Skip to content

Advertisement

  • Research
  • Open Access

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

Diagnostic Pathology201813:36

https://doi.org/10.1186/s13000-018-0715-y

  • Received: 12 January 2018
  • Accepted: 22 May 2018
  • Published:

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

Background

Breast cancer is the most common female malignancy and also the second leading cause of cancer-related death among women worldwide [1]. However, its molecular pathogenesis is largely unknown, and clinically useful prognostic and predictive parameters, apart from human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER), progesterone receptor (PR) and lymph node status, are still insufficient, emphasizing the need for further investigating additional prognostic biomarkers and potential targets for selective therapies.

The forkhead box protein P1 (FOXP1) gene, locating on 3p14.1, is a member of the forkhead/winged helix transcription factor family, and the FOXP1 protein is widely expressed in normal tissues [25]. FOXP1 protein subcellular localization varies between different tissues. A predominant nuclear FOXP1 (N-FOXP1) distribution has been identified in the kidney, thyroid, cerebellum, tonsil, blood, thymus, spleen, skin, pancreas and colon, whereas cytoplasmic FOXP1 (C-FOXP1) labeling was observed in other epithelial tissues, such as the stomach [3]. Altered FOXP1 expression is also associated with various types of tumors [6]. For example, N-FOXP1 protein is up-regulated in diffuse large B-cell lymphoma (DLBCL) and extranodal marginal zone or mucosa-associated lymphoid tissue (MALT) lymphoma [7], while loss of N-FOXP1 expression characterizes malignancy in certain solid tumors, including endometrial and prostate tumors as well as familial and sporadic breast cancer [3, 810]. The presence of N-FOXP1 expression is correlated with ERα and/or ERβ reactivity in invasive breast cancers [8, 11, 12]. A correlation between N-FOXP1 and ERα has also been observed in endometrial adenocarcinoma [9]. Loss of FOXP1 nuclear expression is the most striking observation, and cytoplasmic expression is noted more frequently in endometrial adenocarcinoma according to the literature. However, to date, data regarding C-FOXP1 expression in breast cancer are limited, and its clinicopathological relevance, including its correlation with ER expression, has not been well illustrated.

The oncogenic functions of FOXP1 in tumors, such as DLBCL, MALT lymphoma, and hepatocellular and renal cell carcinoma, have been well documented [4, 13, 14]. On the other hand, FOXP1 might attenuate tumorigenicity to exert a tumor-suppressive effect in other tumors, such as neuroblastoma and prostate cancer [4, 1517]. Therefore, FOXP1 is associated with cancer patient prognosis in a context-dependent manner [4, 18]. Overall, FOXP1 positivity, with either nuclear or an unspecified distribution, is associated with favorable survival in patients with breast cancer [4, 8, 18]. Nevertheless, the prognostic value of C-FOXP1 expression in breast cancer patients has not been discussed in the literature.

The underlying mechanisms of the nucleocytoplasmic shuttling of FOXP1 in breast cancer are largely unknown. The calpains are a family of calcium-dependent cysteine proteases that function in a wide range of important cellular activities [19]. The ubiquitously expressed family members, μ-calpain (calpain I) and m-calpain (calpain II), are the most extensively studied calpains [20, 21]. Calpain II activity is subject to many forms of posttranslational control in vivo, including translocation from the cytosol to the membrane [22]. Calpains are implicated in the cleavage of several apoptosis-associated proteins, notably Bax, Bcl2, JNK and JUN, amongst others [19, 23], and are involved in the regulation of some cell cycle progression-associated proteins, such as p21, cyclin D1, and p27Kip1 [24, 25]. For example, calpains may cleave Bcl-2 and Bid and permit translocation of Bax and Bid to the mitochondria, amplifying the apoptotic signaling pathway in cancer cells [26, 27]. In addition, calpains can mediate p27Kip1 degradation, and nuclear export might be necessary for this process [24]. The PI3K/AKT/mTOR signaling pathway, including its downstream molecules p4E-BP1 and p-p70S6K, plays a crucial role in initiation and progression of breast tumorigenesis and drug resistance [28, 29]. Calpain II might promote breast cancer cell proliferation through the PI3K/AKT signaling pathway [30]. However, whether calpain II plays a role in FOXP1 regulation in breast cancer has not yet been documented.

Herein, we investigated both the cytoplasmic and nuclear expression of FOXP1 protein in cases of invasive ductal cancer (IDC) or ductal carcinoma in situ (DCIS), as well as in atypical ductal hyperplasia (ADH) and usual ductal hyperplasia (UDH) of the breast, and further analyzed the association of C-FOXP1 expression with ER, calpain II and other clinicopathological parameters in IDC, and also evaluated the prognostic value of C-FOXP1.

Methods

Patient selection and tissue microarray (TMA) construction

Altogether, 83 cases of IDC, 41 of DCIS, 20 of ADH, and 20 of UDH were retrieved from the archival files of the Department of Pathology, Fudan University Shanghai Cancer Center (Shanghai, China). The study was approved by the Institutional Review Board of Fudan University Shanghai Cancer Center (Shanghai Cancer Center Ethics Committee). H&E-stained sections for each case were independently reviewed by two of the authors (BHY and BZL) according to the criteria described in the World Health Organization classification of tumors of the breast [31].

Clinical data, including follow-up data, were available for all of the 83 IDC cases. For TMA construction, H&E-stained sections from each formalin-fixed paraffin-embedded block were first observed to define representative tumor cell-rich areas and then 2 representative 0.6 mm cores were obtained from each IDC case and inserted into a recipient paraffin block in a grid pattern using a tissue arrayer (Beecher Instruments, Silver Spring, MD, USA). Four-micrometer-thick sections were then cut from the TMA blocks for routine hematoxylin and eosin (H&E) staining and immunohistochemical procedures. The H&E-stained sections were used to verify the adequate representation of the diagnostic biopsies.

Immunohistochemical staining

Following deparaffinization and heat-mediated antigen retrieval, immunohistochemical staining was carried out using an Envision system (DAKO, Glostrup, Denmark) with primary antibodies against FOXP1 (JC12, AbD Serotec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), calpain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, Roche Tucson), pAKT (736E11, Cell signaling, Danvers, MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 (53H11, Cell signaling) and p-p70S6K (49D7, Cell signaling). The stained sections were then counterstained with hematoxylin. Appropriate positive and negative controls were carried out simultaneously for all stains.

The immunostaining results were reviewed by 2 independent qualified pathologists. Nuclear and cytoplasmic tumor cell staining for FOXP1 protein was analyzed separately. FOXP1 nuclear expression was scored using the following system: negative = 0; weak/focal staining = 1; strong focal/wide spread moderate staining = 2; or strong/widespread staining = 3. Tumors that scored 2 or 3 were considered positive for N-FOXP1 [8]. Scoring of C-FOXP1, calpain II, pAKT, pmTOR, p4E-BP1, p-p70S6K were performed in terms of the staining intensity (intensity score: 0, none; 1, weak; 2, moderate; and 3, strong) and the proportion of positive tumor cells (proportion score: less than 5% positive cells were scored as 0; 5 to 25% as 1; 26 to 50% as 2; 51 to 75% as 3; greater than 75% as 4) according to previously described scoring methods with a slight modification [9, 32, 33]. These two scores were then multiplied to yield the final score. A final score of ≥3 was defined as positive.

The status of ER, PR and HER2 were evaluated using the scoring criteria of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline [34, 35]. Staining was considered positive for ER when nuclear staining was present in more than 1% of the tumor cells. Immunohistochemistry for HER2 as 3+ was defined as positive. For cases of HER2 IHC 2+, Abbott-Vysis HER2 FISH assay was employed to further confirm the status of HER2 gene amplification.

Statistical analysis

All statistical analyses were performed using the SPSS software package (SPSS version 19.0, SPSS Inc., Chicago, IL, USA). Categorical variables were compared with a χ2 test, and measurement data were analyzed using Pearson correlation analysis. Overall survival (OS) was defined as the interval from the initial diagnosis to the time of death or the last contact. Surviving patients were censored at the last known date of contact. Disease-free survival (DFS) was determined according to the time from diagnosis to the time of recurrence or the last contact. Patient survival was estimated using the Kaplan-Meier method and was compared by means of a log-rank test. All p-values were two sided, and a p-value < 0.05 was considered statistically significant.

Results

FOXP1 protein expression patterns in UDH, ADH, DCIS, and IDC of the breast

Most UDH cases (17/20, 85%) showed uniform strong N-FOXP1 staining and the other 3 cases (15.0%) showed both N- and C-FOXP1 staining (Fig. 1). As for ADH group, 40.0% (8/20) of cases demonstrated nuclear positivity and 60.0% (12/20) showed both nuclear and cytoplasmic positivity. Nevertheless, solely cytoplasmic staining was not found in these two groups. In comparison, exclusive N-FOXP1 expression was present only in 12.2% (5/41) of DCIS cases, while both N- and C-FOXP1 expression was observed in the majority of this group (34/41, 82.9%), and the remaining 2 cases (4.9%) revealed exclusive cytoplasmic labeling (Fig. 2). The FOXP1 expression pattern in IDC samples varied. In this group, exclusive cytoplasmic staining (26/83, 31.3%) was more frequently observed than solely nuclear staining (9/83, 10.8%), both nuclear and cytoplasmic staining accounted for 48.2% (40/83) of cases and complete loss of expression was observed in 8 cases (9.6%) (Fig. 3). Moreover, exclusive cytoplasmic FOXP1 expression was more common in IDC than that in DCIS, ADH and UDH (31.3% vs 4.9, 0 and 0%). The FOXP1 expression patterns were significantly different in UDH, ADH, DCIS and IDC (p < 0.001, Table 1). Even within a single case, different lesions showed diverse FOXP1 staining patterns. For example, clear nuclear and cytoplasmic staining was observed in the DCIS region, while solely nuclear staining was seen in the epithelium of adjacent benign ducts.
Fig. 1
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)

Fig. 2
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)

Fig. 3
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)

Table 1

FOXP1 protein expression patterns in different breast lesions

Breast lesions

Total number n = 164

FOXP1 expression patterns

Exclusive nuclear expression n (%)

Both nuclear and cytoplasmic expression n (%)

Exclusive cytoplasmic expression n (%)

Complete loss of 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)

Correlation between C-FOXP1 expression and clinicopathological variables in breast IDC

The identified associations between C-FOXP1 expression and histopathological and clinical variables in IDC are shown in Table 2. ER staining was observed in 47.0% (31/66) of C-FOXP1-positive staining cases, which was lower than that in C-FOXP1-negative cases (13/17, 76.5%) (p = 0.030). Calpain II expression was found in 83.3% (55/66) of C-FOXP1 positive cases, compared with 52.9% (9/17) of C-FOXP1-negative ones, and the difference was statistically significant (p = 0.007, Fig. 4). Nevertheless, there was no significant relevance between C-FOXP1 expression and patient age, tumor size, grade, tumor stage, nodal status, distant metastasis or HER2 expression (all p > 0.05). pAKT, pmTOR, p4E-BP1 and p-p70S6K, as key members in the AKT pathway, was expressed in 72.3% (60/83), 74.7% (62/83), 69.9% (58/83) and 73.5% (61/83) of IDC cases in the current series, respectively. Interestingly, calpain II expression was statistically associated with the expression of pAKT (p = 0.029), pmTOR (p = 0.011), p4E-BP1 (p < 0.001) and p-p70S6K (p = 0.003, Table 3).
Table 2

The correlation between cytoplasmic FOXP1 expression and clinicopathological parameters in IDC cases

Clinicopathological parameters

Cytoplasmic FOXP1 expression

Positive (%) n = 66

Negative (%) n = 17

P value

ER

  

0.030 a

 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 a

 Positive

55 (83.3)

9 (52.9)

 

 Negative

11 (16.7)

8 (47.1)

 

pAKT

  

0.863

 Positive

48 (72.7)

12 (70.6)

 

 Negative

18 (27.3)

5 (29.4)

 

pmTOR

  

0.093

 Positive

52 (78.8)

10 (58.8)

 

 Negative

14 (21.2)

7 (41.2)

 

p4E-BP1

  

0.607

 Positive

47 (71.2)

11 (64.7)

 

 Negative

19 (28.8)

6 (35.3)

 

p-p70S6K

  

0.363

 Positive

50 (75.8)

11 (64.7)

 

 Negative

16 (24.2)

6 (35.3)

 

Stage

  

0.562

 Stage I

7 (10.6)

1 (5.9)

 

 Stage II

38 (57.6)

13 (76.5)

 

 Stage III

21 (31.8)

3 (17.6)

 

Grade

  

0.325

 Grade I

16 (24.2)

3 (17.6)

 

 Grade II

38 (57.6)

9 (52.9)

 

 Grade III

12 (18.2)

5 (29.4)

 

Nodal status

  

0.090

 Positive

49 (74.2)

9 (52.9)

 

 Negative

17 (25.8)

8 (47.1)

 

Distant metastasis

  

0.230

 Positive

26 (39.4)

4 (23.5)

 

 Negative

40 (60.6)

13 (76.5)

 

Tumor size

  

0.907

4 cm

30 (45.5)

8 (47.1)

 

 > 4 cm

36 (54.5)

9 (52.9)

 

Age

  

0.762

55 yrs

44 (66.7)

12 (70.6)

 

 > 55 yrs

22 (33.3)

5 (29.4)

 

aStatistically significant p values are in bold

Fig. 4
Fig. 4

Calpain II-positive staining was found in the cytoplasm of IDC tumor cells (× 400)

Table 3

The correlation between calpain II expression and clinicopathological parameters in IDC cases

Clinicopathological parameters

Calpain II expression

Positive (%) n = 64

Negative (%) n = 19

P value

ER

  

0.580

 Positive

35 (54.7)

9 (47.4)

 

 Negative

29 (45.3)

10 (52.6)

 

HER2

  

0.597

 Positive

21 (32.8)

5 (26.3)

 

 Negative

43 (67.2)

14 (73.7)

 

pAKT

  

0.029 a

 Positive

50 (78.1)

10 (52.6)

 

 Negative

14 (21.9)

9 (47.4)

 

pmTOR

  

0.011 a

 Positive

52 (81.3)

10 (52.6)

 

 Negative

12 (18.8)

9 (47.4)

 

p4E-BP1

  

< 0.001 a

 Positive

52 (81.3)

6 (31.6)

 

 Negative

12 (18.8)

13 (68.4)

 

p-p70S6K

  

0.003 a

 Positive

52 (81.3)

9 (47.4)

 

 Negative

12 (18.8)

10 (52.6)

 

Stage

  

0.773

 Stage I

7 (10.9)

1 (5.3)

 

 Stage II

37(57.8)

14 (73.7)

 

 Stage III

20 (31.3)

4 (21.2)

 

Grade

  

0.568

 Grade I

16 (25.0)

3 (15.8)

 

 Grade II

35 (54.7)

12 (63.2)

 

 Grade III

13 (20.3)

4 (21.1)

 

Nodal status

  

0.876

 Positive

45 (70.3)

13 (68.4)

 

 Negative

19 (29.7)

6 (31.6)

 

Distant metastasis

  

0.122

 Positive

26 (40.6)

4 (21.1)

 

 Negative

38 (59.4)

15 (78.9)

 

Tumor size

  

0.013 a

4 cm

34 (53.1)

4 (21.1)

 

 > 4 cm

30 (46.9)

15 (78.9)

 

Age

  

0.653

55 yrs

44 (68.8)

12 (63.2)

 

 > 55 yrs

20 (31.3)

7 (36.8)

 

aStatistically significant p values are in bold

Correlation between C-FOXP1 expression and the survival of patients with breast IDC

Among the 83 patients with breast IDC, the follow-up period ranged from 2 to 146 months (median, 67 months), and there were 32 relapses and 21 deaths. Twenty out of 66 C-FOXP1-positive patients died of disease, and 28 had relapses, whereas 1 and 4 out of 17 C-FOXP1-negative patients died or relapsed, respectively. 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 that of the C-FOXP1-negative patients (93.3, 75.3%). The OS curve showed that C-FOXP1 status had an impact on outcome (p = 0.045). The DFS curve suggested that patients with C-FOXP1-negative IDCs demonstrated longer DFS than those with C-FOXP1-positive disease, but the result did not reach statistical significance (p = 0.152). Survival curves stratified for C-FOXP1 expression are shown in Fig. 5.
Fig. 5
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

Discussion

Although N-FOXP1 expression in breast cancer has been documented in several studies, the expression patterns of FOXP1 protein at different stages of breast cancer progression, including DCIS and IDC, and in ADH and UDH lesions, have not yet been clearly demonstrated. In the current study, heterogeneous FOXP1 expression patterns were observed in the above-mentioned cases. While FOXP1 staining was predominantly localized in the nuclei in UDH, the FOXP1 nuclear distribution gradually decreased from ADH, DCIS to IDC, and the cytoplasmic staining increased. These results were consistent with the previous reported heterogeneous expression pattern of FOXP1, in terms of the proportion of positive cells, the staining intensity, and subcellular localization [3, 36]. Our observations strongly indicated that FOXP1 expression might shift from the nucleus to the cytoplasm during breast tumorigenesis, and therefore, cytoplasmic mislocalization of FOXP1 is suggested play an important role in breast cancer progression. Similarly, subcellular localization has been suggested to play a distinct role in the pathogenesis of endometrial cancer [9]. Banham et al. also revealed that FOXP1 protein expression levels and compartmentalization varied depending on the cancer stage, although their sample sizes for each tumor were quite small [3].

Studies on the mechanisms of FOXP1 subcellular relocalization in breast cancer are very few. Calpain II has been implicated in mediating cell differentiation, necrosis, migration, and metastasis [19, 22]. Several studies, although limited, have investigated the aberrant expression and role of calpain II in breast cancer [19, 21, 25, 30, 3740]. High calpain II expression has been established in triple-negative and basal-like IDC and calpain II might promote breast cancer cell proliferation through the AKT signaling pathway [21, 30]. Calpain-mediated cleavage of β-catenin and E-cadherin may lead to aberrant stabilization of the proteins and promote tumorigenesis in breast cancer cells [38, 39]. In addition, Ho et al. suggested that the FOXO3a subcellular location was skewed toward nuclear localization in calpain II-deficient cells [20]. To date, we are not aware of any literature establishing the relevance between calpain II and FOXP1 protein in breast cancer. An unexpected but important finding in the current study was that C-FOXP1 expression was remarkably associated with calpain II in IDC. Moreover, in IDC samples in our series, calpain II was strongly correlated with the important molecules in AKT pathway, including pAKT, mTOR, p4E-BP1 and p-p70S6K. The PI3K/AKT/p70S6K signaling pathway, which has been reported to be involved in the nucleus-cytoplasm shuttling of FOXO1, another forkhead protein family member, was previously shown to participate in FOXP1 regulation in breast cancer [30, 41]. Taken together, we speculate that calpain II might play an important role in the subcellular regulation of FOXP1, and the AKT pathway might be involved in this process. Further investigations are merited to confirm this hypothesis and thoroughly explore the underlying mechanisms.

While N-FOXP1 was positively associated with ERα as well as ERβ expression in breast cancer according to the previous studies [8, 11, 12, 30, 42], the clinicopathological relevance of C-FOXP1 positivity in IDC has not been addressed until now. For the first time, we identified an inverse correlation between C-FOXP1 expression and ER expression in IDC. Our results are in line with those of a previous report by Giatromanolaki et al. concerning endometrial carcinoma [9]. They demonstrated that loss of ERα expression was a frequent event in cases with C-FOXP1 expression or loss of FOXP1 expression in endometrial carcinoma. Given that nucleus-cytoplasm shuttling might be an important event in the carcinogenesis, the interaction between ER and C-FOXP1 expression might be more clinically significant than that originally established between ER and N-FOXP1, and its biological significance should be further explored [8, 11, 30, 43, 44].

Previous studies have demonstrated that loss of FOXP1 expression is associated with a poor prognosis in primary invasive and familial breast cancer [11]. For example, both Fox et al. and Ijichi et al. indicated that FOXP1 immunoreactivity predicted better relapse-free survival but not OS in breast cancer patients [6, 8]. Moreover, FOXP1 immunoreactivity may predict a favorable prognosis for breast cancer patients treated with tamoxifen [6, 42, 44]. However, in previous studies, the FOXP1 protein was either located in the nuclei, or its subcellular location was not specified; nonetheless, cytoplasmic FOXP1 localization might play a large role in cancer cell biology because nuclear expression is characteristic of normal breast tissues [9]. Therefore, the prognostic impact of C-FOXP1 overexpression in IDC patients might be meaningful. Our results indicate for the first time that C-FOXP1 immunoreactivity is associated with an unfavorable OS and slightly inferior DFS in patients with breast IDC. Similarly, exclusive C-FOXP1 expression in early endothelial carcinoma has been linked with deep myometrial invasion and conferred a slightly worse outcome, despite an insignificant difference [9]. Hu et al. demonstrated that increased cytoplasmic FOXP1 expression was correlated with increased tumor grade but was not significantly associated with chemotherapy resistance and prognosis [32]. Our results provide reliable evidence regarding the prognostic importance of C-FOXP1 overexpression in breast cancer, which should be further confirmed with a much larger case series.

Conclusions

In summary, cytoplasmic relocalization of the FOXP1 protein is a frequent event in breast cancer. For the first time, we found that C-FOXP1 expression was dramatically associated with ER expression and correlated with reduced OS in patients with breast IDC. Our results indicated that C-FOXP1 might be important in both the pathogenesis and prognosis of breast cancer patients. Another noteworthy finding was that calpain II might be involved in FOXP1 trafficking from the nucleus to the cytoplasm, which might be mediated by the AKT pathway. Further investigations are needed to better understand the biological role of FOXP1 expression in breast cancer development and progression and to provide better strategies for prognosis prediction and therapeutic intervention in breast cancer.

Abbreviations

C-FOXP1: 

Cytoplasmic FOXP1

DCIS: 

Ductal carcinoma in situ

DFS: 

Disease-free survival

DLBCL: 

Diffuse large B-cell lymphoma

ER: 

Estrogen receptor

FOXP1: 

Forkhead box protein P1

HER2: 

Human epidermal growth factor receptor-2

IDC: 

Invasive ductal carcinoma

MALT: 

Mucosa associated lymphoid tissue

N-FOXP1: 

Nuclear FOXP1

OS: 

Overall survival

PR: 

Progesterone receptor

TMA: 

Tissue microarray

Declarations

Funding

This study was supported by grants from the youth project of National Nature Science Funding of China (No. 81700195) and Shanghai Hospital Development Center Emerging Advanced Technology Joint Research Project (SHDC12014105).

Availability of data and materials

Please contact author (xyzhou100@163.com) for data requests.

Authors’ contributions

BHY and XYZ conceived and designed the experiments. BHY performed the experiments. BHY and BZL reviewed the slides and analyzed the data. BHY wrote the manuscript. DRS, XYZ, WTY revised the paper and approved the final version of the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of Fudan University Shanghai Cancer Center (Shanghai Cancer Center Ethical Committee, permission number 050432-4-1212B). Additional patient consent for this retrospective study was not required.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis 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.

Authors’ Affiliations

(1)
Department of Pathology, Fudan University Shanghai Cancer Center, Dong-an Road 270, Xuhui District, Shanghai, 200032, China
(2)
Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China
(3)
Department of Pathology, the Second Affiliated Hospital of Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, China

References

  1. Xu T, He BS, Liu XX, et al. The predictive and prognostic role of stromal tumor-infiltrating lymphocytes in HER2-positive breast cancer with trastuzumab-based treatment: a meta-analysis and systematic review. J Cancer. 2017;8:3838–48.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Maitra A, Wistuba II, Washington C, et al. High-resolution chromosome 3p allelotyping of breast carcinomas and precursor lesions demonstrates frequent loss of heterozygosity and a discontinuous pattern of allele loss. Am J Pathol. 2001;159:119–30.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Banham AH, Beasley N, Campo E, et al. The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p. Cancer Res. 2001;61:8820–9.PubMedGoogle Scholar
  4. Katoh M, Igarashi M, Fukuda H, et al. Cancer genetics and genomics of human FOX family genes. Cancer Lett. 2013;328:198–206.View ArticlePubMedGoogle Scholar
  5. Shu W, Yang H, Zhang L, et al. Characterization of a new subfamily of winged-helix/forkhead (fox) genes that are expressed in the lung and act as transcriptional repressors. J Biol Chem. 2001;276:27488–97.View ArticlePubMedGoogle Scholar
  6. Ijichi N, Ikeda K, Horie-Inoue K, Inoue S. FOXP1 and estrogen signaling in breast cancer. Vitam Horm. 2013;93:203–12.View ArticlePubMedGoogle Scholar
  7. Goatly A, Bacon CM, Nakamura S, et al. FOXP1 abnormalities in lymphoma: translocation breakpoint mapping reveals insights into deregulated transcriptional control. Mod Pathol. 2008;21:902–11.View ArticlePubMedGoogle Scholar
  8. Fox SB, Brown P, Han C, et al. Expression of the forkhead transcription factor FOXP1 is associated with estrogen receptorα and improved survival in primary human breast carcinomas. Clin Cancer Res. 2004;10:3521–7.View ArticlePubMedGoogle Scholar
  9. Giatromanolaki A, Koukourakis MI, Sivridis E, et al. Loss of expression and nuclear/cytoplasmic localization of the FOXP1 forkhead transcription factor are common events in early endometrial cancer: relationship with estrogen receptors and HIF-1α expression. Mod Pathol. 2006;19:9–16.View ArticlePubMedGoogle Scholar
  10. Banham AH, Boddy J, Launchbury R, et al. Expression of the forkhead transcription factor FOXP1 is associated both with hypoxia inducible factors (HIFs) and the androgen receptor in prostate cancer but is not directly regulated by androgens or hypoxia. Prostate. 2007;67:1091–8.View ArticlePubMedGoogle Scholar
  11. Bates GJ, Fox SB, Han C, et al. Expression of the forkhead transcription factor FOXP1 is associated with that of estrogen receptorb in primary invasive breast carcinomas. Breast Cancer Res Treat. 2008;111:453–9.View ArticlePubMedGoogle Scholar
  12. Rayoo M, Yan M, Takano EA, et al. Expression of the forkhead box transcription factor FOXP1 is associated with oestrogen receptor alpha, oestrogen receptor beta and improved survival in familial breast cancers. J Clin Pathol. 2009;62:896–902.View ArticlePubMedGoogle Scholar
  13. Zhang Y, Zhang S, Wang X, et al. Prognostic significance of FOXP1 as an oncogene in hepatocellular carcinoma. J Clin Pathol. 2012;65:528–33.View ArticlePubMedGoogle Scholar
  14. Yu B, Zhou X, Li B, et al. FOXP1 expression and its clinicopathologic significance in nodal and extranodal diffuse large B-cell lymphoma. Ann Hematol. 2011;90:701–8.View ArticlePubMedGoogle Scholar
  15. Koon HB, Ippolito GC, Banham AH, Tucker PW. FOXP1: a potential therapeutic target in cancer. Expert Opin Ther Targets. 2007;11:955–65.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Ackermann S, Kocak H, Hero B, et al. FOXP1 inhibits cell growth and attenuates tumorigenicity of neuroblastoma. BMC Cancer. 2014;14:840.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Takayama K, Suzuki T, Tsutsumi S, et al. Integrative analysis of FOXP1 function reveals a tumor-suppressive effect in prostate cancer. Mol Endocrinol. 2014;28:2012–24.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Xiao J, He B, Zou Y, et al. Prognostic value of decreased FOXP1 protein expression in various tumors: a systematic review and meta-analysis. Sci Rep. 2016;6:30437.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Storr SJ, Thompson N, Pu X, et al. Calpain in breast cancer: role in disease progression and treatment response. Pathobiology. 2015;82:133–41.View ArticlePubMedGoogle Scholar
  20. Ho WC, Pikor L, Gao Y, et al. Calpain 2 regulates Akt-FoxO-p27 (Kip1) protein signaling pathway in mammary carcinoma. J Biol Chem. 2012;287:15458–65.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Storr SJ, Lee KW, Woolston CM, et al. Calpain system protein expression in basal-like and triple-negative invasive breast cancer. Ann Oncol. 2012;23:2289–96.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Xu L, Deng X. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion. J Biol Chem. 2004;279:53683–90.View ArticlePubMedGoogle Scholar
  23. Goll DE, Thompson VF, Li H, et al. The Calpain system. Physiol Rev. 2003;83:731–801.View ArticlePubMedGoogle Scholar
  24. Delmas C, Aragou N, Poussard S, et al. MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. J Biol Chem. 2003;278:12443–51.View ArticlePubMedGoogle Scholar
  25. Libertini SJ, Robinson BS, Dhillon NK, et al. Cyclin E both regulates and is regulated by calpain 2, a protease associated with metastatic breast cancer phenotype. Cancer Res. 2005;65:10700–8.View ArticlePubMedGoogle Scholar
  26. Gil-Parrado S, Fernández-Montalván A, Assfalg-Machleidt I. Ionomycin-activated calpain triggers apoptosis. J Biol Chem. 2002;277:27217–26.View ArticlePubMedGoogle Scholar
  27. Guicciardi ME, Gores GJ. Calpains can do it alone: implications for cancer therapy. Cancer Biol Ther. 2003;2:153–4.View ArticlePubMedGoogle Scholar
  28. Guerrero-Zotano A, Mayer IA, Arteaga CL. PI3K/AKT/mTOR: role in breast cancer progression, drug resistance, and treatment. Cancer Metastasis Rev. 2016;35:515–24.View ArticlePubMedGoogle Scholar
  29. Dey N, De P, Leyland-Jones B. PI3K-AKT-mTOR inhibitors in breast cancers: from tumor cell signaling to clinical trials. Pharmacol Ther. 2017;175:91–106.View ArticlePubMedGoogle Scholar
  30. Halacli SO, Dogan AL. FOXP1 regulation via the PI3K/Akt/p70S6K signaling pathway in breast cancer cells. Oncol Lett. 2015;9:1482–8.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Lakhani SR, Ellis IO, Schnitt SJ. World Health Organization classification of tumours of the breast. 4th ed. Lyon: IARC Press; 2012.Google Scholar
  32. Hu Z, Zhu L, Gao J, et al. Expression of FOXP1 in epithelial ovarian cancer (EOC) and its correlation with chemotherapy resistance and prognosis. Tumour Biol. 2015;36:7269–75.View ArticlePubMedGoogle Scholar
  33. Wu N, Du Z, Zhu Y, et al. The expression and prognostic impact of the PI3K/AKT/mTOR signaling pathway in advanced esophageal squamous cell carcinoma. Technol Cancer Res Treat. 2018;17:1533033818758772. https://doi.org/10.1177/1533033818758772.PubMedPubMed CentralGoogle Scholar
  34. Hammond MEH, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical testing of estrogen and progesterone receptors in breast Cancer. J Clin Oncol. 2010;28:2784–95.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31:3997–4013.View ArticlePubMedGoogle Scholar
  36. Oskay Halacli S. FOXP1 enhances tumor cell migration by repression of NFAT1 transcriptional activity in MDA-MB-231 cell. Cell Biol Int. 2017;41:102–10.View ArticlePubMedGoogle Scholar
  37. Storr SJ, Zhang S, Perren T, et al. The calpain system is associated with survival of breast cancer patients with large but operable inflammatory and non-inflammatory tumours treated with neoadjuvant chemotherapy. Oncotarget. 2016;7:47927–37.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Rios-Doria J, Kuefer R, Ethier SP, Day ML. Cleavage of β-catenin by calpain in prostate and mammary tumor cells. Cancer Res. 2004;64:7237–40.View ArticlePubMedGoogle Scholar
  39. Rios-Doria J, Day KC, Kuefer R, et al. The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells. J Biol Chem. 2003;278:1372–9.View ArticlePubMedGoogle Scholar
  40. Li CL, Yang D, Cao X, et al. Fibronectin induces epithelial-mesenchymal transition in human breast cancer MCF-7 cells via activation of calpain. Oncol Lett. 2017;13:3889–95.View ArticlePubMedPubMed CentralGoogle Scholar
  41. Zhao X, Gan L, Pan H, et al. Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation-and 14-3-3-dependent and-independent mechanisms. Biochem J. 2004;378:839–49.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Shigekawa T, Ijichi N, Ikeda K, et al. FOXP1, an estrogen-inducible transcription factor, modulates cell proliferation in breast cancer cells and 5-year recurrence-free survival of patients with tamoxifen-treated breast cancer. Horm Cancer. 2011;2:286–97.View ArticlePubMedGoogle Scholar
  43. Kim SJ, Kim TW, Lee SY, et al. CpG methylation of the ERalpha and ERbeta genes in breast cancer. Int J Mol Med. 2004;14:289–93.PubMedGoogle Scholar
  44. Ijichi N, Shigekawa T, Ikeda K, et al. Association of double-positive FOXA1 and FOXP1 immunoreactivities with favorable prognosis of tamoxifen-treated breast cancer patients. Horm Cancer. 2012;3:147–59.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2018

Advertisement