Cyclin D as a Retinoid Molecular Target

描述（由申请人提供）：本修订后的新NIH R01申请评估了cyclin D1作为生殖细胞肿瘤（GCT）生长、成熟和化疗反应的分子靶点。全反式维甲酸（RA）在人GCT细胞系NT2/D1中诱导神经元和非致瘤状态。我们发现视黄酸受体（RARgamma）在NT2/D1细胞中调节RA的生长和分化，并赋予RA对NT2/D1- r1细胞的抗性。gct对研究肿瘤分化机制有重要意义。gct的一个独特的临床特征是畸胎瘤的形成，反映了它们成熟的能力。在gct中，分化与致瘤性呈反比关系。为探讨gct的抗肿瘤机制，研究了NT2/D1和NT2/D1- r1细胞。NT2/D1分化再现了畸胎瘤的关键特征，即ra治疗使这些细胞成熟并降低致瘤性。这种反应的一个标志是G1阻滞，至少部分是通过先前未被识别的途径，类维甲酸触发细胞周期蛋白D1蛋白水解。这部分是通过蛋白酶体降解发生的。RA对细胞周期蛋白D1的影响是特异性的，因为细胞周期蛋白D2和D3不受抑制。NT2/D1- r1细胞cyclin D1表达失调。rgamma转染恢复了NT2/D1- r1细胞的类视黄醇反应和cyclin D1抑制。苏氨酸286通过RA调节cyclin D1泛素化和蛋白酶体降解。另一种关系是NT2/D1- r1细胞解除了细胞周期蛋白D1的调控，获得顺铂耐药，表明成熟与化疗反应之间存在联系。转录后和转录类维甲酸机制抑制cyclin D1。这些将被全面研究，并与其他cyclin D1靶向药物进行比较。这与联合治疗有关。有必要揭示细胞周期蛋白D1在GCT生物学中的作用，这些具体目标是：(1)阐明在类视黄醇治疗GCT期间抑制细胞周期蛋白D1的蛋白水解和转录机制，并了解其他细胞周期蛋白D1靶向剂是否参与不同或重叠的机制；(2)验证GCT生长、成熟或顺铂反应是否需要cyclin D1抑制；(3)通过评估cyclin D1作为成熟、未成熟和耐化疗gct的独特组织库中的靶标，将研究结果扩展到临床背景。这些资源结合药理学、细胞和分子生物学方法，将为cyclin D1作为低风险gct的新靶点提供见解。

DESCRIPTION (provided by applicant): This revised new NIH R01 application evaluates cyclin D1 as a molecular target for germ cell tumor (GCT) growth, maturation, and chemotherapy response. All-trans-retinoic acid (RA) induced a neuronal and non-tumorigenic state in the human GCT cell line, NT2/D1. We uncovered retinoic acid receptor (RARgamma) as regulating RA growth and differentiation in NT2/D1 cells and conferring RA resistance to NT2/D1-R1 cells. GCTs are useful to study tumor differentiation mechanisms. A unique clinical feature of GCTs is teratoma formation, reflecting their capacity to mature. An inverse relationship exists in GCTs between differentiation and tumorigenicity. To explore anti-neoplastic mechanisms in GCTs, NT2/D1 and NT2/D1-R1 cells were studied. NT2/D1 differentiation reproduced key features of teratomas in that RA-treatment caused these cells to mature and reduce tumorigenicity. A hallmark of this response is G1 arrest, at least partly through a previously unrecognized pathway, where retinoids trigger cyclin D1 proteolysis. This occurs partly through proteasomal degradation. RA effects on cyclin D1 are specific since cyclins D2 and D3 were not repressed. NT2/D1-R1 cells deregulated cyclin D1 expression. RARgamma transfection restored retinoid response and cyclin D1 repression to NT2/D1-R1 cells. Threonine 286 regulated cyclin D1 ubiquitination and proteasomal degradation by RA. Another relationship was that NT2/D1-R1 cells deregulated cyclin D1 and acquired cisplatin resistance, indicating a link between maturation and chemotherapy responses. Post-transcriptional and transcriptional retinoid mechanisms repressed cyclin D1. These will be studied comprehensively and compared to those of other cyclin D1 targeting agents. This is relevant for combination therapy. There is a need to uncover the cyclin D1 role in GCT biology these specific aims that: (1) elucidate proteolytic and transcriptional mechanisms that repress cyclin D1 during retinoid treatment of GCTs and learn whether other cyclin D1 targeting agents engage distinct or overlapping mechanisms; (2) validate whether cyclin D1 repression is required for GCT growth, maturation, or cisplatin response; and (3) extend findings to the clinical context by evaluating cyclin D1 as a target in a unique tissue bank with mature, immature, and chemotherapy resistant GCTs. These resources with pharmacologic, cell and molecular biological approaches would provide insights into cyclin D1 as a novel target for poor risk GCTs.