The role of adipocytokines in breast cancer

1. The role of adipocytokines in breast cancer 義大醫院醫研部暨婦產部 義守大學生物科技系 袁行修

2. Overview of breast cancer • Breast cancer is a malignant tumor which severely impairs the woman health. According to the statistics of Department of Health from 1995 to 2007, breast cancer has jumped to the first place in the incidence of women-specific malignancies in Taiwan, and growing at a surprising rapidity. • Known breast cancer risk factors include age, family or personal history of breast cancer, high breast tissue density, atypical hyperplasia, a history of chest radiation, early menarche, recent use of oral contraceptive drugs, age of birth of first child over 30, obesity after menopause (Willett et al., 2004; Viswanathan et al., 2009). It is a critical issue to explore the underlying mechanisms for female breast cancer in Taiwan and to discover the prognostic factors and therapeutic targets for Taiwanese breast cancer.

3. The etiology of breast cancer is still poorly understood, but several risk factors are well confirmed, including increasing age, geographic location (USA and western countries), familial history of breast cancer, genetic mutations (BRCA1, BRCA2, p53, ATM NBS1, LKB1), ionizing radiation exposure, history of benign breast disease, increased mammographic density, early menarche and late menopause, nulliparity and old age at first delivery, exogenous hormone usage, lifestyle (alcohol, diet, obesity and physical activity), high IGF-1 and prolactin levels, etc. (Collaborative Group on Hormonal Factors in Breast Cancer, 2001; Dumitrescu and Cotarla, 2005).

4. Adipocytokines • Adipose tissue is no longer considered to be an inert tissue functioning solely as an energy store, but is emerging as an important factor in the regulation of many pathological processes. Various products of adipose tissue have been characterized, and some of the soluble factors produced by this tissue are known as adipocytokines(Calle and Kaaks 2004). • The term adipocytokine is used to describe certain cytokines that are mainly produced by adipose tissue, although it is important to note that they are not all exclusively derived from this tissue (Wellen and Hotamisligil 2005).

5. Adiponectin, leptin, resistin and visfatin are adipocytokines and are thought to provide an important link between obesity, insulin resistance and related inflammatory disorders (Herbert Tilg and Alexander R. Moschen, 2006). • The incidence of obesity and its associated disorders are increasing markedly worldwide. Obesity predisposes individuals to an increased risk of developing many diseases, including atherosclerosis, diabetes, nonalcoholic fatty liver disease, certain cancers and some immune-mediated disorders (Mannino, Mott et al. 2006).

6. Resistin (also known as FIZZ3), which is a 114-amino-acid polypeptide, was originally shown to induce insulin resistance in mice80. It belongs to a family of cysteine-rich proteins, also known as resistin-like molecules (RELMs), that have been implicated in the regulation of inflammatory processes79. Resistin was shown to circulate in two distinct forms: a more prevalent high-molecular-weight hexamer and a substantially more bioactive, but less prevalent, low-molecular-weight complex82. Fig.1 Adipose tissue: cellular components and molecules synthesized. (Herbert Tilg and Alexander R. Moschen, 2006)

7. (Marra and Bertolani 2009)

8. (Herbert Tilg and Alexander R. Moschen, 2006)

9. Adipocytokines function as hormones to influence energy homeostasis and to regulate neuroendocrine function. As cytokines, they affect immune functions and inflammatory processes throughout the body. The field of adipocytokines has attracted tremendous interest recently and the knowledge that has accumulated might lead to the development of new therapeutics (Herbert Tilg and Alexander R. Moschen, 2006).

10. Obesity is a serious health problem in the industrialized world. Also, similar trends have been observed in many developing countries (Shetty and Schmidhuber 2006). Obesity is associated with a number of disorders including cardiovascular disease, hypertension, Type 2 diabetes, dyslipidemia and cancer (Hanif and Kumar 2002). • Probably, a large number of cancers are linked with obesity such as cancers of the colon, breast (postmenopausal), endometrium, kidney, esophagus and gastric cardia (adenocarcinoma), gall bladder, liver, pancreas, prostate (advanced malignancy), ovary and hematopoietic tissues like non-Hodgkin's lymphoma (NHL), multiple myeloma and leukemia(Calle 2007).

11. Furthermore, it is worthy to mention that obesity may influence prognosis through various mechanisms, including co-morbidities and endocrine factors (McTiernan 2005). In addition to serve as an energy depot, adipose tissue or fat mass releases several hormone-like chemicals or adipokines, which perhaps provide a link among cancer, insulin resistance, inflammation and oxidative stress (Ruan and Lodish 2004). • Here, we provide an overview of recent advances in our view of the role of adipocytokines in breast cancers.

12. Figure 1: Effects of obesity on the pathological processes that favor carcinogenesis (Murthy, Mukherjee et al. 2009)

14. Increased resistin and its association with positive ER status in breast cancer is associated with a poor overall survival

15. Resistin • Resistin, a 12.5 kDa protein and named for resistance to insulin, is a signaling molecule secreted from adipocytes. It is expressed in adipocytes, muscle, pancreatic cells, and mRNA displays an even wide range, having been found in white fat, spleen, hypothalamus, adrenal gland, skeletal muscle, gastrointestinal tract, and pancreas (Wozniak et al., 2009). • Resistin serves as a hormone (Steppan et al., 2001), that could decrease the sensitivities of insulin in the adipose cells so that insulin resistance is formed (Steppan and Lazar, 2004).

16. Circulating resistin levels are decreased by the anti-diabetic drug, increased in diet-induced and genetic forms of obesity. Insulin-stimulated glucose uptake by adipocytes is enhanced by neutralization of resistin and is reduced by resistin treatment (Kang et al., 2006). • In cancer studies, human serum resistin levels are significantly increased in breast cancer patients as compared to controls, especially after menopause, and correlated with the size of tumor. In addition, resistin concentration in lymph node metastasis group is higher than that in the group without lymph node metastasis, suggest that resistin may promote metastasis of breast cancer cells (Hou et al., 2007).

17. Fig.3 Effects of various adipocytokines on the monocyte–macrophage system. c | The receptor for resistin is unknown, but this adipocytokine induces the activation of p38, ERK and phosphatidylinositol 3-kinase (PI3K). Resistin increases the production of TNF, IL-1β, IL-6 and IL-12. Its effect on monocyte and macrophage functions is not known. Whereas adiponectin can be considered an anti-inflammatory strategy of the ‘adipose organ’, leptin and resistin have dominant pro-inflammatory features (Herbert Tilg and Alexander R. Moschen, 2006).

18. To determine the expression profiles of resistin in breast cancer and their correlation with prognosis and other clinico-pathological parameters. Positive > 30%

19. Table 1. Correlation of resistin expression in the serum of breast cancer and non breast cancer. Breast cancer (n=81) Non breast cancer (n=81) P value * Resistin (Mean±SD) 30.78 ±8.59 ng/ml 26.91±5.00 ng/ml 0.001 *The P value was calculated by the t-test.

21. *The P value was calculated by the chi-square test.

22. Fig. 2 The average tumor size (cm in diameter) for breast patients with low and high resistin expression. The average tumor size was 2.71±0.27 cm for low resistin expression (n=19) and 2.86±0.18 cm for high resistin expression (n=62) in breast cancer, P=0.689. Values were expressed as Mean±SEM determined by independent-samples t test.

23. Low(≦27 ng/ml) High(＞27 ng/ml) P=0.033 Fig. 3 Kaplan–Meier survival curves are shown for the low and high resistin expression groups in breast cancer.

24. Low (≦27 ng/ml) resistin expression and ER negative Low (≦27 ng/ml) resistin expression and ER positive High(＞27 ng/ml) resistin expression and ER negative High(＞27 ng/ml) resistin expression and ER positive P =0.108 Fig. 4 Kaplan–Meier survival curves are shown for the low and high resistin expression groups combined with positive and negative ER status in breast cancer.

26. A B C D Fig. 1 Immunohistochemistry showing high (A) and low (B) expression of resistin in breast cancer tissue. (C) normal breast tissue, (D) negative control. Original magnification was X100.

27. Table 1. Immunohistochemistry of resistin expressions in breast cancer and adjacent normal breast tissue. Breast cancer tissue (n=37) Adjacent normal breast tissue (n=37) P value * Resistin Low-expression High-expression 18 (48.6%) 19 (51.4%) 33 (89.2%) <0.001 4 (10.8%) *The P value was calculated by the chi-square test.

29. *The P value was calculated by the chi-square test.

30. * Fig. 2 The average tumor size (cm in diameter) for breast patients with low and high resistin expression. The average tumor size was 1.79±0.15 cm for low resistin expression (n=34) and 2.29±0.14 cm for high resistin expression (n=74) in immunohistochemistry analysis groups, *P=0.017. Values were expressed as Mean±SEM determined by independent-samples t test.

31. Low High P=0.003 Fig. 3 Kaplan–Meier survival curves are shown for the low and high resistin expression groups, determined by immunohistochemistry analysis.

32. Low resistin expression and ER negative Low resistin expression and ER positive High resistin expression and ER negative High resistin expression and ER positive P=0.004 Fig. 4 Kaplan–Meier survival curves are shown for the low and high resistin expression groups combined with positive and negative ER, determined by immunohistochemistry analysis.

34. Conclusion • Altered resistin expression may be involved in the pathogenesis of breast cancer in an ER-dependent manner. • Adipocytokines could be attractive candidates as the missing link between obesity and cancer.

35. Altered visfatin expression in breast cancer tissue is associated with a poor overall survival

36. Visfatin • Visfatin, a 52 kDa protein and produced by lymphocytes (Fukuhara et al., 2005), has nicotinamide adenine dinucleotide (NAD) biosynthetic activity, which is essential for B-cell maturation and function (Samal et al., 1994; 2003; Revollo et al., 2007). • Recently, visfatin has been identified as an adipocytokine hormone that could make adipose cells to increase the sensitivity of insulin (Fukuhara et al., 2005) and associated with obesity, type II diabetes and rheumatoid arthritis (Brentano et al., 2007).

37. In human studies, a positive correlation between visfatin gene expression in visceral adipose tissue and body mass index (BMI) is noted, along with negative correlation between BMI and subcutaneous fat visfatin (Berndt et al., 2005), suggesting that visfatin regulation in these different depots is different, and adipose depot ratios are highly dependent on the obesity of the subjects. • In breast cancer studies, visfatin is reported to be expressed in doxorubicin-responsive breast cancer (Folgueira et al., 2005), and have demonstrated that visfation mRNA and protein expressed in MCF-7 breast cancer cells. Furthermore, visfatin is present in bovine mammary epithelial cells, lactating mammary gland and milk (Yonezawa et al., 2006). These studies suggest that visfatin may be plays some important role in the mammary epithelial cells and mammary gland.

38. (Hausenloy 2009)

39. To determine the expression profiles of visfatin in breast cancer and their correlation with prognosis and other clinico-pathological parameters. Positive > 30%

40. Table 1. Correlation of visfatin expression in the serum of breast cancer and non breast cancer. *The P value was calculated by the T-test.

43. Fig. 1 The average tumor size (cm in diameter) for breast patients with low and high resistin expression. The average tumor size was 2.61±0.17 cm for low resistin expression (n=30) and 2.88±0.20 cm for high resistin expression (n=38) in serum analysis groups, P=0.341. Values were expressed as Mean±SEM determined by independent-samples t test.

44. Low expression (≦32 ng/ml) High expression (＞32 ng/ml) P=0.021 Fig. 2 Kaplan–Meier survival curves generated by the low and high visfatin expression groups.

45. Low expression(≦32 ng/ml) and ER positive High expression(＞32 ng/ml) and ER positive Low expression(≦32 ng/ml) and ER negative High expression(＞32 ng/ml) and ER negative P=0.002 Fig. 3 Kaplan–Meier survival curves generated by the low and high visfatin expression groups combined with positive and negative ER status.

46. Low expression(≦32 ng/ml) and PR positive High expression(＞32 ng/ml) and PR positive Low expression(≦32 ng/ml) and PR negative High expression(＞32 ng/ml) and PR negative P=0.004 Fig. 4 Kaplan–Meier survival curves generated by the low and high visfatin expression groups combined with positive and negative PR status.

47. Table 4. Cox regression multivariate analysis of overall survival for breast cancer

48. A B C D Fig. 1 Immunohistochemistry showing high (A) and low (B) expression of visfatin in breast cancer tissue. (C) normal breast tissue, (D) negative control. Original magnification was X100.

49. Table 1. Immunohistochemistry of visfatin expressions in breast cancer and adjacent normal breast tissue (n=45). *The P value was calculated by the chi-square test.