Med Oncol (2014) 31:210 DOI 10.1007/s12032-014-0210-6
ORIGINAL PAPER
HBXIP expression predicts patient prognosis in breast cancer Daye Cheng • Bin Liang • Yunhui Li
Received: 6 August 2014 / Accepted: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Emerging evidence demonstrated that hepatitis B virus X-interacting protein (HBXIP) has broad roles in cancers. The aim of the study is to investigate the association between HBXIP expression and clinicopathological features of breast cancer patients so as to determine whether HBXIP protein may be correlated with poor prognosis in breast cancer patients. HBXIP protein expression was assessed in a well-characterized series of breast cancer (n = 196) with long-term follow-up, using immunohistochemistry method. Correlation between HBXIP expression and clinicopathological factors was analyzed. The effects of several variables on survival were tested by a Cox proportional hazards regression analysis. High HBXIP expression was predominantly observed in breast cancer tissues but not the adjacent normal breast tissues. The expression of HBXIP was high in 125 (63.8 %) of the 196 cancer patients and low in 71 (36.2 %) of the 196 patients, respectively. High HBXIP expression was positively correlated with TNM stage (P = 0.001), lymph node metastasis (P \ 0.001), and Ki67 expression (P = 0.002). The patients with high HBXIP expression had lower 5-year overall survival (OS) and disease-free survival (DFS) rates
D. Cheng (&) Department of Transfusion, The First Hospital of China Medical University, North Nanjing Street, No. 155, Shenyang People’s Republic of China e-mail: [email protected] B. Liang High Vocational Technological College, China Medical University, Shenyang, People’s Republic of China Y. Li Department of Clinical Laboratory, No. 202 Hospital, Shenyang People’s Republic of China
than those with low HBXIP expression as determined using the Kaplan–Meier method (OS: P = 0.006; DFS: P = 0.022). In Cox regression analysis, both HBXIP expression (P = 0.002 and P = 0.009, respectively) and lymph node metastasis (P \ 0.001 and P = 0.008, respectively) were associated with poor OS and DFS. Our study suggested that high HBXIP is associated with the progression of breast cancer. HBXIP could be a valuable prognostic marker as well as a potential molecular therapy target for breast cancer patients. Keywords Breast cancer Hepatitis B virus X-interacting protein Prognosis Immunohistochemistry
Introduction Breast cancer is the most common cancer, and the main cause of death among women, with an estimated 170,000 new cases, in 2008 in China alone [1]. In spite of advances in cancer management and treatment, only a limited progress has been accomplished in recent years in the mortality of patients and the control of metastatic disease. Breast cancer, like other cancers, is a disease that occurs as a result of multiple genetic alterations, including the upregulation of growth-promoting oncogenes and the downregulation of growth-inhibiting tumor suppressor genes [2]. To date, clinical characteristics in breast cancer, such as age, menstrual status, tumor size, lymph node status, and morphological characteristics, are the most important prognostic factors [3]. However, a better understanding of the molecular mechanisms involved in breast cancer initiation and progression will likely contribute to provide useful prognostic biomarkers and therapeutic targets for breast cancer therapy.
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The hepatitis B virus X-interacting protein (HBXIP), originally cloned by Melegari et al. from HepG2 cells [4], is specifically associated with the C-terminal of the X protein of the hepatitis B virus (HBX) and negatively regulates the activity of HBX [4]. Emerging evidence demonstrated that HBXIP has broad roles in cancers. In hepatoma cells, HBXIP functions as a cofactor for survivin, and serves as a link between the cellular apoptosis machinery and a viral pathogen involved in hepatocellular carcinogenesis [5]. HBXIP was overexpressed in clinical breast cancer tissues and could promote proliferation and migration through NF-jB, MAPK/ERK, or PI3K/AKT pathways in breast cancer cells [6–8]. Moreover, it was also shown that HBXIP is a regulator of centrosome duplication and is required for bipolar spindle formation in HeLa human carcinoma cells and primary mouse embryonic fibroblasts [9]. Although HBXIP has been implicated in several types of cancers, the frequency and clinical significance of HBXIP in breast cancer has yet to be determined. Therefore, our aim in this study was to investigate whether or not HBXIP expression has a pathological significance in the development or progression of breast cancer. We performed immunohistochemistry (IHC) of HBXIP in breast cancer tissue, and examined the association of HBXIP protein expression with clinical outcome in terms of survival in 196 breast cancer patients.
Materials and methods Patients From March 2002 to May 2008, 196 cases of newly diagnosed and surgically treated breast cancer patients at the Department of Breast Surgery, No. 202 Hospital, were included in this study. Paired cancer and adjacent noncancerous breast tissues, located at least 1 cm away from the tumor, were collected from the surgically treated breast cancer patients. For all specimens, histopathological workup was performed by two experienced, board-certified breast pathologists. The histological subtype was determined according to the World Health Organization classification [10], and the TNM stage was determined postoperatively according to the American Joint Committee on Cancer [11]. None of the patients received chemotherapy, radiotherapy, or immunotherapy prior to surgery. The clinicopathological data of patients are summarized in Table 1. The length of follow-up ranged from 1 to 66 months, with a mean of 42 months. Informed consent was obtained from all patients for the use of their samples to detect HBXIP expression. The present study conformed to the ethical standards of the
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Med Oncol (2014) 31:210 Table 1 Clinicopathological data in breast cancer patients Numbers of patients Total number of patients
%
196
Age (years) B50
124
63.3
[50
72
36.7
Premenopausal
141
71.9
Postmenopausal
55
28.1
Ductal
138
70.4
Lobular
58
29.6
Menopausal status
Histological subtype
Tumor size B2 cm
87
44.4
109
55.6
119
60.7
77
39.3
101
51.5
95
48.5
Negative
117
59.7
Positive
79
40.3
Negative
110
56.1
Positive
86
43.9
104 92
53.1 46.9
Negative
78
39.8
Positive
118
60.2
[2 cm Grade G1, G2 G3 TNM stage I ? II III Lymph node metastasis
ER status
PR status Negative Positive Her-2 status
Ki67 Negative
58
29.6
Positive
138
70.4
HBXIP expression Low
71
36.2
High
125
63.8
World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of No. 202 Hospital. Immunohistochemistry Immunohistochemical analysis of breast tissue was performed as described previously before [12]. Briefly, paraffin sections were cut at 4 lm thickness, mounted on silane-coated slides and incubated overnight at 37 °C.
Med Oncol (2014) 31:210
Sections were washed with distilled water after two changes of xylene and three changes of ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0), and sections were held in Tris buffered saline (TBS). Endogenous peroxidase activity was blocked by incubation in 3 % hydrogen peroxide. The sections were incubated overnight in mouse monoclonal HBXIP primary antibody (Santa Cruz Biotechnology, Inc., CA, USA) diluted with 1/100 in 1 % BSA in Tris buffer (100 mM, pH 7.6) at room temperature. Antibody binding was amplified using horseradish peroxidase- conjugated goat anti-rabbit IgG for 15 min each and the complex was visualized using DAB Horseradish Peroxidase Color Development Kit (Maixin Co., Fuzhou, China). Estrogen receptor (ER), progesterone receptor (PR), and HER2 status, and Ki67 status were analyzed by immunohistochemical staining (DAKO Company, Shanghai, China). Immunohistochemistry scoring Immunohistochemical evaluation was performed by counting at least 1,000 tumor cells in each case by two independent observers blinded to the clinical data, with complete observer agreement. The expression of HBXIP was evaluated for the percentage of positively stained tumor cells and the staining intensity. The percent positivity was scored as ‘‘0’’ (\5 %, negative), ‘‘1’’ (5–25 %, sporadic), ‘‘2’’ (25–50 %, focal), or ‘‘3’’ ([50 %, diffuse). The staining intensity was scored as ‘‘0’’ (negative), ‘‘1’’ (weak staining), ‘‘2’’ (moderate staining), or ‘‘3’’ (strong staining). The total score, ranged from 0 to 9, was calculated by multiplying the intensity and percentage scores. Based on the HBXIP expression levels, the breast cancer patients were divided into two groups: the low HBXIP expression group (score 1–3) and the high HBXIP expression group (score 4–9). Statistical analysis All statistical analyses were performed using the SPSS statistical package (version 15.0, SPSS Inc, Chicago, IL, USA). Statistical significances between HBXIP expression and clinicopathological parameters were assessed using the v2 or Mann–Whitney U tests. Kaplan–Meier curves were used to assess the effect of HBXIP expression on diseasefree survival (DFS) and overall survival (OS). OS was defined as the time from surgery to death from breast cancer. Patients known to be still alive at time of analysis were censored at time of their last follow-up. DFS was calculated from the time of surgery until the first progression. A Cox proportional hazards regression model was developed to evaluate the association between the potential
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prognostic marker and patients’ survival. Cox regression analysis was conducted at both univariate and multivariate levels. All P values were based on a two-tailed statistical analysis, and P \ 0.05 was considered statistically significant.
Results The clinicopathological features of the 196 patients with breast cancer are summarized in Table 1. All patients were female between the ages of 26 and 77 years (50.7 ± 11.3 years). Of the paraffin-embedded breast cancer specimens examined, the expression of HBXIP was high in 125 (63.8 %) of the 196 cancer patients and low in 71 (36.2 %) of the 196 patients, respectively. Further analysis revealed HBXIP staining was predominantly in breast cancer tissue but not the adjacent normal breast tissues/non-cancerous tissues (P \ 0.001). As shown in Fig. 1, HBXIP was detected in cancer cells either in the nucleus, nucleus and cytoplasm or cytoplasm, with the combined pattern of staining predominating. Table 2 summarizes the associations between HBXIP expression and clinicopathological variables. High expression of HBXIP was positively correlated with TNM stage (P = 0.001), lymph node metastasis (P \ 0.001), and Ki67 expression (P = 0.002). In contrast, there was no significant correlation between the HBXIP expression and the clinicopathological parameters including age (P = 0.360), menopausal status (P = 0.247), histological subtype (P = 0.748), tumor size (P = 0.134), tumor grade (P = 0.226), ER status (P = 0.136), PR status (P = 0.372), and Her-2 status (P = 0.130). To further validate the potential clinical utility of HBXIP expression, we evaluated the prognostic power of HBXIP protein on OS and DFS in 196 breast cancer patients. The patients with high HBXIP expression had lower 5-year OS and DFS rates than those with low HBXIP expression as determined using the Kaplan–Meier method (OS: P = 0.006; DFS: P = 0.022) (Fig. 2a, b). To evaluate the impact of each variable on OS and DFS, univariate and multivariate Cox regression were used, as shown in Tables 3 and 4. In the univariate analysis, the significant factors associated with OS included TNM stage (HR = 2.347, 95 % CI = 1.696–4.026, P = 0.006), lymph node metastasis (HR = 2.865, 95 % CI = 1.702–5.252, P \ 0.001), and HBXIP expression (HR = 2.777, 95 % CI = 1.847–5.009, P = 0.002; Table 3), whereas the significant factors associated with DFS included tumor grade (HR = 1.812, 95 % CI = 1.303–3.018, P = 0.041), TNM stage (HR = 2.564, 95 % CI = 1.707–4.210, P = 0.010), lymph node
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Fig. 1 Immunohistochemical staining of HBXIP and Ki67 in breast cancer tissues (9400). a, b Negative expression of HBXIP and Ki67 in the matched adjacent normal breast tissues/non-cancerous tissues.
c, d Weak staining of HBXIP and Ki67 in breast cancer tissue. e, f Moderate staining of HBXIP and Ki67 in breast cancer tissue. g, h Strong staining of HBXIP and Ki 67 in breast cancer tissue
metastasis (HR = 2.722, 95 % CI = 1.630–4.127, P = 0.008), and HBXIP expression (HR = 2.583, 95 % CI = 1.710–4.805, P = 0.009; Table 4). In multivariate analyses, both HBXIP expression (P = 0.002 and P = 0.009, respectively) and lymph node metastasis (P \ 0.001 and P = 0.008, respectively) were associated with poor OS and DFS (Tables 3, 4).
Discussion
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HBXIP, a recently identified nucleolar protein, is involved in the regulation of centrosome duplication, causing excessive centrosome production and multipolar mitotic spindles in HeLa cells [9, 13]. Accumulating evidence indicates that HBXIP expression is closely associated with
Med Oncol (2014) 31:210
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Table 2 Correlation of HBXIP immunoreactivity with clinicopathological variables in breast cancer patients Low HBXIP expression
High HBXIP expression
48
76
23
49
Age (years) B50 [50 Menopausal status Premenopausal
55
86
Postmenopausal
16
39
Histological subtype Ductal
49
89
Lobular
22
36
Tumor size B2 cm
37
50
34
75
G1 ? G2
39
80
G3
32
45
I ? II
48
53
III
23
74
[2 cm Grade
TNM stage
Positive ER status
P value
0.902
0.360
1.684
0.247
0.104
0.748
2.691
0.134
1.562
0.226
12.199
0.001
17.020 \0.001
Lymph node metastasis Negative
v2 value
56
61
15
64
Negative
45
65
Positive
26
60
Negative
41
63
Positive
30
62
Negative
23
55
Positive
48
70
Negative
31
27
Positive
40
98
PR status
Her-2 status
Ki67
2.381
0.136
0.981
0.372
2.546
0.130
10.578
0.002
tumor development and progression [6–8, 14]. In this study, we incorporated staining intensity and percentage of immunopositive tumor cells for the scoring of HBXIP expression and examined a more diverse set of breast tumors. The results indicated that high HBXIP expression was significantly higher in cancerous tissues than adjacenttumor tissues. Moreover, HBXIP protein was found to be related to tumor stage, lymph node metastasis, and Ki67 status in the 196 breast cancers studied. The Cox regression analysis showed HBXIP protein was detected as an independent prognostic factor for OS and DFS. HBXIP gene, encoding a 9.6 kDa protein, located at human chromosome 1p13.3 [4]. It contained a putative leucine zipper motif and 2 consensus phosphorylation sites at threonines 12 and 36 for protein kinase C and casein kinase II [15]. Importantly, previous studies showed that HBXIP plays crucial roles in the development of breast cancer, serving as a key oncoprotein in cancer [7, 8, 16]. HBXIP functions as a novel oncogenic coactivator of some transcription factors, such as STAT4, Sp1 and E2F1, to transactivate S100A4, LMO4, and Skp2 in promotion of cell proliferation and migration of breast cancer cells [8, 17, 18]. Cui WJ et al. also reported that HBXIP is able to regulate CD46, CD55, and CD59 through p-ERK1/2/NFjB signaling to protect breast cells from complement attack [6]. Nan’s result reported that 75 and 94 % of breast cancer tissues and metastatic lymph tissues, respectively, were positive for HBXIP expression [7]. Similar with Nan’s, our study indicated that HBXIP staining was predominantly in breast cancer tissue but not the adjacent normal breast tissues/non-cancerous tissues. Therefore, the abnormality of HBXIP expression is oncogenic and may facilitate carcinogenesis and tumor progression by transactivating more important genes to promote proliferation of cancer cells as a transcriptional coactivator. Breast cancer is a highly heterogeneous disease in terms of morphology, molecular characteristics, and response to treatment, which suggests the existence of molecular subsets. Thus, the identification of molecular predictive signatures is necessary and will allow for the characterization of breast cancer and the design of optimal treatment
Fig. 2 Kaplan–Meier survival curves for overall survival (OS) and disease-free survival (DFS) according to HBXIP expression, P values were obtained by logrank test. a OS curves of breast cancer patients according to HBXIP expression (P = 0.006); b DFS curves of breast cancer patients according to HBXIP expression (P = 0.022)
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210 Page 6 of 7 Table 3 Univariate and multivariate analysis of overall survival in breast cancer
Table 4 Univariate and multivariate analysis of diseasefree survival in breast cancer
Med Oncol (2014) 31:210
Variables
Univariate analysis HR
95 % CI
Age (B50 vs. [50)
1.125
1.008–1.564
Menopausal status (Pre- vs. Post-)
1.202
1.100–1.963
0.444
Histological subtype (ductal vs. lobular)
1.187
1.024–1.744
0.480
Tumor size (B2 vs. [2 cm)
1.304
1.009–2.001
0.416
Grade (G1 ? G2 vs. G3)
1.522
1.134–2.017
0.250
TNM stage (I ? II vs. III)
2.347
1.696–4.025
0.006
1.639
1.222–2.520
0.112
Lymph node metastasis (negative vs. positive) ER status (negative vs. positive)
2.865
1.702–5.252 \0.001
2.020
1.547–3.863
0.010
1.310
1.112–1.856
0.411
PR status (negative vs. positive)
1.477
1.131–2.222
0.327
Her-2 status (negative vs. positive)
1.239
1.007–1.830
0.423
Ki 67 status (negative vs. positive)
1.585
1.113–2.311
0.236
HBXIP expression (low vs. high)
2.777
1.847–5.009
0.002
1.814
1.119–3.241
0.032
Variables
Univariate analysis
Age (B50 vs. [50)
P value
HR
95 % CI
P value
0.517
Univariate analysis
HR
95 % CI
P value
1.102
1.004–1.600
0.623
Menopausal status (Pre- vs. Post-)
1.312
1.115–2.054
0.328
Histological subtype (ductal vs. lobular)
1.207
1.016–1.835
0.520
HR
95 % CI
P value
Tumor size (B2 vs. [2 cm)
1.403
1.136–2.101
0.333
Grade (G1 ? G2 vs. G3)
1.812
1.303–3.018
0.041
1.411
1.128–2.054
0.213
TNM stage (I ? II vs. III)
2.564
1.707–4.210
0.010
1.540
1.205–2.310
0.117
Lymph node metastasis (negative vs. positive) ER status (negative vs. Positive)
2.722
1.630–4.127
0.008
1.986
1.448–3.245
0.035
1.436
1.110–1.963
0.325
PR status (negative vs. positive)
1.515
1.213–2.301
0.286
Her-2 status (negative vs. positive)
1.325
1.019–1.867
0.384 1.724
1.253–3.052
0.038
Ki 67 status (negative vs. positive)
1.696
1.212–2.477
0.089
HBXIP expression (low vs. high)
2.583
1.710–4.805
0.009
modalities. Current established prognostic indicators for breast cancer include parameters such as tumor size, histological subtype, grade, lymph node metastases, and lymphovascular invasion, hormonal receptor status, Her2 gene amplification, and Ki67 status [19, 20]. In the present study, we analyzed the association of HBXIP expression with the clinicopathological factors in breast cancer patients, which was the first study to demonstrate in detail an association among them. We found that increased HBXIP expression was associated with higher TNM stage, lymph node metastasis, and Ki67 status and thus with the acquisition of invasive, proliferation, and metastatic potential. The current study investigated the prognostic value of HBXIP expression in patients with breast cancer, because few studies have discussed the relationship between HBXIP expression and the long-term prognosis in these
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Multivariate analysis
patients. The present study found that the long-term prognosis for OS and DFS was significantly poorer in breast cancer patients with high HBXIP expression than in those with low HBXIP expression in breast cancer. Cox regression analysis revealed that lymph node metastasis and HBXIP expression were independent prognostic factors in patients with breast cancer. This data indicated that HBXIP is an important biomarker for the detection of breast cancer.
Conclusion In summary, our study provides evidence the high HBXIP expression is associated with worse prognosis in breast cancer. In addition, HBXIP is correlated with development of breast cancer. This is the first study evaluating the
Med Oncol (2014) 31:210
relationship between HBXIP expression and prognosis in breast cancer patients, and a larger prospective study is currently underway to confirm the prognostic value of HBXIP in breast cancer. However, the exact mechanism by which HBXIP enhances the proliferation of breast cancer cells remains poorly understood and requires further investigation. Acknowledgments This study was supported by Liaoning Natural Science Fund, Liaoning Province, China (Topic No: 201102279). Conflict of interest of interest.
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9.
10.
11.
12.
The authors declare that there are no conflicts
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migration of breast cancer cells. J Biol Chem. 2012;287(36): 30228–39. Fujii R, Zhu C, Wen Y, et al. HBXIP, cellular target of hepatitis B virus oncoprotein, is a regulator of centrosome dynamics and cytokinesis. Cancer Res. 2006;66(18):9099–107. Bocker W. WHO classification of breast tumors and tumors of the female genital organs: pathology and genetics. Verh Dtsch Ges Pathol. 2002;86:116–9. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol. 2010;17(6):1471–4. Liang B, Wu M, Bu Y, Zhao A, Xie F. Prognostic value of TMPRSS4 expression in patients with breast cancer. Med Oncol. 2013;30(2):497. Wen Y, Golubkov VS, Strongin AY, Jiang W, Reed JC. Interaction of hepatitis B viral oncoprotein with cellular target HBXIP dysregulates centrosome dynamics and mitotic spindle formation. J Biol Chem. 2008;283(5):2793–803. Zhang Y, Zhao Y, Li L, et al. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells. Biochem Biophys Res Commun. 2013;434(2):305–10. Wang FZ, Sha L, Zhang WY, et al. Involvement of hepatitis B X-interacting protein (HBXIP) in proliferation regulation of cells. Acta Pharmacol Sin. 2007;28(3):431–8. Wang FZ, Fei HR, Lian LH, Wang JM, Qiu YY. Hepatitis B x-interacting protein induces HepG2 cell proliferation through activation of the phosphatidylinositol 3-kinase/Akt pathway. Exp Biol Med (Maywood). 2011;236(1):62–9. Yue L, Li L, Liu F, et al. The oncoprotein HBXIP activates transcriptional coregulatory protein LMO4 via Sp1 to promote proliferation of breast cancer cells. Carcinogenesis. 2013;34(4):927–35. Xu F, You X, Liu F, et al. The oncoprotein HBXIP up-regulates Skp2 via activating transcription factor E2F1 to promote proliferation of breast cancer cells. Cancer Lett. 2013;333(1):124–32. Weigel MT, Dowsett M. Current and emerging biomarkers in breast cancer: prognosis and prediction. Endocr Relat Cancer. 2010;17(4):R245–62. Carlson RW, Allred DC, Anderson BO, et al. Breast cancer. Clinical practice guidelines in oncology. J Natl Compr Cancer Netw. 2009;7(2):122–92.
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ORIGINAL PAPER
HBXIP expression predicts patient prognosis in breast cancer Daye Cheng • Bin Liang • Yunhui Li
Received: 6 August 2014 / Accepted: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Emerging evidence demonstrated that hepatitis B virus X-interacting protein (HBXIP) has broad roles in cancers. The aim of the study is to investigate the association between HBXIP expression and clinicopathological features of breast cancer patients so as to determine whether HBXIP protein may be correlated with poor prognosis in breast cancer patients. HBXIP protein expression was assessed in a well-characterized series of breast cancer (n = 196) with long-term follow-up, using immunohistochemistry method. Correlation between HBXIP expression and clinicopathological factors was analyzed. The effects of several variables on survival were tested by a Cox proportional hazards regression analysis. High HBXIP expression was predominantly observed in breast cancer tissues but not the adjacent normal breast tissues. The expression of HBXIP was high in 125 (63.8 %) of the 196 cancer patients and low in 71 (36.2 %) of the 196 patients, respectively. High HBXIP expression was positively correlated with TNM stage (P = 0.001), lymph node metastasis (P \ 0.001), and Ki67 expression (P = 0.002). The patients with high HBXIP expression had lower 5-year overall survival (OS) and disease-free survival (DFS) rates
D. Cheng (&) Department of Transfusion, The First Hospital of China Medical University, North Nanjing Street, No. 155, Shenyang People’s Republic of China e-mail: [email protected] B. Liang High Vocational Technological College, China Medical University, Shenyang, People’s Republic of China Y. Li Department of Clinical Laboratory, No. 202 Hospital, Shenyang People’s Republic of China
than those with low HBXIP expression as determined using the Kaplan–Meier method (OS: P = 0.006; DFS: P = 0.022). In Cox regression analysis, both HBXIP expression (P = 0.002 and P = 0.009, respectively) and lymph node metastasis (P \ 0.001 and P = 0.008, respectively) were associated with poor OS and DFS. Our study suggested that high HBXIP is associated with the progression of breast cancer. HBXIP could be a valuable prognostic marker as well as a potential molecular therapy target for breast cancer patients. Keywords Breast cancer Hepatitis B virus X-interacting protein Prognosis Immunohistochemistry
Introduction Breast cancer is the most common cancer, and the main cause of death among women, with an estimated 170,000 new cases, in 2008 in China alone [1]. In spite of advances in cancer management and treatment, only a limited progress has been accomplished in recent years in the mortality of patients and the control of metastatic disease. Breast cancer, like other cancers, is a disease that occurs as a result of multiple genetic alterations, including the upregulation of growth-promoting oncogenes and the downregulation of growth-inhibiting tumor suppressor genes [2]. To date, clinical characteristics in breast cancer, such as age, menstrual status, tumor size, lymph node status, and morphological characteristics, are the most important prognostic factors [3]. However, a better understanding of the molecular mechanisms involved in breast cancer initiation and progression will likely contribute to provide useful prognostic biomarkers and therapeutic targets for breast cancer therapy.
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210 Page 2 of 7
The hepatitis B virus X-interacting protein (HBXIP), originally cloned by Melegari et al. from HepG2 cells [4], is specifically associated with the C-terminal of the X protein of the hepatitis B virus (HBX) and negatively regulates the activity of HBX [4]. Emerging evidence demonstrated that HBXIP has broad roles in cancers. In hepatoma cells, HBXIP functions as a cofactor for survivin, and serves as a link between the cellular apoptosis machinery and a viral pathogen involved in hepatocellular carcinogenesis [5]. HBXIP was overexpressed in clinical breast cancer tissues and could promote proliferation and migration through NF-jB, MAPK/ERK, or PI3K/AKT pathways in breast cancer cells [6–8]. Moreover, it was also shown that HBXIP is a regulator of centrosome duplication and is required for bipolar spindle formation in HeLa human carcinoma cells and primary mouse embryonic fibroblasts [9]. Although HBXIP has been implicated in several types of cancers, the frequency and clinical significance of HBXIP in breast cancer has yet to be determined. Therefore, our aim in this study was to investigate whether or not HBXIP expression has a pathological significance in the development or progression of breast cancer. We performed immunohistochemistry (IHC) of HBXIP in breast cancer tissue, and examined the association of HBXIP protein expression with clinical outcome in terms of survival in 196 breast cancer patients.
Materials and methods Patients From March 2002 to May 2008, 196 cases of newly diagnosed and surgically treated breast cancer patients at the Department of Breast Surgery, No. 202 Hospital, were included in this study. Paired cancer and adjacent noncancerous breast tissues, located at least 1 cm away from the tumor, were collected from the surgically treated breast cancer patients. For all specimens, histopathological workup was performed by two experienced, board-certified breast pathologists. The histological subtype was determined according to the World Health Organization classification [10], and the TNM stage was determined postoperatively according to the American Joint Committee on Cancer [11]. None of the patients received chemotherapy, radiotherapy, or immunotherapy prior to surgery. The clinicopathological data of patients are summarized in Table 1. The length of follow-up ranged from 1 to 66 months, with a mean of 42 months. Informed consent was obtained from all patients for the use of their samples to detect HBXIP expression. The present study conformed to the ethical standards of the
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Med Oncol (2014) 31:210 Table 1 Clinicopathological data in breast cancer patients Numbers of patients Total number of patients
%
196
Age (years) B50
124
63.3
[50
72
36.7
Premenopausal
141
71.9
Postmenopausal
55
28.1
Ductal
138
70.4
Lobular
58
29.6
Menopausal status
Histological subtype
Tumor size B2 cm
87
44.4
109
55.6
119
60.7
77
39.3
101
51.5
95
48.5
Negative
117
59.7
Positive
79
40.3
Negative
110
56.1
Positive
86
43.9
104 92
53.1 46.9
Negative
78
39.8
Positive
118
60.2
[2 cm Grade G1, G2 G3 TNM stage I ? II III Lymph node metastasis
ER status
PR status Negative Positive Her-2 status
Ki67 Negative
58
29.6
Positive
138
70.4
HBXIP expression Low
71
36.2
High
125
63.8
World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of No. 202 Hospital. Immunohistochemistry Immunohistochemical analysis of breast tissue was performed as described previously before [12]. Briefly, paraffin sections were cut at 4 lm thickness, mounted on silane-coated slides and incubated overnight at 37 °C.
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Sections were washed with distilled water after two changes of xylene and three changes of ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0), and sections were held in Tris buffered saline (TBS). Endogenous peroxidase activity was blocked by incubation in 3 % hydrogen peroxide. The sections were incubated overnight in mouse monoclonal HBXIP primary antibody (Santa Cruz Biotechnology, Inc., CA, USA) diluted with 1/100 in 1 % BSA in Tris buffer (100 mM, pH 7.6) at room temperature. Antibody binding was amplified using horseradish peroxidase- conjugated goat anti-rabbit IgG for 15 min each and the complex was visualized using DAB Horseradish Peroxidase Color Development Kit (Maixin Co., Fuzhou, China). Estrogen receptor (ER), progesterone receptor (PR), and HER2 status, and Ki67 status were analyzed by immunohistochemical staining (DAKO Company, Shanghai, China). Immunohistochemistry scoring Immunohistochemical evaluation was performed by counting at least 1,000 tumor cells in each case by two independent observers blinded to the clinical data, with complete observer agreement. The expression of HBXIP was evaluated for the percentage of positively stained tumor cells and the staining intensity. The percent positivity was scored as ‘‘0’’ (\5 %, negative), ‘‘1’’ (5–25 %, sporadic), ‘‘2’’ (25–50 %, focal), or ‘‘3’’ ([50 %, diffuse). The staining intensity was scored as ‘‘0’’ (negative), ‘‘1’’ (weak staining), ‘‘2’’ (moderate staining), or ‘‘3’’ (strong staining). The total score, ranged from 0 to 9, was calculated by multiplying the intensity and percentage scores. Based on the HBXIP expression levels, the breast cancer patients were divided into two groups: the low HBXIP expression group (score 1–3) and the high HBXIP expression group (score 4–9). Statistical analysis All statistical analyses were performed using the SPSS statistical package (version 15.0, SPSS Inc, Chicago, IL, USA). Statistical significances between HBXIP expression and clinicopathological parameters were assessed using the v2 or Mann–Whitney U tests. Kaplan–Meier curves were used to assess the effect of HBXIP expression on diseasefree survival (DFS) and overall survival (OS). OS was defined as the time from surgery to death from breast cancer. Patients known to be still alive at time of analysis were censored at time of their last follow-up. DFS was calculated from the time of surgery until the first progression. A Cox proportional hazards regression model was developed to evaluate the association between the potential
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prognostic marker and patients’ survival. Cox regression analysis was conducted at both univariate and multivariate levels. All P values were based on a two-tailed statistical analysis, and P \ 0.05 was considered statistically significant.
Results The clinicopathological features of the 196 patients with breast cancer are summarized in Table 1. All patients were female between the ages of 26 and 77 years (50.7 ± 11.3 years). Of the paraffin-embedded breast cancer specimens examined, the expression of HBXIP was high in 125 (63.8 %) of the 196 cancer patients and low in 71 (36.2 %) of the 196 patients, respectively. Further analysis revealed HBXIP staining was predominantly in breast cancer tissue but not the adjacent normal breast tissues/non-cancerous tissues (P \ 0.001). As shown in Fig. 1, HBXIP was detected in cancer cells either in the nucleus, nucleus and cytoplasm or cytoplasm, with the combined pattern of staining predominating. Table 2 summarizes the associations between HBXIP expression and clinicopathological variables. High expression of HBXIP was positively correlated with TNM stage (P = 0.001), lymph node metastasis (P \ 0.001), and Ki67 expression (P = 0.002). In contrast, there was no significant correlation between the HBXIP expression and the clinicopathological parameters including age (P = 0.360), menopausal status (P = 0.247), histological subtype (P = 0.748), tumor size (P = 0.134), tumor grade (P = 0.226), ER status (P = 0.136), PR status (P = 0.372), and Her-2 status (P = 0.130). To further validate the potential clinical utility of HBXIP expression, we evaluated the prognostic power of HBXIP protein on OS and DFS in 196 breast cancer patients. The patients with high HBXIP expression had lower 5-year OS and DFS rates than those with low HBXIP expression as determined using the Kaplan–Meier method (OS: P = 0.006; DFS: P = 0.022) (Fig. 2a, b). To evaluate the impact of each variable on OS and DFS, univariate and multivariate Cox regression were used, as shown in Tables 3 and 4. In the univariate analysis, the significant factors associated with OS included TNM stage (HR = 2.347, 95 % CI = 1.696–4.026, P = 0.006), lymph node metastasis (HR = 2.865, 95 % CI = 1.702–5.252, P \ 0.001), and HBXIP expression (HR = 2.777, 95 % CI = 1.847–5.009, P = 0.002; Table 3), whereas the significant factors associated with DFS included tumor grade (HR = 1.812, 95 % CI = 1.303–3.018, P = 0.041), TNM stage (HR = 2.564, 95 % CI = 1.707–4.210, P = 0.010), lymph node
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Fig. 1 Immunohistochemical staining of HBXIP and Ki67 in breast cancer tissues (9400). a, b Negative expression of HBXIP and Ki67 in the matched adjacent normal breast tissues/non-cancerous tissues.
c, d Weak staining of HBXIP and Ki67 in breast cancer tissue. e, f Moderate staining of HBXIP and Ki67 in breast cancer tissue. g, h Strong staining of HBXIP and Ki 67 in breast cancer tissue
metastasis (HR = 2.722, 95 % CI = 1.630–4.127, P = 0.008), and HBXIP expression (HR = 2.583, 95 % CI = 1.710–4.805, P = 0.009; Table 4). In multivariate analyses, both HBXIP expression (P = 0.002 and P = 0.009, respectively) and lymph node metastasis (P \ 0.001 and P = 0.008, respectively) were associated with poor OS and DFS (Tables 3, 4).
Discussion
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HBXIP, a recently identified nucleolar protein, is involved in the regulation of centrosome duplication, causing excessive centrosome production and multipolar mitotic spindles in HeLa cells [9, 13]. Accumulating evidence indicates that HBXIP expression is closely associated with
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Table 2 Correlation of HBXIP immunoreactivity with clinicopathological variables in breast cancer patients Low HBXIP expression
High HBXIP expression
48
76
23
49
Age (years) B50 [50 Menopausal status Premenopausal
55
86
Postmenopausal
16
39
Histological subtype Ductal
49
89
Lobular
22
36
Tumor size B2 cm
37
50
34
75
G1 ? G2
39
80
G3
32
45
I ? II
48
53
III
23
74
[2 cm Grade
TNM stage
Positive ER status
P value
0.902
0.360
1.684
0.247
0.104
0.748
2.691
0.134
1.562
0.226
12.199
0.001
17.020 \0.001
Lymph node metastasis Negative
v2 value
56
61
15
64
Negative
45
65
Positive
26
60
Negative
41
63
Positive
30
62
Negative
23
55
Positive
48
70
Negative
31
27
Positive
40
98
PR status
Her-2 status
Ki67
2.381
0.136
0.981
0.372
2.546
0.130
10.578
0.002
tumor development and progression [6–8, 14]. In this study, we incorporated staining intensity and percentage of immunopositive tumor cells for the scoring of HBXIP expression and examined a more diverse set of breast tumors. The results indicated that high HBXIP expression was significantly higher in cancerous tissues than adjacenttumor tissues. Moreover, HBXIP protein was found to be related to tumor stage, lymph node metastasis, and Ki67 status in the 196 breast cancers studied. The Cox regression analysis showed HBXIP protein was detected as an independent prognostic factor for OS and DFS. HBXIP gene, encoding a 9.6 kDa protein, located at human chromosome 1p13.3 [4]. It contained a putative leucine zipper motif and 2 consensus phosphorylation sites at threonines 12 and 36 for protein kinase C and casein kinase II [15]. Importantly, previous studies showed that HBXIP plays crucial roles in the development of breast cancer, serving as a key oncoprotein in cancer [7, 8, 16]. HBXIP functions as a novel oncogenic coactivator of some transcription factors, such as STAT4, Sp1 and E2F1, to transactivate S100A4, LMO4, and Skp2 in promotion of cell proliferation and migration of breast cancer cells [8, 17, 18]. Cui WJ et al. also reported that HBXIP is able to regulate CD46, CD55, and CD59 through p-ERK1/2/NFjB signaling to protect breast cells from complement attack [6]. Nan’s result reported that 75 and 94 % of breast cancer tissues and metastatic lymph tissues, respectively, were positive for HBXIP expression [7]. Similar with Nan’s, our study indicated that HBXIP staining was predominantly in breast cancer tissue but not the adjacent normal breast tissues/non-cancerous tissues. Therefore, the abnormality of HBXIP expression is oncogenic and may facilitate carcinogenesis and tumor progression by transactivating more important genes to promote proliferation of cancer cells as a transcriptional coactivator. Breast cancer is a highly heterogeneous disease in terms of morphology, molecular characteristics, and response to treatment, which suggests the existence of molecular subsets. Thus, the identification of molecular predictive signatures is necessary and will allow for the characterization of breast cancer and the design of optimal treatment
Fig. 2 Kaplan–Meier survival curves for overall survival (OS) and disease-free survival (DFS) according to HBXIP expression, P values were obtained by logrank test. a OS curves of breast cancer patients according to HBXIP expression (P = 0.006); b DFS curves of breast cancer patients according to HBXIP expression (P = 0.022)
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210 Page 6 of 7 Table 3 Univariate and multivariate analysis of overall survival in breast cancer
Table 4 Univariate and multivariate analysis of diseasefree survival in breast cancer
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Variables
Univariate analysis HR
95 % CI
Age (B50 vs. [50)
1.125
1.008–1.564
Menopausal status (Pre- vs. Post-)
1.202
1.100–1.963
0.444
Histological subtype (ductal vs. lobular)
1.187
1.024–1.744
0.480
Tumor size (B2 vs. [2 cm)
1.304
1.009–2.001
0.416
Grade (G1 ? G2 vs. G3)
1.522
1.134–2.017
0.250
TNM stage (I ? II vs. III)
2.347
1.696–4.025
0.006
1.639
1.222–2.520
0.112
Lymph node metastasis (negative vs. positive) ER status (negative vs. positive)
2.865
1.702–5.252 \0.001
2.020
1.547–3.863
0.010
1.310
1.112–1.856
0.411
PR status (negative vs. positive)
1.477
1.131–2.222
0.327
Her-2 status (negative vs. positive)
1.239
1.007–1.830
0.423
Ki 67 status (negative vs. positive)
1.585
1.113–2.311
0.236
HBXIP expression (low vs. high)
2.777
1.847–5.009
0.002
1.814
1.119–3.241
0.032
Variables
Univariate analysis
Age (B50 vs. [50)
P value
HR
95 % CI
P value
0.517
Univariate analysis
HR
95 % CI
P value
1.102
1.004–1.600
0.623
Menopausal status (Pre- vs. Post-)
1.312
1.115–2.054
0.328
Histological subtype (ductal vs. lobular)
1.207
1.016–1.835
0.520
HR
95 % CI
P value
Tumor size (B2 vs. [2 cm)
1.403
1.136–2.101
0.333
Grade (G1 ? G2 vs. G3)
1.812
1.303–3.018
0.041
1.411
1.128–2.054
0.213
TNM stage (I ? II vs. III)
2.564
1.707–4.210
0.010
1.540
1.205–2.310
0.117
Lymph node metastasis (negative vs. positive) ER status (negative vs. Positive)
2.722
1.630–4.127
0.008
1.986
1.448–3.245
0.035
1.436
1.110–1.963
0.325
PR status (negative vs. positive)
1.515
1.213–2.301
0.286
Her-2 status (negative vs. positive)
1.325
1.019–1.867
0.384 1.724
1.253–3.052
0.038
Ki 67 status (negative vs. positive)
1.696
1.212–2.477
0.089
HBXIP expression (low vs. high)
2.583
1.710–4.805
0.009
modalities. Current established prognostic indicators for breast cancer include parameters such as tumor size, histological subtype, grade, lymph node metastases, and lymphovascular invasion, hormonal receptor status, Her2 gene amplification, and Ki67 status [19, 20]. In the present study, we analyzed the association of HBXIP expression with the clinicopathological factors in breast cancer patients, which was the first study to demonstrate in detail an association among them. We found that increased HBXIP expression was associated with higher TNM stage, lymph node metastasis, and Ki67 status and thus with the acquisition of invasive, proliferation, and metastatic potential. The current study investigated the prognostic value of HBXIP expression in patients with breast cancer, because few studies have discussed the relationship between HBXIP expression and the long-term prognosis in these
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Multivariate analysis
patients. The present study found that the long-term prognosis for OS and DFS was significantly poorer in breast cancer patients with high HBXIP expression than in those with low HBXIP expression in breast cancer. Cox regression analysis revealed that lymph node metastasis and HBXIP expression were independent prognostic factors in patients with breast cancer. This data indicated that HBXIP is an important biomarker for the detection of breast cancer.
Conclusion In summary, our study provides evidence the high HBXIP expression is associated with worse prognosis in breast cancer. In addition, HBXIP is correlated with development of breast cancer. This is the first study evaluating the
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relationship between HBXIP expression and prognosis in breast cancer patients, and a larger prospective study is currently underway to confirm the prognostic value of HBXIP in breast cancer. However, the exact mechanism by which HBXIP enhances the proliferation of breast cancer cells remains poorly understood and requires further investigation. Acknowledgments This study was supported by Liaoning Natural Science Fund, Liaoning Province, China (Topic No: 201102279). Conflict of interest of interest.
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9.
10.
11.
12.
The authors declare that there are no conflicts
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