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。结直肠癌是美国第二大致死性癌症，主要是由于侵袭和转移到肝脏和淋巴结。在这个提议中，我们打算了解一种新的肿瘤抑制因子Pdcd 4对结肠肿瘤转移的影响以及参与这一过程的分子机制。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. PUBLIC HEALTH RELEVANCE Colorectal cancer is the second most fatal cancer in the United States, largely due to invasion and metastasis to the liver and lymph nodes. In this proposal, we intend to understand the effects of a novel tumor suppressor, Pdcd4, on colon tumor metastasis and the molecular mechanism involved in this process. 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.