The role of Pdcd4 in colorectal tumor progression

描述(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，对结肠癌的侵袭/转移有深入的了解对于预防和治疗结肠癌是必要的。程序性细胞死亡4(Pdcd4)是一种新的肿瘤抑制因子，过表达可抑制结肠癌细胞侵袭和AP-1依赖的转录。与癌旁正常组织相比，结肠癌组织中Pdcd4的表达经常下调。本研究的目的是明确Pdcd4在肿瘤侵袭/转移中的作用，并阐明参与这些过程的分子机制。我们下调了Pdcd4在结肠癌GEO和HT29细胞中的表达，发现Pdcd4下调可触发细胞形态变化，诱导侵袭，并激活依赖于2-catenin/Tcf和AP-1的转录。我们的初步结果进一步表明，E-钙粘蛋白表达的减少与E-钙粘蛋白表达的转录抑制因子slug的蛋白水平增加有关。因此，我们推测，Pdcd4基因敲除通过下调E-钙粘蛋白、激活2-catenin/Tcf和AP-1依赖的转录来促进结肠癌细胞的侵袭/转移。为了验证我们的假设，我们提出了四个具体目标。目的1：确定Pdcd4在结肠癌细胞中是否具有转移抑制作用。小鼠上皮到间充质的转变和转移将使用Pdcd4基因敲除和过表达的细胞进行分析。目的：研究E-钙粘素下调诱导的Pdcd4细胞2-catenin/Tcf依赖转录的激活是否促进肿瘤的侵袭和转移。我们将通过敲除E-钙粘素来检测2-连环蛋白/Tcf依赖转录的激活，通过抑制2-连环蛋白/Tcf依赖转录逆转侵袭/转移能力，验证uPAR和c-Myc作为2-连环蛋白/Tcf依赖转录的靶基因，并通过过表达Pdcd4来抑制2-连环蛋白/TCF4依赖的转录及其靶基因的表达。目的：研究Pdcd4基因敲除细胞中依赖2-catenin/TCF4的转录激活是否通过c-Myc上调MAP4K1的表达和AP-1的活性。我们将建立c-Myc作为MAP4K1表达的调节因子，并激活依赖于2-catenin/Tcf的转录，导致MAP4K1表达上调、JNK激活和AP-1依赖的转录。目的：确定Pdcd4是否在翻译上抑制转录抑制因子slug的表达，从而上调E-钙粘蛋白，抑制肿瘤的侵袭/转移。翻译抑制和抑制机制将通过体外和体内翻译试验进行分析，然后检测E-钙粘蛋白在Slug基因敲除和Pdcd4表达细胞中的表达水平和侵袭/转移能力。这项工作将提供对Pdcd4如何调控结肠癌进展的全面了解，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.