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 （Pdcd4）是一种新的肿瘤抑制因子，其过表达可抑制结肠肿瘤细胞的侵袭和AP-1依赖性转录。与邻近正常组织相比，Pdcd4在结肠肿瘤组织中的表达经常下调。本研究的目的是明确Pdcd4在肿瘤侵袭/转移中的功能，并阐明这些过程中的分子机制。我们在结肠肿瘤GEO和HT29细胞中敲低Pdcd4的表达，发现Pdcd4敲低可引起形态学改变，诱导侵袭，激活2-catenin/Tcf和AP-1依赖性转录。我们的初步结果进一步表明，E-cadherin表达的减少与E-cadherin表达的转录抑制因子Slug蛋白水平的增加相关。因此，我们假设Pdcd4敲低通过下调E-cadherin和激活2-catenin/Tcf和AP-1依赖性转录来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Pdcd4在结肠肿瘤细胞中是否具有转移抑制剂的功能。利用Pdcd4敲低和过表达细胞检测小鼠上皮到间质转化和转移。目的2：研究Pdcd4敲低细胞中E-cadherin下调导致的2-catenin/Tcf依赖性转录激活是否促进侵袭/转移。我们将通过敲除E-cadherin来测试2-catenin/Tcf依赖性转录的激活，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是2-catenin/Tcf依赖性转录的靶基因，以及通过过表达Pdcd4来抑制2-catenin/Tcf4依赖性转录及其靶基因表达。目的3：确定Pdcd4敲除细胞中2-catenin/Tcf4依赖性转录的激活是否通过c-Myc上调MAP4K1表达和AP-1活性。我们将建立c-Myc作为MAP4K1表达的调节因子，并激活2- catenin/Tcf依赖性转录，导致MAP4K1表达上调、JNK激活和AP-1依赖性转录。目的4：确定Pdcd4是否通过翻译抑制转录抑制因子Slug的表达从而导致E-cadherin的上调和侵袭/转移的抑制。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，随后检测E-cadherin表达水平和Slug敲除和Pdcd4表达细胞的侵袭/转移能力。这项工作将提供对Pdcd4如何调节结肠肿瘤进展的全面理解，并为预防和治疗结肠癌的新干预措施提供巨大的希望。结直肠癌是美国第二大致命癌症，主要是由于其侵袭和转移到肝脏和淋巴结。在本研究中，我们试图了解一种新的肿瘤抑制因子Pdcd4在结肠肿瘤转移中的作用及其分子机制。这项工作将提供对Pdcd4如何调节结肠肿瘤进展的全面理解，并为预防和治疗结肠癌的新干预措施提供巨大的希望。

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.