High Visfatin Expression in Breast Cancer Tissue Is Associated with Poor Survival

This article is featured in Highlights of This Issue, p. 1807

Breast cancer is the most common malignancy in women worldwide, and its prevalence is increasing at a surprisingly rapid rate. Therefore, it is critical to discover prognostic factors as well as therapeutic targets for breast cancer. Previous studies clearly showed that obesity is correlated with the risk and prognosis of breast cancer, particularly postmenopausal breast cancer (1–3). Adipocytokines constitute a group of polypeptide growth factors and cytokines produced exclusively, or substantially, by preadipocytes and mature adipocytes in white adipose tissue (4, 5). These adipocytokines include adiponectin, leptin, resistin, and visfatin (6–9). In our previous study, low serum adiponectin and high serum leptin levels were observed in breast cancer patients and were associated with an increased risk for developing breast cancer (5).

Visfatin, also known as Nampt/pre-B-cell–enhancing factor, is expressed in normal, inflamed, and tumor tissues (10, 11). It possesses NAD biosynthetic activity and regulates growth, apoptosis, and angiogenesis in mammalian cells (12, 13). It is also a cytokine-like molecule secreted from human peripheral blood lymphocytes and is essential for B-cell maturation and function (14, 15). Recent studies showed that the expression of visfatin was correlated with various cancers including colorectal, prostatic, gastric, and esophageal cancers (16–19). In human endothelial cells, exogenous visfatin increases matrix metalloproteinase (MMP)-2/9 activity as well as endothelial cell proliferation via phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling pathways (20). The overexpression of visfatin in cultured human vascular smooth muscle cells and endothelial cells confers resistance to acute oxidative stress, delayed senescence, and an increased replicative life span (21, 22). Visfatin is expressed in breast cancer tissue (23) and MCF-7 breast cancer cells (10, 24). Furthermore, visfatin is present in bovine mammary epithelial cells, lactating mammary glands, and milk (24). Although accumulating evidence suggests that visfatin plays some important roles in mammary epithelial cells and the mammary gland, its role in breast cancer is still poorly understood.

In this study, the involvement of visfatin in breast cancer in Taiwanese patients was determined. Herein, we report that elevated visfatin expression was observed in breast cancer tissues and was associated with poor prognosis, particularly when combined with estrogen receptor (ER) and progesterone receptor (PR) expression status.

From 2003 to 2008, samples from 105 cases of newly diagnosed and surgically treated breast cancer were collected at the Cancer Center of Kaohsiung Medical University Hospital. Paired cancer and adjacent noncancerous breast tissues were obtained from the surgically treated breast cancer patients. None of the patients had undergone radiotherapy or chemotherapy before surgery. This study was approved by the ethics committee of Kaohsiung Medical University Hospital, and informed consent was obtained from each patient. The clinicopathologic features of the patients were also recorded. The histologic types and grades of the primary tumors were determined according to a system based on a modification of the WHO classification (25) and the modified Bloom-Richardson grading scheme (26), respectively, whereas the staging of breast cancer was defined according to the tumor-node-metastasis (TNM) system (27). ER and PR status was analyzed by immunohistochemical staining, and HER2/neu oncoprotein staining was evaluated by the standard HercepTest procedure (Dako 5204). Chemotherapy consisted of 6 cycles of fluorouracil, epirubicin, and Endoxan (cyclophosphamide) or 6 cycles of Taxotere (docetaxel), epirubicin, and Endoxan. Hormonal therapy, usually tamoxifen and Arimidex (anastrozole), was administered to patients who were ER positive.

All tissues for tissue microarray were obtained from formalin-fixed, paraffin-embedded tissue blocks. Slides from hematoxylin-eosin–stained sections were reviewed by a pathologist to select representative areas of tumor and normal regions to be cored. The construction of the tissue microarray was done using Booster Arrayer & TMA designer software. This instrument was designed to produce sample spots that were 1.5 mm in diameter at a space of 3.0 mm and ranging in length from 2.0 to 5.0 mm depending on the depth of tissue in the donor block. Small cores were retrieved from selected regions of donor tissue and transferred to a new paraffin block (recipient block). Forty-five such cylindrical cores were precisely arrayed in the recipient block. Five-micrometer sections were cut from this recipient tissue microarray block with a microtome with an adhesive-coated tape sectioning system (Instrumedics). The initial slide was prepared for hematoxylin-eosin staining to verify histology and immunohistochemical staining.

The detailed protocol for immunohistochemical analysis was reported previously (28). For immunohistochemical staining, sections of tissue microarray were deparaffinized with xylene rinse, rehydrated with a graded alcohol series (100%, 95%, 85%, and 75%) for 5 minutes each, and then rinsed with distilled water. Antigen retrieval was enhanced by autoclaving slides in sodium citrate buffer (10 mmol/L, pH 6.0) for 30 minutes. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide/methanol buffer for 30 minutes. The slides were incubated overnight at 4°C in humidified chambers with rabbit polyclonal anti-visfatin antibody (Santa Cruz) at a dilution of 1:200. The slides were washed 3 times in phosphate-buffered solution and further incubated with a biotinylated secondary antibody for 30 minutes at room temperature. Antigen–antibody complexes were detected by the avidin-biotin-peroxidase method, using 3,3′-diaminobenzidine as a chromogenic substrate (Dako). Finally, the slides were counterstained with hematoxylin and then examined under a light microscope. Notably, only the staining in tumor cells (approximately 1,000 cells in 3–4 hpf) was calculated. The photographs were captured by a Nikon E-800M microscope and then processed using Kodak MGDS330 and Adobe Photoshop 6.0.

The results for visfatin staining were scored on the basis of the percentage of positively stained cells in 4 quantitative categories: score 1, 25% or less positive cells; score 2, 26% to 50% positive cells; score 3, 51% to 75% positive cells; and score 4, 76% or more positive cells. The staining was determined separately for each specimen by 2 independent experts simultaneously under the same condition. The rare cases with discordant scores were reevaluated and scored on the basis of consensual opinion.

All statistical analyses were done using the SPSS 14.0 statistical package for PC (SPSS, Inc.). For visfatin expression, scores 1 and 2 were categorized as low expression and scores 3 and 4 were categorized as high expression. Comparisons between the high visfatin expression group and the low expression group regarding tumor stage, tumor grade, age at diagnosis, body mass index (BMI), tumor size, lymph nodes status, recurrence, ER status, PR status, and Her2/neu status were done using the χ² test. Fisher's exact test was used when a χ² test was conducted with cells that had an expected frequency of 5 or less. Survival curves were generated using Kaplan–Meier estimates, and the significance of differences between curves was evaluated by the log-rank test. Furthermore, HRs and 95% CIs computed from univariate and multivariate Cox regression models were used for investigating the relationship between clinicopathologic characteristics and survival. P < 0.05 was considered statistically significant.

The expression of visfatin in breast cancer tissues was further analyzed by immunohistochemistry and correlated with the clinicopathologic characteristics of the breast cancer patients as shown in Supplementary Table S1. As shown in Supplementary Table S1, 47.6% of the cancer tissues exhibited high visfatin expression (52.4% exhibited low visfatin expression). Further analysis revealed that the visfatin staining was predominantly observed in breast cancer tissue but not the adjacent normal breast tissues/noncancerous tissues (P = 0.002; Fig. 1A and Supplementary Table S2). However, weak to absent visfatin expression was observed in the neighboring stromal cells, but its significance remains to be determined.

The expression patterns of visfatin in breast cancer tissues were correlated to clinicopathologic variables including tumor stage, tumor grade, age at diagnosis, BMI, tumor size, lymph node status, ER status, PR status, and Her2/neu status (Table 1). High visfatin expression in breast cancer tissues was significantly associated with increased tumor size (P = 0.025; Table 1). In the high visfatin expression group, 66.7% and 62.2% of patients were ER negative (P = 0.005) and PR negative (P = 0.009), respectively (Table 1). There was no significant correlation between visfatin expression and Her2/neu status. The immunohistochemistry result for the localization of visfatin, ER, PR, and Her2/neu in cancer tissues is shown in Figure 1B; the expression of visfatin was negatively correlated with the expression of ER and PR.

The association of visfatin expression with disease recurrence after radiotherapy, chemotherapy, and hormone therapy was further analyzed. Interestingly, the high visfatin expression group, which received hormone therapy, had a significantly lower risk of disease recurrence (P = 0.005) than the group that did not receive hormone therapy (P = 0.572; Table 2). There was no significant difference in disease recurrence regarding the administration of radiotherapy or chemotherapy in the low and high visfatin expression groups.

The expression patterns of visfatin in breast cancer tissues were further correlated with the disease-free and overall survival of the patients by Kaplan–Meier estimates. Increased disease-free and overall survival rates were observed in low visfatin-expressing cells (score 1; P < 0.001 and P = 0.001, respectively; Fig. 2A) and the low visfatin expression group (score 1 + 2; P < 0.001 and P = 0.005, respectively) as determined by the log-rank test (Fig. 2B). Regarding ER and PR status, the group with high visfatin expression (score 3 + 4) and ER-negative status had significantly worse disease-free and overall survival rates (P < 0.001 and P = 0.001, respectively; Fig. 2C). Similarly, the group with high visfatin expression and PR-negative status had significantly poorer disease-free and overall survival rates (P < 0.001 and P = 0.001, respectively; Fig. 2D).

We also studied the effect of visfatin expression on patient survival after radiotherapy, chemotherapy, and hormone therapy. There were no significant differences in disease-free and overall survival among the patients after radiotherapy and chemotherapy in the low and high visfatin expression groups (Fig. 3A–D). Interestingly, the disease-free and overall survival rates were improved after hormone therapy (P = 0.002 and P = 0.007, respectively; Fig. 3E and F).

To evaluate the factors related to visfatin expression in breast cancer, HRs were estimated by univariate and multivariate Cox regression analyses as shown in Tables 3 and 4. In the univariate analysis, the significant factors associated with disease-free survival included tumor stage (HR = 5.33, 95% CI = 2.25–12.62, P < 0.001), age at diagnosis (HR = 4.27, 95% CI = 1.56–11.66, P = 0.005), ER status (HR = 2.45, 95% CI = 1.04–5.77, P = 0.041), hormone therapy (HR = 0.18, 95% CI = 0.07–0.47, P < 0.001), and visfatin expression (HR = 5.73, 95% CI = 1.92–17.14, P = 0.002; Table 3), whereas the significant factors associated with overall survival included tumor stage (HR = 5.00, 95% CI = 1.81–18.83, P = 0.002), age at diagnosis (HR = 8.22, 95% CI = 1.85–36.43, P = 0.006), ER status (HR = 4.34, 95% CI = 1.48–12.70, P = 0.007), PR status (HR = 3.97, 95% CI = 1.26–12.46, P = 0.018), hormone therapy (HR = 0.18, 95% CI = 0.06–0.57, P = 0.003), and visfatin expression (HR = 5.05, 95% CI = 1.42–17.98, P = 0.012; Table 4). However, after adjusting for tumor grade, age at diagnosis, BMI, ER status, PR status, Her2/neu status, radiotherapy, chemotherapy, and hormone therapy in multivariate Cox regression analysis, only tumor stage (HR = 6.08, 95% CI = 1.83–20.18, P = 0.003 and HR = 10.17, 95% CI = 2.41–42.94, P = 0.002, respectively) and visfatin expression (HR = 4.11, 95% CI = 1.15–14.70, P = 0.030 and HR = 6.20, 95% CI = 1.18–32.50, P = 0.031, respectively) were significantly independent predictors of disease-free and overall survival in breast cancer patients (Tables 3 and 4).

To the best of our knowledge, this is the first study to evaluate the prognostic significance of visfatin expression in breast cancer. A significant increase in visfatin expression was observed in breast cancer tissues (Fig. 1 and Supplementary Table S2), and visfatin is an indicator of poor prognosis in breast cancer (Figs. 2 and 3). Our observation is in agreement with previous reports that higher visfatin expression is observed in primary colorectal cancer than in nonneoplastic mucosa and that it serves as a biomarker for gastric cancer (16, 29).

In preadipocytes, visfatin expression is suppressed significantly by treatment with 1 μmol/L insulin, 1 to 15 nmol/L T3, 10 to 1 μmol/L progesterone, or 10 to 1 μmol/L testosterone (30), suggesting that visfatin expression is regulated by multiple hormones. Interestingly, in our study, low visfatin expression in breast cancer tissue was associated significantly with ER and PR positivity, an indicator for good prognosis (Table 1). Taken together, our results clearly showed that visfatin is a novel and promising prognostic factor for breast cancer.

Visfatin upregulates VEGF and MMPs through MAPK and PI3K/Akt signaling pathways and promotes angiogenesis and prostate cancer progression (19, 20). Alternatively, visfatin stimulates an interleukin-6/STAT3–mediated cell survival pathway in macrophages through a nonenzymatic mechanism, which may account for alterations in macrophage physiology and mediate the effects of obesity in inflammation, atherogenesis, and tumorigenesis (14, 31–33). The visfatin inhibitor APO866 selectively inhibited tumor growth through the depletion of intracellular NAD, the product catalyzed by visfatin, in murine gastric and bladder tumor models (34). In addition, the addition of APO866 synergistically increases the caspase-dependent apoptotic activity of TNF-related apoptosis-inducing ligand in leukemia cells by enhancing NAD⁺ depletion, mitochondrial transmembrane potential dissipation, and ATP depletion (35–37). In our study, visfatin expression was higher in breast cancer tissues and significantly associated with poor survival and disease prognosis (Figs. 2 and 3 and Tables 3 and 4), suggesting that targeted visfatin inhibition may be a promising approach for cancer treatment in the future.

The overexpression of visfatin increases cellular NAD⁺ content, enhances SIRT1-mediated p53 degradation, and induces resistance to hydrogen peroxide–induced cell damage in human endothelial cells (38). Furthermore, modest overexpression of visfatin endows aging endothelial cells with increases in proliferative capacity, replicative life span, and functional regenerative capacity in vitro (22). Interestingly, visfatin-homozygous mutant mice die during the embryonic period, suggesting that visfatin is required for viability (39, 40). In agreement with these studies, we observed increased malignant behavior in high visfatin-expressing breast cancer cells (Figs. 2 and 3 and Tables 1–4). Further studies are required to elucidate the detailed mechanisms of how the regulatory effect of visfatin on aging and longevity is incorporated into malignant breast cancer cell behavior.

In conclusion, using a cohort of 105 breast cancer patients, we observed a positive association between visfatin expression and malignant breast cancer behavior. High visfatin expression was found to be independently associated with poor disease-free and overall survival in the multivariate Cox regression analysis. Further investigations are required to explore the detailed mechanisms of visfatin signaling in breast cancer development and to establish new diagnostic and therapeutic strategies using visfatin as the target.

No potential conflicts of interest were disclosed.

We thank Dr. Chun-Chieh Wu (Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan) for critical comments on the manuscript.

This work was supported by National Science Council grant NSC 99-2320-B-214-002-MY3 to S.S.F. Yuan and Department of Health, Executive Yuan grant (DOH100-TD-C-111-002), Taiwan.

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