J Cancer Res Clin Oncol DOI 10.1007/s00432-014-1676-5
Original Article – Cancer Research
Aberrant expression of CD227 is correlated with tumor characteristics and invasiveness of breast carcinoma Ya‑Wen Wang · Duan‑Bo Shi · Ya‑Min Liu · Yan‑Lin Sun · Xu Chen · Shuai Xiang · Qiang Fu · Jun‑Min Wei · Peng Gao
Received: 17 February 2014 / Accepted: 8 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Increasing evidences demonstrate that CD227 plays a crucial role in the development and progression of breast cancer. However, the function of CD227 in breast carcinoma was still controversial and the investigation on CD227 in Asian race was scarce. Methods To investigate the relationship between CD227 and tumor characteristics of breast carcinoma, CD227, estrogen receptor (ER), progesterone receptor (PR), Her2⁄neu and Ki-67 were detected by immunohistochemistry in a series of 227 patients. The Kaplan–Meier method and log-rank tests were used to estimate the correlation between CD227 expression and patients’ prognosis. Furthermore, in vitro invasion assay was performed to examine the effect of CD227 on the invasiveness of breast carcinoma cells after transfection with CD227 cDNA or antisense
Electronic supplementary material The online version of this article (doi:10.1007/s00432-014-1676-5) contains supplementary material, which is available to authorized users. Y.-W. Wang · D.-B. Shi · Y.-M. Liu · Y.-L. Sun · X. Chen · S. Xiang · Q. Fu · J.-M. Wei (*) · P. Gao (*) Department of Pathology, School of Medicine, Shandong University, Wen Hua Xi Road 44, Jinan 250012, People’s Republic of China e-mail: [email protected] P. Gao e-mail: [email protected] Y.-M. Liu Department of Pathology, Laiwu Steel Affiliated Hospital of Taishan Medical College, Laiwu, People’s Republic of China J.-M. Wei Department of Chemotherapy, Qi Lu Hospital, Shandong University, Jinan, People’s Republic of China
phosphorothioate oligodeoxynucleotides (ASODN) against CD227 mRNA. Results Our data demonstrate that the cytoplasm staining and high expression of CD227 were positively related to the aggressiveness of breast cancer. Both circumferential membrane staining and cytoplasm staining were associated with lymph node metastasis. Moreover, the cytoplasm staining and overexpression of CD227 were found to be related to Her-2/neu positivity, higher Ki-67 positivity and poorer survival of patients. We further demonstrated that the invasion ability of breast carcinoma cells could be enhanced or inhibited by CD227 cDNA or ASODN, respectively. Conclusions We conclude that the aberrant expression of CD227, especially cytoplasm staining could be predictive for tumor aggressiveness, lymph node metastasis, poorer outcome of patients with breast cancers. And CD227 could promote the invasion ability of breast cancer cells, suggesting a potential role of CD227 as an oncogene in breast carcinoma. Keywords Breast carcinoma · CD227 · Tumor characteristics · Invasiveness · Antisense phosphorothioate oligodeoxynucleotides · Prognosis
Introduction Breast carcinoma is one of the most frequently occurring cancers and the leading cause of cancer death in females worldwide, responsible for 23 % of the total new cancer cases and 14 % of the total cancer deaths in 2008 (Jemal et al. 2011). Invasion and metastasis are the major features of malignant tumors and result in poor prognosis of patients. The invasive abilities of cancer cells are the critical parameters of the metastatic cascade (Yilmaz and
13
Christofori 2010). Thus, better understanding of the prognosis and metastasis associated factors and the underlying mechanisms would help to improve patients’ prognosis. CD227 belongs to the family of high molecular weight, heavily O-glycosylated proteins. Accumulating evidences suggest that CD227 is overexpressed in various kinds of carcinomas including breast, ovarian, lung, pancreatic, prostate, gastric and colorectal cancers (Yonezawa et al. 2011). Although burgeoning studies showed a significant correlation between CD227 upregulation and tumor progression and worse prognosis in patients with breast carcinoma (Yonezawa et al. 2008; McGuckin et al. 1995), others demonstrated that overexpression of CD227 was associated with improved survival or longer recurrence free stage (Rahn et al. 2001; Rakha et al. 2005). Several studies, however, found no correlation between CD227 expression and patients’ outcome (Parham et al. 1988; Arnerlov et al. 1988). These findings make the function of CD227 in breast carcinoma controversial. Moreover, most of the previous researches were carried among the white (Rakha et al. 2005; Rahn et al. 2001; Arnerlov et al. 1988; Parham et al. 1988; McGuckin et al. 1995), and the investigation on Asia race was scarce. In this study, expression of CD227 and some wellknown tumor markers including estrogen receptor (ER), progesterone receptor (PR), Her2⁄neu and Ki-67 were determined by immunohistochemistry in a spectrum of breast lesions, including usual ductal hyperplasia (UDH), benign breast tumor (intraductal papilloma), ductal carcinoma in situ (DCIS), primary invasive breast carcinoma, secondary lymph node metastasis loci and invasive micropapillary carcinoma (IMPC). Correlation between CD227 expression and clinicopathological parameters and patient prognosis was analyzed. Invasion assay was performed to examine the effect of CD227 on the invasiveness of breast carcinoma cells. We suggested that positive aberrant expression of CD227 in cytoplasm and circumferential membrane was related to higher grade and increased aggressiveness of breast carcinoma. And cytoplasmic staining and overexpression of CD227 were indicated of poorer survival for patients with breast cancers. In addition, our data showed that the invasion ability of breast carcinoma cells could be enhanced or inhibited by CD227 cDNA or antisense phosphorothioate oligodeoxynucleotides (ASODN), respectively.
Materials and methods Tissue samples Five cases of normal breast tissues were used as normal control. Five cases of intraductal papilloma, 21 cases of UDH, 22 cases of DCIS, 13 cases of DCIS with
13
J Cancer Res Clin Oncol
microinvasion, 131 cases of primary invasive breast carcinoma (101 cases of invasive ductal carcinoma and 30 cases of invasive lobular carcinoma), 20 cases of secondary lymph node metastasis loci and 10 cases of IMPC were collected from Qi Lu Hospital of Shandong University from 1997 to 2012. All samples were fixed in 40 g/L formaldehyde and embedded in paraffin for immunohistochemistry analysis. Ethical statement The study was approved by the Ethics Committee of School of Medicine, Shandong University, Shandong, China (approval code: MECSDUM2012028). We obtained written informed consent from all participants involved in our study. The principles outlined in the Declaration of Helsinki have been followed. Immunohistochemistry Paraffin-embedded breast tissue sections (4 μm thickness) were dewaxed and subjected to antigen retrieval by microwave in 0.01 M citric buffer (PH 6.0) for 10 min, followed by incubation in 3 % H2O2 for 10 min to quench endogenous peroxidase. Non-specific binding was prevented by incubation in 5 % normal horse serum for 20 min in humid chamber. Then, the slides were incubated with the primary antibody for CD227, ER, PR, Her2⁄neu or Ki-67 overnight at 4 °C. Information about the primary antibodies used in this study was summarized in Supplementary Table S1. The 139H2 antibody (Santa Cruz, mouse monoclonal antibody, 1:100) was used to determine CD227 expression of breast tissues. After washing with PBS (phosphate buffered saline), the primary antibodies were detected with appropriate secondary antibodies for 30 min at 37 °C. Following washes, slides were incubated in streptavidin biotin complex (DAKO, Carpinteria, CA) for 20 min at 37 °C, washed three times, visualized using DAB, rinsed in distilled water and counterstained with hematoxylin. Evaluation of immunohistochemical staining Based on the subcellular localization, CD227 staining patterns were divided as apical membrane staining, circumferential membrane staining, cytoplasm staining and reversed apical staining. Cytoplasmic expression mixed with circumferential membrane staining was simply classified as cytoplasm staining. As normal breast tissues were reported to show apical membrane staining (Rahn et al. 2001; McGuckin et al. 1995), circumferential membrane staining, cytoplasm staining and reversed apical staining were all identified as aberrant expression in our study. The expression level of CD227 was divided into high expression and
J Cancer Res Clin Oncol
low expression by proportions of positive cells (>50 vs. ≤50 %) as previously described (Rahn et al. 2001). ER, PR and Ki-67 were considered positive if >10 % of tumor cell nuclei are immunoreactive. Staining intensity for Her2/neu was graded as “0” to “+++” according to the original FDA criteria (Birner et al. 2001). FISH was not available for evaluating Her2/neu (++), so we defined Her2/neu (+++) as positive and Her2/neu (0) or (+) as negative. And cases with Her2/neu (++) were excluded when we analyzed the relationship between CD227 expression and Her2/neu status. Evaluation of immunohistochemical staining was done by two experienced pathologists independently.
random 9 mers (TaKaRa, Japan). The reaction was carried out at 30 °C for 1 min, 45 °C for 30 min, 99 °C for 5 min and 5 °C for 5 min. PCR was performed using a thermal cycler (M J Research, USA), in which 30 cycles were carried out at 94 °C for 2 min, 55 °C for 45 s, 72 °C for 90 s. The sequence of the forward and reverse primers for CD227 mRNA is as follows: 5′-AGCACCGACTACTACCAAGAG-3′ and 5′-CAGCCAAGGCAATGAGATA-3′, respectively. β-actin was used as the control set. All reactions were run in duplicate, and data were presented as mean ± SD.
Cell culture
The amount of CD227 protein in breast carcinoma cells was quantified by fluorescence-activated cell sorting as previously reported (Suwa et al. 1998). Data were obtained from two independent experiments and presented as mean ± SD.
The non-metastatic breast carcinoma cell line MCF-7 and metastatic breast carcinoma cell line MDA-MB-231 were obtained from the American National Cancer Institute. They were maintained in RPMI-1640 culture medium (Gibco, Los Angeles, USA) containing 10 % FBS (fetal bovine serum) at 37 °C in a humidified atmosphere containing 5 % CO2. Transfection of breast carcinoma cell lines The pCI-Neo vector containing the full-length CD227 cDNA used for cell transfection was a kind gift from Dr. Sandra Gendler and Dr. Cathy Madsen (Mayo Clinic Arizona, Scottsdale, Arizona 85259, USA). For transfection of metastatic breast carcinoma cell line MDA-MB-231, the ASODN against CD227 mRNA was synthesized with the sequences as follows: 5′-ACCTCCAGTTTAATTC CTC-3′. A nonsense antisense oligonucleotide (5′-ACCTG CAATTCAACTCGTC-3′) was used as negative control. For each well, 5 μl (1 mg/ml) of lipofectamine (Invitrogen, Carlsbad, USA) and 5 μl (1 mg/ml) of pCI-Neo or ASODN were diluted into 250 μl of culture medium without serum, respectively, and incubated for 5 min at room temperature. Before adding the complexes directly to the cells, the growth medium was removed and replaced with 0.5 ml of medium without serum. Six hours later, the medium was replaced with 1 ml of complete culture medium. For selection of positive CD227 cDNA transfected cells, G418 (Geneticin, GIBCO-BRL) was added in the culture medium at a concentration of 800 μg/ml. Determination of CD227 mRNA by real‑time quantitative PCR (RT‑qPCR) Total RNA was prepared as described (Chomczynski and Sacchi 1987) and converted to single-strand cDNA using
Determination of CD227 expression by flow cytometry
Invasion assay The invasion assay in vitro was performed in 24-well Bicoat Matrigel Invasive Chambers (Collaborative Research) as described (Gao et al. 2013). The chambers contain an 8-μm pore size polycarbonate membrane coated with the solubilized basement membrane Matrigel (Collaborative Research). Cells (2 × 105) were added into each upper compartment and cultured for 24 h. The non-invasive cells and Matrigel were removed with a cotton swab. Cells migrated through the Matrigel and membrane and stuck to the lower surface of the polycarbonate membrane were fixed with methanol and stained with Giemsa (Gibco, Los Angeles, USA) for observation. The migrated cells were counted by choosing five fields of each chamber randomly at magnification of 100× and the average number was calculated. Each experiment was performed in duplicate, and data were presented as mean ± SD. Statistical analysis Analyses were performed using the statistical package Prism 5 software (GraphPad Software, San Diego, CA, USA). Differences were analyzed with the Student’s t test between two groups or with one-way analysis of variance (ANOVA) among three groups. χ2 tests were used to evaluate the association of CD227 staining patterns or expression levels with clinicopathological parameters and biological markers. Fisher’s exact test was used where applicable. Kaplan–Meier curves were constructed and log-rank test was used to address the correlation between CD227 expression and patients’ prognosis. P values less than 0.05 were considered statistically significant.
13
J Cancer Res Clin Oncol
Fig. 1 CD227 expression in normal breast tissue, breast benign tumor and UDH. In our immunohistochemistry analysis, apical membrane staining of CD227 expression was observed in normal breast acinus (a immunoperoxidase, original magnification ×200), normal
breast duct (b immunoperoxidase, original magnification ×200), intraductal papilloma (c immunoperoxidase, original magnification ×100) and UDH (d immunoperoxidase, original magnification ×200)
Table 1 CD227 staining patterns in normal breast tissues and breast lesions Staining patterns
Normal
UDH
Intraductal
DCIS
Papilloma
Primary invasive
Secondary lymph node
Carcinoma
Metastasis loci
IMPC
Apical membrane Circumferential membrane Cytoplasm staining Reversed apical staining Negative
5 (100 %) 0 0
21 (100 %) 0 0
5 (100 %) 0 0
3 (8.57 %) 8 (22.86 %) 24 (68.57 %)
0 18 (13.74 %) 101 (77.10 %)
0 3 (15 %) 17 (85 %)
0
0
0
0
12 (9.16 %)
0
0 0 0 10 (100 %) 0
Total of positive staining
5
21
5
35
119
20
10
UDH usual ductal hyperplasia, DCIS ductal carcinoma in situ, IMPC invasive micropapillary carcinoma DCIS with microinvasion was included
Results Immunohistochemical staining of human breast lesions The immunohistochemical results showed that only apical membrane staining of CD227 was seen in normal breast
13
tissue (Fig. 1a, b), intraductal papilloma (Fig. 1c) and UDH (Fig. 1d). In DCIS, three cases were observed apical membrane expression of CD227 (Table 1). Thirty-two cases of DCIS showed aberrant localization of CD227 (Fig. 2a, b) including 8 cases (22.86 %) of circumferential membrane staining and 24 cases (68.57 %) of cytoplasm
J Cancer Res Clin Oncol
Fig. 2 Expression patterns of CD227 in breast carcinoma detected by immunohistochemistry. Twenty of 22 cases of DCIS (a low-grade DCIS; b high-grade DCIS, immunoperoxidase, original magnification ×100) showed positive aberrant localization represented by circumferential membrane staining or cytoplasm staining, or both, with low expression level. In 13 cases of DCIS with microinvasion (c immunoperoxidase, original magnification ×200), circumferential membrane staining combined with cytoplasm staining was observed. And most of the DCIS cases (10/13) showed high expression level.
Table 2 CD227 expression levels in different stages of breast lesions
UDH usual ductal hyperplasia, DCIS ductal carcinoma in situ, IMPC invasive micropapillary carcinoma
In invasive ductal carcinoma (d immunoperoxidase, original magnification ×200) and invasive lobular carcinoma (e immunoperoxidase, original magnification ×200), positive aberrant localization of CD227 including circumferential membrane staining (13.74 %) and cytoplasm staining (77.10 %) with high expression level were detected. Seventeen of the 20 cases of lymph node metastasis loci also showed cytoplasm staining with high expression level (f immunoperoxidase, original magnification ×200)
Groups
Number
Expression levels High expression (%)
Low expression (%)
UDH Intraductal papilloma DCIS DCIS with microinvasion Primary invasive carcinoma Secondary lymph node metastasis loci IMPC
21 5 22 13 131 20 10
8 (38.10) 2 (40.00) 12 (54.55) 10 (76.92) 109 (83.21) 17 (85.00) 9 (90.00)
13 (61.90) 3 (60.00) 10 (45.45) 3 (23.18) 22 (16.79) 3 (15.00) 1 (10.00)
Total
222
167 (75.23)
55 (24.77)
staining. In 13 cases of DCIS with microinvasion, cytoplasm staining was observed (Fig. 2c). In breast primary invasive carcinoma (Fig. 2d, e), 119 cases (90.84 %) were observed aberrant localization of CD227 including 18 cases (13.74 %) of circumferential membrane staining and 101 cases (77.10 %) of cytoplasm staining. The other 12 cases of breast invasive carcinoma showed negative expression of CD227, and no one was observed apical membrane staining. Eighty-five percent (17/20) of the secondary lymph
node metastasis loci showed cytoplasm staining (Fig. 2f). Specially, frequent reversed apical pattern and decreased cytoplasmic immunoreactivity were found in IMPC cases (Supplementary Fig. S1). The expression level of CD227 was variable in terms of the proportion of positive cells in individual case. Compared with DCIS, the cases of DCIS with microinvasion and invasive carcinoma showed significantly increased proportion of high expression (Table 2, P
Original Article – Cancer Research
Aberrant expression of CD227 is correlated with tumor characteristics and invasiveness of breast carcinoma Ya‑Wen Wang · Duan‑Bo Shi · Ya‑Min Liu · Yan‑Lin Sun · Xu Chen · Shuai Xiang · Qiang Fu · Jun‑Min Wei · Peng Gao
Received: 17 February 2014 / Accepted: 8 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Increasing evidences demonstrate that CD227 plays a crucial role in the development and progression of breast cancer. However, the function of CD227 in breast carcinoma was still controversial and the investigation on CD227 in Asian race was scarce. Methods To investigate the relationship between CD227 and tumor characteristics of breast carcinoma, CD227, estrogen receptor (ER), progesterone receptor (PR), Her2⁄neu and Ki-67 were detected by immunohistochemistry in a series of 227 patients. The Kaplan–Meier method and log-rank tests were used to estimate the correlation between CD227 expression and patients’ prognosis. Furthermore, in vitro invasion assay was performed to examine the effect of CD227 on the invasiveness of breast carcinoma cells after transfection with CD227 cDNA or antisense
Electronic supplementary material The online version of this article (doi:10.1007/s00432-014-1676-5) contains supplementary material, which is available to authorized users. Y.-W. Wang · D.-B. Shi · Y.-M. Liu · Y.-L. Sun · X. Chen · S. Xiang · Q. Fu · J.-M. Wei (*) · P. Gao (*) Department of Pathology, School of Medicine, Shandong University, Wen Hua Xi Road 44, Jinan 250012, People’s Republic of China e-mail: [email protected] P. Gao e-mail: [email protected] Y.-M. Liu Department of Pathology, Laiwu Steel Affiliated Hospital of Taishan Medical College, Laiwu, People’s Republic of China J.-M. Wei Department of Chemotherapy, Qi Lu Hospital, Shandong University, Jinan, People’s Republic of China
phosphorothioate oligodeoxynucleotides (ASODN) against CD227 mRNA. Results Our data demonstrate that the cytoplasm staining and high expression of CD227 were positively related to the aggressiveness of breast cancer. Both circumferential membrane staining and cytoplasm staining were associated with lymph node metastasis. Moreover, the cytoplasm staining and overexpression of CD227 were found to be related to Her-2/neu positivity, higher Ki-67 positivity and poorer survival of patients. We further demonstrated that the invasion ability of breast carcinoma cells could be enhanced or inhibited by CD227 cDNA or ASODN, respectively. Conclusions We conclude that the aberrant expression of CD227, especially cytoplasm staining could be predictive for tumor aggressiveness, lymph node metastasis, poorer outcome of patients with breast cancers. And CD227 could promote the invasion ability of breast cancer cells, suggesting a potential role of CD227 as an oncogene in breast carcinoma. Keywords Breast carcinoma · CD227 · Tumor characteristics · Invasiveness · Antisense phosphorothioate oligodeoxynucleotides · Prognosis
Introduction Breast carcinoma is one of the most frequently occurring cancers and the leading cause of cancer death in females worldwide, responsible for 23 % of the total new cancer cases and 14 % of the total cancer deaths in 2008 (Jemal et al. 2011). Invasion and metastasis are the major features of malignant tumors and result in poor prognosis of patients. The invasive abilities of cancer cells are the critical parameters of the metastatic cascade (Yilmaz and
13
Christofori 2010). Thus, better understanding of the prognosis and metastasis associated factors and the underlying mechanisms would help to improve patients’ prognosis. CD227 belongs to the family of high molecular weight, heavily O-glycosylated proteins. Accumulating evidences suggest that CD227 is overexpressed in various kinds of carcinomas including breast, ovarian, lung, pancreatic, prostate, gastric and colorectal cancers (Yonezawa et al. 2011). Although burgeoning studies showed a significant correlation between CD227 upregulation and tumor progression and worse prognosis in patients with breast carcinoma (Yonezawa et al. 2008; McGuckin et al. 1995), others demonstrated that overexpression of CD227 was associated with improved survival or longer recurrence free stage (Rahn et al. 2001; Rakha et al. 2005). Several studies, however, found no correlation between CD227 expression and patients’ outcome (Parham et al. 1988; Arnerlov et al. 1988). These findings make the function of CD227 in breast carcinoma controversial. Moreover, most of the previous researches were carried among the white (Rakha et al. 2005; Rahn et al. 2001; Arnerlov et al. 1988; Parham et al. 1988; McGuckin et al. 1995), and the investigation on Asia race was scarce. In this study, expression of CD227 and some wellknown tumor markers including estrogen receptor (ER), progesterone receptor (PR), Her2⁄neu and Ki-67 were determined by immunohistochemistry in a spectrum of breast lesions, including usual ductal hyperplasia (UDH), benign breast tumor (intraductal papilloma), ductal carcinoma in situ (DCIS), primary invasive breast carcinoma, secondary lymph node metastasis loci and invasive micropapillary carcinoma (IMPC). Correlation between CD227 expression and clinicopathological parameters and patient prognosis was analyzed. Invasion assay was performed to examine the effect of CD227 on the invasiveness of breast carcinoma cells. We suggested that positive aberrant expression of CD227 in cytoplasm and circumferential membrane was related to higher grade and increased aggressiveness of breast carcinoma. And cytoplasmic staining and overexpression of CD227 were indicated of poorer survival for patients with breast cancers. In addition, our data showed that the invasion ability of breast carcinoma cells could be enhanced or inhibited by CD227 cDNA or antisense phosphorothioate oligodeoxynucleotides (ASODN), respectively.
Materials and methods Tissue samples Five cases of normal breast tissues were used as normal control. Five cases of intraductal papilloma, 21 cases of UDH, 22 cases of DCIS, 13 cases of DCIS with
13
J Cancer Res Clin Oncol
microinvasion, 131 cases of primary invasive breast carcinoma (101 cases of invasive ductal carcinoma and 30 cases of invasive lobular carcinoma), 20 cases of secondary lymph node metastasis loci and 10 cases of IMPC were collected from Qi Lu Hospital of Shandong University from 1997 to 2012. All samples were fixed in 40 g/L formaldehyde and embedded in paraffin for immunohistochemistry analysis. Ethical statement The study was approved by the Ethics Committee of School of Medicine, Shandong University, Shandong, China (approval code: MECSDUM2012028). We obtained written informed consent from all participants involved in our study. The principles outlined in the Declaration of Helsinki have been followed. Immunohistochemistry Paraffin-embedded breast tissue sections (4 μm thickness) were dewaxed and subjected to antigen retrieval by microwave in 0.01 M citric buffer (PH 6.0) for 10 min, followed by incubation in 3 % H2O2 for 10 min to quench endogenous peroxidase. Non-specific binding was prevented by incubation in 5 % normal horse serum for 20 min in humid chamber. Then, the slides were incubated with the primary antibody for CD227, ER, PR, Her2⁄neu or Ki-67 overnight at 4 °C. Information about the primary antibodies used in this study was summarized in Supplementary Table S1. The 139H2 antibody (Santa Cruz, mouse monoclonal antibody, 1:100) was used to determine CD227 expression of breast tissues. After washing with PBS (phosphate buffered saline), the primary antibodies were detected with appropriate secondary antibodies for 30 min at 37 °C. Following washes, slides were incubated in streptavidin biotin complex (DAKO, Carpinteria, CA) for 20 min at 37 °C, washed three times, visualized using DAB, rinsed in distilled water and counterstained with hematoxylin. Evaluation of immunohistochemical staining Based on the subcellular localization, CD227 staining patterns were divided as apical membrane staining, circumferential membrane staining, cytoplasm staining and reversed apical staining. Cytoplasmic expression mixed with circumferential membrane staining was simply classified as cytoplasm staining. As normal breast tissues were reported to show apical membrane staining (Rahn et al. 2001; McGuckin et al. 1995), circumferential membrane staining, cytoplasm staining and reversed apical staining were all identified as aberrant expression in our study. The expression level of CD227 was divided into high expression and
J Cancer Res Clin Oncol
low expression by proportions of positive cells (>50 vs. ≤50 %) as previously described (Rahn et al. 2001). ER, PR and Ki-67 were considered positive if >10 % of tumor cell nuclei are immunoreactive. Staining intensity for Her2/neu was graded as “0” to “+++” according to the original FDA criteria (Birner et al. 2001). FISH was not available for evaluating Her2/neu (++), so we defined Her2/neu (+++) as positive and Her2/neu (0) or (+) as negative. And cases with Her2/neu (++) were excluded when we analyzed the relationship between CD227 expression and Her2/neu status. Evaluation of immunohistochemical staining was done by two experienced pathologists independently.
random 9 mers (TaKaRa, Japan). The reaction was carried out at 30 °C for 1 min, 45 °C for 30 min, 99 °C for 5 min and 5 °C for 5 min. PCR was performed using a thermal cycler (M J Research, USA), in which 30 cycles were carried out at 94 °C for 2 min, 55 °C for 45 s, 72 °C for 90 s. The sequence of the forward and reverse primers for CD227 mRNA is as follows: 5′-AGCACCGACTACTACCAAGAG-3′ and 5′-CAGCCAAGGCAATGAGATA-3′, respectively. β-actin was used as the control set. All reactions were run in duplicate, and data were presented as mean ± SD.
Cell culture
The amount of CD227 protein in breast carcinoma cells was quantified by fluorescence-activated cell sorting as previously reported (Suwa et al. 1998). Data were obtained from two independent experiments and presented as mean ± SD.
The non-metastatic breast carcinoma cell line MCF-7 and metastatic breast carcinoma cell line MDA-MB-231 were obtained from the American National Cancer Institute. They were maintained in RPMI-1640 culture medium (Gibco, Los Angeles, USA) containing 10 % FBS (fetal bovine serum) at 37 °C in a humidified atmosphere containing 5 % CO2. Transfection of breast carcinoma cell lines The pCI-Neo vector containing the full-length CD227 cDNA used for cell transfection was a kind gift from Dr. Sandra Gendler and Dr. Cathy Madsen (Mayo Clinic Arizona, Scottsdale, Arizona 85259, USA). For transfection of metastatic breast carcinoma cell line MDA-MB-231, the ASODN against CD227 mRNA was synthesized with the sequences as follows: 5′-ACCTCCAGTTTAATTC CTC-3′. A nonsense antisense oligonucleotide (5′-ACCTG CAATTCAACTCGTC-3′) was used as negative control. For each well, 5 μl (1 mg/ml) of lipofectamine (Invitrogen, Carlsbad, USA) and 5 μl (1 mg/ml) of pCI-Neo or ASODN were diluted into 250 μl of culture medium without serum, respectively, and incubated for 5 min at room temperature. Before adding the complexes directly to the cells, the growth medium was removed and replaced with 0.5 ml of medium without serum. Six hours later, the medium was replaced with 1 ml of complete culture medium. For selection of positive CD227 cDNA transfected cells, G418 (Geneticin, GIBCO-BRL) was added in the culture medium at a concentration of 800 μg/ml. Determination of CD227 mRNA by real‑time quantitative PCR (RT‑qPCR) Total RNA was prepared as described (Chomczynski and Sacchi 1987) and converted to single-strand cDNA using
Determination of CD227 expression by flow cytometry
Invasion assay The invasion assay in vitro was performed in 24-well Bicoat Matrigel Invasive Chambers (Collaborative Research) as described (Gao et al. 2013). The chambers contain an 8-μm pore size polycarbonate membrane coated with the solubilized basement membrane Matrigel (Collaborative Research). Cells (2 × 105) were added into each upper compartment and cultured for 24 h. The non-invasive cells and Matrigel were removed with a cotton swab. Cells migrated through the Matrigel and membrane and stuck to the lower surface of the polycarbonate membrane were fixed with methanol and stained with Giemsa (Gibco, Los Angeles, USA) for observation. The migrated cells were counted by choosing five fields of each chamber randomly at magnification of 100× and the average number was calculated. Each experiment was performed in duplicate, and data were presented as mean ± SD. Statistical analysis Analyses were performed using the statistical package Prism 5 software (GraphPad Software, San Diego, CA, USA). Differences were analyzed with the Student’s t test between two groups or with one-way analysis of variance (ANOVA) among three groups. χ2 tests were used to evaluate the association of CD227 staining patterns or expression levels with clinicopathological parameters and biological markers. Fisher’s exact test was used where applicable. Kaplan–Meier curves were constructed and log-rank test was used to address the correlation between CD227 expression and patients’ prognosis. P values less than 0.05 were considered statistically significant.
13
J Cancer Res Clin Oncol
Fig. 1 CD227 expression in normal breast tissue, breast benign tumor and UDH. In our immunohistochemistry analysis, apical membrane staining of CD227 expression was observed in normal breast acinus (a immunoperoxidase, original magnification ×200), normal
breast duct (b immunoperoxidase, original magnification ×200), intraductal papilloma (c immunoperoxidase, original magnification ×100) and UDH (d immunoperoxidase, original magnification ×200)
Table 1 CD227 staining patterns in normal breast tissues and breast lesions Staining patterns
Normal
UDH
Intraductal
DCIS
Papilloma
Primary invasive
Secondary lymph node
Carcinoma
Metastasis loci
IMPC
Apical membrane Circumferential membrane Cytoplasm staining Reversed apical staining Negative
5 (100 %) 0 0
21 (100 %) 0 0
5 (100 %) 0 0
3 (8.57 %) 8 (22.86 %) 24 (68.57 %)
0 18 (13.74 %) 101 (77.10 %)
0 3 (15 %) 17 (85 %)
0
0
0
0
12 (9.16 %)
0
0 0 0 10 (100 %) 0
Total of positive staining
5
21
5
35
119
20
10
UDH usual ductal hyperplasia, DCIS ductal carcinoma in situ, IMPC invasive micropapillary carcinoma DCIS with microinvasion was included
Results Immunohistochemical staining of human breast lesions The immunohistochemical results showed that only apical membrane staining of CD227 was seen in normal breast
13
tissue (Fig. 1a, b), intraductal papilloma (Fig. 1c) and UDH (Fig. 1d). In DCIS, three cases were observed apical membrane expression of CD227 (Table 1). Thirty-two cases of DCIS showed aberrant localization of CD227 (Fig. 2a, b) including 8 cases (22.86 %) of circumferential membrane staining and 24 cases (68.57 %) of cytoplasm
J Cancer Res Clin Oncol
Fig. 2 Expression patterns of CD227 in breast carcinoma detected by immunohistochemistry. Twenty of 22 cases of DCIS (a low-grade DCIS; b high-grade DCIS, immunoperoxidase, original magnification ×100) showed positive aberrant localization represented by circumferential membrane staining or cytoplasm staining, or both, with low expression level. In 13 cases of DCIS with microinvasion (c immunoperoxidase, original magnification ×200), circumferential membrane staining combined with cytoplasm staining was observed. And most of the DCIS cases (10/13) showed high expression level.
Table 2 CD227 expression levels in different stages of breast lesions
UDH usual ductal hyperplasia, DCIS ductal carcinoma in situ, IMPC invasive micropapillary carcinoma
In invasive ductal carcinoma (d immunoperoxidase, original magnification ×200) and invasive lobular carcinoma (e immunoperoxidase, original magnification ×200), positive aberrant localization of CD227 including circumferential membrane staining (13.74 %) and cytoplasm staining (77.10 %) with high expression level were detected. Seventeen of the 20 cases of lymph node metastasis loci also showed cytoplasm staining with high expression level (f immunoperoxidase, original magnification ×200)
Groups
Number
Expression levels High expression (%)
Low expression (%)
UDH Intraductal papilloma DCIS DCIS with microinvasion Primary invasive carcinoma Secondary lymph node metastasis loci IMPC
21 5 22 13 131 20 10
8 (38.10) 2 (40.00) 12 (54.55) 10 (76.92) 109 (83.21) 17 (85.00) 9 (90.00)
13 (61.90) 3 (60.00) 10 (45.45) 3 (23.18) 22 (16.79) 3 (15.00) 1 (10.00)
Total
222
167 (75.23)
55 (24.77)
staining. In 13 cases of DCIS with microinvasion, cytoplasm staining was observed (Fig. 2c). In breast primary invasive carcinoma (Fig. 2d, e), 119 cases (90.84 %) were observed aberrant localization of CD227 including 18 cases (13.74 %) of circumferential membrane staining and 101 cases (77.10 %) of cytoplasm staining. The other 12 cases of breast invasive carcinoma showed negative expression of CD227, and no one was observed apical membrane staining. Eighty-five percent (17/20) of the secondary lymph
node metastasis loci showed cytoplasm staining (Fig. 2f). Specially, frequent reversed apical pattern and decreased cytoplasmic immunoreactivity were found in IMPC cases (Supplementary Fig. S1). The expression level of CD227 was variable in terms of the proportion of positive cells in individual case. Compared with DCIS, the cases of DCIS with microinvasion and invasive carcinoma showed significantly increased proportion of high expression (Table 2, P
