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，并假设促甲状腺激素(TSH)在甲状腺功能减退症患者中以高浓度循环，促进黑色素瘤的生长。这一概念得到了黑色素瘤细胞表达功能性TSH受体(TSHR)以及TSH诱导培养的黑色素瘤细胞增殖的初步数据的支持。这项建议中描述的研究将检验这样一种假设，即TSH通过其受体在人类黑色素瘤细胞上的信号传递促进了肿瘤的生长和进展；这一有丝分裂过程是通过循环中TSH水平的升高和黑色素瘤TSHR的过度表达来增强的。有三个具体目标。特定目标1利用培养的黑色素瘤细胞进行饱和放射配基结合实验，以检测黑色素瘤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TSHR基因的扩增情况。还将进行实验，以确定黑色素瘤TSHR是否激活甲状腺细胞TSHR信号的三条典型通路。这些通路包括蛋白激酶A(PKA)、丝裂原活化蛋白激酶(MAPK)和磷脂酰肌醇3-激酶(PI3K)级联通路，将通过蛋白质印迹法检测对TSH的反应中的磷酸蛋白和信号蛋白转位。具体目标2包括一项对150名已切除原发肿瘤的高危II期黑色素瘤患者的临床研究。这些患者有30%的复发率，并常规接受前哨淋巴结活检(SNB)以检测早期淋巴转移。TSH水平将在SNB之前在研究患者中进行测量，预测有肿瘤前哨淋巴结阳性的患者的TSH水平将高于那些肿瘤前哨淋巴结阴性的患者。在特定的目标3中，来自目标2研究队列的石蜡包埋的原发肿瘤将用于制备组织微阵列。阵列的部分将通过TSHR密度的免疫染色进行检查，结果将与疾病进展相关。与目标1类似，如果在黑色素瘤组织中发现高密度的TSHR，将使用FISH来确定是否存在TSHR基因扩增。如果我们的假设被证明是正确的，这项研究将开辟一个涉及TSH促进黑色素瘤生长的新的、完全未知的领域，对筛查黑色素瘤患者的甲状腺功能减退以及积极处理甲状腺功能减退患者的可疑皮肤损害具有广泛的意义。此外，黑色素瘤TSHR最终可能被证明是一个直接的治疗靶点，或者被用作传递抗体或激素复合药物的一种手段。 这个项目研究了黑色素瘤与甲状腺功能减退之间的联系的生物学机制。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病史的个人的监测来预防。

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.