Malignant melanoma: Regulation by AP-2 and PAR-1

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)的转变以及转移表型的获得相关的分子变化还不是很清楚。该实验室最近的工作表明，这种转变与转移性黑色素瘤细胞中AP-2a核表达的丧失有关，从而导致c-kit、MCAM/MUC18、MMP-2和VEGF的失控，所有这些都参与了人类黑色素瘤的发展。然而，AP-2a在转移性黑色素瘤细胞中缺乏表达的机制尚不清楚。利用黑色素瘤组织微阵列结合LSC和AQUA技术，我们进一步证实了在转移性黑色素瘤细胞中缺乏核AP-2a的表达。为了寻找AP-2a调控的其他靶基因，我们鉴定了凝血酶受体(PAR-1)，并为AP-2a和PAR-1在转移性黑色素瘤细胞中的表达负相关提供了证据。AP-2a对PAR-1的调节在体外和体内都得到了证实，从而在凝血系统和人类黑色素瘤的进展之间提供了独特的联系。通过这项持续研究的努力，PAR-1在人类黑色素瘤进展中的作用现在得到了证实。我们利用慢病毒shRNA结合基因芯片阵列和细胞因子膜阵列稳定沉默PAR-1，以寻找可能受凝血酶/PAR-1轴调控的下游基因。这些基因包括MCAM/MUC18、连接蛋白43、Maspin、RUNX3、IL-8、Ang-2、Gro、ACRP30和GITR配体。在这项建议中要检验的假设是，核AP-2a的丢失导致PAR-1的上调，并有助于获得人类黑色素瘤的恶性表型。为了验证这一假说，我们现在建议：1)确定在黑色素瘤进展过程中核AP-2a表达缺失的机制(S)；2)研究凝血酶/PAR-1轴在黑色素瘤转移表型中的作用；以及3)通过传递包装在中性脂质体纳米粒中的小干扰RNA来抑制体内PAR-1的表达，作为一种可能的新的治疗方式。预期这项研究的结果将有助于更好地了解AP-2a和PAR-1在人类黑色素瘤进展中的作用，从而可能导致抑制黑色素瘤转移的新方法。公共卫生相关性：与人类黑色素瘤从径向生长阶段进入垂直生长阶段(转移表型)相关的分子变化在很大程度上是未知的。此前，我们发现转录因子AP-2a的缺失与这一转变有关。在这项拨款申请中，我们将继续研究AP-2a及其下游靶基因凝血酶受体(PAR-1)在人类黑色素瘤进展中的作用。

DESCRIPTION (provided by applicant): The molecular changes associated with the transition of melanoma cells from Radial Growth Phase (RGP) to Vertical Growth Phase (VGP) and the acquisition of the metastatic phenotype is not very well-defined. Recent work from this laboratory demonstrated that this transition is associated with loss of nuclear expression of AP-2a in metastatic melanoma cells which resulted in deregulation of c-KIT, MCAM/MUC18, MMP-2, and VEGF, all of which are involved in the progression of human melanoma. However, the mechanisms for the lack of AP-2a expression in metastatic melanoma cells are not known. Using melanoma tissue microarrays combined with LSC and AQUA technologies, here, we further validated the lack of nuclear AP-2a expression in metastatic melanoma cells. In an effort to search for other target genes regulated by AP-2a, we have identified the thrombin receptor (PAR-1) and provided evidence for inverse correlation between AP-2a and PAR-1 expression in metastatic melanoma cells. Regulation of PAR-1 by AP-2a was demonstrated in vitro and in vivo, thus providing a unique link between the coagulation system and the progression of human melanoma. The role of PAR-1 in the progression of human melanoma is now established through the efforts of this continued research. We have used stable silencing of PAR-1 by lentiviral shRNA combined with cDNA chip arrays and cytokine membrane arrays to identify possible downstream genes regulated by the axis of thrombin/PAR-1. These genes include MCAM/MUC18, Connexin 43, Maspin, RUNX3, IL-8, Ang-2, GRO, ACRP30, and GITR-ligand. The hypothesis to be tested in this proposal is that loss of nuclear AP-2a results in upregulation of PAR-1 and contributes to the acquisition of the malignant phenotype in human melanoma. To test this hypothesis, we now propose: 1) To determine the mechanism(s) for the loss of nuclear AP-2a expression during melanoma progression; 2) To investigate how the axis of thrombin/PAR-1 contributes to the metastatic phenotype; and 3) To inhibit PAR-1 expression in vivo via delivery of siRNA packaged in neutral liposome nanoparticles as a possible new therapeutic modality. It is expected that the results obtained from this study will provide a better understanding of the role of AP-2a and PAR-1 in the progression of human melanoma that could thereby lead to new modalities to inhibit melanoma metastasis. PUBLIC HEALTH RELEVANCE: The molecular changes that are associated with the progression of human melanoma from radial growth phase into a vertical growth phase (metastatic phenotype) are largely unknown. Previously, we identified that the loss of the transcription factor AP-2a is associated with this transition. In this grant application, we will continue to investigate the role of AP-2a and its downstream target gene, the thrombin receptor (PAR-1), in the progression of human melanoma.