Chinese Medical Journal 2007;120(18):1592-1596
Adipocytokines and breast cancer risk

HOU Wei-kai,  XU Yu-xin,  YU Ting,  ZHANG Li,  ZHANG Wen-wen,  FU Chun-li,  SUN Yu,  WU Qing,  CHEN Li

HOU Wei-kai (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

XU Yu-xin (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

YU Ting (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

ZHANG Li (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

ZHANG Wen-wen (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

FU Chun-li (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

SUN Yu (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

WU Qing (Department of Breast Surgery, Qilu Hospital of Shandong University, Jinan 250012, China)

CHEN Li (Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China)

Correspondence to:CHEN Li,Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China (Tel: 86-531-82169452. Fax:. E-mail:wkhou@sina.com)
Keywords
obesity; cytokines; breast neoplasms
Abstract

Background  Many researches suggested that obesity increased the risk of breast cancer, but the mechanism was currently unknown. Adipocytokines might mediate the relationship. Our study was aimed to investigate the relationship between serum levels of resistin, adiponectin and leptin and the onset, invasion and metastasis of breast cancer.
Methods  Blood samples were collected from 80 newly diagnosed, histologically confirmed breast cancer patients and 50 age-matched healthy controls. Serum levels of resistin, adiponectin and leptin were determined by enzyme-linked immunosorbent assays (ELISA); fasting blood glucose (FBG), lipids, body mass index (BMI), and waist circumference (WC) were assayed simultaneously.
Results  Serum levels of adiponectin ((8.60±2.92) mg/L vs (10.37±2.81) mg/L, P=0.001) and HDL-c were significantly decreased in breast cancer patients in comparison to controls. Serum levels of resistin ((26.35±5.36) μg/L vs (23.32±4.75) μg/L, P=0.000), leptin ((1.35±0.42) μg/L vs (1.06±0.39) μg/L, P=0.003), FBG and triglyceride (TG) in breast cancer patients were increased in contrast to controls, respectively. However, we did not find the significant difference of the serum levels of resistin, adiponectin and leptin between premenopausal breast cancer patients and healthy controls (P=0.091, 0.109 and 0.084, respectively). The serum levels of resistin, adiponectin and leptin were significantly different between patients with lymph node metastasis (LNM) and those without LNM (P=0.001, 0.000 and 0.006, respectively). The stepwise regression analysis indicated that the tumor size had the close correlation with leptin (R2=0.414, P=0.000) and FBG (R2=0.602, P=0.000). Logistic regression analysis showed that reduced serum levels of adiponectin (OR: 0.805; 95%CI: 0.704–0.921; P=0.001), HDL (OR: 0.087; 95%CI: 0.011–0.691, P=0.021), elevated leptin (OR: 2.235; 95%CI:1.898–4.526; P=0.004) and resistin (OR: 1.335; 95%CI: 1.114–2.354; P=0.012) increased the risk for breast cancer; Reduced serum levels of adiponectin (OR: 0.742; 95%CI: 0.504–0.921; P=0.003) and elevated leptin (OR: 2.134; 95%CI:1.725–3.921; P= 0.001) were associated with lymph node metastasis of breast cancer.
Conclusions  The decreased serum adiponectin levels and increased serum resistin and leptin levels are risk factors of breast cancer. The low serum adiponectin levels and high serum leptin levels are independent risk factors for metastasis of cancer. The association between obesity and breast cancer risk might be explained by adipocytokines.

Breast cancer is a malignant tumor which severely harms the female health. Researches have shown that obesity possibly correlates with risk for breast cancer, especially with onset and prognosis of postmenopausal breast cancer, but the concrete mechanism is in nubibus. Recent researches indicate adipose tissue as an endocrine organ, which secretes adiponectin, resistin, leptin and other cytokines.1 Although many recent researches find that some adipocytokines may play an important role in the onset of breast cancer in obese people, the relationship between adipocytokines and breast cancer need further study. We measured serum levels of adiponectin, resistin, leptin and obesity-related metabolic factors of Chinese breast cancer patients, in order to investigate the correlation between adipocytokines and onset, development and metastasis of breast cancer.

METHODS

Subjects
Patients
We selected eighty women who were newly diagnosed as having breast cancer and treated surgically at Qilu Hospital of Shandong University from January 2005 to January 2006. Of whom forty-three were premenopausal and thirty-seven postmenopausal. The median age was 48 years (19–87 years). Diagnosis of breast cancer was confirmed histologically in each case (Infiltrating ductal carcinomas, 46 cases; Infiltrating lobular carcinoma, 24; Medullary carcinomas or other types of carcinoma,10). Of the 80 patients, 49 were estrogen receptor (ER)-positive and 41 were progesterone (PR)-positive; The staging of breast cancer was determined according to the TNM system. The distribution of the TNM staging was 13 patients in TNM I, 43 in TNM II, 24 in TNM III. All patients had never accepted radiotherapy or chemotherapy before operation.

Controls
We chose 50 healthy women (26 premenopausal women and 24 postmenopausal women) as controls, who attended routine physical examination in the same hospital in the corresponding period. The median age of controls was 49 years (38–63 years), matched with that of patients. All of the 50 women were confirmed free from benign or malignant breast diseases by physical examination and mammography. Woman with history or family history of any tumor was excluded.

Measurements
Blood samples after a 12-hour overnight fasting were obtained to evaluate fasting blood glucose (FBG), triglyceride (TG), high density lipoprotein (HDL-c), low density lipoprotein (LDL-c), total cholesterol (TC). Another 3-ml ulnar venous blood samples were collected and then the serum was immediately separated and stored at –20˚C to measure adiponectin, reistin and leptin levels. Meanwhile, we inquired history, age at first live birth, age at menarche, marriage age, childbearing history, smoking, alcohol drinking, and family history of breast cancer. Body mass index (BMI) and waist circumference (WC) were evaluated according to the WHO standard; FBG, TG, HDL-c, and LDL-c were measured by automatic biochemistry analyzer.

Resitin, adiponectin, and leptin were determined by immunosorbent assays (ELISA, R&D systems Inc., USA and Phoenix Biotech, USA) conducted according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were 5.2% and 7.8% for resistin, 7.2% and 9.2% for adiponectin, and 6.2% and 8.3% for letpin respectively.

Statistical analysis
Data were analyzed with the Statistical Package of the SPSS 13.0. Descriptive data were given as mean ± standard deviation (SD). Comparison between the two groups was performed with t-test and covariance analysis, and comparisons among groups were performed with the least significance of difference (LSD) t-test. The relationships between tumor size and variables were analyzed by linear correlation and stepwise regression. The Logistic multi-variant regression analysis was used to analyze the relationship between adipocytokines, metabolic factors and risk of onset and metastasis of breast cancer. A P value less than 0.05 was considered statistically significant.

RESULTS

Baseline characteristics
There was no significant difference between breast cancer patients and healthy controls with respect to age, age at menarche, age at first live birth, marriage age, family history of breast cancer in the first-degree relatives, smoking, alcohol consuming, menopausal status. BMI was increased in postmenopausal breast cancer patients compared with healthy controls (P=0.027); WC was increased in breast cancer patients in comparison to healthy controls (P=0.017).

Comparison of adipocytokines among groups
The serum adiponectin level of patients was significantly lower than controls (P=0.001); There was significant difference in serum adiponectin levels between post- menopausal patients and controls (P=0.002), but no difference was found between premenopausal patients and controls (P=0.109); The serum levels of leptin and resistin were increased in patients in comparison to controls (P=0.003 and 0.000), and the difference was more significant in the postmenopausal patients and controls (P=0.008 and 0.002). The result was the same after adjusting BMI and WC by covariance analysis. The serum levels of resistin, adiponectin and leptin were significantly different between lymph node positive and negative patients (P=0.001, 0.000 and 0.006, respectively); the serum levels of adiponectin and leptin in patients of TNM I, TNM II and TNM III stage were significantly different (P=0.000 and 0.007), but the difference of resistin among groups was not significant (P=0.178, Tables 1 and 2).


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Table 1. Serum levels of adipocytokines and metabolic factors among groups


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Table 2. Serum levels of adipocytokines and metabolic factors among groups

Correlation between adipocytokines and metabolic factors
Serum adiponectin levels showed negative correlation with BMI, WC and TG (r=–0.458, P=0.000; r=–0.357, P=0.000; r=–0.332, P=0.003, respectively) and positive correlation with HDL-c (r=0.426, P=0.004). Serum leptin levels were positively correlated to WC and TG (r=0.342, P=0.000 and r=0.625, P=0.003, respectively). Serum resistin concentrations were positively correlated to BMI and FBG (r=0.424, P=0.005 and r=0.386, P=0.001, respectively) but negatively to HDL-c (r=–0.524, P=0.003).

Correlation between tumor size and adipocytokines, metabolic factors
Tumor size was correlated with serum resistin concentration (r=0.385, P=0.023), leptin concentration (r=0.416, P=0.000), BMI (r=0.586, P=0.005), TG (r=0.327, P=0.000), WC (r=0.465, P=0.004), FBG (r=0.635, P=0.000) and HDL-c (r=–0.304, P=0.001). Stepwise regression analysis in which the dependent variable was the tumor size and resistin, adiponectin, leptin, BMI, TG, WC, TC, HDL-c, LDL-c and FBG were employed as independent variables showed that the tumor size had the close correlation with leptin (R2=0.414, P=0.000) and FBG (R2=0.602, P=0.000) (Tables 3 and 4).
 

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Table 3. Correlation between tumor size and adipocytokines, metabolic factors


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Table 4. Analysis of stepwise regression analysis of tumor size

Multivariate Logistic regression with risk for breast cancer and lymph node metastasis as a dependent variable respectively
Multivariate Logistic regression analysis, with the risk for breast cancer as the dependent variable and resistin, adiponectin, leptin, BMI, WC, TG, TC, HDL-c, LDL-c and FBG as independent variables, showed that the variables including adiponectin, HDL-c, leptin and resistin were accepted by the final model. The final model suggested that adiponectin, HDL-c, leptin and resistin were risk factors for breast cancer, and the OR for adiponectin, HDL-c, leptin and resistin was 0.805 (95%CI: 0.704–0.921, P=0.001), 0.087 (95%CI: 0.011–0.691, P=0.021), 2.235 (95%CI: 1.898–4.526, P=0.004), 1.335 (95%CI: 1.114–2.354, P=0.012), respectively (Table 5). In addition, the dependent variable was the risk for lymph node metastasis and the same independent variables mentioned above were employed as independent variables. The variables including adiponectin and leptin were accepted by the final model, and the OR was 0.742 (95%CI: 0.504–0.921, P=0.003), 2.134 (95%CI: 1.725–3.921, P=0.001), respectively (Table 6).
 

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Table 5. Analysis of multivariate Logistic regression with risk for breast cancer as a dependent variable


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Table 6. Analysis of multivariate Logistic regression with risk for lymph node metastasis as a dependent variable

DISCUSSION

Breast cancer is a malignant tumor that threats women’s health. Adiposity or abnormality of body fat distribution is considered to be one of major risk factors for breast cancer in postmenopausal women.2 The adipose tissue of postmenopausal obese women secretes more biologically active estrogen to stimulate mammary epithelial cell mitosis and to promote the development of tumor.3 But this cannot explain why obesity mediates incidence of not only estrogen receptor-positive breast cancer but also the estrogen receptor-negative breast cancer. Therefore, we need further researches on the underlying connection between obesity and breast cancer. In recent years, the endocrine function of adipose tissue has already been a hot spot, especially white adipose tissue can secrete adipocytokines such as resistin, adiponectin and leptin, which mainly regulate energy metabolism. However, recent studies have revealed that adipocytokines might have other pathophysiological functions. Some studies indicate that hyperleptinemia may be associated with breast cancer especially with the onset and metastasis of breast cancer after menopause, suggesting that hyperleptinemia may mediate the relationship between obesity and breast cancer. Similar to leptin, serum resistin level is positively associated with BMI, and adiponectin is negatively correlated with BMI. We need to continue research whether resistin and adiponectin are also closely correlated with breast cancer. In this study we analyze the serum levels of adipocytokines including leptin, adiponectin and resistin in Chinese breast cancer patients with different grades to probe their relationship with risk for onset and metastasis of breast cancer.

We determined serum resistin levels in patients with breast cancer and the healthy controls, and found that serum resistin levels were significantly increased in patients as compared to controls, especially after menopause, and were correlated with the size of tumor. Multivariate analysis showed that there might be a close correlation between resistin and breast cancer. However, the underlying mechanisms of this association are not clear. Steppan et al4 searched for genes that were downregulated by TZDs and discovered a new mRNA coding a novel protein. The protein which can induce glucose intolerance and insulin resistance was called resistin. The biological activity of resistin is not very clear now. To our knowledge, there was no report about the association between breast cancer and resistin. In recent years, studies show that resistin is strongly associated with tumors of gastrointestinal system and hematological system. Some studies reveal resistin-like molecules β (RELMβ), one member of the resistin family, was expressed and secreted in gastrointestinal tract, especially in colon. Epithelial cell gene expression and resistin secretion increase, especially in carcinogenesis. Pamuk et al5 researched resistin levels in patients with various hematological malignancies, and found that serum resistin level was significantly increased in lymphoma group when compared to the control group and leukemia group, although the underlying mechanisms of this association are not clear. But those two studies at least suggested that resistin might be correlated with the generation of some tumors. In our study, we found that resistin can increase the risk of breast cancer, and its concentration in lymph node metastasis group was higher than that in the group without lymph node metastasis, suggesting that resistin might promote metastasis of tumor cells. Recently some studies showed that there might be a correlation between resistin and angiogenesis. Di Simone et al6 found that resistin enhanced both matrix metalproteinase 2 (MMP-2) mRNA expression and protein synthesis, significantly reduced TIMP-1 and TIMP-2 synthesis and increased trophoblast-like cell invasiveness. Additionally, resistin induced production of vascular endothelium growth factor (VEGF) and stimulated formation of endothelial cell tube in vitro. Our studies found that resistin might be correlated with generation and metastasis of breast carcinoma. However, because resistin expression in mammary gland is rare, and it is not known whether resistin receptors exist on mammary gland epithelium, relationship between resistin and breast carcinoma, and the mechanisms need further study.

Our study showed that hypoadiponectinemia might increase the risk of onset and lymph node metastasis of breast cancer, and be associated with high histological grade. Adiponectin is a biologically active polypeptide which is exclusively produced by white adipose tissue. It is inverse proportion to BMI and may improve insulin sensitivity. Takahata et al7 showed that AdipoR1 and AdipoR2 were expressed in both normal breast epithelial cells and breast cancer cells; Dieudonne et al8 reported that MCF-7 cells expressed adiponectin receptors and responded to adiponectin by reducing their growth, AMPkinase activation, and p42/p44 MAPkinase inactivation. Those findings indicated that adiponectin might inhibit the proliferation of breast cancer cells directly through binding to adiponectin receptors. We demonstrated that decreased serum levels of adiponectin were found in breast cancer patients compared with healthy controls after correcting BMI and WC, especially in postmenopausal patients, which was in keeping with previous studies.9 Serum adiponectin level was reported to be associated with tumor size, and patients with lower serum levels of adiponectin tended to have larger tumors. But in our study no such association was found. We also found that patients with high histological grade and lymph node metastasis had lower serum adiponectin levels in comparison to ones with low grade and negative lymph node metastasis, which indicated that hypoadiponectin might be a risk factor of lymph node metastasis of tumor. However, Miyoshi et al found no significant difference of serum adiponcetin levels between negative lymph node metastasis patients and positive ones in their study. The disparity might be relevant to failing to control the confounding factors such as age. The report by Brakenhielm et al10 showed that adiponectin possessed antiangiogenic activity. Adiponectin remarkably prevented new blood vessel growth in the chick chorioallantoic membrane and the mouse cornea, and inhibited the proliferation and migration of endothelial cells. Adiponectin could also prevent the vascular smooth muscle cell proliferation induced by growth factors.11 The findings implied that adiponectin might prevent neovascularization and inhibit the metastasis of tumor.

We also found that the serum leptin levels of breast cancer patients were significantly higher than those of controls after adjustment of BMI and WC, which was concordant with the findings of Ozet et al12 and Tessitore et al.13 Mantzoros et al14 and Petridou et al15 reported that leptin increased risk for breast cancer in postmenopausal women, but had no relationship with onset of premenopausal breast cancer, which were accordant with our research. However Ozet et al12 did not find the variability resulted from menopausal status. Dieudonne et al16 found that MCF-7 cells expressed leptin receptor and leptin could influence the growth of human mammary cancer MCF-7 cells; Okumura et al17 investigated the effects of leptin on human mammary cancer MCF-7 cells by evaluating cell doubling time, DNA synthesis, levels of cell cycle related proteins and protein kinase C (PKC) isozyme expression, and implied that hyper-leptinemia increased breast cancer cell proliferation through accelerated cell cycle progression. Our research also found leptin positively correlated with the size of tumor, the same as research of Chen et al.9 Park et al18 discovered leptin could stimulate vascular endothelial cell proliferation and promote blood vessel generation in experimental model, which suggested leptin probably increased invasive ability of tumor through stimulating blood vessel generation. Our research further supported this viewpoint, because the serum leptin levels of high TNM staging and lymph metastasis positive patients were significantly higher than low TNM staging and lymph metastasis negative ones. However, Ozet et al12did not find significant difference of serum leptin levels among groups. This discrepancy might be related to the difference of races and pathological types of tumor.

In addition, we found that FBG and lipids had intimate relationship with risk for breast cancer. The blood glucose of breast cancer patients was higher and stepwise regression analysis showed hyperglycemia might be correlated with tumor size. Many researches discovered breast cancer supervened insulin resistance and abnormal glucose metabolism. Because obesity often complicated with hyperinsulinemia and insulin could stimulate cellular mitosis, insulin resistance probably might be one of the mechanisms underlying the relationship between obesity and breast cancer risk.

In conclusion, our results seemed to suggest a possibility that the serum adipocytokines levels could be new risk factors for breast cancer and to provide a new insight into understanding of the association between obesity and breast cancer risk.

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