Adiponectin effects on leptin signaling in hepatocellular carcinoma

描述（由申请人提供）： 肥胖是肝细胞癌（HCC）的独立危险因素。肥胖患者表现出更大的肿瘤负荷、增加的转移和不良预后的更高风险。流行病学研究已经建立了肥胖和HCC之间的联系。这种相关性可能代表肥胖患者的基础非酒精性脂肪性肝病（NAFLD）进展为肝硬化和肝细胞癌，尽管没有研究明确将这些变量联系起来。肥胖通过两种主要的脂肪细胞因子瘦素和脂联素介导的自分泌和旁分泌作用影响肝癌的发生。我们最近研究瘦素的致癌作用的研究显示：i）瘦素通过Stat 3、Akt和ERK激活诱导增殖，ii）瘦素诱导的ERK和Akt磷酸化依赖于Stat 3激活，iii）瘦素诱导HCC细胞的侵袭和迁移。尽管有相反的作用，脂联素减少HCC细胞的侵袭和迁移。脂联素以LKB 1依赖的方式激活AMPK，并抑制S6 K激活，表明LKB 1-AMPK-S6 K轴参与其中。最重要的是，脂联素治疗阻断瘦素诱导的i）Stat 3的活化，ii）细胞周期蛋白D1的过表达，和iii）细胞增殖。这些数据有力地表明，脂联素拮抗瘦素对肝癌细胞的促癌作用。脂联素抑制瘦素信号传导的分子机制将通过基于电细胞基质阻抗传感（ECIS）的侵袭和迁移测定来分析。将使用免疫荧光、co-IP、蛋白质印迹分析和激酶测定来研究脂联素对瘦素信号传导的各种调节步骤的影响。为了确定脂联素对瘦素信号传导的抑制是否涉及LKB 1-AMPK轴，我们将使用特异性化学抑制剂、siRNA和过表达、显性负性和/或激酶缺陷构建体靶向LKB 1-AMPK轴，并确定脂联素对瘦素的拮抗作用是否通过该途径介导。为了确定脂联素是否阻断瘦素诱导的HCC肿瘤生长和转移，将在无胸腺裸鼠中进行HCC肿瘤发生测定。将通过microPET成像监测肿瘤生长和转移。瘦素和脂联素的关键信号分子将在肿瘤和转移样本中进行检查。我们将全面分析瘦素和脂联素信号传导的关键分子在不同肝癌组中的表达。 与公众的相关性：肥胖是美国的一个主要健康问题，是肝细胞癌的一个重要危险因素，其内分泌效应是由脂肪细胞因子介导的。本研究旨在建立瘦素和脂联素作为HCC新的生物标志物。此外，我们将阐明脂联素对瘦素信号转导在肝细胞癌中的保护作用，从而提出脂联素作为肝细胞癌患者新的预防和治疗选择的作用。 公共卫生相关性：肥胖是肝细胞癌（HCC）的独立危险因素。肥胖患者表现出更大的肿瘤负荷、增加的转移和不良预后的更高风险。流行病学研究已经建立了肥胖和HCC之间的联系。我们将分析脂联素如何拮抗瘦素对肝癌细胞的促癌作用。

DESCRIPTION (provided by applicant): Obesity is an independent risk factor for hepatocellular carcinoma (HCC). Obese patients exhibit a higher risk for larger tumor burden, increased metastasis and poor prognosis. Epidemiological studies have established a link between obesity and HCC. This association might represent the progression of underlying nonalcoholic fatty liver disease (NAFLD) to cirrhosis and hepatocellular carcinoma in obese patients although no study has clearly linked these variables. Obesity affects hepatocellular carcinogenesis by autocrine and paracrine actions mediated by two major adipocytokines, leptin and adiponectin. Our recent studies investigating the oncogenic actions of leptin revealed that - i) leptin induces proliferation via Stat3, Akt and ERK activation, ii) leptin-induced phosphorylation of ERK and Akt was dependent on Stat3 activation, and iii) leptin induces invasion and migration of HCC cells. Displaying opposing effects, adiponectin reduces invasion and migration of HCC cells. Adiponectin activates AMPK in an LKB1-dependent manner, and inhibits S6K activation demonstrating the involvement of LKB1-AMPK-S6K axis. Most importantly, adiponectin treatment blocks leptin-induced i) activation of Stat3, ii) overexpression of cyclin D1, and iii) cell proliferation. These data strongly suggest that adiponectin antagonizes the cancer-promoting effects of leptin on hepatocellular carcinoma cells. The molecular mechanism by which adiponectin inhibits leptin signaling will be analyzed by Electric Cell-substrate Impedance Sensing (ECIS) based invasion and migration assays. The effect of adiponectin on various regulatory steps of leptin signaling will be studied using immunofluorescence, co-IP, western blot analysis and kinase assays. To determine whether suppression of leptin signaling by adiponectin involves LKB1-AMPK axis, we will use specific chemical inhibitors, siRNA and overexpression, dominant negative and/or kinase deficient constructs to target LKB1-AMPK axis, and determine if the antagonistic effects of adiponectin against leptin are mediated through this pathway. To determine whether adiponectin blocks leptin-induced HCC tumor growth and metastasis, HCC tumorigenesis assays in athymic nude mice will be conducted. Tumor growth and metastasis will be monitored by microPET imaging. Key signaling molecules of leptin and adiponectin will be examined in tumor and metastasis samples. We will analyze expression of the key molecules of leptin and adiponectin signaling in different groups of HCC in a comprehensive manner. Relevance to public: Obesity, a major health problem in US is a significant risk factor for hepatocellular carcinoma and its endocrine effects are mediated by adipocytokines. The studies proposed here are designed to establish leptin and adiponectin as novel biomarkers for HCC. Also we will delineate the protective role of adiponectin against leptin signaling in hepatocellular carcinoma which presents role of adiponectin as new preventive and therapeutic option for HCC patients. PUBLIC HEALTH RELEVANCE: Obesity is an independent risk factor for hepatocellular carcinoma (HCC). Obese patients exhibit a higher risk for larger tumor burden, increased metastasis and poor prognosis. Epidemiological studies have established a link between obesity and HCC. We will analyze how adiponectin antagonizes the cancer-promoting effects of leptin on hepatocellular carcinoma cells.