Human Cancer Biology Proangiogenic Contribution of Adiponectin toward Mammary Tumor Growth In vivo

Purpose: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors (“adipokines”) is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model. Experimental Design: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds. Results: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells. Conclusions: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic microenvironment. The physiologic role of adipose tissue as a dynamic organ has shown its crucial role in maintaining normal systemic energy balance, glucose homeostasis, and the immune response (1, 2). In the context of the mammary gland, the adipocytes are an abundant cell type in the stroma and are vital for ductal development and survival. This is due largely to their secretory profile of adipokines such as leptin, adiponectin, hepatocyte growth factor, collagen VI, interleukin 6, and tumor necrosis factor α (3). Mammary tumor growth is determined by cell-autonomous effects of epithelial cancer cells as well as by contributions of the stromal compartment (4–6). Here, we focus on the adipocyte microenvironment of the mammary gland. Our previous work characterized a broad spectrum of effects mediated by soluble adipokines on the proliferative, invasive, and angiogenic capacity of ductal epithelial cells (5, 7), suggestive of major contributions of adipokines to the malignant progression of breast cancer. A widely studied adipokine in the area of metabolism is adiponectin (APN; ref. 8). APN is an adipocyte-specific secretory protein that enhances hepatic insulin sensitivity by suppressing Authors' Affiliations: Departments of Cell Biology, Developmental and Molecular Biology, Center of Reproductive Biology and Womens' Health, Albert Einstein Cancer Center, and Department of Nuclear Medicine, M. Donald Blaufox Laboratory for Molecular Imaging, Albert Einstein College of Medicine, Bronx, New York; Departments of Molecular Profiling and Metabolic Disorders, Merck Research Laboratories, Rahway, New Jersey; and Touchstone Diabetes Center, Departments of Internal Medicine, Cell Biology and Simmons Cancer, University of Texas Southwestern Medical Center, Dallas, Texas Received 10/14/08; revised2/10/09; accepted 2/24/09; published online 5/15/09. Grant support: NIH grant R01-CA112023 (P.E. Scherer) and Training Program in Cellular and Molecular Biology and Genetics Grant T32-GM04791 (S. Landskroner-Eiger). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Current address for A.R. Nawrocki: Department of Metabolic Disorders, Merck Research Laboratories, Rahway, NJ 07065. Requests for reprints: Philipp E. Scherer, Touchstone Diabetes Center, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8549. Phone: 214-648-8715; Fax: 214-648-8720; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2649 3265 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Research. on June 9, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from hepatic glucose output from gluconeogenesis (9, 10) and also affects glucose uptake in the muscle (11). In addition, it has potent protective effects against inflammation, adverse lipid profiles, and atherosclerosis. As a result, it is thought to be potently cardioprotective (11, 12). Recently, a great deal of attention has been given to the study of the epidemiologic association between APN levels in circulation and breast cancer incidence. It is generally accepted that obesity is a risk factor for breast cancer in postmenopausal but not premenopausal women (13). Because APN levels are inversely correlated with obesity (11), it has been suggested that the decreased levels of APN may explain the increased risk of breast cancer in obesity (13, 14). The epidemiologic association between APN levels and breast cancer incidence suggested an inverse correlation between APN and breast cancer risk, an association that seems to be stronger for postmenopausal women (14, 15). In vitro assays have studied the APN-mammary cancer axis, suggesting an inhibitory role for APN in mammary tumor growth (16, 17). Similar results were obtained after intratumoral injection of APN into fibrosarcoma tumors (18). In the majority of these cases, bacterially produced forms of APN were used either in vitro or in the xenograft models, conditions with limited relevance for the physiologic action of endogenous, full-length APN, and autochthonous tumors. Due to its highly complex tertiary and quaternary structure, APN has to be synthesized in a mammalian production system to recapitulate the complex nature of its endogenous counterpart (9). In addition, xenograft models may not accurately predict the antiangiogenic and antitumor responses in human tumors (19, 20). Thus, we took a direct in vivo approach, using our previously generated APN knockout (KO) mice (21), to decipher the role of APN and its effect on mammary tumor growth with the widely used mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) model, a spontaneous mammary tumor model (22). This tumor model has been shown to recapitulate many processes found in human breast cancer progression both morphologically and in the pattern of expression of biomarkers associated with poor prognosis (23). The APN KO mice develop dietand obesity-induced insulin resistance (21, 24) and an impaired angiogenic response to ischemia (25), but have, in an unchallenged state, minimal phenotypic manifestations. We observed that in early stages of tumor progression, lack of APN delays tumor growth by inhibiting angiogenesis in a paracrine and/or endocrine fashion. Despite the defects in angiogenesis in early stages of tumorigenesis in APN KO mice, tumor growth persisted, leading to rapid tumor growth at later stages of carcinoma. This involved increased levels of vascular endothelial growth factor (VEGF)-A in the tumor, the mobilization of circulating endothelial progenitor cells (CEP), and the emergence of a vasculature with normal appearance as opposed to the dilated and distorted shape characteristic of tumor vessels. We hypothesized that in mice lacking APN, the antiangiogenic stress at early stages leads to an adaptation mechanism during which CEPs are mobilized to contribute to tumor growth, similar to other tumor models undergoing antiangiogenic stress (26–28). Combined, our observations indicate that APN has potent angio-mimetic properties in the early steps of tumor vascularization where mice lacking APN have a lagging angiogenic response. However, this antiangiogenic stress triggers an adaptive reaction that fuels tumor growth at later stages. The present data suggest a possible mechanism for the increased risk and poorer prognosis seen in breast cancer patients with low serum APN levels (such as obese women) due to acquired adaptation of tumors to antiangiogenic stress. Materials and Methods Animals. All animal experimental protocols were approved by the Institute for Animal Studies of the Albert Einstein College of Medicine and by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center at Dallas. Mice were maintained as described previously (29). Experiments presented were done on a mixed C57/Bl6 and FVB background or a pure FVB background (an excess of nine backcrosses; Figs. 1D, 2, 3, 5, and 6; Supplementary Figs. S1C-D and S2). The majority of experiments were done on both genetic backgrounds with similar results. Double heterozygous PyMT/ APN males were crossed with APN heterozygous females to obtain littermates that are deficient for APN or wild type (WT). Further experimental cohorts were generated by crossing the heterozygous PyMT/APN KO males to APN KO females, and male heterozygous PyMT to WT females (21). Intermittently, animals were bred as male PyMT/APN heterozygous at both loci crossed to APN heterozygous females. Mammary tumor development was compared between age-matched females of PyMT and PyMT/APN null mice. For detection of mammary tumor volume, mice were monitored biweekly. Tumor diameter was measured with a caliper, and tumor volume calculated as (H × W)/2. APN-overexpressing mice were previously obtained and characterized (29). Whole-mount staining and histology of mammary gland. For histology, inguinal mammary glands were fixed for 24 h in 10% normal buffered formalin (Fisher) and embedded in paraffin. Five-micrometer sections were stained with H&E; two to three sections per mouse were examined for tumor staging in a blind fashion and staged as described previously (23). Whole-mount staining was done as described, imaged on a Stemi SV11 stereo dissection microscope, and analyzed for lesion area using Image J software. Translational Relevance The contributions of stromal adipocytes toward mammary tumor growth have been appreciated for decades. The effects of adiponectin (APN) on tumor cells are not well understood, but a number of cancer epidemiologic studies have focused on APN as a predictive marker. APN levels are becoming a popular and widely measured clinical parameter, including in the context of breast cancer studies. Here, we present the first in vivo data on the role of APN on mammary tumor growth and highlight its function as a potent proangiogenic factor. APN is a key mediator of the antidiabetic actions of the widely used peroxisome proliferator–activated receptor γ agonists (thiazolindinediones). These compounds lead to an increased tumor mass in the present studies. Our data suggest a possible mechanism for the increased risk and poorer prognosis seen in breast cancer patients with low serum APN levels (such as obese women) due to acquired adaptation of tumors to antiangiogenic stress. 7 http://mammary.nih.gov/ 3266 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Human Cancer Biology Research. on June 9, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Morphologic analysis of mammary gland development. To determine ductal lengths, mammary gland whole-mount preparations were analyzed by measuring the distance of the three longest ducts from the edge of the 4th mammary gland at the interface with the 5th mammary gland to the leading edge of the mammary gland. Terminal end buds were counted in the whole mammary gland. Peroxisome proliferator–activated receptor-γ agonist regimen. The peroxisome proliferator–activated receptor-γ (PPARγ) agonist 2-(2-(4phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH) was a kind gift from Merck Research Laboratories. The drug was mixed with a standard chow diet powder at 10 mg/kg body weight/d and mice were treated from 4 until 9 wk of age. The mice were subjected to tail bleeding before and during the course of the treatment. Immunohistochemistry analysis—CD31 and terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling. Inguinal mammary glands were evaluated for the presence of microvessels on sections fixed 24 h in Zinc Fixative (BD Pharmingen) and stained with antimouse platelet endothelial cell adhesion molecule/CD31 antibody (BD Pharmingen) overnight at 4°C (1:30). Biotinylated secondary antibodies were detected with a DAB Substrate Chromagen System (DakoCytomation). The CD31-positive blood vessels in PyMT and PyMT/APN KO derived specimens were counted in four fields having the maximal number of CD31-positive staining per unit area of a section [modification of Weidner et al. (30)]. These were averaged to calculate the microvessel density. Vessel imaging on frozen sections was carried out with a 1:50 dilution of antimouse platelet endothelial cell adhesion molecule/ Fig. 1. PyMT/APN KO mice display delayed mammary tumor progression. A, top, representative whole-mount preparations of inguinal mammary gland derived from age-matched 7-wk-old mice. Bar, 1 mm. Bottom, total lesion area of PyMT and PyMT/APN KO at 5 wk (n = 4-6 per group) and 7 wk (n = 11-12 per group; bars, SE; *, P < 0.05). B, summary of tumor staging distribution derived from H&E of 7-wk-old inguinal mammary glands sections (n = 13 per group). C, a representative experiment is shown at the top. Western blot quantification of circulating APN levels in mice treated with monoclonal antibodies neutralizing APN (bars, SE; *, P < 0.001, two-way ANOVA). Bottom, APN neutralizing antibody–treated mice (n = 6) show a trend of a 2-fold decrease in lesion area compared with IgG-treated mice (n = 3) as measured in whole mounts of inguinal mammary glands (bars, SE; P = 0.2). D, mice were treated with either vehicle or the PPARγ agonist COOH from 4 to 9 wk of age. Total lesion area from whole mounts was measured. PyMT mice treated with the PPARγ agonist have a 35% increase in lesion area compared with vehicle-treated PyMT, whereas PyMT/APN KO mice treated with the PPARγ agonist show a more modest increase in lesion area compared with the untreated PyMT/APN KO group (n = 9-10 per group; bars, SE; *, P < 0.05). 3267 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Adiponectin and Tumor Angiogenesis Research. on June 9, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from CD31 antibody (BD Pharmingen) and a secondary antibody labeled with Alexa Fluor 488 at 1:200 (Molecular Probes). Nuclei were detected with 4′,6-diamidino-2-phenylindole. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining was done according to the manufacturer's protocol (Chemicon), and 10 random fields from areas of lesions were imaged per mouse for further analysis. Sections were imaged using the Zeiss Axioskop Plus with the AxioCam MRc camera or Olympus IX81 microscope. In vivo vessel labeling. Texas red–conjugated dextran (MW 70,000; Molecular Probes) was prepared at 6.2 mg/mL in PBS. One hundred microliters were injected i.v., and 5 min after the injection, mice were sacrificed and inguinal mammary glands were fixed in 10% buffered formalin to be processed by standard procedures (6). For analysis, samples were further stained with 4′,6-diamidino-2-phenylindole and imaged using the Olympus IX81 microscope. Every second field of tissue section was imaged, of which 10 random images per mouse were further quantified using Image J software. APN, leptin, insulin, and glucose measurements. APN was measured from tail blood with a mouse adiponectin RIA kit (LINCO Research) or by Western blot analysis with rabbit polyclonal anti-mouse APN antibody. The bands were detected by the Odyssey Infrared Imaging System (LI-COR) and band intensity was quantified with Odyssey v1.2 software (LI-COR Biotechnology). Leptin levels were measured using the mouse leptin Elisa kit (LINCO Research). Insulin, glucose, and oral glucose tolerance tests were done as described (29). APN neutralization and APN injections. For APN neutralization experiments, 5-wk-old PyMT females were injected i.p. for a 2-wk period with either antimouse APN monoclonal antibodies (clones 14 and 45) or purified mouse IgGs (Sigma) at 50 μg per mouse every 3rd day (31). APN protein levels were monitored from tail blood by Western blot