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

描述（由申请人提供）：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不是很明确。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的失调，所有这些都与人类黑色素瘤的进展有关。然而，转移性黑色素瘤细胞中AP-2a表达缺失的机制尚不清楚。利用黑色素瘤组织微阵列结合LSC和AQUA技术，我们进一步验证了转移性黑色素瘤细胞中核AP-2a表达的缺失。为了寻找AP-2a调控的其他靶基因，我们确定了凝血酶受体（PAR-1），并提供了转移性黑色素瘤细胞中AP-2a和PAR-1表达负相关的证据。AP-2a在体外和体内对PAR-1的调节已被证实，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。通过这项持续的研究，PAR-1在人类黑色素瘤进展中的作用现在已经确定。我们利用慢病毒shRNA结合cDNA芯片阵列和细胞因子膜阵列稳定沉默PAR-1来鉴定凝血酶/PAR-1轴可能调控的下游基因。这些基因包括MCAM/MUC18、Connexin 43、Maspin、RUNX3、IL-8、Ang-2、GRO、ACRP30和gitr -配体。本提案需要验证的假设是，核AP-2a的缺失导致PAR-1的上调，并有助于人类黑色素瘤恶性表型的获得。为了验证这一假设，我们现在提出：1)确定黑色素瘤进展过程中核AP-2a表达缺失的机制；2)探讨凝血酶/PAR-1轴对转移表型的影响；3)通过递送中性脂质体纳米颗粒包装的siRNA来抑制PAR-1在体内的表达，这可能是一种新的治疗方式。预计本研究的结果将更好地了解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模式。公共卫生相关性：与人类黑色素瘤从放射状生长阶段进展到垂直生长阶段（转移表型）相关的分子变化在很大程度上是未知的。之前，我们发现转录因子AP-2a的缺失与这种转变有关。在本次拨款申请中，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.