d ×400) Yu et al. Diagnostic Pathology (2018) 13:36 Page 5 of 9 Fig. 3 FOXP1 expression patterns in IDC. The FOXP1 protein expression patterns in IDC tumor cells ranged from exclusive cytoplasmic (a, TMA; b ×400) to mixed nuclear/cytoplasmic (c, TMA; d ×400) and to exclusive nuclear (e, TMA; f ×400) patients with C-FOXP1-negative IDCs demonstrated lon- Discussion ger DFS than those with C-FOXP1-positive disease, but Although N-FOXP1 expression in breast cancer has the result did not reach statistical significance (p =0.152). been documented in several studies, the expression Survival curves stratified for C-FOXP1 expression are patterns of FOXP1 protein at different stages of breast shown in Fig. 5. cancer progression, including DCIS and IDC, and in Table 1 FOXP1 protein expression patterns in different breast lesions Breast lesions Total FOXP1 expression patterns number n = 164 Exclusive nuclear Both nuclear and cytoplasmic Exclusive cytoplasmic Complete loss of expression n (%) expression n (%) expression n (%) expression n (%) UDH 20 17 (85.0) 3 (15.0) 0 (0) 0 (0) ADH 20 8 (40.0) 12 (60.0) 0 (0) 0 (0) DCIS 41 5 (12.2) 34 (82.9) 2 (4.9) 0 (0) IDC 83 9 (10.8) 40 (48.2) 26 (31.3) 8 (9.6) Yu et al. Diagnostic Pathology (2018) 13:36 Page 6 of 9 Table 2 The correlation between cytoplasmic FOXP1 expression and clinicopathological parameters in IDC cases Clinicopathological Cytoplasmic FOXP1 expression parameters Positive (%) n = 66 Negative (%) n =17 P value ER 0.030 Positive 31 (47.0) 13 (76.5) Negative 35 (53.0) 4 (23.5) HER2 0.443 Positive 22 (33.3) 4 (23.5) Negative 44 (66.7) 13 (76.5) Calpain II 0.007 Positive 55 (83.3) 9 (52.9) Negative 11 (16.7) 8 (47.1) pAKT 0.863 Fig. 4 Calpain II-positive staining was found in the cytoplasm of IDC Positive 48 (72.7) 12 (70.6) tumor cells (× 400) Negative 18 (27.3) 5 (29.4) pmTOR 0.093 ADH and UDH lesions, have not yet been clearly dem- Positive 52 (78.8) 10 (58.8) onstrated. In the current study, heterogeneous FOXP1 Negative 14 (21.2) 7 (41.2) expression patterns were observed in the above-mentioned p4E-BP1 0.607 cases. While FOXP1 staining was predominantly localized Positive 47 (71.2) 11 (64.7) in the nuclei in UDH, the FOXP1 nuclear distribution grad- Negative 19 (28.8) 6 (35.3) ually decreased from ADH, DCIS to IDC, and the cytoplas- mic staining increased. These results were consistent with p-p70S6K 0.363 the previous reported heterogeneous expression pattern of Positive 50 (75.8) 11 (64.7) FOXP1, in terms of the proportion of positive cells, the Negative 16 (24.2) 6 (35.3) staining intensity, and subcellular localization [3, 36]. Our Stage 0.562 observations strongly indicated that FOXP1 expression Stage I 7 (10.6) 1 (5.9) might shift from the nucleus to the cytoplasm during breast Stage II 38 (57.6) 13 (76.5) tumorigenesis, and therefore, cytoplasmic mislocalization of FOXP1 is suggested play an important role in breast can- Stage III 21 (31.8) 3 (17.6) cer progression. Similarly, subcellular localization has been Grade 0.325 suggested to play a distinct role in the pathogenesis of Grade I 16 (24.2) 3 (17.6) endometrial cancer [9]. Banham et al. also revealed that Grade II 38 (57.6) 9 (52.9) FOXP1 protein expression levels and compartmentalization Grade III 12 (18.2) 5 (29.4) varied depending on the cancer stage, although their sam- Nodal status 0.090 ple sizes for each tumor were quite small [3]. Studies on the mechanisms of FOXP1 subcellular relo- Positive 49 (74.2) 9 (52.9) calization in breast cancer are very few. Calpain II has Negative 17 (25.8) 8 (47.1) been implicated in mediating cell differentiation, necro- Distant metastasis 0.230 sis, migration, and metastasis [19, 22]. Several studies, Positive 26 (39.4) 4 (23.5) although limited, have investigated the aberrant expres- Negative 40 (60.6) 13 (76.5) sion and role of calpain II in breast cancer [19, 21, 25, Tumor size 0.907 30, 37–40]. High calpain II expression has been estab- lished in triple-negative and basal-like IDC and calpain ≦4 cm 30 (45.5) 8 (47.1) II might promote breast cancer cell proliferation through > 4 cm 36 (54.5) 9 (52.9) the AKT signaling pathway [21, 30]. Calpain-mediated Age 0.762 cleavage of β-catenin and E-cadherin may lead to aber- ≦55 yrs 44 (66.7) 12 (70.6) rant stabilization of the proteins and promote tum- > 55 yrs 22 (33.3) 5 (29.4) origenesis in breast cancer cells [38, 39]. In addition, Ho Statistically significant p values are in bold et al. suggested that the FOXO3a subcellular location was skewed toward nuclear localization in calpain Yu et al. Diagnostic Pathology (2018) 13:36 Page 7 of 9 Table 3 The correlation between calpain II expression and important finding in the current study was that clinicopathological parameters in IDC cases C-FOXP1 expression was remarkably associated with Clinicopathological Calpain II expression calpain II in IDC. Moreover, in IDC samples in our parameters series, calpain II was strongly correlated with the im- Positive (%) n = 64 Negative (%) n =19 P value portant molecules in AKT pathway, including pAKT, ER 0.580 mTOR, p4E-BP1 and p-p70S6K. The PI3K/AKT/p70S6K Positive 35 (54.7) 9 (47.4) signaling pathway, which has been reported to be in- Negative 29 (45.3) 10 (52.6) volved in the nucleus-cytoplasm shuttling of FOXO1, HER2 0.597 another forkhead protein family member, was previously Positive 21 (32.8) 5 (26.3) shown to participate in FOXP1 regulation in breast can- Negative 43 (67.2) 14 (73.7) cer [30, 41]. Taken together, we speculate that calpain II might play an important role in the subcellular regula- pAKT 0.029 tion of FOXP1, and the AKT pathway might be involved Positive 50 (78.1) 10 (52.6) in this process. Further investigations are merited to Negative 14 (21.9) 9 (47.4) confirm this hypothesis and thoroughly explore the pmTOR 0.011 underlying mechanisms. Positive 52 (81.3) 10 (52.6) While N-FOXP1 was positively associated with ERα as Negative 12 (18.8) 9 (47.4) well as ERβ expression in breast cancer according to the previous studies [8, 11, 12, 30, 42], the clinicopathological p4E-BP1 < 0.001 relevance of C-FOXP1 positivity in IDC has not been ad- Positive 52 (81.3) 6 (31.6) dressed until now. For the first time, we identified an in- Negative 12 (18.8) 13 (68.4) verse correlation between C-FOXP1 expression and ER p-p70S6K 0.003 expression in IDC. Our results are in line with those of a Positive 52 (81.3) 9 (47.4) previous report by Giatromanolaki et al. concerning endo- Negative 12 (18.8) 10 (52.6) metrial carcinoma [9]. They demonstrated that loss of ERα expression was a frequent event in cases with Stage 0.773 C-FOXP1 expression or loss of FOXP1 expression in Stage I 7 (10.9) 1 (5.3) endometrial carcinoma. Given that nucleus-cytoplasm Stage II 37(57.8) 14 (73.7) shuttling might be an important event in the carcinogen- Stage III 20 (31.3) 4 (21.2) esis, the interaction between ER and C-FOXP1 expression Grade 0.568 might be more clinically significant than that originally Grade I 16 (25.0) 3 (15.8) established between ER and N-FOXP1, and its biological significance should be further explored [8, 11, 30, 43, 44]. Grade II 35 (54.7) 12 (63.2) Previous studies have demonstrated that loss of FOXP1 Grade III 13 (20.3) 4 (21.1) expression is associated with a poor prognosis in primary Nodal status 0.876 invasive and familial breast cancer [11]. For example, both Positive 45 (70.3) 13 (68.4) Fox et al. and Ijichi et al. indicated that FOXP1 immunore- Negative 19 (29.7) 6 (31.6) activity predicted better relapse-free survival but not OS in Distant metastasis 0.122 breast cancer patients [6, 8]. Moreover, FOXP1 immunore- activity may predict a favorable prognosis for breast cancer Positive 26 (40.6) 4 (21.1) patients treated with tamoxifen [6, 42, 44]. However, in pre- Negative 38 (59.4) 15 (78.9) vious studies, the FOXP1 protein was either located in the Tumor size 0.013 nuclei, or its subcellular location was not specified; none- ≦4 cm 34 (53.1) 4 (21.1) theless, cytoplasmic FOXP1 localization might play a large > 4 cm 30 (46.9) 15 (78.9) role in cancer cell biology because nuclear expression is Age 0.653 characteristic of normal breast tissues [9]. Therefore, the prognostic impact of C-FOXP1 overexpression in IDC pa- ≦55 yrs 44 (68.8) 12 (63.2) tients might be meaningful. Our results indicate for the first > 55 yrs 20 (31.3) 7 (36.8) time that C-FOXP1 immunoreactivity is associated with an Statistically significant p values are in bold unfavorable OS and slightly inferior DFS in patients with breast IDC. Similarly, exclusive C-FOXP1 expression in II-deficient cells [20]. To date, we are not aware of any early endothelial carcinoma has been linked with deep literature establishing the relevance between calpain II myometrial invasion and conferred a slightly worse out- and FOXP1 protein in breast cancer. An unexpected but come, despite an insignificant difference [9]. Hu et al. Yu et al. Diagnostic Pathology (2018) 13:36 Page 8 of 9 Fig. 5 Kaplan-Meier survival curves of patients with IDC according to C-FOXP1 expression. Patients with positive C-FOXP1 immunoreactivity showed inferior OS (a) and DFS (b) compared with C-FOXP1-negative patients, although the difference in DFS was not statistically significant demonstrated that increased cytoplasmic FOXP1 expres- Availability of data and materials Please contact author (xyzhou100@163.com) for data requests. sion was correlated with increased tumor grade but was not significantly associated with chemotherapy resistance Authors’ contributions and prognosis [32]. Our results provide reliable evidence BHY and XYZ conceived and designed the experiments. BHY performed the experiments. BHY and BZL reviewed the slides and analyzed the data. BHY regarding the prognostic importance of C-FOXP1 overex- wrote the manuscript. DRS, XYZ, WTY revised the paper and approved the pression in breast cancer, which should be further con- final version of the manuscript. All authors read and approved the final firmed with amuchlargercaseseries. manuscript. Ethics approval and consent to participate Conclusions This study was approved by the Institutional Review Board of Fudan University In summary, cytoplasmic relocalization of the FOXP1 Shanghai Cancer Center (Shanghai Cancer Center Ethical Committee, permission number 050432-4-1212B). Additional patient consent for this retro- protein is a frequent event in breast cancer. For the first spective study was not required. time, we found that C-FOXP1 expression was dramatic- ally associated with ER expression and correlated with Competing interests The authors declare that they have no competing interests. reduced OS in patients with breast IDC. Our results in- dicated that C-FOXP1 might be important in both the pathogenesis and prognosis of breast cancer patients. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published Another noteworthy finding was that calpain II might be maps and institutional affiliations. involved in FOXP1 trafficking from the nucleus to the cytoplasm, which might be mediated by the AKT path- Author details Department of Pathology, Fudan University Shanghai Cancer Center, way. Further investigations are needed to better under- Dong-an Road 270, Xuhui District, Shanghai 200032, China. Department of stand the biological role of FOXP1 expression in breast Oncology, Shanghai Medical College, Fudan University, Shanghai, China. cancer development and progression and to provide bet- Department of Pathology, the Second Affiliated Hospital of Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China. ter strategies for prognosis prediction and therapeutic intervention in breast cancer. Received: 12 January 2018 Accepted: 22 May 2018 Abbreviations C-FOXP1: Cytoplasmic FOXP1; DCIS: Ductal carcinoma in situ; DFS: Disease-free References survival; DLBCL: Diffuse large B-cell lymphoma; ER: Estrogen receptor; 1. Xu T, He BS, Liu XX, et al. The predictive and prognostic role of stromal FOXP1: Forkhead box protein P1; HER2: Human epidermal growth factor tumor-infiltrating lymphocytes in HER2-positive breast cancer with receptor-2; IDC: Invasive ductal carcinoma; MALT: Mucosa associated lymphoid trastuzumab-based treatment: a meta-analysis and systematic review. 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Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

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Copyright © 2018 by The Author(s).
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Medicine & Public Health; Pathology
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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: xyzhou100@163.com Department of Pathology, Fudan University Shanghai Cancer Center, Dong-an Road 270, Xuhui District, Shanghai 200032, China Department 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 entlyreviewedbytwo of theauthors (BHY andBZL) 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). Oncology (ASCO)/College of American Pathologists Four-micrometer-thick sections were then cut from the (CAP) guideline [34, 35]. Staining was considered TMA blocks for routine hematoxylin and eosin (H&E) positive for ER when nuclear staining was present in staining and immunohistochemical procedures. The more than 1% of the tumor cells. Immunohistochemistry H&E-stained sections were used to verify the adequate for HER2 as 3+ was defined as positive. For cases of representation of the diagnostic biopsies. HER2 IHC 2+, Abbott-Vysis HER2 FISH assay was employed to further confirm the status of HER2 gene amplification. Immunohistochemical staining Following deparaffinization and heat-mediated antigen re- Statistical analysis trieval, immunohistochemical staining was carried out All statistical analyses were performed using the SPSS soft- using an Envision system (DAKO, Glostrup, Denmark) ware package (SPSS version 19.0, SPSS Inc., Chicago, IL, with primary antibodies against FOXP1 (JC12, AbD Sero- USA). Categorical variables were compared with a χ test, tec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), cal- and measurement data were analyzed using Pearson correl- pain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, ation analysis. Overall survival (OS) was defined as the Roche Tucson), pAKT (736E11, Cell signaling, Danvers, interval from the initial diagnosis to the time of death or MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 the last contact. Surviving patients were censored at the (53H11, Cell signaling) and p-p70S6K (49D7, Cell signal- last known date of contact. Disease-free survival (DFS) ing). The stained sections were then counterstained with was determined according to the time from diagnosis to hematoxylin. Appropriate positive and negative controls the time of recurrence or the last contact. Patient survival were carried out simultaneously for all stains. was estimated using the Kaplan-Meier method and was The immunostaining results were reviewed by 2 inde- compared by means of a log-rank test. All p-values were pendent qualified pathologists. Nuclear and cytoplasmic two sided, and a p-value < 0.05 was considered statistically tumor cell staining for FOXP1 protein was analyzed separ- significant. ately. FOXP1 nuclear expression was scored using the following system: negative = 0; weak/focal staining = 1; Results strong focal/wide spread moderate staining = 2; or strong/ FOXP1 protein expression patterns in UDH, ADH, DCIS, widespread staining = 3. Tumors that scored 2 or 3 were and IDC of the breast considered positive for N-FOXP1 [8]. Scoring of C-FOXP1, Most UDH cases (17/20, 85%) showed uniform strong calpain II, pAKT, pmTOR, p4E-BP1, p-p70S6K were per- N-FOXP1 staining and the other 3 cases (15.0%) showed formed in terms of the staining intensity (intensity score: 0, both N- and C-FOXP1 staining (Fig. 1). As for ADH none; 1, weak; 2, moderate; and 3, strong) and the propor- group, 40.0% (8/20) of cases demonstrated nuclear tion of positive tumor cells (proportion score: less than 5% positivity and 60.0% (12/20) showed both nuclear and positive cells were scored as 0; 5 to 25% as 1; 26 to 50% as cytoplasmic positivity. Nevertheless, solely cytoplasmic 2; 51 to 75% as 3; greater than 75% as 4) according to previ- staining was not found in these two groups. In compari- ously described scoring methods with a slight modification son, exclusive N-FOXP1 expression was present only in [9, 32, 33]. These two scores were then multiplied to yield 12.2% (5/41) of DCIS cases, while both N- and C-FOXP1 the final score. A final score of ≥3was definedas positive. expression was observed in the majority of this group Fig. 1 Representative cases of FOXP1 expression in UDH and ADH. FOXP1 positive staining was located in the nuclei of ductal cells in UDH (a ×400). In ADH, FOXP1 positivity was observed in the nuclei of tumor cells (b ×400) or in both the nuclei and the cytoplasm (c ×400) Yu et al. Diagnostic Pathology (2018) 13:36 Page 4 of 9 (34/41, 82.9%), and the remaining 2 cases (4.9%) revealed with 52.9% (9/17) of C-FOXP1-negative ones, and the exclusive cytoplasmic labeling (Fig. 2). The FOXP1 ex- difference was statistically significant (p = 0.007, Fig. 4). pression pattern in IDC samples varied. In this group, ex- Nevertheless, there was no significant relevance between clusive cytoplasmic staining (26/83, 31.3%) was more C-FOXP1 expression and patient age, tumor size, grade, frequently observed than solely nuclear staining (9/83, tumor stage, nodal status, distant metastasis or HER2 10.8%), both nuclear and cytoplasmic staining accounted expression (all p > 0.05). pAKT, pmTOR, p4E-BP1 and for 48.2% (40/83) of cases and complete loss of expression p-p70S6K, as key members in the AKT pathway, was was observed in 8 cases (9.6%) (Fig. 3). Moreover, exclu- expressed in 72.3% (60/83), 74.7% (62/83), 69.9% (58/83) sive cytoplasmic FOXP1 expression was more common in and 73.5% (61/83) of IDC cases in the current series, IDC than that in DCIS, ADH and UDH (31.3% vs 4.9, 0 respectively. Interestingly, calpain II expression was and 0%). The FOXP1 expression patterns were signifi- statistically associated with the expression of pAKT cantly different in UDH, ADH, DCIS and IDC (p <0.001, (p = 0.029), pmTOR (p = 0.011), p4E-BP1 (p < 0.001) Table 1). Even within a single case, different lesions and p-p70S6K (p = 0.003, Table 3). showed diverse FOXP1 staining patterns. For example, clear nuclear and cytoplasmic staining was observed in Correlation between C-FOXP1 expression and the survival the DCIS region, while solely nuclear staining was seen in of patients with breast IDC the epithelium of adjacent benign ducts. Among the 83 patients with breast IDC, the follow-up period ranged from 2 to 146 months (median, 67 months), Correlation between C-FOXP1 expression and and there were 32 relapses and 21 deaths. Twenty out of clinicopathological variables in breast IDC 66 C-FOXP1-positive patients died of disease, and 28 had The identified associations between C-FOXP1 expres- relapses, whereas 1 and 4 out of 17 C-FOXP1-negative sion and histopathological and clinical variables in IDC patients died or relapsed, respectively. The 10-year OS are shown in Table 2. ER staining was observed in 47.0% and DFS rates of the C-FOXP1-positive patients were 60.5 (31/66) of C-FOXP1-positive staining cases, which was and 48.7%, respectively, both of which were lower than lower than that in C-FOXP1-negative cases (13/17, that of the C-FOXP1-negative patients (93.3, 75.3%). The 76.5%) (p = 0.030). Calpain II expression was found in OS curve showed that C-FOXP1 status had an impact on 83.3% (55/66) of C-FOXP1 positive cases, compared outcome (p = 0.045). The DFS curve suggested that Fig. 2 FOXP1 expression patterns in DCIS. FOXP1 immunostaining was observed in the nuclei of tumor cells (a ×200, b ×400) or both the nuclei and cytoplasm (c ×200, d ×400) Yu et al. Diagnostic Pathology (2018) 13:36 Page 5 of 9 Fig. 3 FOXP1 expression patterns in IDC. The FOXP1 protein expression patterns in IDC tumor cells ranged from exclusive cytoplasmic (a, TMA; b ×400) to mixed nuclear/cytoplasmic (c, TMA; d ×400) and to exclusive nuclear (e, TMA; f ×400) patients with C-FOXP1-negative IDCs demonstrated lon- Discussion ger DFS than those with C-FOXP1-positive disease, but Although N-FOXP1 expression in breast cancer has the result did not reach statistical significance (p =0.152). been documented in several studies, the expression Survival curves stratified for C-FOXP1 expression are patterns of FOXP1 protein at different stages of breast shown in Fig. 5. cancer progression, including DCIS and IDC, and in Table 1 FOXP1 protein expression patterns in different breast lesions Breast lesions Total FOXP1 expression patterns number n = 164 Exclusive nuclear Both nuclear and cytoplasmic Exclusive cytoplasmic Complete loss of expression n (%) expression n (%) expression n (%) expression n (%) UDH 20 17 (85.0) 3 (15.0) 0 (0) 0 (0) ADH 20 8 (40.0) 12 (60.0) 0 (0) 0 (0) DCIS 41 5 (12.2) 34 (82.9) 2 (4.9) 0 (0) IDC 83 9 (10.8) 40 (48.2) 26 (31.3) 8 (9.6) Yu et al. Diagnostic Pathology (2018) 13:36 Page 6 of 9 Table 2 The correlation between cytoplasmic FOXP1 expression and clinicopathological parameters in IDC cases Clinicopathological Cytoplasmic FOXP1 expression parameters Positive (%) n = 66 Negative (%) n =17 P value ER 0.030 Positive 31 (47.0) 13 (76.5) Negative 35 (53.0) 4 (23.5) HER2 0.443 Positive 22 (33.3) 4 (23.5) Negative 44 (66.7) 13 (76.5) Calpain II 0.007 Positive 55 (83.3) 9 (52.9) Negative 11 (16.7) 8 (47.1) pAKT 0.863 Fig. 4 Calpain II-positive staining was found in the cytoplasm of IDC Positive 48 (72.7) 12 (70.6) tumor cells (× 400) Negative 18 (27.3) 5 (29.4) pmTOR 0.093 ADH and UDH lesions, have not yet been clearly dem- Positive 52 (78.8) 10 (58.8) onstrated. In the current study, heterogeneous FOXP1 Negative 14 (21.2) 7 (41.2) expression patterns were observed in the above-mentioned p4E-BP1 0.607 cases. While FOXP1 staining was predominantly localized Positive 47 (71.2) 11 (64.7) in the nuclei in UDH, the FOXP1 nuclear distribution grad- Negative 19 (28.8) 6 (35.3) ually decreased from ADH, DCIS to IDC, and the cytoplas- mic staining increased. These results were consistent with p-p70S6K 0.363 the previous reported heterogeneous expression pattern of Positive 50 (75.8) 11 (64.7) FOXP1, in terms of the proportion of positive cells, the Negative 16 (24.2) 6 (35.3) staining intensity, and subcellular localization [3, 36]. Our Stage 0.562 observations strongly indicated that FOXP1 expression Stage I 7 (10.6) 1 (5.9) might shift from the nucleus to the cytoplasm during breast Stage II 38 (57.6) 13 (76.5) tumorigenesis, and therefore, cytoplasmic mislocalization of FOXP1 is suggested play an important role in breast can- Stage III 21 (31.8) 3 (17.6) cer progression. Similarly, subcellular localization has been Grade 0.325 suggested to play a distinct role in the pathogenesis of Grade I 16 (24.2) 3 (17.6) endometrial cancer [9]. Banham et al. also revealed that Grade II 38 (57.6) 9 (52.9) FOXP1 protein expression levels and compartmentalization Grade III 12 (18.2) 5 (29.4) varied depending on the cancer stage, although their sam- Nodal status 0.090 ple sizes for each tumor were quite small [3]. Studies on the mechanisms of FOXP1 subcellular relo- Positive 49 (74.2) 9 (52.9) calization in breast cancer are very few. Calpain II has Negative 17 (25.8) 8 (47.1) been implicated in mediating cell differentiation, necro- Distant metastasis 0.230 sis, migration, and metastasis [19, 22]. Several studies, Positive 26 (39.4) 4 (23.5) although limited, have investigated the aberrant expres- Negative 40 (60.6) 13 (76.5) sion and role of calpain II in breast cancer [19, 21, 25, Tumor size 0.907 30, 37–40]. High calpain II expression has been estab- lished in triple-negative and basal-like IDC and calpain ≦4 cm 30 (45.5) 8 (47.1) II might promote breast cancer cell proliferation through > 4 cm 36 (54.5) 9 (52.9) the AKT signaling pathway [21, 30]. Calpain-mediated Age 0.762 cleavage of β-catenin and E-cadherin may lead to aber- ≦55 yrs 44 (66.7) 12 (70.6) rant stabilization of the proteins and promote tum- > 55 yrs 22 (33.3) 5 (29.4) origenesis in breast cancer cells [38, 39]. In addition, Ho Statistically significant p values are in bold et al. suggested that the FOXO3a subcellular location was skewed toward nuclear localization in calpain Yu et al. Diagnostic Pathology (2018) 13:36 Page 7 of 9 Table 3 The correlation between calpain II expression and important finding in the current study was that clinicopathological parameters in IDC cases C-FOXP1 expression was remarkably associated with Clinicopathological Calpain II expression calpain II in IDC. Moreover, in IDC samples in our parameters series, calpain II was strongly correlated with the im- Positive (%) n = 64 Negative (%) n =19 P value portant molecules in AKT pathway, including pAKT, ER 0.580 mTOR, p4E-BP1 and p-p70S6K. The PI3K/AKT/p70S6K Positive 35 (54.7) 9 (47.4) signaling pathway, which has been reported to be in- Negative 29 (45.3) 10 (52.6) volved in the nucleus-cytoplasm shuttling of FOXO1, HER2 0.597 another forkhead protein family member, was previously Positive 21 (32.8) 5 (26.3) shown to participate in FOXP1 regulation in breast can- Negative 43 (67.2) 14 (73.7) cer [30, 41]. Taken together, we speculate that calpain II might play an important role in the subcellular regula- pAKT 0.029 tion of FOXP1, and the AKT pathway might be involved Positive 50 (78.1) 10 (52.6) in this process. Further investigations are merited to Negative 14 (21.9) 9 (47.4) confirm this hypothesis and thoroughly explore the pmTOR 0.011 underlying mechanisms. Positive 52 (81.3) 10 (52.6) While N-FOXP1 was positively associated with ERα as Negative 12 (18.8) 9 (47.4) well as ERβ expression in breast cancer according to the previous studies [8, 11, 12, 30, 42], the clinicopathological p4E-BP1 < 0.001 relevance of C-FOXP1 positivity in IDC has not been ad- Positive 52 (81.3) 6 (31.6) dressed until now. For the first time, we identified an in- Negative 12 (18.8) 13 (68.4) verse correlation between C-FOXP1 expression and ER p-p70S6K 0.003 expression in IDC. Our results are in line with those of a Positive 52 (81.3) 9 (47.4) previous report by Giatromanolaki et al. concerning endo- Negative 12 (18.8) 10 (52.6) metrial carcinoma [9]. They demonstrated that loss of ERα expression was a frequent event in cases with Stage 0.773 C-FOXP1 expression or loss of FOXP1 expression in Stage I 7 (10.9) 1 (5.3) endometrial carcinoma. Given that nucleus-cytoplasm Stage II 37(57.8) 14 (73.7) shuttling might be an important event in the carcinogen- Stage III 20 (31.3) 4 (21.2) esis, the interaction between ER and C-FOXP1 expression Grade 0.568 might be more clinically significant than that originally Grade I 16 (25.0) 3 (15.8) established between ER and N-FOXP1, and its biological significance should be further explored [8, 11, 30, 43, 44]. Grade II 35 (54.7) 12 (63.2) Previous studies have demonstrated that loss of FOXP1 Grade III 13 (20.3) 4 (21.1) expression is associated with a poor prognosis in primary Nodal status 0.876 invasive and familial breast cancer [11]. For example, both Positive 45 (70.3) 13 (68.4) Fox et al. and Ijichi et al. indicated that FOXP1 immunore- Negative 19 (29.7) 6 (31.6) activity predicted better relapse-free survival but not OS in Distant metastasis 0.122 breast cancer patients [6, 8]. Moreover, FOXP1 immunore- activity may predict a favorable prognosis for breast cancer Positive 26 (40.6) 4 (21.1) patients treated with tamoxifen [6, 42, 44]. However, in pre- Negative 38 (59.4) 15 (78.9) vious studies, the FOXP1 protein was either located in the Tumor size 0.013 nuclei, or its subcellular location was not specified; none- ≦4 cm 34 (53.1) 4 (21.1) theless, cytoplasmic FOXP1 localization might play a large > 4 cm 30 (46.9) 15 (78.9) role in cancer cell biology because nuclear expression is Age 0.653 characteristic of normal breast tissues [9]. Therefore, the prognostic impact of C-FOXP1 overexpression in IDC pa- ≦55 yrs 44 (68.8) 12 (63.2) tients might be meaningful. Our results indicate for the first > 55 yrs 20 (31.3) 7 (36.8) time that C-FOXP1 immunoreactivity is associated with an Statistically significant p values are in bold unfavorable OS and slightly inferior DFS in patients with breast IDC. Similarly, exclusive C-FOXP1 expression in II-deficient cells [20]. To date, we are not aware of any early endothelial carcinoma has been linked with deep literature establishing the relevance between calpain II myometrial invasion and conferred a slightly worse out- and FOXP1 protein in breast cancer. An unexpected but come, despite an insignificant difference [9]. Hu et al. Yu et al. Diagnostic Pathology (2018) 13:36 Page 8 of 9 Fig. 5 Kaplan-Meier survival curves of patients with IDC according to C-FOXP1 expression. Patients with positive C-FOXP1 immunoreactivity showed inferior OS (a) and DFS (b) compared with C-FOXP1-negative patients, although the difference in DFS was not statistically significant demonstrated that increased cytoplasmic FOXP1 expres- Availability of data and materials Please contact author (xyzhou100@163.com) for data requests. sion was correlated with increased tumor grade but was not significantly associated with chemotherapy resistance Authors’ contributions and prognosis [32]. Our results provide reliable evidence BHY and XYZ conceived and designed the experiments. BHY performed the experiments. BHY and BZL reviewed the slides and analyzed the data. BHY regarding the prognostic importance of C-FOXP1 overex- wrote the manuscript. DRS, XYZ, WTY revised the paper and approved the pression in breast cancer, which should be further con- final version of the manuscript. All authors read and approved the final firmed with amuchlargercaseseries. manuscript. Ethics approval and consent to participate Conclusions This study was approved by the Institutional Review Board of Fudan University In summary, cytoplasmic relocalization of the FOXP1 Shanghai Cancer Center (Shanghai Cancer Center Ethical Committee, permission number 050432-4-1212B). Additional patient consent for this retro- protein is a frequent event in breast cancer. For the first spective study was not required. time, we found that C-FOXP1 expression was dramatic- ally associated with ER expression and correlated with Competing interests The authors declare that they have no competing interests. reduced OS in patients with breast IDC. Our results in- dicated that C-FOXP1 might be important in both the pathogenesis and prognosis of breast cancer patients. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published Another noteworthy finding was that calpain II might be maps and institutional affiliations. involved in FOXP1 trafficking from the nucleus to the cytoplasm, which might be mediated by the AKT path- Author details Department of Pathology, Fudan University Shanghai Cancer Center, way. Further investigations are needed to better under- Dong-an Road 270, Xuhui District, Shanghai 200032, China. Department of stand the biological role of FOXP1 expression in breast Oncology, Shanghai Medical College, Fudan University, Shanghai, China. cancer development and progression and to provide bet- Department of Pathology, the Second Affiliated Hospital of Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China. ter strategies for prognosis prediction and therapeutic intervention in breast cancer. Received: 12 January 2018 Accepted: 22 May 2018 Abbreviations C-FOXP1: Cytoplasmic FOXP1; DCIS: Ductal carcinoma in situ; DFS: Disease-free References survival; DLBCL: Diffuse large B-cell lymphoma; ER: Estrogen receptor; 1. Xu T, He BS, Liu XX, et al. The predictive and prognostic role of stromal FOXP1: Forkhead box protein P1; HER2: Human epidermal growth factor tumor-infiltrating lymphocytes in HER2-positive breast cancer with receptor-2; IDC: Invasive ductal carcinoma; MALT: Mucosa associated lymphoid trastuzumab-based treatment: a meta-analysis and systematic review. 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Diagnostic Pathology – Springer Journals

Published: May 30, 2018

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