Circulating Adiponectin Is Inversely Associated with Risk of Thyroid ...

J C E M B r i e f

R e p o r t — E n d o c r i n e

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Circulating Adiponectin Is Inversely Associated with Risk of Thyroid Cancer: In Vivo and in Vitro Studies Nicholas Mitsiades,* Kalliopi Pazaitou-Panayiotou,* Konstantinos N. Aronis,* Hyun-Seuk Moon,* John P. Chamberland, Xiaowen Liu, Kalliope N. Diakopoulos, Vasileios Kyttaris, Vasiliki Panagiotou, Geetha Mylvaganam, Sofia Tseleni-Balafouta, and Christos S. Mantzoros Department of Internal Medicine (N.M., K.P.-P., K.N.A., H.-S.M., J.C., X.L., K.N.D., V.K., G.M., C.S.M.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; Division of Endocrinology-Endocrine Oncology (K.P.-P., V.P.), Theagenio Cancer Hospital, Thessaloniki 54630, Greece; Department of Pathology (S.T.-B.), University of Athens, Athens 10671, Greece; and Department of Internal Medicine (C.S.M.), VA Boston Healthcare System, Harvard Medical School, Boston, Massachusetts 02130

Context: Circulating adiponectin has been inversely associated with risk for several malignancies. Its association with thyroid cancer has not yet been evaluated. Objective/Methods: We measured circulating adiponectin levels in 175 thyroid carcinoma patients and 107 controls. We also examined the expression of adiponectin receptors (AdipoR1 and AdipoR2) using immunohistochemistry in 82 thyroid carcinoma tissues and using RT-qPCR in 40 human thyroid carcinoma tissues (32 papillary, six follicular/Hurthle, one anaplastic, one medullary), four normal human thyroid tissue specimens, and the BHP7 and SW579 thyroid cancer cell lines. We then utilized these thyroid cancer cell lines to investigate whether adiponectin could directly regulate cell cycle or apoptosis. Results: Thyroid cancer patients had lower circulating adiponectin levels than controls (17.00 ⫾ 6.32 vs. 19.26 ⫾ 6.28 ␮g/ml; P ⬍ 0.001). Subjects in the highest tertile of circulating adiponectin concentrations had significantly lower odds of developing any type of thyroid carcinoma (odds ratio ⫽ 0.29; 95% confidence interval, 0.16 – 0.55), or papillary thyroid carcinoma (odds ratio ⫽ 0.27; 95% confidence interval, 0.14 – 0.55), before and after adjustment for potential confounders. Both thyroid carcinoma cell lines and tissues expressed AdipoR1 and AdipoR2. Recombinant adiponectin did not exert a clinically significant direct effect on cell cycle, proliferation, or apoptosis in thyroid cancer cell lines in vitro. Conclusions: Circulating adiponectin is independently and inversely associated with the risk of thyroid cancer. Human thyroid carcinomas and cell lines express adiponectin receptors. However, in the absence of a major direct effect of adiponectin on thyroid cancer cell lines in vitro, the negative association observed herein may be attributed to the metabolic effects of adiponectin. (J Clin Endocrinol Metab 96: E2023–E2028, 2011)

everal epidemiological studies have suggested that increased adiposity (especially central adiposity) is associated with increased incidence and/or mortality from several malignancies, such as colon, breast (in

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postmenopausal women), endometrium, renal cell, esophageal (adenocarcinoma), gastric, pancreatic, gallbladder, and liver cancer (1). Adiponectin (acrp30, adipoQ, apM1 gene product), a 30-kDa adipokine

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/jc.2010-1908 Received August 13, 2010. Accepted August 30, 2011. First Published Online September 20, 2011

* N.M., K.P.-P., K.N.A., and H.-S.M. contributed equally to this work. Abbreviations: ATC, Anaplastic thyroid carcinoma; CI, confidence interval; FTC, follicular thyroid carcinoma; IGFBP-3, IGF binding protein-3; MTC, medullary thyroid carcinoma; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetra-zolium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OR, odds ratio; PTC, papillary thyroid carcinoma; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

J Clin Endocrinol Metab, December 2011, 96(12):E2023–E2028

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Circulating Adiponectin in Thyroid Cancer

closely and inversely associated with insulin resistance, has been proposed to be a biological link between obesity and increased cancer risk (2). Adiponectin circulates in serum as trimers, hexamers, and high molecular weight forms. In healthy adults, it is among the most abundant proteins in serum, with levels up to 30 ␮g/ml (1 ␮M) in lean subjects (3, 4). Contrary to other adipokines, circulating adiponectin levels are decreased in obesity, insulin resistance, and type 2 diabetes (2). Adiponectin acts through two different cell surface adiponectin receptors, AdipoR1 and AdipoR2. Adiponectin stimulates phosphorylation and activation of AMP kinase in liver and skeletal muscle, thus suppressing gluconeogenesis in the liver and promoting fatty-acid oxidation and glucose uptake in muscle. This reduces glucose levels in vivo and improves insulin sensitivity. Obesity decreases expression levels of AdipoR1/R2, thereby reducing adiponectin sensitivity (5). In addition to its effects on glucose and lipid metabolism, adiponectin may also have antiatherogenic, antiinflammatory, antiangiogenic, and possibly anticancer properties. Circulating adiponectin concentrations are lower in patients with endometrial (6), prostate (7), colorectal (8), renal (9), and postmenopausal breast (10, 11) carcinomas than controls. The antineoplastic activity of adiponectin may be indirect, possibly by decreasing insulin resistance, hyperinsulinemia, and free IGF-I levels or by modulating neovascularization and inflammation. It is also possible that adiponectin can act on tumor cells directly (2). Several cancer cell types express adiponectin receptors that may mediate the direct effects of adiponectin on cellular proliferation (2, 12, 13). An association between obesity and increased thyroid cancer incidence has been reported in women (14). Conversely, patients with differentiated thyroid carcinoma have an increased prevalence of insulin resistance (15). However, the role of adiponectin and its receptors in the pathophysiology of thyroid carcinoma has not been investigated. In this study, we investigated the role of adiponectin in patients with thyroid carcinomas. We found that patients with thyroid carcinomas have decreased plasma adiponectin levels compared with controls. We observed using immunohistochemistry that both adiponectin receptors (AdipoR1 and AdipoR2) are expressed in human thyroid cancer specimens. We also confirmed the expression of both adiponectin receptors in thyroid cancer cell lines (BHP7 and SW579) and in human PTC tissue using RT-qPCR. Finally, we observed that although both cell lines express AdipoR1 and AdipoR2, treatment with recombinant adiponectin at low and high physiological concentrations had no major effect on cell proliferation or cell death of thyroid carcinoma cell lines in vitro.


Patients and Methods Measurement of circulating hormone levels Serum was available for analysis from 175 thyroid carcinoma patients and 107 controls who participated in this study. Detailed information on patient enrollment and hormone measurements is provided in the Supplemental Data (published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).

Tissues for immunostaining analysis A panel of 82 formalin-fixed, paraffin-embedded thyroid carcinoma tissue specimens, including 62 papillary thyroid carcinoma (PTC), 18 follicular thyroid carcinoma (FTC), one anaplastic thyroid carcinoma (ATC), and one medullary thyroid carcinoma (MTC), were obtained in the form of tissue array slides mounted to standard silanized slides (Imgenex, San Diego, CA).

Immunohistochemistry Detection of AdipoR1 and AdipoR2 in the above-described thyroid carcinoma specimens was performed and evaluated as described in the Supplemental Data.

Human thyroid cDNA material for RT-qPCR A panel of 40 normalized cDNA prepared from human thyroid carcinoma tissues, including 32 PTC, six FTC/Hurthle, one ATC, and one MTC, as well as four normal thyroid tissue specimens, was purchased from OriGene Technologies (Rockville, MD).

Relative expression of AdipoR1/R2 mRNA Adiponectin receptors were quantified using RT-qPCR with human-specific gene expression assays (Applied Biosystems Inc., La Jolla, CA) as previously described (11).

Cell line culture and treatments The SW579 and BHP7 thyroid carcinoma cell lines were cultured as described in detail in the Supplemental Data. Quantification of cell number after treatment with recombinant adiponectin [by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)/3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay], assessment of cell cycle (by propidium iodide labeling), and apoptosis [by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and Annexin V labeling], and immunoblotting analysis were performed as described in detail in the Supplemental Data.

Statistical analysis Statistical analysis of these data was performed using IBM PASW 18 with the regression add-on module (SPSS Inc. Chicago, IL). Descriptive characteristics of thyroid cancer cases and controls are presented as proportions or as mean ⫾ SD. Descriptive characteristics were compared between cases and controls using ␹2 tests and independent-samples t tests. Nonparametric Spearman correlation coefficients were calculated to examine linear trends between the variables and associations between hormones and anthropometric characteristics, to assess for potential confounding. Subjects were stratified by



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TABLE 1. Circulating adiponectin levels: crude and adjusted OR Adiponectin tertiled Control vs. all cancer subtypes

1st 2nd 3rd

Control vs. papillary 1st 2nd 3rd Control vs. follicular, medullary, and Hurthle Papillary vs. follicular, medullary, and Hurthle

1st 2nd 3rd 1st 2nd 3rd

Model 1a P trende ⬍ 0.01 1.00 0.54 (0.31– 0.96) 0.29 (0.16 – 0.55) P trende ⬍ 0.01 1.00 0.54 (0.29 –1.00) 0.27 (0.14 – 0.55) P trende ⫽ 0.02 1.00 0.54 (0.02–1.19) 0.35 (0.15– 0.9) P trende ⫽ 0.64 1.00 0.99 (0.46 –2.12) 1.31 (0.52–3.2)

P value

0.04 ⬍0.01 0.05 ⬍0.01 0.13 0.02 0.99 0.57

Model 2b P trende ⬍ 0.01 1.00 0.73 (0.37–1.43) 0.34 (0.165– 0.70) P trende ⬍ 0.01 1.00 0.745 (0.36 –1.53) 0.29 (0.13– 0.65) P trende ⫽ 0.09 1.00 0.72 (0.29 –1.73) 0.46 (0.17–1.24) P trende ⫽ 0.40 1.00 0.96 (0.41–2.21) 1.61 (0.58 – 4.39)

P value

0.35 ⬍0.01 0.42 ⬍0.01 0.46 0.13 0.93 0.35

Model 3c P trende ⬍ 0.01 1.00 0.82 (0.41–1.66) 0.33 (0.16 – 0.69) P trende ⬍ 0.01 1.00 0.74 (0.35–1.57) 0.26 (0.11– 0.61) P trende ⫽ 0.27 1.00 1.04 (0.41–2.65) 0.55 (0.19 –1.53) P trende ⫽ 0.21 1.00 0.14 (0.57–3.47) 2.09 (0.72– 6.03)

P value

0.57 ⬍0.01 0.43 ⬍0.01 0.93 0.25 0.45 0.17

Data are expressed as OR (95% CI). a

Unadjusted crude OR.

b

OR adjusted for TSH, free T4, age, gender, height, weight, diabetes, and smoking status.

c

OR adjusted for TSH, free T4, age, gender, height, weight, diabetes, and smoking status (model 2) plus IGF-I and IGFBP-3.

d

Adiponectin tertiles: 1st, ⬍17.99 ng/ml; 2nd, 17.99 –21.40 ng/ml; 3rd, ⱖ21.40 ng/ml.

e

P value for linear trend across the tertiles.

control-based tertiles of adiponectin, IGF-I, and IGF binding protein-3 (IGFBP-3) to investigate associations of the hormone levels with thyroid cancer risk. Simple and multivariate, stepwise, unconditional logistic regression models were used to produce crude and adjusted odds ratios (OR). The analyses of expression of adiponectin receptors were conducted by applying Fisher exact test because the size of controls was small, using SPSS version 11.5 (SPSS Inc.). A level of ␣ ⫽ 0.05 was set to determine statistical significance.

Results Circulating adiponectin levels are decreased in patients with thyroid carcinoma compared with controls Descriptive characteristics of study subjects with thyroid carcinoma and controls who provided sera are presented in Supplemental Table 1. Circulating adiponectin levels were lower in thyroid carcinoma patients compared with controls (17.00 ⫾ 6.32 vs. 19.26 ⫾ 6.28 ␮g/ml, respectively; P ⬍ 0.001). The Spearman’s correlations between hormone levels and anthropometric characteristics are shown in Supplemental Tables 2a (all subjects) and 2b (controls only). Subjects in the highest tertile of serum adiponectin concentration had significantly lower odds of developing any thyroid carcinoma [OR ⫽ 0.29; 95% confidence interval (CI), 0.16 – 0.55], PTC (OR ⫽ 0.27; 95% CI, 0.14 – 0.55), or follicular, Hurthle cell, or medullary carcinoma (collectively, OR ⫽ 0.35; 95% CI, 0.15– 0.9) compared with

subjects in the lowest tertile (Table 1). The significance of these associations persisted for the odds of developing any thyroid carcinoma, as well as for the PTC diagnosis after sequential covariate adjustment for potential confounders including free T4, TSH, age, gender, diabetes status, smoking status, BMI, height, and weight, whereas it disappeared for the FTC/Hurthle/MTC diagnosis. Supplemental Table 3 summarizes the OR for thyroid cancer by control-defined tertiles of IGF-I concentrations, with and without sequential covariate adjustment. Subjects in the highest tertile of IGF-I concentration presented with significantly higher odds of papillary cancer compared with those in the lowest tertile (OR ⫽ 2.25; 95% CI, 1.14 – 4.42). However, after adjustment for covariates, these associations were no longer statistically significant. OR for thyroid cancer by control-defined tertiles of IGFBP-3 concentrations are shown in Supplemental Table 4. We found no significant associations between IGFBP-3 and the odds of thyroid cancer, before and after adjustment for covariates. Presence of adiponectin receptor mRNA in thyroid carcinoma cell lines and human tissue specimens Both adiponectin receptor 1 and 2 mRNA were detected in the two cell lines studied (SW579 and BHP7) by RT-qPCR (Fig. 1A) and in human thyroid carcinoma tissues by RT-qPCR (Fig. 1B). Immunohistochemistry analysis revealed the presence of adiponectin receptors 1 and 2 on the surface of human thyroid carcinoma tissues (Sup-

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(Supplemental Fig. 3) and cell cycle analysis with propidium iodide and annexin 4 labeling (Supplemental Figs. 4 and 5) and found that adiponectin at 20 ␮g/ml does not induce cell death or cell cycle arrest in human thyroid cancer cell lines in vitro. We also performed immunoblotting analysis for p53 and p21 and observed that adiponectin does not modulate the expression levels of p53 and p21 when compared with controls in human thyroid cancer cell lines in vitro (data not shown).

Discussion Adiponectin is an adipokine inversely associated with obesity and insulin resistance, and it has been proposed to be FIG. 1. A and B, Presence of adiponectin receptor mRNA in thyroid carcinoma cell lines and a biological link between adiposity and human specimens. A, Adiponectin receptor 1 (black bars) and 2 (empty bars) mRNA were increased cancer risk (2). However, its detected in the two PTC cell lines studied (SW579 and BHP7) by RT-qPCR. Results were association with thyroid cancer has not normalized to 18S RNA and presented as relative expression (adipose tissue served as positive been studied to date. We now report control). B, Adiponectin receptor 1 (black bars) and 2 (empty bars) mRNA were detected in human thyroid carcinoma tissues (32 PTC, six FTC/Hurthle, one anaplastic, one medullary) and that patients with thyroid carcinoma four normal thyroid tissue specimens by RT-qPCR. Results were normalized to ␤-actin and (papillary thyroid carcinoma in parpresented as relative expression (average ⫾ SE). PTC expressed lower levels of both receptor ticular) have decreased circulating mRNA compared with normal tissue. *, P ⬍ 0.02 in PTC vs. control for both receptors, adjusted for age and gender. C and D, Effect of recombinant adiponectin on human thyroid adiponectin levels. Additionally, we carcinoma cell proliferation in vitro. BHP7 (C) and SW579 (D) thyroid carcinoma cells were demonstrated with both immunohistocultured as described in detail in the Supplemental Data. The cells were treated with chemistry and RT-qPCR that both adiadiponectin (10, 20, or 50 ␮g/ml) for 72 h, and cell proliferation was measured by MTS assay as described in detail in the Supplemental Data. All data were analyzed using one-way ponectin receptors are expressed in huANOVA followed by post hoc test for multiple comparisons. Values represent average man thyroid cancer tissues. However, (n ⫽ 3) ⫾ SD. recombinant adiponectin did not have any major or clinically significant effect plemental Fig. 1). PTC expressed lower levels of AdipoR1 on cell proliferation or apoptosis in thyroid carcinoma cell and AdipoR2 mRNA than control thyroid tissue (P ⬍ 0.02 lines in vitro. for both, adjusted for age and gender). The results of this case-control study demonstrate an inverse association of adiponectin with the risk of develEffect of recombinant adiponectin on human oping thyroid carcinoma, and particularly PTC, and are in thyroid carcinoma cell cycle and apoptosis in vitro agreement with prior studies in breast, endometrial, coloWe assessed the impact of recombinant adiponectin on rectal, gastric, and renal carcinomas, multiple myeloma, human thyroid carcinoma cell proliferation and survival. and myelodysplastic syndrome (2, 6, 8 –10, 16 –19). They Using the MTT and MTS assays, we demonstrated that are also in agreement with a recent study of nondiabetic treatment of BHP7 and SW579 cells with adiponectin at low (10 and 20 ␮g/ml) and high (50 ␮g/ml) physiological peritoneal dialysis patients, where plasma adiponectin levdoses had no significant effect on cell proliferation (Fig. 1, els were significantly lower in patients that developed any C and D; results from experiments performed at Beth Is- malignancy, with colon cancer being the most common rael Deaconess Medical Center). Further independent ex- site, followed by thyroid carcinoma (20). In healthy periments performed in parallel at Baylor College of Med- adults, adiponectin is one of the most abundant proteins icine confirmed the absence of a clinically meaningful in serum (3, 4), and in our study, adiponectin levels in decrease in proliferation upon treatment of thyroid cancer control subjects were 19.26 ⫾ 6.28 ␮g/ml. Contrary to cells with adiponectin (Supplemental Fig. 2). To confirm other adipokines, circulating adiponectin levels are deand extend these results, we performed TUNEL staining creased in obesity, insulin resistance, and type 2 diabetes


(2). Circulating adiponectin levels are inversely related to BMI and visceral fat accumulation, and this was confirmed in our study. Interestingly, the strength of the association between adiponectin levels and thyroid cancer was somewhat attenuated after adjusting for TSH, diabetes status, smoking status, BMI, height, weight, age, free T4, and gender, suggesting that metabolic parameters (possibly related to BMI) mediate at least a part of the association between adiponectin levels and risk of thyroid cancer (2). We also demonstrated that subjects in the highest tertile of IGF-I concentrations had a statistically significantly higher OR of PTC compared with subjects in the first tertile, yet the statistical significance of this association was completely lost after adjustment for covariates, including BMI. These data suggest that the association between the IGF-I axis and risk of thyroid carcinoma is probably completely mediated by other metabolic covariates. Hyperinsulinemia or abnormal lipid metabolism, both of which are associated with hyperadiponectinemia (4), may also contribute to thyroid cancer (21). Adiponectin has been reported to have direct anticancer effects in several malignant cell lines (2). Adiponectin, particularly the high molecular weight forms, inhibits prostate cancer cell growth; suppresses leptin-, IGF-I-, and dihydrotestosterone-stimulated prostate carcinoma cell growth; and enhances the anticancer activity of doxorubicin in vitro (3, 12). In addition, adiponectin treatment has resulted in a significant dose-dependent growth inhibition of breast cancer cells in vitro (13, 22). However, we observed that treatment with recombinant adiponectin, at low and high physiological concentrations, does not have any major growth-suppressive effect on thyroid cancer cell lines in vitro. We also confirmed using TUNEL and flow cytometry analysis with propidium iodide and, separately, Annexin V labeling, that adiponectin does not directly induce apoptosis or cell cycle arrest in human thyroid cancer cell lines in vitro. In the absence of a clinically significant suppressive growth effect of recombinant adiponectin on thyroid cancer cell lines in vitro, we propose that the observed negative association in humans may be mainly due to indirect effects of adiponectin through regulation of insulin sensitivity and metabolism. Limitations of this study include the fact that patient sera were collected after thyroidectomy and therefore reflect the metabolic status of the patients in the absence of tumor-host interactions. Future studies of prospective cohorts should assess the levels of adiponectin before and after thyroidectomy, and more in depth mechanistic studies are needed to address causality. Furthermore, due to the small number of nonpapillary histologies in our present study, additional future studies will be necessary to


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address the role of the adiponectin axis in the entire spectrum of thyroid carcinomas. We have used state-of-the-art methodology, but random error in responses to questionnaires and hormonal measurements cannot be excluded. In any case, such errors would have probably resulted in depressed effect estimates and thus would have attenuated statistical significance. We have adjusted for known confounding factors, but unknown confounding factors may have remained. Although prior studies have reported variable and conflicting effects of the thyroid axis on adiponectin levels (23–25), we cannot attribute the difference in adiponectin levels between cancer patients and controls in our study to any thyroid hormone effect because both groups had TSH, free T4, and free T3 values in the normal range and we adjusted for these factors as well in our analysis. Finally, a recent report indicates that low adiponectin is associated with thyroid cancer in patients with renal failure (20). In summary, we report a significant inverse association between circulating adiponectin levels and risk of thyroid carcinoma, and particularly PTC. Although human thyroid tumor tissues express adiponectin receptors, recombinant adiponectin did not have any substantial effect on cell proliferation or survival in thyroid cancer cell lines in vitro. Our results suggest that the observed inverse association between circulating adiponectin levels and risk of thyroid cancer in humans could be attributed to indirect effects of adiponectin, possibly through regulation of metabolism and insulin resistance.

Acknowledgments We thank Dr. Jerome M. Hershman (West Los Angeles VA Medical Center, Los Angeles, CA) for the generous gift of the BHP7 papillary cancer cell line. Address all correspondence and requests for reprints to: Christos S. Mantzoros, M.D., D.Sc., Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, FD 875, Boston, Massachusetts 02215. E-mail: [email protected]. This work was supported by a discretionary grant from Beth Israel Deaconess Medical Center (to C.S.M.). The Mantzoros Lab is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (Grants DK058785, DK079929, and DK081913), the National Institute on Aging (Grant AG032030), and a discretionary grant from Beth Israel Deaconess Medical Center. Current affiliation for N.M.: Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030 Disclosure Summary: The authors have nothing to disclose.

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