Leptin is an adipocyte-derived hormone that regulates energy expenditure and food intake. A significant role for leptin in breast cancer has also been indicated by the resistance of leptin knockout mice in development of mammary tumors. In vitro, leptin induces proliferation of MCF-7 cells by activating cellular signaling pathways (1, 11, 12, 16, 17, 56). As leptin is emerging as an important factor for tumor growth, and hormones can exert their actions via autocrine/paracrine mechanisms, we hypothesized leptin may act by regulating epithelial-derived proteins. To test this hypothesis, leptin-regulated proteins secreted from MCF-7 mammary tumor cells were identified using proteomics techniques. Treatment of MCF-7 cells with 500 ng/ml leptin for 24 hours resulted in a 40% increase in cell number and a 5-fold increase in protein secretion as compared to controls. Establishing the significance of leptin-induced secreted factors, the addition of conditioned media from leptin-treated MCF-7 cells to synchronized MCF-7 cells resulted in 40% increase in cell number. Identification of leptin-regulated secreted proteins was done by 2D gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteins identified using Pro Found software and NCBI database included KF10 Collagen Precursor, Serologically Defined Breast Cancer Antigen NY-BR-62 and Cortactin Isoform a. A Human Cytokine Antibody Array system was used to identify low abundant proteins in the media of control and 500 ng/ml leptin-stimulated MCF-7 cells. In leptin treated cells, levels of FGF-9 were increased while IGFBP-3 and TGF-β3 levels were decreased. Many previous studies have focused on the regulation of distinct cellular proteins by leptin during mammary tumor cell proliferation. However, ours is the first study to identify leptin-regulated secreted proteins, many of which are known to play important roles in cancer. Our data support that leptin can influence mammary tumor growth and progression through regulation of autocrine/paracrine factors and by modulating the extracellular matrix composition.
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References
1.
Garofalo C, Surmacz E. Leptin and cancer. J Cell Physiol207:12–22, 2006.
2.
Lorincz AM, Sukumar S. Molecular links between obesity and breast cancer. Endocr Relat Cancer13:279–292, 2006.
3.
Barnett JB. The relationship between obesity and breast cancer risk and mortality. Nutr Rev61:73–76, 2003.
4.
Rose PD, Gilhooly EM, Nixon DW. Adverse effects of obesity on breast cancer prognosis and the biological actions of leptin. Internat J Oncology21:1285–1292, 2002.
5.
Hu X, Juneja SC, Maihle NJ, Cleary MP. Leptin—a growth factor in normal and malignant breast cells and for normal mammary gland development. J Natl Cancer Inst94:1704–1711, 2002.
O’Brien SN, Welter BH, Price TM. Presence of leptin in breast cell lines and breast tumors. Biochem Biophys Res Commun259:695–698, 1999.
8.
Tessitore L, Vizio B, Jenkins O, De Stefano I, Ritossa C, Argiles JM, Benedetto C, Mussa A. Leptin expression in colorectal and breast cancer patients. Int J Mol Med5:421–426, 2000.
9.
Cleary MP, Juneja SC, Phillips FC, Hu X, Grande JP, Maihle NJ. Leptin receptor-deficient MMTV-TGF-alpha/Lepr(db)Lepr(db) female mice do not develop oncogene-induced mammary tumors. Exp Biol Med229:182–193, 2004.
10.
Somasundar P, Yu AK, Vona-Davis L, McFadden DW. Differential effects of leptin on cancer in vitro. J Surg Res113:50–55, 2003.
11.
Dieudonne MN, Machinal-Quelin F, Serazin-Leroy V, Leneveu MC, Pecquery R, Giudicelli Y. Leptin mediates a proliferative response in human MCF7 breast cancer cells. Biochem Biophys Res Commun293: 622–628, 2002.
12.
Okumura M, Yamamoto M, Sakuma H, Kojima T, Maruyama T, Jamali M, Cooper DR, Yasuda K. Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: reciprocal involvement of PKC-alpha and PPAR expression. Biochim Biophys Acta1592:107–116, 2002.
13.
Hegyi K, Fulop K, Kovacs K, Toth S, Falus A. Leptin-induced signal transduction pathways. Cell Biol Internat28:159–169, 2004.
14.
Sweeney G. Leptin signalling. Cellular Signalling14:655–663, 2002.
15.
Fruhbeck G. Intracellular signalling pathways activated by leptin. Biochem J393:7–20, 2006.
16.
Catalano S, Marsico S, Giordano C, Mauro L, Rizza P, Panno ML, Ando S. Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line. J Biol Chem278:28668–28676, 2003.
17.
Catalano S, Mauro L, Marsico S, Giordano C, Rizza P, Rago V, Montanaro D, Maggiolini M, Panno ML, Andó S. Leptin induces, via ERK1/ERK2 signal, functional activation of estrogen receptor alpha in MCF-7 cells. J Biol Chem279:19908–19915, 2004.
18.
Vogel WF, Aszodi A, Alves F, Pawson T. Discoidin domain receptor 1 tyrosine kinase has an essential role in mammary gland development. Mol Cell Biol21:2906–2917, 2001.
19.
Campbell DH, deFazio A, Sutherland RL, Daly RJ. Expression and tyrosine phosphorylation of EMS1 in human breast cancer cell lines. Int J Cancer68:485–492, 1996.
20.
Van Rossum AG, Moolenaar WH, Schuuring E. Cortactin affects cell migration by regulating intercellular adhesion and cell spreading. Journal Exp Cell Res312:1658–1670, 2006.
21.
Scanlan MJ, Gout I, Gordon CM, Williamson B, Stockert E, Gure AO, Jäger D, Chen YT, Mackay A, O’Hare MJ, Old LJ. Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression. Cancer Immun1:4, 2001.
22.
Calle EE, Kaaks R. Overweight, obesity and cancer: epidemiological evidence and proposed mechanisums. Nature Reviews Cancer4:579–591, 2004.
23.
Sexena NK. Leptin induces increased α2(1) collagen gene expression in cultured rat hepatic stellate cells. J Cell Biochem89:311–320, 2003.
24.
Han DC, Isono M, Chen S, Casaretto A, Hong SW, Wolf G, Ziyadeh FN. Leptin stimulates type I collagen production in db/db mesangial cells: glucose uptake and TGF-beta type II receptor expression. Kidney Int59:1315–1323, 2001.
25.
Castellucci M, De Matteis R, Meisser A, Cancello R, Monsurrò V, Islami D, Sarzani R, Marzioni D, Cinti S, Bischof P. Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion. Mol Hum Reprod6:951–958, 2000.
26.
Eckhardt BL, Parker BS, van Laar RK, Restall CM, Natoli AL, Tavaria MD, Stanley KL, Sloan EK, Moseley JM, Anderson RL. Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix. Mol Cancer Res3:1–13, 2005.
27.
Miyagi N, Kato S, Terasaki M, Aoki T, Sugita Y, Yamaguchi M, Shigemori M, Morimatsu M. Fibroblast growth factor-9 (glia-activating factor) stimulates proliferation and production of glial fibrillary acidic protein in human gliomas either in the presence or in the absence of the endogenous growth factor expression. Oncol Rep6:87–92, 1999.
Barcellos-Hoff MH, Ewan KB. Transforming growth factor-beta and breast cancer: mammary gland development. Breast Cancer Res2:92–99, 2000.
30.
Ren Z, Shin A, Cai Q, Shu XO, Gao YT, Zheng W. IGFBP3 mRNA expression in benign and malignant breast tumors. Breast Cancer Res9(1):R2, 2007.
31.
Vestey SB, Perks CM, Sen C, Calder CJ, Holly JM, Winters ZE. Immunohistochemical expression of insulin-like growth factor binding protein-3 in invasive breast cancers and ductal carcinoma in situ: implications for clinicopathology and patient outcome. Breast Cancer Res7:119–129, 2005.
32.
Ogunwobi OO, Beales IL. The anti-apoptotic and growth stimulatory actions of leptin in human colon cancer cells involves activation of JNK mitogen activated protein kinase, JAK2 and PI3 kinase/Akt. Int J Colorectal Dis22:401–409, 2007.
33.
Saxena NK, Titus MA, Ding X, Floyd J, Srinivasan S, Sitaraman SV, Anania FA. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. FASEB J3:1612–1614, 2004.
34.
Bouloumie A, Drexler HC, Lafontan M, Busse R. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res83:1059–1066, 1998.
35.
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell100:57–70, 2000.
36.
Garofalo C, Koda M, Cascio S, Sulkowska M, Kanczuga-Koda L, Golaszewska J, Russo A, Sulkowski S, Surmacz E. Increased expression of leptin and the leptin receptor as a marker of breast cancer progression: possible role of obesity-related stimuli. Clin Cancer Res12:1447–1453, 2006.
37.
Surmacz E. Obesity hormone leptin: a new target in breast cancer? Breast Cancer Res9:301–320, 2007.
38.
Tlsty TD, Coussens LM. Tumor stroma and regulation of cancer development. Annu Rev Pathol1:119–150, 2006
39.
Le T, Rehrer C, Huff B, Nichols M, Camarillo IG, Cheng J. Multimodal multiphoton imaging to evaluate the impact of diet and obesity on mammary tumor stroma. Molecular Imaging6:205–211, 2007.
40.
Perera CN, Chin HG, Camarillo IG. Leptin regulated gene expression in human breast cancer cells: insights into mechanisms underlying leptin influenced mammary tumor formation. 2008. (In Review).
41.
Kondo S, Shimo T, Nishida T, Yosimichi G, Eguchi T, Sugahara T, Takigawa M. Connective tissue growth factor increased by hypoxia may initiate angiogenesis in collaboration with matrix metalloproteinases. Carcinogenesis23:769–776, 2002.
42.
Minn AJ, Kang Y, Serganova I, Gupta GP, Giri DD, Doubrovin M, Ponomarev V, Gerald WL, Blasberg R, Massague J. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest115:44–55, 2005.
43.
Perera CN, Mohammed SI, Camarillo IG. Profiling of Leptin Regulated Gene and Protein Expression in Human Breast Cancer Cells: Insights into Pathways Underlying Leptin Induced Mammary Tumor Formation. Endocrine Society’s 88th Annual Meeting, Boston, MA, June 24–27, 2007.
44.
Steiling H, Werner S. Fibroblast growth factors: key players in epithelial morphogenesis, repair and cytoprotection. Curr Opin Biotechnol14:533–537, 2003.
45.
Kwabi-Addo B, Ozen M, Ittmann M. The role of fibroblast growth factors and their receptors in prostate cancer. Endocr Relat Cancer11: 709–724, 2004.
46.
Buck MB, Knabbe C. TGF-beta signaling in breast cancer. Ann N Y Acad Sci1089:119–126, 2006.
47.
Kaaks R. 2001 Plasma insulin, IGF-I and breast cancer. Gynecol Obstet Fertil29:185–191, 2001.
48.
Chong YM, Subramanian A, Sharma AK, Mokbel K. The potential clinical applications of insulin-like growth factor-1 ligand in human breast cancer. Anticancer Res27:1617–1624, 2007.
49.
Nam SY, Lee EJ, Kim KR, Cha BS, Song YD, Lim SK, Lee HC, Huh KB. Effect of obesity on total and free insulin-like growth factor (IGF)-1, and their relationship to IGF-binding protein (BP)-1, IGFBP-2, IGFBP-3, insulin, and growth hormone. Int J Obes Relat Metab Disord21:355–359, 1997.
50.
Burger AM, Leyland-Jones B, Banerjee K, Spyropoulos DD, Seth AK. Essential roles of IGFBP-3 and IGFBP-rP1 in breast cancer. Eur J Cancer41:1515–1527, 2005.
51.
Carmichael AR. 2006 Obesity as a risk factor for development and poor prognosis of breast cancer. BJOG113:1160–1166, 2006.
52.
Hirsch DS, Wu WJ. Cdc42: an effector and regulator of ErbB1 as a strategic target in breast cancer therapy. Expert Rev Anticancer Ther7: 147–157, 2007.
53.
Ouhtit A, Abd Elmageed ZY, Abdraboh ME, Lioe TF, Raj MH. In vivo evidence for the role of CD44s in promoting breast cancer metastasis to the liver. Am J Pathol171:2033–2039, 2007.
54.
Niu L, Wang X, Li J, Huang Y, Yang Z, Chen F, Ni H, Jin Y, Lu X, Cao Q. Leptin stimulates alpha1(I) collagen expression in human hepatic stellate cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. Liver Int27:1265–1272, 2007.
55.
Chen HN, Fan S, Weng CF. Down-regulation of TGFbeta1 and leptin meliorates thioacetamide-induced liver injury in lipopolysaccharide-primed rats. J Endotoxin Res13:176–188, 2007.
56.
Saxena NK, Vertino PM, Anania FA. Sharma D. Leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3. J Biol Chem282:13316–13325, 2007.
