The role of Pdcd4 in colorectal tumor progression

描述（由申请人提供）：结直肠癌是美国癌症相关死亡的第二大原因，主要是由于肝脏和淋巴结的侵袭和转移。因此，深入了解结肠癌的侵袭/转移是预防和治疗结肠癌的必要条件。程序性细胞死亡4（Pdcd 4）是一种新的肿瘤抑制因子，过表达Pdcd 4可抑制结肠癌细胞的侵袭和AP-1依赖性转录。与邻近的正常组织相比，结肠肿瘤组织中pdcd 4的表达经常下调。本研究的目的是明确Pdcd 4在肿瘤侵袭/转移中的功能，并阐明参与这些过程的分子机制。我们敲低了结肠肿瘤GEO和HT 29细胞中的Pdcd 4表达，发现Pdcd 4敲低触发了形态学变化，诱导了侵袭，并激活了2-catenin/Tcf和AP-1依赖的转录。我们的初步结果进一步表明，E-cadherin表达的减少与Slug（E-cadherin表达的转录抑制因子）蛋白水平的增加相关。因此，我们推测Pdcd 4基因敲低通过下调E-钙粘蛋白和激活2-catenin/Tcf和AP-1依赖性转录促进结肠肿瘤细胞的侵袭/转移。为了验证我们的假设，提出了四个具体目标。目的1：确定Pdcd 4是否在结肠肿瘤细胞中作为转移抑制剂发挥作用。将使用Pdcd 4敲低和过表达的细胞测定小鼠中的上皮向间充质转化和转移。目标二：检查Pdcd 4敲低细胞中由E-钙粘蛋白下调导致的2-连环蛋白/Tcf依赖性转录的激活是否促进侵袭/转移。我们将测试通过敲低E-cadherin来激活2-catenin/Tcf依赖性转录，通过抑制2-catenin/Tcf依赖性转录来逆转侵袭/转移能力，验证uPAR和c-Myc作为2-catenin/Tcf依赖性转录的靶基因，以及通过过表达Pdcd 4来抑制2-catenin/Tcf 4依赖性转录及其靶基因表达。目标3：确定Pdcd 4敲低细胞中2-连环蛋白/Tcf 4依赖性转录的激活是否通过c-Myc上调MAP 4K 1表达和AP-1活性。我们将建立c-Myc作为MAP 4K 1表达的调节器，并激活2-连环蛋白/Tcf依赖性转录，导致MAP 4K 1表达上调，JNK激活和AP-1依赖性转录。目标4：确定Pdcd 4是否抑制转录阻遏物Slug表达，从而导致E-钙粘蛋白的上调和侵袭/转移的抑制。将使用体外和体内翻译测定分析翻译抑制和抑制机制，然后检查Slug敲低和Pdcd 4表达细胞中的E-钙粘蛋白表达水平和侵袭/转移能力。这项工作将全面了解Pdcd 4如何调节结肠肿瘤进展，并为预防和治疗结肠癌的新干预措施带来巨大希望。

DESCRIPTION (provided by applicant): Colorectal cancer is the second leading cause of cancer-related deaths in the United States, largely due to invasion and metastasis to the liver and lymph nodes. Thus, a thorough understanding of invasion/metastasis is necessary for prevention and treatment of colon cancer. Over- expression of programmed cell death 4 (Pdcd4), a novel tumor suppressor, inhibits invasion and AP-1 dependent transcription in colon tumor cells. Pdcd4 expression is frequently down-regulated in colon tumor tissues as compared to adjacent normal tissues. The objective of the proposed study is to define the function of Pdcd4 in tumor invasion/metastasis and elucidate the molecular mechanisms involved in these processes. We knocked down Pdcd4 expression in colon tumor GEO and HT29 cells and found that Pdcd4 knock-down triggers morphological changes, induces invasion, and activates 2-catenin/Tcf and AP-1 dependent transcription. Our preliminary results further demonstrated that a decrease in E-cadherin expression correlates with an increase in the protein level of Slug, a transcription repressor for E-cadherin expression. We, therefore, hypothesize that Pdcd4 knock-down promotes colon tumor cell invasion/metastasis through down-regulation of E-cadherin and activation of 2-catenin/Tcf and AP-1 dependent transcription. To test our hypothesis, four Specific Aims are proposed. Aim 1: determine whether Pdcd4 functions as a metastasis inhibitor in colon tumor cells. Epithelial-to-mesenchymal transition and metastasis in mice will be assayed using Pdcd4 knock- down and over-expressed cells. Aim 2: examine whether activation of 2-catenin/Tcf dependent transcription led by E-cadherin down-regulation in Pdcd4 knock-down cells promotes invasion/metastasis. We will test activation of 2-catenin/Tcf dependent transcription by knocking down E-cadherin, reversion of the invasive/metastatic ability by inhibiting 2-catenin/Tcf dependent transcription, verification of uPAR and c-Myc as the target genes of 2-catenin/Tcf dependent transcription, and inhibition of 2-catenin/Tcf4 dependent transcription and its target gene expression by over-expressing Pdcd4. Aim 3: determine whether activation of 2-catenin/Tcf4 dependent transcription in Pdcd4 knock-down cells up-regulates MAP4K1 expression and AP-1 activity through c-Myc. We will establish the c-Myc as a regulator of MAP4K1 expression, and activation of 2- catenin/Tcf dependent transcription causing up-regulation of MAP4K1 expression, JNK activation, and AP-1 dependent transcription. Aim 4: determine whether Pdcd4 translationally inhibits transcription repressor Slug expression resulting in up-regulation of E-cadherin and suppression of invasion/metastasis. Translation inhibition and the inhibitory mechanism will be analyzed using in vitro and in vivo translation assays followed by an examination of E-cadherin expression level and the ability of invasion/metastasis in Slug knock-down and Pdcd4 expressing cells. This work will provide a comprehensive understanding of how Pdcd4 modulates colon tumor progression and holds great promise for novel interventions for preventing and treating colon cancer.