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Cancer Biomarkers 13 (2013) 261–267 DOI 10.3233/CBM-130356 IOS Press
Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer Hong Yina,b , Cheng Lub , Yongfeng Tangb , Haiyan Wangc , Hai Wanga and Jiandong Wanga,c,∗ a
Department of Pathology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, China Department of Surgery, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu, China c Department of Pathology, Southern Medical University, Guangzhou, Guangdong, China b
Abstract. BACKGROUND: The Eph family of receptor tyrosine kinases and their ephrin ligands are membrane-bound cell-signaling proteins and they play critical regulatory roles in embryonic development and carcinogenesis. Eph receptors require direct cell to cell interaction for activation and they are divided into EphA and EphB receptor classes, depending on their preferential binding affinity for EphrinA or EphrinB ligands. Eph receptors have been documented in breast cancer, but the Ephrin ligands have not been thoroughly investigated. MATERIALS AND METHODS: We conducted a systematic assessment of EphrinB1 expression in a set of 75 formalin-fixed paraffin-embedded breast cancers, using immunohistochemical staining (IHC) with a specific antibody, and we examined the relationship between EphrinB1 expression, histopathological parameters, and the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. RESULTS: High level expression of EphrinB1 is positively associated with lymph node metastasis (P < 0.0001) and with the presence of HER-2 receptor (P = 0.041) and it is more often detected in triple-negative breast carcinomas (P = 0.038). No relation was apparent between EphrinB1 expression level and other histopathological parameters, but enhanced EphrinB1 expression is associated with shorter overall survival (P = 0.015). CONCLUSION: Our analysis demonstrates that EphrinB1 expression is related to the metastasis of breast cancer and its enhanced expression confers a poor prognosis, suggesting that EphrinB1 may be a relevant therapeutic target in breast cancers. Keywords: EphrinB1, breast cancer, metastasis, triple-negative breast cancer
1. Introduction The large family of Eph receptor tyrosine kinases and their Ephrin ligands play important cell-signaling roles in embryonic development and carcinogenesis. Eph receptors and Ephrin ligands are divided into A and B classes based on their sequence similarity, common structural features and binding affinities; EphA and EphB receptors interact with and preferentially ∗ Corresponding author: Jiandong Wang, Department of Pathology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, China. E-mail: [email protected].
bind to their respective EphrinA and EphrinB ligands. The receptors and ligands interact through cell-cell contact, with the potential to transduce forward signals from the receptors and reverse signals from the ligands and referred to as bidirectional signaling. EphEphrin signaling modulates a variety of biological activities, including cell-cell interaction, cell migration, cell segregation, cell attachment, and shape. Eph receptors and Ephrin ligands have been implicated in diverse developmental functions, especially neurological development [1–5]. Eph and Ephrin are involved in oncogenesis of many types of cancer, including colorectal, lung, prostate, and breast cancers [6–8]. Therefore,
c 2013 – IOS Press and the authors. All rights reserved ISSN 1574-0153/13/$27.50
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H. Yin et al. / Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer
they might be valuable candidates for cancer diagnosis and prognosis and they might be new therapeutic targets. Eph receptors and their ligands have been reported in mammary gland development. In mice, EphB4 is expressed on myoepithelial cells, whereas EphrinB2 is expressed on the luminal cells. Moreover, EphB4 and EphrinB2 expression is dependent on estrogen and regulated during the estrus cycle [9]. Of all Eph receptors, the roles of EphA2, EphB4 and EphB6 have been most extensively studied in breast cancer and those particular Eph receptors and their ligands appear to have dual roles in tumor suppression and tumor promotion for breast carcinomas. EphA2 is expressed at a low level in normal mammary gland epithelium, but it is at a high level in breast cancer [10] and increased expression of EphA2 is associated with poor patient prognosis [11]. High levels of EphB4 receptor protein were also found in breast cancer cell lines and its EphrinB2 ligand was in a low level; however, mounting evidence suggests that the EphB4 receptor can also function as a tumor suppressor in breast cancer. For example, EphB4 could act as a tumor suppressor through Abl tyrosine kinase and the Crk adaptor protein in a mouse xenograft model of breast cancer [12]. However, contradicting this, EphB4 protein was detected in 58% (7/12 cases) breast cancer specimens and in four breast cancer cell lines, where overexpression of EphB4 in breast cancer cells was driven by gene amplification and by the ErbB family of receptors via activation of Janus tyrosine kinase-signal transducers and activators of transcription and protein kinase B. Therefore, biologically active EphB4 may function as a survival factor in breast cancer [13]. EphB6 is an Eph receptor lacking catalytic capacity. Increasing data show that EphB6 acts as a suppressor of cancer aggressiveness in breast cancer [14–16]. Until now, no detailed expression profile of the EphrinB1 ligand has been reported in breast cancers. In the present study, we detected EphrinB1 in a set of 75 breast cancer samples and analyzed the relation between its expression profile and clinicopathological parameters.
of a study approved by Nanjing Maternity and Child Health Care Hospital, China. These patients had undergone surgery without any preoperative therapy between 2008 and 2012 in Nanjing Maternity and Child Health Care Hospital, China. After surgery, 71 patients were confirmed pathologically to express invasive ductal carcinomas (IDC) and 4 patients expressed invasive lobular carcinomas (ILC). All samples were diagnosed and classified according to the World Health Organization (WHO) grading system. 2.2. Immunohistochemical staining Sections from surgical specimens had been fixed in 10% formalin and embedded in paraffin and they were used here for immunohistochemical staining according to a standard method. Briefly, each 4-µm tissue section was deparaffinized and rehydrated. After rehydration through a graded ethanol series, the sections were autoclaved in 10 mM citrate buffer (pH 6.0) at 120◦C for 2 min for antigen retrieval, then cooled to 30◦ C and washed with phosphate-buffered saline (PBS, pH 7.3). After endogenous peroxidase had been quenched with aqueous 3% H2 O2 for 10 minutes and washed with PBS, the sections were incubated at 4◦ C overnight with a polyclonal antibody (Abgent, San Diego, CA, USA) at a 1:100 dilution in antibody diluent solution (Zymed, Invitrogen) and then washed with PBS. Next, the sections were incubated with secondary antibody (Dako REAL EnVision Detection System, Dako, UK) for 30 min at room temperature. Color development was performed with 3, 3’-diaminobenzidine (DAB). Nuclei were lightly counterstained with hematoxylin. Two pathologists independently assessed the immunostained slides. Any difference in immunohistochemical scores was resolved by a consensus. Immunohistochemical staining of cancer cells was semiquantitatively assessed according to the staining intensity and percentage of positive cells. EphrinB1 expression was assessed for intensity (0 = no staining, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (0 = 0%, 1 10%, 2 = 10% to 50%, 3 = 51% to 80%, 4 80% positive cells) as defined previously. The scores for intensity and percentage were added and a cut-off of 6 was used.
2. Materials and methods 2.3. Statistical analysis 2.1. Tissue samples The breast cancer samples were collected from 75 patients (mean age 53.2 years, range 21–76) as part
The statistical significance of intergroup differences was evaluated by a chi-square test. Kaplan-Meier survival curve was calculated, and the differences in the
H. Yin et al. / Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer
263
Fig. 1. Expression of EphrinB1 in breast cancers. (a) Negative staining of EphrinB1 and H&E staining (b); (c) weak staining EphrinB1 and H&E staining (d); (e) moderate EphrinB1 staining and H&E staining (f); (g) strong EphrinB1 staining and H&E staining (h). (Magnification × 100). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-130356)
curves were using the log-rank test. The breast cancerrelated death (overall survival) were used as the endpoints. All statistical analyses were performed using SPSS software (SPSS 16.0, Chicago, IL). A two-sided P value of less than 0.05 was considered statistically significant.
3. Results 3.1. Expression of EphrinB1 was detected in breast cancers Expression of EphrinB1 was investigated in the set of 75 breast cancer tissue samples by using a spe-
264
H. Yin et al. / Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer Table 1 Relationship between expression of EphrinB1 and histopathological parameters Parameters Total number Age (years) < 50 50 Tumor diameter (cm)
Cancer Biomarkers 13 (2013) 261–267 DOI 10.3233/CBM-130356 IOS Press
Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer Hong Yina,b , Cheng Lub , Yongfeng Tangb , Haiyan Wangc , Hai Wanga and Jiandong Wanga,c,∗ a
Department of Pathology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, China Department of Surgery, Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu, China c Department of Pathology, Southern Medical University, Guangzhou, Guangdong, China b
Abstract. BACKGROUND: The Eph family of receptor tyrosine kinases and their ephrin ligands are membrane-bound cell-signaling proteins and they play critical regulatory roles in embryonic development and carcinogenesis. Eph receptors require direct cell to cell interaction for activation and they are divided into EphA and EphB receptor classes, depending on their preferential binding affinity for EphrinA or EphrinB ligands. Eph receptors have been documented in breast cancer, but the Ephrin ligands have not been thoroughly investigated. MATERIALS AND METHODS: We conducted a systematic assessment of EphrinB1 expression in a set of 75 formalin-fixed paraffin-embedded breast cancers, using immunohistochemical staining (IHC) with a specific antibody, and we examined the relationship between EphrinB1 expression, histopathological parameters, and the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. RESULTS: High level expression of EphrinB1 is positively associated with lymph node metastasis (P < 0.0001) and with the presence of HER-2 receptor (P = 0.041) and it is more often detected in triple-negative breast carcinomas (P = 0.038). No relation was apparent between EphrinB1 expression level and other histopathological parameters, but enhanced EphrinB1 expression is associated with shorter overall survival (P = 0.015). CONCLUSION: Our analysis demonstrates that EphrinB1 expression is related to the metastasis of breast cancer and its enhanced expression confers a poor prognosis, suggesting that EphrinB1 may be a relevant therapeutic target in breast cancers. Keywords: EphrinB1, breast cancer, metastasis, triple-negative breast cancer
1. Introduction The large family of Eph receptor tyrosine kinases and their Ephrin ligands play important cell-signaling roles in embryonic development and carcinogenesis. Eph receptors and Ephrin ligands are divided into A and B classes based on their sequence similarity, common structural features and binding affinities; EphA and EphB receptors interact with and preferentially ∗ Corresponding author: Jiandong Wang, Department of Pathology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, China. E-mail: [email protected].
bind to their respective EphrinA and EphrinB ligands. The receptors and ligands interact through cell-cell contact, with the potential to transduce forward signals from the receptors and reverse signals from the ligands and referred to as bidirectional signaling. EphEphrin signaling modulates a variety of biological activities, including cell-cell interaction, cell migration, cell segregation, cell attachment, and shape. Eph receptors and Ephrin ligands have been implicated in diverse developmental functions, especially neurological development [1–5]. Eph and Ephrin are involved in oncogenesis of many types of cancer, including colorectal, lung, prostate, and breast cancers [6–8]. Therefore,
c 2013 – IOS Press and the authors. All rights reserved ISSN 1574-0153/13/$27.50
262
H. Yin et al. / Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer
they might be valuable candidates for cancer diagnosis and prognosis and they might be new therapeutic targets. Eph receptors and their ligands have been reported in mammary gland development. In mice, EphB4 is expressed on myoepithelial cells, whereas EphrinB2 is expressed on the luminal cells. Moreover, EphB4 and EphrinB2 expression is dependent on estrogen and regulated during the estrus cycle [9]. Of all Eph receptors, the roles of EphA2, EphB4 and EphB6 have been most extensively studied in breast cancer and those particular Eph receptors and their ligands appear to have dual roles in tumor suppression and tumor promotion for breast carcinomas. EphA2 is expressed at a low level in normal mammary gland epithelium, but it is at a high level in breast cancer [10] and increased expression of EphA2 is associated with poor patient prognosis [11]. High levels of EphB4 receptor protein were also found in breast cancer cell lines and its EphrinB2 ligand was in a low level; however, mounting evidence suggests that the EphB4 receptor can also function as a tumor suppressor in breast cancer. For example, EphB4 could act as a tumor suppressor through Abl tyrosine kinase and the Crk adaptor protein in a mouse xenograft model of breast cancer [12]. However, contradicting this, EphB4 protein was detected in 58% (7/12 cases) breast cancer specimens and in four breast cancer cell lines, where overexpression of EphB4 in breast cancer cells was driven by gene amplification and by the ErbB family of receptors via activation of Janus tyrosine kinase-signal transducers and activators of transcription and protein kinase B. Therefore, biologically active EphB4 may function as a survival factor in breast cancer [13]. EphB6 is an Eph receptor lacking catalytic capacity. Increasing data show that EphB6 acts as a suppressor of cancer aggressiveness in breast cancer [14–16]. Until now, no detailed expression profile of the EphrinB1 ligand has been reported in breast cancers. In the present study, we detected EphrinB1 in a set of 75 breast cancer samples and analyzed the relation between its expression profile and clinicopathological parameters.
of a study approved by Nanjing Maternity and Child Health Care Hospital, China. These patients had undergone surgery without any preoperative therapy between 2008 and 2012 in Nanjing Maternity and Child Health Care Hospital, China. After surgery, 71 patients were confirmed pathologically to express invasive ductal carcinomas (IDC) and 4 patients expressed invasive lobular carcinomas (ILC). All samples were diagnosed and classified according to the World Health Organization (WHO) grading system. 2.2. Immunohistochemical staining Sections from surgical specimens had been fixed in 10% formalin and embedded in paraffin and they were used here for immunohistochemical staining according to a standard method. Briefly, each 4-µm tissue section was deparaffinized and rehydrated. After rehydration through a graded ethanol series, the sections were autoclaved in 10 mM citrate buffer (pH 6.0) at 120◦C for 2 min for antigen retrieval, then cooled to 30◦ C and washed with phosphate-buffered saline (PBS, pH 7.3). After endogenous peroxidase had been quenched with aqueous 3% H2 O2 for 10 minutes and washed with PBS, the sections were incubated at 4◦ C overnight with a polyclonal antibody (Abgent, San Diego, CA, USA) at a 1:100 dilution in antibody diluent solution (Zymed, Invitrogen) and then washed with PBS. Next, the sections were incubated with secondary antibody (Dako REAL EnVision Detection System, Dako, UK) for 30 min at room temperature. Color development was performed with 3, 3’-diaminobenzidine (DAB). Nuclei were lightly counterstained with hematoxylin. Two pathologists independently assessed the immunostained slides. Any difference in immunohistochemical scores was resolved by a consensus. Immunohistochemical staining of cancer cells was semiquantitatively assessed according to the staining intensity and percentage of positive cells. EphrinB1 expression was assessed for intensity (0 = no staining, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (0 = 0%, 1 10%, 2 = 10% to 50%, 3 = 51% to 80%, 4 80% positive cells) as defined previously. The scores for intensity and percentage were added and a cut-off of 6 was used.
2. Materials and methods 2.3. Statistical analysis 2.1. Tissue samples The breast cancer samples were collected from 75 patients (mean age 53.2 years, range 21–76) as part
The statistical significance of intergroup differences was evaluated by a chi-square test. Kaplan-Meier survival curve was calculated, and the differences in the
H. Yin et al. / Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer
263
Fig. 1. Expression of EphrinB1 in breast cancers. (a) Negative staining of EphrinB1 and H&E staining (b); (c) weak staining EphrinB1 and H&E staining (d); (e) moderate EphrinB1 staining and H&E staining (f); (g) strong EphrinB1 staining and H&E staining (h). (Magnification × 100). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-130356)
curves were using the log-rank test. The breast cancerrelated death (overall survival) were used as the endpoints. All statistical analyses were performed using SPSS software (SPSS 16.0, Chicago, IL). A two-sided P value of less than 0.05 was considered statistically significant.
3. Results 3.1. Expression of EphrinB1 was detected in breast cancers Expression of EphrinB1 was investigated in the set of 75 breast cancer tissue samples by using a spe-
264
H. Yin et al. / Enhanced expression of EphrinB1 is associated with lymph node metastasis and poor prognosis in breast cancer Table 1 Relationship between expression of EphrinB1 and histopathological parameters Parameters Total number Age (years) < 50 50 Tumor diameter (cm)
