Rap1Gap and Tumor Progression

描述(由申请人提供)：癌变过程中最关键的步骤之一是肿瘤细胞获得转移的能力。在这项建议中要检验的假设是，Rap1GAP的下调有助于甲状腺肿瘤的进展。这一概念是基于数据显示，Rap1GAP在正常人类甲状腺细胞中高表达，而在大多数浸润性乳头状甲状腺癌中其表达显著降低。对人甲状腺癌细胞株的研究表明，Rap1GAP的缺失与上皮结构的丧失之间存在显著的相关性。Rap1GAP基因缺陷的肿瘤细胞缺乏E-钙粘附素，并获得波形蛋白的表达，提示上皮向间充质转化。与保留Rap1GAP的肿瘤细胞相比，这些细胞表现出更强的迁移和侵袭特性。将Rap1GAP恢复到Rap1GAP缺陷细胞可抑制细胞的迁移、侵袭和锚定非依赖性增殖。在保持上皮形态的甲状腺癌细胞中，沉默Rap1GAP的表达导致细胞分散，增强细胞集合体的解离，并调节失调的E-钙粘附素，提示细胞/细胞黏附缺陷。由于细胞/细胞连接的破坏与严重的病理后果相关，我们认为Rap1GAP的下调通过减弱细胞/细胞间的黏附而参与甲状腺肿瘤的发病。体外研究将探索Rap1GAP表达减少减轻细胞/细胞黏附的分子机制，并评估消除Rap1GAP是否赋予肿瘤细胞迁移特性和侵袭潜力的改变。由于Rap1GAP的TSH调节在人甲状腺肿瘤细胞系中丢失，但可能在原发甲状腺肿瘤中保留，因此将在分化的大鼠甲状腺细胞中进行补充研究。这些研究将在三维环境中的细胞中进行，这种环境更接近地再现人体组织的基质顺应性，并使细胞/细胞接触最大化。体外研究与对人类肿瘤标本的研究相辅相成，将确定甲状腺肿瘤中Rap1GAP表达降低的亚型，并探讨Rap1GAP缺失的临床意义。总之，这项建议提出了一个高度连贯的计划，以调查Rap1GAP缺失对人类甲状腺肿瘤进展的贡献和临床意义。公共卫生相关性：这项建议调查了Rap1GAP表达降低对人类甲状腺肿瘤进展的贡献和临床意义。对人甲状腺肿瘤细胞系的研究将阐明Rap1GAP下调减弱细胞/细胞黏附的机制，并研究Rap1GAP缺失是否增强了迁移和侵袭潜力。对人类肿瘤标本的研究将确定显示Rap1GAP下调的甲状腺肿瘤的亚型，评估Rap1GAP表达降低是否与出现转移疾病相关，检查与原发肿瘤相比，淋巴结转移患者Rap1GAP表达是否进一步降低，以及评估Rap1GAP表达是否与血管侵袭或淋巴侵袭相关，后者是预后不良的标志物。

DESCRIPTION (provided by applicant): One of the most critical steps in the progression to malignancy is the acquisition by tumor cells of the ability to metastasize. The hypothesis to be tested in this proposal is that downregulation of Rap1GAP contributes to thyroid tumor progression. This notion is based on data showing that Rap1GAP is highly expressed in normal human thyroid cells, and that its expression is dramatically reduced in the majority of invasive papillary thyroid carcinomas. Studies in human thyroid carcinoma cell lines revealed a striking correlation between loss of Rap1GAP and loss of epithelial structure. Rap1GAP-deficient tumor cells lacked E-cadherin and acquired the expression of vimentin, indicative of epithelial-to-mesenchymal transition. These cells exhibited enhanced migratory and invasive properties compared to tumor cells that retained Rap1GAP. Restoring Rap1GAP to Rap1GAP-deficient cells inhibited cell migration, invasion and anchorage-independent proliferation. Silencing Rap1GAP expression in thyroid carcinoma cells that retain an epithelial morphology caused the cells to disperse, enhanced the dissociation of cell aggregates, and dysregulated E-cadherin, suggestive of defects in cell/cell adhesion. As disruption of cell/cell junctions is associated with serious pathological consequences, we propose that downregulation of Rap1GAP contributes to the pathogenesis of thyroid tumors by attenuating cell/cell adhesion. In vitro studies will explore the molecular mechanism through which decreased Rap1GAP expression attenuates cell/cell adhesion and assess whether eliminating Rap1GAP endows tumor cells with altered migratory properties and invasive potential. As TSH regulation of Rap1GAP is lost from the human thyroid tumor cell lines, but is likely to be retained in primary thyroid tumors, complementary studies will be performed in differentiated rat thyroid cells. These studies will be conducted in cells in three-dimensional environments, conditions that more closely reproduce the matrix compliance of human tissues and where cell/cell contacts are maximized. The in vitro studies are complemented with studies in human tumor specimens that will identify the subtypes of thyroid tumors in which Rap1GAP expression is decreased and probe the clinical significance of loss of Rap1GAP. In summary, this proposal presents a highly cohesive plan to investigate the contribution and clinical significance of Rap1GAP depletion to the progression of human thyroid tumors. PUBLIC HEALTH RELEVANCE: This proposal investigates the contribution and clinical significance of decreased Rap1GAP expression to the progression of human thyroid tumors. Studies in human thyroid tumor cell lines will elucidate the mechanism through which downregulation of Rap1GAP attenuates cell/cell adhesion and investigate whether loss of Rap1GAP enhances migratory and invasive potential. Studies in human tumor specimens will identify the subtypes of thyroid tumors that exhibit Rap1GAP downregulation, assess whether decreased expression of Rap1GAP is correlated with the presence of metastatic disease at presentation, examine whether Rap1GAP expression is further decreased in lymph node metastases compared to primary tumors and assess whether Rap1GAP expression is correlated with the presence of angioinvasion or lymphoinvasion, markers of poor prognosis.