Sphingolipid Signaling in Wilms' Tumor Cells

肾母细胞瘤细胞中的鞘脂信号传导

• 批准号: 6984335
• 负责人: FERNANDO A FERRER
• 金额: $ 12.66万
• 依托单位: CONNECTICUT CHILDREN'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6984335
• 关键词: G protein; Wilms' tumor; athymic mouse; biological signal transduction; cell line; cell surface receptors; clinical research; guanosinetriphosphatases; human tissue; immunocytochemistry; metastasis; neoplastic growth; phosphorylation; polymerase chain reaction; protein kinase; receptor binding; receptor coupling; receptor expression; renal cell carcinoma; small interfering RNA; sphingolipids; sphingosine; western blottings

DESCRIPTION (provided by applicant): Pediatric and adult renal tumors are typically, angiogenic, invasive and potentially metatastic. Morbidity and mortality associated with Wilm's tumor is usually associated with metastases. Recently, the lysophospholipid, sphingosine-1-phosphate (S1P), and its associated G-protein-coupled receptors have emerged as putative regulators of angiogenesis, tumor proliferation, migration and metastasis. S1P receptor isotypic expression may be a determinant of tumor cell migration. We propose to gain mechanistic insights into the role of S1P receptor signaling in Wilm's tumor cells. In Aim 1 the expression of S1P receptors and S1P metabolizing enzymes in Wilm's tumor cell lines and clinical specimens will be evaluated using quantitative RT-PCR, western blot analysis, and immunohistochemical techniques. In Aim 2 the mechanisms of S1P signaling will be studied by S1P receptor overexpression and RNA interference. Cell lines that overexpress key SIP-metabolizing enzyme will be used to assess cell behavior in response to changes in S1P. S1P-receptor coupling to G proteins (Gi, GS, Gq and G12/13) and subsequent activation of Rho family GTPases (Rho, Rac, Cdc42) and Rho kinases have been implicated in S1P regulation of tumor cell motility. We will study the importance of these pathways and of the effectors downstream from Rho kinase, such as LIM-kinase (LIMK) and cofilin, by studying Rho and Rac activation, inhibition of Rho kinase, and the phosphorylation status of LIMK and cofilin. In Aim 3 growth, invasion and metastasis of naive cell lines and of S1P receptor-modified Wilm's cell lines in an established nude mouse model will be studied, thereby confirming and validating our ex vivo and in vitro results. Renal cell carcinoma cell lines from adult patients will be used for comparison. The results of these studies will contribute to our understanding of the processes of Wilm's tumor migration/metastasis and may offer novel therapeutic approaches for renal tumors.

描述（由申请人提供）： 儿童和成人肾肿瘤通常具有血管生成性、侵袭性和潜在转移性。肾母细胞瘤的发病率和死亡率通常与转移有关。最近，溶血磷脂、1-磷酸鞘氨醇 (S1P) 及其相关的 G 蛋白偶联受体已成为血管生成、肿瘤增殖、迁移和转移的假定调节剂。 S1P受体同种型表达可能是肿瘤细胞迁移的决定因素。我们建议深入了解 S1P 受体信号传导在肾母细胞瘤细胞中的作用。在目标 1 中，将使用定量 RT-PCR、蛋白质印迹分析和免疫组织化学技术评估 Wilm 肿瘤细胞系和临床标本中 S1P 受体和 S1P 代谢酶的表达。在目标 2 中，将通过 S1P 受体过度表达和 RNA 干扰来研究 S1P 信号传导机制。过度表达关键 SIP 代谢酶的细胞系将用于评估细胞响应 S1P 变化的行为。 S1P 受体与 G 蛋白（Gi、GS、Gq 和 G12/13）的偶联以及随后 Rho 家族 GTPases（Rho、Rac、Cdc42）和 Rho 激酶的激活与肿瘤细胞运动的 S1P 调节有关。我们将通过研究 Rho 和 Rac 的激活、Rho 激酶的抑制以及 LIMK 和 cofilin 的磷酸化状态，研究这些途径以及 Rho 激酶下游效应器（例如 LIM 激酶 (LIMK) 和 cofilin）的重要性。在Aim 3中，将在已建立的裸鼠模型中研究幼稚细胞系和S1P受体修饰的Wilm细胞系的生长、侵袭和转移，从而证实和验证我们的离体和体外结果。来自成年患者的肾细胞癌细胞系将用于比较。这些研究的结果将有助于我们了解肾母细胞瘤迁移/转移的过程，并可能为肾肿瘤提供新的治疗方法。