Malignant Melanoma: Regulation by AP-2 and PAR-2

描述(申请人提供)：与黑色素瘤细胞从径向生长期(RGP)到垂直生长期(VGP，转移表型)的转变相关的分子变化还不是很清楚。该实验室最近的工作表明，这种转变与AP-2转录因子的表达缺失有关。我们发现，在转移性黑色素瘤细胞中，AP-2的表达缺失导致c-kit和MCAM/MUC18基因的失控，这两个基因都参与了人类黑色素瘤的发展。此外，W.T.的失活。通过显性负性AP-2(AP-2B基因)将AP-2导入原代皮肤黑色素瘤细胞中，由于上调了基质金属蛋白酶-2的表达，导致其体内致瘤性和转移潜能增加。在晚期原发黑色素瘤患者中也观察到AP-2的表达缺失，这表明AP-2的缺失是人类黑色素瘤进展过程中的一个关键事件。为了确定AP-2调控的其他靶基因，我们提供了在转移性黑色素瘤细胞中AP-2的表达与凝血酶受体(PAR-1)之间负相关的证据。AP-2在体外和体内对黑色素瘤细胞中PAR-1的调节被证实，从而在凝血系统和人类黑色素瘤进展之间提供了独特的联系。近年来的研究表明，凝血酶受体在黑色素瘤的转移中起重要作用。PAR-1是一种独特的G偶联蛋白受体，属于蛋白水解酶激活受体家族。PAR-1的激活可导致参与黏附(整合素)、侵袭(MMPs)和血管生成(IL-8、VEGF、uPA、PDGF和bFGF)的基因产物上调。在这项建议中要检验的假设是，AP-2的缺失导致PAR-1的上调，这与人类黑色素瘤的恶性表型相关。为了验证这一假说，我们现在建议：1)确定AP-2如何调节PAR-1的表达；2)分析AP-2和PAR-1在黑色素瘤患者肿瘤标本中的表达；3)研究PAR-1的激活如何与人类黑色素瘤的转移表型有关。了解PAR-1被激活的机制可能会导致抑制黑色素瘤侵袭和转移的新技术。

DESCRIPTION (provided by applicant): The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP, metastatic phenotype) are not very well-defined. Recent work from this laboratory demonstrated that this transition is associated with loss of expression of the AP-2 transcription factor. We showed that lack of expression of AP-2 in metastatic melanoma cells resulted in deregulation of the c-KIT and MCAM/MUC18 genes, both of which are involved in the progression of human melanoma. Moreover, inactivation of w.t. AP-2 in primary cutaneous melanoma cells by dominant-negative AP-2 (AP-2B gene) resulted in an increase in their tumorigenicity and metastatic potential in vivo due to upregulation of MMP-2. Loss of AP-2 expression was also observed in advanced primary of melanoma patients indicating that loss of AP-2 is a crucial event in the progression of human melanoma. In an effort to identify other target genes regulated by AP-2, here we provide evidence of an inverse correlation between the expression of AP-2 and the thrombin receptor (PAR-1) in metastatic melanoma cells. Regulation of PAR-1 by AP-2 in melanoma cells is demonstrated in vitro and in vivo, thus providing a unique link between the coagulation system and the progression of human melanoma. Recent evidence suggests that thrombin receptor plays an important role in metastasis of human melanoma. PAR-1 is a unique G-coupled protein receptor that belongs to the protease activated receptor family. Activation of PAR-1 can result in upregulation of gene products involved in adhesion (integrins), invasion (MMP's) and angiogenesis (IL-8, VEGF, uPA, PDGF and bFGF). The hypothesis to be tested in this proposal is that loss of AP-2 results in upregulation of PAR-1 which correlates with the malignant phenotype of human melanoma. To test this hypothesis we now propose to: 1) determine how AP-2 regulates PAR-1 expression; 2) to analyze AP-2 and PAR-1 expression in tumor specimens from melanoma patients and; 3) to study how activation of PAR-1 contributes to the metastatic phenotype in human melanoma. Understanding the mechanisms by which PAR-1 is activated may lead to new techniques to inhibit melanoma invasion and metastasis.