Wnt antagonist genes in kidney tumor progression and metastasis

描述（由申请人提供）：肾肿瘤进展和转移中的Wnt拮抗剂基因。该项目的主要目标是研究Wnt拮抗基因在肾癌进展和转移中的作用。其原理是通过CpG甲基化途径使Wnt拮抗基因失活，从而激活Wnt/b-catenin途径并诱导多种器官的恶性转化。然而，肾癌方面缺乏此类研究。将测试三个具体假设以确定：1）Wnt 拮抗剂基因的失活是否与肾癌的进展和转移有关。 2）Wnt拮抗剂基因失活的机制是通过DNA甲基化、组蛋白修饰和染色质重塑等表观遗传途径。 3)转染Wnt拮抗基因抑制肾癌体内外生长和转移。为了检验这些假设，我们将追求以下具体目标。具体目标#1：研究Wnt拮抗剂基因失活是否与人肾细胞癌的进展和转移有关。根据初步数据，我们筛选了多个Wnt拮抗基因，并鉴定出6个在肾癌中沉默的基因。这些基因是：分泌型卷曲相关蛋白-1 (sFRP-1)、sFRP-2、sFRP-4、sFRP-5、Wnt 抑制因子-1 (Wif-1) 和 DICKKOPF-3 (DKK-3)。在此特定目标下，我们将测定正常肾癌和不同阶段和级别肾癌中Wnt拮抗剂基因的mRNA和蛋白表达水平。将通过实时 RT-PCR 分析 mRNA 表达，并通过免疫组织化学和蛋白质印迹分析蛋白质表达。具体目标#2：研究肾癌中 Wnt 拮抗剂基因失活的机制。在此目标下，我们将使用亚硫酸氢钠甲基化技术分析Wnt拮抗剂基因启动子区域中CpG岛的高甲基化，并通过直接DNA测序进行确认。我们还将通过分析肾癌组织中的这些参数来研究Wnt拮抗剂基因失活的机制是否是由于DNA甲基转移酶（DNMT-1、DNMT3a、DNMT3b）、去甲基化酶（MBD2）基因、组蛋白乙酰化和染色质重塑引起的。具体目标#3：研究 Wnt 拮抗剂基因在肾癌中的功能作用。在此特定目标下，我们将全长Wnt拮抗剂基因cDNA转染至显性失活的肾癌细胞中，建立稳定的转染细胞。我们将分析转染肾癌细胞的体外和体内生长。还分析转染细胞和亲代细胞的体外侵袭性（细胞外基质结合测定、侵袭测定和软琼脂集落形成效率）。这些实验的成功完成将证明Wnt拮抗基因在抑制肾癌生长、进展和转移中的功能作用，以及这些基因在肾癌中失活的机制。未来，这些结果可能为肾癌进展和转移的管理提供更好的策略。肾细胞癌（RCC）是泌尿生殖系统第三大常见恶性肿瘤。根据已发表的文献，很明显Wnt拮抗剂基因与多种癌症相关。然而，肾癌方面缺乏此类研究。我们建议使用体外和体内模型研究 Wnt 拮抗剂基因在肾细胞癌进展和转移中的功能作用。成功完成所提出的实验可能为我们提供更好的肾癌治疗策略。

DESCRIPTION (provided by applicant): Wnt antagonist genes in kidney tumor progression and metastasis. Main goal of this project is to investigate the role of Wnt antagonist genes in the progression and metastasis of renal cancer. The rationale is that Wnt antagonist genes are inactivated through CpG methylation pathways with the result that Wnt/ b-catenin pathways are activated and induce malignant transformation of various organs. However, such studies are lacking in kidney cancer. Three specific hypotheses will be tested to determine if: 1) Inactivation of Wnt antagonist genes is involved in the progression and metastasis of kidney cancer. 2) The mechanisms of inactivation of the Wnt antagonist genes are through epigenetic pathways such as DNA methylation, histone modification and chromatin remodeling. 3) Transfection of Wnt antagonist genes suppresses the in vitro and in vivo growth and metastasis of kidney cancer. To test these hypotheses, we will pursue the following specific aims. Specific Aim # 1: To investigate whether inactivation of Wnt antagonist genes is involved in the progression and metastasis of human renal cell carcinoma. Based on the preliminary data, we have screened several Wnt antagonist genes and have identified six genes that are silenced in kidney cancer. These genes are: secreted frizzled-related protein-1 (sFRP-1), sFRP-2, sFRP-4, sFRP-5, Wnt inhibitory factor-1 (Wif-1) and DICKKOPF-3 (DKK-3). Under this specific aim, we will determine the levels of mRNA and protein expression of Wnt antagonist genes in normal and different stages and grades of kidney cancer. The mRNA expression will be analyzed by real-time RT- PCR and protein expression by immunohistochemistry and Western blotting. Specific Aim # 2: To investigate the mechanisms of inactivation of Wnt antagonist genes in kidney cancer. Under this aim, we will analyze hypermethylation of CpG Islands in promoter regions of Wnt antagonist genes using sodium bisulfite methylation techniques and confirm by direct DNA sequencing. We will also investigate whether the mechanisms of inactivation of Wnt antagonist genes are due to DNA methyltransferase (DNMT-1, DNMT3a, DNMT3b), demethylase (MBD2) genes, histone acetylation and chromatin remodeling through analysis of these parameters in kidney cancer tissues. Specific Aim # 3: To investigate the functional role of Wnt antagonist genes in kidney cancer. Under this specific aim, we will transfect full-length Wnt antagonist gene cDNA in dominant-negative kidney cancer cells and establish the stable transfectant cells. We will analyze in vitro and in vivo growth of transfected kidney cancer cells. Also analyze in vitro invasiveness (extra-cellular matrix binding assay, invasion assay and soft agar colony forming efficiency) of transfected and parental cells. Successful completion of these experiments will demonstrate the functional role of Wnt antagonist genes in the suppression of kidney cancer growth, progression and metastasis and also the mechanism of inactivation of these genes in kidney cancer. In the future, these results may provide better strategies for the management of kidney cancer progression and metastasis. Renal cell carcinoma (RCC) is the third most common malignancy of the genitourinary system. Based on the published literature, it is clear that Wnt antagonist genes are associated with various cancers. However, such studies are lacking in kidney cancer. We have proposed to investigate the functional role of Wnt antagonist genes in the progression and metastasis of renal cell carcinoma using both in vitro and in vivo models. Successful completion of proposed experiments may provide us with the better strategies for the management of kidney cancer.