Overexpression of macrophage migration inhibitory factor induces angiogenesis in human breast cancer

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Cancer Letters 261 (2008) 147–157 www.elsevier.com/locate/canlet

Overexpression of macrophage migration inhibitory factor induces angiogenesis in human breast cancer Xiangdong Xu a, Bo Wang b, Caisheng Ye a, Chen Yao a, Ying Lin a, Xueling Huang a, Yunjian Zhang a, Shenming Wang a,* a

b

Department of Center of Breast Disease, The First Affiliated Hospital, Sun Yat-Sen University, 58 Zhongshan Road II, Guangzhou 510080, China Department of Laboratory Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China Received 14 April 2007; received in revised form 9 September 2007; accepted 6 November 2007

Abstract Macrophage migration inhibitory factor (MIF) is known to be an important contributor to tumor progression. Overexpression of MIF has been reported in different types of tumors. However, the correlation between MIF expression and tumor pathologic features in patients with breast cancer has not been elucidated. In this study, we examined the expression of MIF, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in human tissues with or without tumor. In addition, we investigated the expression of MIF in MDA-MB-231, MCF-7 (breast cancer cell lines) and MCF-10A (epithelial cell line) cells, and its effect on VEGF and IL-8. We found that MIF was overexpressed in breast cancer tissues compared with normal ones. The level of MIF showed the positive correlation between the expression of IL-8 and tumor microvessel density (MVD). The patients with positive MIF expression in tumor tissues showed a significantly worse disease-free survival compared with negative ones. Increased MIF serum levels were also found to correlate with higher levels of IL-8 in the sera of the patients with breast cancer. In vitro experiments successfully detected MIF in breast cell lines. However, the expression level of it by normal epithelial cells was much less than that of cancer cells. Exogenous MIF did not cause endothelial tube formation and migration but induced a dose dependent increase in VEGF and IL-8 secretion in breast cancer cell lines. In summary, our studies show that human breast cancer tissue expresses MIF. Its in vitro effect on VEGF and IL-8 indicates that MIF may contribute to tumor in angiogenesis and thus play an important role in the pathogenesis of breast cancer.  2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Breast cancer; Angiogenesis; MIF; VEGF; IL-8; Interleukin-8

1. Introduction It is well established that tumor growth beyond the size of 1–2 mm whether at the primary or meta* Corresponding author. Tel.: +86 20 87335886; fax: +86 20 87338198. E-mail address: [email protected] (S. Wang).

static site is angiogenesis dependent [1]. This process is dependent on the induction of angiogenesis mediated by angiogenic factors secreted by the tumor cells. Tumor cells are reported to secrete a wide variety of angiogenic factors [2,3]. Among those, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) have received much attention because of their roles in tumor angiogenesis. VEGF

0304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.11.028

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expression has been detected in a large variety of malignant human tumors including breast cancer [4]. IL-8 is a member of the CXC chemokine family [5] and found to be a potent angiogenic factor in several carcinomas including breast cancer [6]. Apart from that, IL-8 and VEGF are also considered as important angiogenic factors in promoting tumor growth and metastasis in numerous studies [7,8]. Although there are a number of studies showing a significant correlation between high serum VEGF level and advanced stage or metastatic disease in breast cancer, the mechanisms on expression of angiogenic factors in breast cancer remains unclear. Macrophage migration inhibitory factor (MIF) is a conserved 12.5 kDa mediator that functionally belongs to the class of inflammatory cytokines [9]. It has been found to play a pivotal role in the host response to acute and chronic inflammatory diseases including septic shock [10], transplantation rejection [11], rheumatoid arthritis and atherosclerosis [12]. Overexpression of MIF was reported in numerous tumors such as prostate tumors, breast cancer, melanomas, colon carcinomas, hepatocellular carcinomas, neuroblastoma, gastric cancer and gliomas [13–18]. In addition, MIF is shown to promote cell proliferation and tumor angiogenesis [19– 21]. Bando et al. [14] detected the expression MIF protein in 93 breast cancer tissues and demonstrated that cytoplasmic MIF staining was correlated significantly with microvessel density (MVD) in human breast carcinoma. However, the function of MIF in tumor biology, still remains unclear. To explore the role of MIF in human breast cancer progression, we analyzed the expression of MIF and its relationship with IL-8, VEGF, and MVD in human breast cancer tissue. Then, we compared the disease-free survival between MIF positive and negative group. Furthermore, we performed in vitro experiment to analyze the expression of MIF and its association with IL-8, VEGF in human breast cancer cell lines and epithelial cell lines. The role of MIF in angiogenesis is also investigated.

purchased from BD Pharmingen. The material and reagents of cell culture were purchased from Sigma Co. ELISA kits were purchased from R&D (Minneapolis, MN). 2.2. Study design Normal tissues derived from breast reductions (n = 20) and cancer tissues from 121 patients with primary breast cancer undergoing surgery at the First Affiliated Hospital of Sun Yat-Sen University were collected from March 1994 to July 2002. Additionally, benign tumor tissue (n = 20) from patients with fibroadenoma of breast were also examined. Cancer patients with distant metastases were excluded from the analysis. The patients were treated by simple mastectomy or wide local excision with axillary node sampling. Grading of ductal carcinomas was performed by breast pathologists. Repeated follow-up was performed every 3 months for the first 18 months and then every 12 months, and clinical parameters, relapsefree survival, and overall survival were recorded from the date of surgery. In the patients aged less than 50 years, adjuvant chemotherapy regimen CMF (cyclophosphamide, methotrexate and 5-fluorouracil) or CAF (cyclophosphamide, doxorubicin and 5-fluorouracil) was administered if tumors were found to be node-positive, or estrogen receptor (ER) negative and/or >3 cm. Patients aged over 50 years with ER negative, node-positive tumors also received CMF or CAF, regardless of tumor size Tamoxifen was given to the patients with ER-positive tumors for 5 years. The median follow-up was 6.25 years (range 2.5–10.58 years) with 18 deaths recorded because of breast cancer. 2.3. Tissue microarrays

2. Materials and methods

Tissue microarray (TMA) from 20 normal tissue samples, 20 tissue samples of fibroadenoma of breast and 121 tissue samples of primer breast cancer was generated. Haematoxylin and eosin-stained sections of each tumor were examined to determine representative areas of the tumor from which core biopsies were taken. Duplicate 1 mm cores were then taken from designated donor blocks using a precision instrument (Beecher Instruments, Silver Spring, MD, USA) and arrayed on a recipient paraffin wax block using techniques originally developed by Kononen et al. [22]. Five-micrometre sections were cut, placed on polylysine-coated slides, and used for immunohistochemical analysis.

2.1. Materials

2.4. Immunohistochemical analysis

Most of antibodies and cytokines were purchased either from R&D (Minneapolis, MN) or from BD Pharmingen (San Diego, CA) unless stated specifically. Horse radish peroxidase (HRP)-conjugated streptavidin was

Sections were first placed in an oven at 60 C for 30 min prior to being deparaffinized and hydrated by sequential immersion in xylene and graded alcohol solutions. Then slides were immersed in sodium citrate buffer

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(pH 6.0), heated on a microwave oven for 5 min and allowed to cool down for 30 min. And the sections were incubated in 3% H2O2 for 30 min to block the endogenous peroxidase activity. Sections were then treated in phosphate-buffered saline (PBS) 0.3% Triton for 10 min and rinsed in PBS. Slides were treated with normal serum obtained from the same species in which the secondary antibody was developed for 15 min to block the nonspecific staining. Subsequently, slides were incubated with the primary antibodies (1) biotin-conjugated antihuman MIF goat IgG at 1:200 dilution for 16 h at 4 C and (2) antihuman CD34 mouse IgG (Dakopatts, Glostrup, Denmark) at 1:50 dilution for 90 min at room temperature. The slides were then rinsed and overlaid with secondary biotinylated antibody (Vectastain ABC kit) and incubated for 30 min. After washing twice with PBS, slides were overlaid with ABC reagent, and incubated for 30 min. 3,3 0 -Diaminobenzidine substrate (Vector) was used to localize antigenic MIF. After optimal color development, sections were immersed in sterile water, counterstained with Mayer’s hematoxylin for 0.5–3 min, and coverslipped with aqueous mounting solution. Washes in PBS followed all steps. For the negative controls, biotinylated normal goat IgG was substituted for the anti-MIF antibody. For CD34 staining, counterstaining was not done. VEGF and IL-8 expression were assessed using purified mouse anti-human monoclonal antibodies diluted 1:150 and 1:100, respectively, in an overnight incubation, following standard immunohistochemistry procedures. The sections were photographed with a Spot RT color camera coupled to a Nikon microscope. The tumors were scored using a semi-quantitative system based on cytoplasmic expression of chromogen intensity: 0 equals negative; 1 equals weak; 2 equals moderate and 3 equals strong, with the proportion of cells staining also recorded for the whole tissue sections. Two observers over a conference microscope performed the scoring separately. In the final the definition for positiveness is that more than 10% of the tumor cells exhibited moderate or strong cytoplasmic immunoreaction in the sample. The same definition applied to MIF, VEGF and IL-8. Similarly, ER and progesterone receptor (PR) status were defined as positiveness only if more than 10% moderate or strong nuclei stained positively. Human epidermal growth factor receptor 2 (HER-2) staining was scored on a scale ranging from 0 (absent) to 3+ (maximum cytomembranous staining), with a score above 2+ considered to be HER-2 positive. The MVD was counted by two independent investigators who were blinded to the whole experiment. The tissue sections were scanned by light microscopy at low power field (40·) to identify vascular hot spots and the vessel count in areas with the highest number of capillaries was assessed at high power field (HPF, 200·). Any stained endothelial cell or endothelial cell cluster clearly separated from adjacent vessels or other stromal elements was considered a single countable micro-

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vessel. The presence of a vessel lumen was not necessary for an element to be defined as a microvessel [23]. MVD for each patient tumor used in the statistics was expressed as the average number of vessels per high power (200·) field from three nonoverlapping microscopic fields. 2.5. Blood sample findings Blood samples were collected from the patients (n = 54) prior to operation and healthy controls (n = 41) at the First Affiliated Hospital of Sun Yat-Sen University between October 2006 and March 2007. Blood was collected in a serum separator tube and allowed to stand for 30 min at room temperature to ensure full clotting. All samples were subsequently centrifuged at 3000g for 5 min, and the supernatant was aliquoted and stored at 80 C until further analysis. Serum IL-8, VEGF and MIF concentration was determined with an enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). The measurements were conducted according to the methods recommended by the manufacturers. The half of the detection limit value of the patient samples was used for statistical analysis in case the measured values did not reach the detection limit of the assay. The detection limits of the assays were 1.5 pg/mL for IL8, 1.6 ng/mL for MIF, and 9 pg/mL for VEGF. 2.6. Cell culture Human breast cancer cell lines (MDA-MB-231 and MCF-7), normal epithelial cell line (MCF-10A) and human umbilical cord vein endothelial cell line (HUVECs) were originally from American Tissue Culture Collection. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), streptomycin 100 ng/ml, and penicillin 100 U/ml. Cultures were maintained at 37 C in a humidified incubator in an atmosphere of 95% air plus 5% CO2. HUVECs were cultured for studying angiogenesis activity. For stimulation experiments, cells were seeded at 1 · 105/ml in 24-well plates and then cultured in DMEM with 10% FBS for 24 h. The cells were washed with PBS and treated with DMEM and different concentrations of recombinant human MIF (ranging from 1 to 200 ng/ml) or anti-MIF, isotype control monoclonal antibody (mab) (100 lg/ml) for 24 h. The levels of MIF, VEGF and IL-8 in supernatants of tumor cell lines and normal epithelial cell were measured using ELISA kits. Tumor cells and normal epithelial cells 1 · 105 were cultured in 24-well plates in DMEM with 10% FBS for 24 h and then washed twice with PBS, and further cultured for 24 h in medium without sera. The culture supernatants of cells were collected after each experiment, and centrifuged at 1000 rpm for 10 min to remove cells and debris. ELISA was then performed according to the methods recommended by the manufacturers.

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2.7. Western blot analysis

2.9. Statistical analysis

Total proteins from cell lines were extracted by using prechilled radioimmunoprecipitation (RIPA) buffer (Roche, Mannheim, Germany). Their contents were measured with the Bradford method, were separated by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to a polyvinylidene fluoride (PVDF) membrane (Gene Co. Ltd., USA). The membrane was blocked with TBST (Tris-buffered saline containing 0.01% Tween 20) containing 5% non-fat dry milk at 4 C overnight and then was incubated with the diluted primary antibodies (anti-human MIF mouse IgG; 1:500 dilution). After 3 h it was washed with TBST and then probed with the secondary antibodies (antimouse goat IgG, horseradish peroxidase [HRP]; 1:500 dilution). The membrane was left at room temperature for 1 h and then was washed again. The target protein band was detected whether to be existed by enhanced chemiluminescence (ECL) using the ECL Western blot detection system (Amersham, Arlington Heights, IL). bActin (Sigma Chemical Company) was used as an internal control.

Data are expressed as means ± standard deviation (SD). Chi-square tests or Fisher exact tests were conducted to assess the differences in covariate distributions between variables (age, grade, lymph nodes, etc. list all the variables to replace etc.) and expression of MIF. The results of MIF, IL-8 and VEGF in sera were compared between patients and healthy controls by using a nonparametric analysis, the Mann–Whitney U test. The relation between continuous variables was analyzed with a Spearman rank correlation analysis. Survival was estimated by the Kaplan–Meier method, and the differences were analyzed by the Log-rank test. A p value of less than 0.05 indicated that the difference was significant. All statistical analyses were performed using the computer software Statistical Package for the Social Sciences 13.0 (SPSS Inc., Chicago, IL).

2.8. In vitro angiogenesis assays

MIF was primarily detected in the cytoplasm of the carcinoma cells (Fig. 1A). Expression of MIF was observed in 36 cases out of 121 (29.8%). MIF protein was also detected in the vessel endothelia and some infiltrating cells such as macrophages and lymphocytes in non-tumor regions. MIF expression was only found positive in three fibroadenoma of breast and one normal tissue samples. An association of MIF expression was noted between breast cancer tissues and non-tumor specimens (p = 0.031, Fisher’s exact test). The correlation between MIF expression in breast cancer tissues and clinicopathologic features are shown in Table 1. No statistically significant association is found between MIF expression and age, tumor grade, or ER, PR. However MIF expression correlated with HER-2 (p = 0.030). There was a trend of MIF expression and lymph node metastasis (p = 0.063).

2.8.1. Migration assay Migration assay was done by using 24-well Transwell culture plates inserted with polycarbonate filters. HUVECs (1 · 105) were placed on the upper chambers precoated with 30 ll matrigel. The bottom plates were fed with different test medium (concentrations of MIF from 1 to 100 ng/ml). VEGF (100 ng/ml) was used as a positive control and nonconditioned medium was used as a negative control. After 24 h, the matrigel was removed from the plate. The cells that migrated to the lower side of the filter were fixed in 4% formaldehyde and stained with Giemsa solution. The number of matrigel-invading cells was counted under microscope. Each experiment was carried out three times. 2.8.2. Collagen tube formation Three-dimensional collagen gel plates (12 plates) were prepared by adding 0.8 ml of chilled rat tail collagen into each well and adjusting to neutral pH with NaHCO3 as described previously [6]. Collagen was allowed to solidify at 37 C for 30 min. HUVEC cells (1 · 105) per well were plated on collagen gel in DMEM media containing 15% FBS and endothelial cell growth factor. When the cells were confluent, the media was replaced by test media (VEGF 50 ng/ml, MIF 1 ng/ml, 50 ng/ml, 100 ng/ml or negative control medium). The wells were monitored and the pictures were taken 24 h after the addition of reagents. The experiments were repeated three times and the results were viewed by three independent individuals.

3. Results 3.1. MIF expression in breast tissues and clinicopathologic features

3.2. Correlations among MIF and VEGF, IL8, MVD in the breast tissues Tumor tissues sections were also stained with VEGF and IL-8. VEGF was primarily detected in the cytoplasm or on the membranes of the carcinoma cells. Expression of VEGF was observed in 81 cases out of 121 (66.9%). IL-8 was detected in 35 cases out of 121 (28.9%) (the cytoplasm of the carcinoma cells). However, there was no correlation between the expression of VEGF and any clinicopathologic features and expression of IL-8 (data not shown). MIF expression positively correlated with IL-8 expression (p = 0.014). Specific staining of capillary vessels with anti-CD34 was observed in all tumor speci-

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Fig. 1. MIF, MVD, VEGF and IL-8 expression in breast cancer tissue. Immunostaining demonstrated strong cytoplasmic expression (brown colour) (A) and negative expression (B) of MIF in breast cancer, high expression of MVD (C) and low expression of MVD (D), positive expression of VEGF (E) and IL-8 (F) (magnification, 200·).

mens (Fig. 1C). The median tumor MVD was 11.83/HPF (range, 4–35). According to the median tumor MVD levels, patients were divided into high (n = 67) and low (n = 54) groups. The MVD was significantly higher in tumors with expression of MIF than in tumors without expression of MIF (p = 0.043). 3.3. MIF expression and survival The patients were divided into negative and positive groups based on MIF expression in their tissues. Kaplan–Meier survival curves for these two groups were shown in Figs. 2 and 3. The mean survival time for MIF negative and positive groups was 115.6 months and 108.2 months, respectively (p = 0.172) (Fig. 2). In the course of follow-up (median, 75 months; range 2.5– 10.58 years), 21 of 121 patients had recurrence. MIF positive group had a significantly worse disease-free survival than MIF negative group (p = 0.029) (Fig. 3).

3.4. Serum levels of MIF, IL-8 and VEGF The levels of MIF, IL-8 and VEGF in the sera of patients with breast cancer were significantly increased compared to healthy controls. In the 41 healthy controls, serum IL-8 concentration was 5.0 ± 1.3 pg/mL (median, 4.7 pg/mL) with a range from 2.9 to 8.1 pg/mL. In the 54 patients with operable disease, serum IL-8 increased to 12.3 ± 7.7 pg/mL (median, 10 pg/mL). The difference in serum IL-8 between the controls and the patients with breast cancer was significant (p < 0.01). Serum MIF (91.1 ± 43.2 versus 8.3 ± 3.9 ng/mL, p < 0.01) and VEGF (318.7 ± 83.1 versus 151.9+36.0 pg/mL, p < 0.01) levels of patients with breast cancer were significantly higher that those of healthy controls (Fig. 4). Furthermore, serum MIF levels correlated positively with serum IL-8 levels (r = 0.612, p < 0.01). The correlation between serum MIF and VEGF levels was not statistically significant (r = 0.211, p = 0.281).

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Table 1 Patients grouping and the correlation between MIF and clinical features Clinical feature

Total

MIF expression

X2

p

VEGF (1355 ± 171.4 pg/mL; 306.8 ± 46.5 pg/mL) than MCF-10A (IL-8, 6.8 ± 1.3 pg/mL; VEGF, 101.1 ± 26.9 pg/mL) in the absence of exogenous MIF stimulation. MIF at concentrations from 1 up to 200 ng/mL in MDA-MB-231 and MCF-7 cells induced a significant dose-dependent increase in IL-8 and VEGF secretion. However, MIF had no effect on stimulating the production of IL-8 and VEGF in MCF-10A cells. Furthermore, the production of IL-8 and VEGF by breast cancer cell lines was inhibited by neutralizing MIF mAb, but not by control antibody (Figs. 6 and 7).

Positive

Negative

63 58

17 19

46 39

0.482

0.488

91 30

25 11

66 19

0.913

0.339

No. of lymph nodes involved 0–4 79 18 5–8 34 14 9 8 4

61 20 4

5.527

0.063

Estrogen receptor Positive 58 Negative 63

14 22

44 41

1.680

0.195

Progesterone receptor Positive 68 Negative 53

23 13

45 40

1.231

0.267

We tested the role of rMIF in angiogenesis by performing two in vitro assays. The two assays were carried out to assess the effects of rMIF on migration, formation of new capillary tubes of HUVECs. These two events are required in the angiogenic response. MIF did not demonstrate any angiogenic bioactivity in vitro either in HUVEC chemotaxis assays or in tube formation of HUVECs.

HER-2 Positive Negative

14 22

17 68

4.735

0.030

4. Discussion

Age (median) 650y >50y Tumor grade I/II III

31 90

MVD High Low

67 54

25 11

42 43

4.107

0.043

VEGF Positive Negative

81 40

25 11

56 29

0.145

0.703

IL-8 Positive Negative

35 86

16 20

19 66

6.003

0.014

3.5. Expression of MIF in breast cancer cell lines and normal epithelial cells The levels of secreted MIF protein in the supernatants of cultured breast cancer cell lines (MDA-MB-231 and MCF-7) and epithelial cells (MCF-10A) were determined by ELISA after incubation for 24 h. The concentration of MIF in the supernatants of MCF-10A, MDA-MB-231 and MCF-7 cultures was 1.1 ± 0.9, 15.3 ± 3.5 and 6.7 ± 2.1 ng/mL, respectively. Although normal epithelial cells expressed MIF, the level of secreted MIF by normal epithelial cells was much less than that of cancer cells. The expression of MIF in cell lines was confirmed by Western blot (Fig. 5). The expression level of MIF by normal epithelial cells was much less than that of cancer cells. 3.6. Effect of MIF on stimulation of VEGF and IL-8 secretion MDA-MB-231 and MCF-7 cells secreted higher levels of IL-8 (204.1 ± 21.4 pg/mL; 19.8 ± 6.8 pg/mL) and

3.7. Induction of in vitro angiogenesis by rMIF

In the present study, overexpression of MIF was found in breast cancer tissues comparing with nontumor tissues, which correlated with levels of IL-8 and MVD shown by immunohistology. Breast cancer patients with positive MIF expression had a significantly worse disease-free survival. Consistently, serum levels of VEGF, MIF and IL-8 in those patients increased significantly compared with healthy volunteers. Among these cytokines, MIF was found to correlate with IL-8 levels in the sera of patients with breast cancer. In vitro MIF stimulation of breast cancer cells induced a dose dependent increase in VEGF and IL-8 secretion. There are several novel findings in our study. First, this is the first report to demonstrate its relationship with IL-8 in human breast cancer. Second, the positive MIF expression was shown to correlate with a significantly worse disease-free survival. Finally, our in vitro study showed that MIF stimulation of breast cancer cell lines induced a dose dependent increase in VEGF and IL-8 secretion. MIF is considered to play an important role in carcinogenesis by promoting cell proliferation, tumor angiogenesis and metastasis [19,24–27]. He et al. [18] reported epithelial and serum MIF expression was progressively increased in H pylori induced gastritis, intestinal metaplasia and gastric cancer, suggesting that MIF was involved in gastric carcinogenesis and may be a valuable biomarker for the

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Fig. 2. Kaplan–Meier survival curves illustrating overall survival for 121 patients who had breast cancer with negative expression levels of MIF versus positive expression levels of MIF (p = 0.171, Log-rank test).

Fig. 3. Kaplan–Meier survival curves illustrating disease-free survival for 121 patients who had breast cancer with negative expression levels of MIF versus positive expression levels of MIF (p = 0.029, Log-rank test).

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Fig. 4. Serum IL-8 (pg/ml), VEGF (pg/ml), and MIF (ng/ml) levels in breast cancer patients and controls. Serum levels of IL-8, VFGF, and MIF in healthy controls (n = 41) and in patients (n = 54) were measured by ELISA. The difference in three cytokines between breast cancer patients and controls is significant (**p < 0.01).

Fig. 5. Western blot analysis of MIF protein produced by breast cancer cells line (MDA-MB-231 and MCF-7) and epithelial cells (MCF-10A). The level of expression MIF by normal epithelial cells was much less than that of tumor cells.

early detection for gastric cancer sequentially. Bando et al. [14] reported MIF was overexpressed in 93 breast cancer tissues detected by ELISA and had a significant positive correlation with IL-1b. They demonstrated that its levels correlated with MVD in the 20 patients with breast cancer. These are consistent with our findings. MIF was found strongly expressed in human breast cancer specimens, as well as in the serum. Moreover, MDAMB-231 and MCF-7 cells secreted large amounts of MIF compared with MCF-10A in vitro. However, the mechanism by which tumor cells up-regulate MIF expression remains unknown. Our study also showed that patients with positive MIF had a significantly worse disease-free survival than those counterparts. The association between

Fig. 6. MIF induced the secretion of IL-8 by breast cancer cells line (MDA-MB-231 and MCF-7), which was neutralized by antiMIF in breast cancer cells. However it could not increase the secretion of IL-8 by normal epithelial cell (MCF-10A). (A) Cells were seeded at 1 · 105/ml in 24-well plates and then cultured in DMEM with 10% FBS for 24 h. Cells were washed with PBS and treated with DMEM and different concentrations of recombinant human MIF (ranging from 1 to 200 ng/ml). Cell-free supernatants were collected to determine the IL-8 content using ELISA. (B) In same condition, the cells were treated with anti-MIF neutralizing mAb (100 lg/mL) or isotype control mAb (100 lg/ mL) for 24 h. IL-8 in the supernatants were measured by ELISA (means ± SD, n = 3) *p < 0.05.

overexpression of HER-2 and MIF was observed. The relationship of them would be examined in our future studies. There is also a trend of association between nodal metastasis and MIF (p = 0.063). Bando reported that intratumoral MIF protein concentrations correlated inversely with nodal involvement detected by ELSIA, which was different from ours. Their study did not sort patients according to the tumor stage as we did. The expression of MIF and its role in tumor growth might vary according to different tumor stages. The TMA technique we applied to our study mainly focused on representative tumor areas. All our study objects were based on tumor cells while Bando studied on whole tumor tissues including

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Fig. 7. MIF induces the secretion of VEGF by breast cancer cells line (MDA-MB-231 and MCF-7), which is neutralized by antiMIF in breast cancer cells. However it cannot increase the secretion of VEGF by normal epithelial cell (MCF-10A). (A) Cells were seeded at 1 · 105/ml in 24-well plates and then cultured in DMEM with 10% FBS for 24 h. Cells were washed with PBS and treated with DMEM and different concentrations of recombinant human MIF (ranging from 1 to 200 ng/ml). Cellfree supernatants were collected to determine the VEGF content using ELISA. (B) In same condition, the cells were treated with anti-MIF neutralizing mAb (100 lg/mL) or isotype control mAb (100 lg/mL) for 24 h. VEGF in the supernatants were measured by ELISA (means ± SD, n = 3) *p < 0.05.

the stromal cells, another source of MIF. In esophageal squamous cell carcinoma, MIF expression was found correlated with lymph node status [28]; in hepatocellular carcinoma, MIF expression was found correlated with intrahepatic recurrence [29]. However, we did not find a relation between MIF and hormone receptor status. Although our results showed the significant correlations between the overexpression of MIF and metastasis and cancer recurrence, MIF expression was not found correlated with poor overall patient survival (p = 0.173). Angiogenesis, the formation of new vessels from a preexisting network of vessels, is essential for the growth and progression of solid tumors [30]. Many

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factors are involved in tumor angiogenesis including VEGF [31] and IL-8 [32]. Our study showed that MIF expression positively correlated with IL-8 expression and MVD in tumor tissues in breast cancer patients. Serum MIF, VEGF and IL-8 levels were also found more significantly increased than those in healthy controls. There was a positive correlation between serum MIF and IL-8 levels. However, the correlation between serum levels of MIF and VEGF was not statistically significant, suggesting some factors other than MIF may also be involved in modulating serum VEGF production in breast cancer. We suspect that MIF overexpression results in the upregulation of angiogenic factors IL-8, thus creating the subsequent increase in tumor MVD and the worsening prognosis. Then we investigated whether MIF induced the production of IL-8 and VEGF in breast cancer cells. The study demonstrated that rMIF could induce two different breast cancer tumor cell lines to increase expression of IL-8 and VEGF in vitro in a dose-dependent manner. However, the production of VEGF and IL-8 of breast cancer cell lines was inhibited by anti-MIF, indicating that rMIF resulted in the increased expression of IL-8 and VEGF. Furthermore, MIF did not demonstrate any angiogenic bioactivity in vitro either in HUVECs chemotaxis assays or in tube formation of HUVECs. Hira et al. [29] reported that rMIF could induce angiogenesis directly in an in vitro model after administration of rMIF in a co-culture of HUVECs and fibroblasts. However, the study did not show any dose-dependent effect of rMIF on in vitro angiogenesis in this model. Fibroblasts can produce many cytokines such as FGF2 and cxcl12 [33], which are angiogenically bioactive. That helps to explain the result of our study with fibroblasts excluded. Ren et al. [16] found that rMIF augments VEGF and IL-8 expression in hepatocellular carcinoma cell lines and then promoted angiogenesis. In addition, Munaut et al. [34] showed a close correlation between MIF expression and VEGF expression in human glioblastoma. These results implied that MIF promoted angiogenesis by inducing expression of angiogenic factors in tumor cells. In addition to promoting angiogenesis, MIF also plays a role in regulating p53 activity in tumors by virtue of its ability. Some studies have identified MIF in inhibiting p53 activity [35,36]. Lue et al. [37] found MIF can directly promote cell survival through activation of the PI3K/Akt pathway which is critical to maintain life in cancer cells. These stud-

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ies suggest that MIF could provide both angiogenic and antiapoptotic activity within a tumor and may contribute to tumor progression. In conclusion, MIF was overexpressed in breast cancer patients in our study and its expression correlated with IL-8, HER-2, MVD and worse diseasefree survival. MIF stimulation of breast cancer cell lines (MDA-MB-231 and MCF-7) induced a dose dependent increase in VEGF and IL-8 secretion in vitro, which contribute to angiogenesis and tumor growth. Therefore, MIF might play an important role in progression, invasion and angiogenesis in breast cancer and it could be a therapeutic target in patients with breast cancer. Acknowledgements This project was supported by the National Natural Science Foundation of China (NSFC) (30600588). We thank Prof. Wen Li and Dr. Jiong Bi, Laboratory of Surgery, Sun Yat-Sen University, Guangzhou, China, for technical assistance. We also thank Dr. Song Jin and Dr. Lanlan Ma for them assistance in this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2007.11.028. References [1] A.C. Hartford, T. Gohongi, D. Fukumura, R.K. Jain, Irradiation of a primary tumor, unlike surgical removal, enhances angiogenesis suppression at a distal site: potential role of host–tumor interaction, Cancer Res. 60 (2000) 2128– 2131. [2] P. Carmeliet, R.K. Jain, Angiogenesis in cancer and other diseases, Nature 407 (2000) 249–257. [3] I.J. Fidler, L.M. Ellis, The implications of angiogenesis for the biology and therapy of cancer metastasis, Cell 79 (1994) 185–188. [4] J.A. Foekens, H.A. Peters, N. Grebenchtchikov, et al., High tumor levels of vascular endothelial growth factor predict poor response to systemic therapy in advanced breast cancer, Cancer Res. 61 (2001) 5407–5414. [5] K. Xie, Interleukin-8 and human cancer biology, Cytokine Growth Factor Rev. 12 (2001) 375–391. [6] Y. Lin, R. Huang, L. Chen, et al., Identification of interleukin-8 as estrogen receptor-regulated factor involved in breast cancer invasion and angiogenesis by protein arrays, Int. J. Cancer 109 (2004) 507–515. [7] R. Aalinkeel, M.P. Nair, G. Sufrin, et al., Gene expression of angiogenic factors correlates with metastatic potential of prostate cancer cells, Cancer Res. 64 (2004) 5311–5321.

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