Thyroid Stimulating Hormone Promotes the Growth and Progression of Human Melanoma

描述（由申请人提供）：我们报道了黑色素瘤人群中甲状腺功能减退的高发率，并假设促甲状腺激素（TSH）在甲状腺功能减退个体中高浓度循环，促进黑色素瘤的生长。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）， TSH诱导培养的黑色素瘤细胞增殖。本提案中描述的研究将验证TSH通过其受体在人类黑色素瘤细胞上的信号传导促进肿瘤生长和进展的假设；这种有丝分裂过程会因循环中TSH水平的升高和黑色素瘤TSHR的过度表达而增强。有三个具体目标。特异性目的1采用培养黑色素瘤细胞的饱和放射配体结合实验来检测黑色素瘤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）检查细胞是否扩增TSHR基因。实验还将确定黑色素瘤TSHR是否激活了甲状腺细胞TSHR信号的三个典型途径。这些途径，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，将通过western blotting检测响应TSH的磷酸化蛋白和信号蛋白易位。Specific Aim 2包含了150例原发肿瘤切除的高风险II期黑色素瘤患者的临床研究。这些患者复发率为30%，常规接受前哨淋巴结活检（SNB）以检测早期淋巴转移。在接受SNB治疗之前，将测量研究患者的TSH水平，预测肿瘤前哨淋巴结阳性患者的TSH水平将高于淋巴结阴性患者。在Specific Aim 3中，来自Aim 2研究队列的石蜡包埋原发肿瘤将用于制备组织微阵列。切片将通过免疫染色检查TSHR密度，结果将与疾病进展相关。与Aim 1类似，如果在黑色素瘤组织中发现高密度的TSHR，则将使用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新的和完全未开发的领域，涉及TSH促进黑色素瘤的生长，对黑色素瘤患者甲状腺功能减退的筛查和甲状腺功能减退患者可疑皮肤病变的积极治疗具有广泛的意义。此外，黑色素瘤TSHR可能最终被证明是一个直接的治疗靶点，或者被利用作为递送抗体或激素复合物药物的手段。

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.