Thyroid Stimulating Hormone Promotes the Growth and Progression of Human Melanoma

描述（由申请人提供）：我们报告了黑色素瘤人群中甲状腺功能减退症的高患病率，并假设甲状腺功能减退个体中高浓度循环的促甲状腺激素（TSH）促进黑色素瘤生长。这一概念得到了初步数据的支持，这些数据显示黑色素瘤细胞表达功能性TSH受体（TSHR），并且TSH诱导培养的黑色素瘤细胞增殖。本提案中描述的研究将测试以下假设：TSH通过其在人黑色素瘤细胞上的受体发出信号促进肿瘤生长和进展;并且这种促有丝分裂过程通过TSH的循环水平升高和黑色素瘤TSHR的过度表达而增强。有三个具体目标。具体目标1使用培养的黑素瘤细胞的饱和放射性配体结合实验来检查黑素瘤TSHR的密度和亲和力，并将结果与甲状腺细胞进行比较。如果发现高密度的黑色素瘤TSHR，将通过荧光原位杂交（FISH）检查细胞的TSHR基因扩增。还将进行实验以确定黑色素瘤TSHR是否激活甲状腺细胞TSHR信号传导的三种典型途径。这些途径，其中包括蛋白激酶A（PKA），促分裂原活化蛋白激酶（MAPK），和磷脂酰肌醇3-激酶（PI 3 K）级联，将通过蛋白质印迹法检测磷蛋白和信号蛋白易位响应TSH。具体目标2包括对150名已切除原发性肿瘤的高风险II期黑色素瘤患者的临床研究。这些患者有30%的复发率，常规进行前哨淋巴结活检（SNB）以检测早期淋巴转移。将在SNB之前测量研究患者的TSH水平，预测肿瘤阳性前哨淋巴结患者的TSH水平将高于阴性淋巴结患者。在特定目标3中，来自目标2研究队列的石蜡包埋的原发性肿瘤将用于制备组织微阵列。将通过TSHR密度的免疫染色来检查阵列的切片，并且发现将与疾病进展相关。与目标1相似，如果在黑素瘤组织中发现高密度TSHR，则将使用FISH来确定TSHR基因扩增的存在。如果我们的假设被证明是正确的，这项研究将开辟一个全新的、完全未探索的领域，涉及促甲状腺激素促进黑色素瘤生长，对黑色素瘤患者甲状腺功能减退症的筛查以及对可疑皮肤病变的积极治疗具有广泛的影响甲状腺功能减退症个体。此外，黑色素瘤TSHR可能最终被证明是一个直接的治疗靶点，或被利用作为一种手段，提供抗体或抗体复合物的药物。 这个项目研究的生物学机制，以解释与甲状腺功能减退症的黑色素瘤。用于治疗甲状腺功能减退症的药物实际上可能对黑色素瘤有治疗作用。此外，黑色素瘤病例可以通过加强对有甲状腺功能减退症病史的个体的监测来预防。

DESCRIPTION (provided by applicant): We have reported a high prevalence of hypothyroidism within the melanoma population and hypothesize that thyroid stimulating hormone (TSH), which circulates at high concentrations in hypothyroid individuals, promotes melanoma growth. This concept is supported by preliminary data showing that melanoma cells express functional TSH receptors (TSHR) and that TSH induces proliferation of cultured melanoma cells. The research described in this proposal will test the hypothesis that signaling of TSH through its receptor on human melanoma cells promotes tumor growth and progression; and that this mitogenic process is enhanced by elevated circulating levels of TSH and over-expression of melanoma TSHR. There are three Specific Aims. Specific Aim 1 uses saturation radioligand binding experiments with cultured melanoma cells to examine the density and affinity of melanoma TSHR, and compare the findings to thyrocytes. If a high density of melanoma TSHR is found, the cells will be examined for amplification of the TSHR gene by fluorescent in situ hybridization (FISH). Experiments will also be conducted determine if melanoma TSHR activate three pathways typical of thyrocyte TSHR signaling. These pathways, which include the protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) cascades, will be examined by western blotting for phosphoproteins and signal protein translocation in response to TSH. Specific Aim 2 encompasses a clinical study of 150 high risk Stage II melanoma patients who have had their primary tumors excised. These patients have a 30% rate of relapse and routinely undergo sentinel node biopsy (SNB) to detect early lymphatic metastases. TSH levels will be measured in the study patients prior to SNB, with the prediction that patients with tumor-positive sentinel nodes will have higher TSH levels than those with negative nodes. In Specific Aim 3, paraffin embedded primary tumors from the Aim 2 study cohort will be used to prepare a tissue microarray. Sections of the array will be examined by immunostaining for TSHR density, and findings will be correlated with disease progression. Similar to Aim 1, if a high density of TSHR are found in the melanoma tissues, FISH will be used to determine the presence of TSHR gene amplification. If our hypothesis is proven to be correct, this research will open a new and completely unexplored area involving the promotion of melanoma growth by TSH, with wide implications for screening of melanoma patients for hypothyroidism, and for aggressive management of suspicious cutaneous lesions in hypothyroid individuals. Moreover, melanoma TSHR may ultimately prove to be a direct therapeutic target, or be exploited as a means of delivering antibody- or hormone-complexed drugs. This project examines the biologic mechanisms to account for the association of melanoma with hypothyroidism. Medications used to treat hypothyroidism may actually be therapeutic for melanoma. Furthermore, melanoma cases may be prevented by heightened surveillance of individuals with a history of hypothyroidism.