Prevention of Tumor Progression by a Novel Approach

描述（由申请人提供）：新出现的证据表明，癌症进展通常与上皮-间充质转化（EMT）表型的获得有关，这让人想起“癌症干细胞样细胞（CSCs）”，这在一定程度上负责胰腺癌（PC）细胞获得侵袭性的能力，促进肿瘤转移。我们发表的数据显示，在吉西他滨耐药的EMT-型细胞中，miR-200表达降低，导致ZEB1上调，从而导致E-cadherin下调，miR-21表达增加，导致PTEN表达失活。我们还发现，与正常志愿者相比，PC患者血浆中的miR-21水平明显更高，这与生存恶化有关。基于这些结果，我们推断发现一种新的无毒途径，miR- 200可以上调，miR-21可以下调，这将是一种预防肿瘤进展的新方法。为此，我们首次发表了一种无毒天然姜黄素的合成类似物，命名为CDF，其靶组织（胰腺）生物利用度优越，没有任何不良副作用，并且CDF在消除EMT表型细胞方面非常有效，部分原因是miR-200上调，miR-21下调，导致ZEB1下调，E-cadherin和PTEN的表达增加。因此，我们似乎发现了一种新的方法，通过对耐药emt型细胞的生物环境进行“调节”，可以有效地消除emt型细胞或CSCs，这可能有助于通过消除肿瘤复发的“根本”原因来预防肿瘤进展。根据我们的初步结果，我们假设miR-21的激活在吉西他滨耐药（GR） PC细胞获得EMT表型的过程中是至关重要的，并且这些细胞可以通过CDF单独或与常规疗法联合消除。我们将验证我们的假设，以获得miR-21和PTEN在GR细胞中获得EMT表型过程中的作用机制，并通过操纵miR-21 （Aim-1）的表达来研究这些细胞的生物学后果。我们还将研究CDF如何下调miR- 21并上调其靶蛋白的表达。这可能导致PC细胞的生物环境发生“调节”，导致细胞对常规药物敏感（Aim-2；体外研究）。最后，我们将利用异种移植物和K-ras转基因动物模型，确定与单独使用CDF相比，CDF与体内常规治疗药物联合使用时是否能提高抗肿瘤活性（Aim-3）。我们的研究结果将提供miR-21在耐药emt型细胞（CSCs）中的作用机制，并将提供临床前数据，支持CDF在预防肿瘤进展和/或治疗PC方面的作用。因此，我们的研究结果将对预防肿瘤复发具有显著的高影响，从而使诊断为PC的患者获得更好的生存结果。

DESCRIPTION (provided by applicant): Emerging evidence suggests that cancer progression is often associated with the acquisition of Epithelial-to- Mesenchymal Transition (EMT) phenotype that is reminiscent of "Cancer Stem-like Cells (CSCs)", which is partly responsible for the ability of pancreatic cancer (PC) cells to acquire aggressiveness, contributing to tumor metastasis. Our published data showed decreased expression of miR-200 in gemcitabine resistant EMT- type cells, resulting in the up-regulation of ZEB1 and thereby down-regulation of E-cadherin, and also showed increased expression of miR-21, resulting in the inactivation of PTEN expression. We also found significantly higher levels of miR-21 in the plasma of PC patients compared to normal volunteers, which was correlated with worsened survival. Based on these results, we reasoned that finding a novel non-toxic avenue by which miR- 200 could be up-regulated and miR-21 could be down-regulated would be a novel approach for the prevention of tumor progression. To that end, we have published, for the first time, that a synthetic analog of a non-toxic natural agent curcumin, named CDF is superior in its target tissue (pancreas) bioavailability without any adverse side effects, and the CDF was very effective in the elimination of EMT phenotypic cells, which was in part due to up-regulation of miR-200 and down-regulation of miR-21, resulting in the down-regulation of ZEB1, and increased expression of E-cadherin and PTEN. Therefore, it appears that we found a novel approach by which "conditioning" of the biological milieu of drug-resistant EMT-type cells could be achieved toward effective elimination of EMT-type cells or CSCs, which could be useful for the prevention of tumor progression by eliminating the "root" cause of tumor recurrence. Based on our preliminary results, we hypothesize that the activation of miR-21 is critical during the acquisition of EMT phenotype in gemcitabine-resistant (GR) PC cells, and these cells could be eliminated by CDF alone or in combination with conventional therapeutics. We will test our hypothesis to gain mechanistic insight on the role of miR-21 and PTEN during the acquisition of EMT phenotype in GR cells, and investigate the biological consequence of these cells by manipulating the expression of miR-21 (Aim-1). We will also investigate how CDF could down-regulate miR- 21 and up-regulate the expression of its targets. This may lead to the "conditioning" of the biological milieu of PC cells, resulting in sensitization of cells to conventional agents (Aim-2; in vitro studies). Finally, we will determine whether CDF could cause increased anti-tumor activity when combined with conventional therapeutics in vivo using xenograft and K-ras transgenic animal models compared to CDF alone (Aim-3). The results of our studies will provide mechanistic insight as to the role of miR-21 in drug-resistant EMT-type cells (CSCs), and will also provide pre-clinical data in support of the role of CDF for the prevention of tumor progression and/or treatment of PC. Therefore, our results will have significantly high impact toward preventing tumor recurrence, which will lead to achieve better survival outcome of patients diagnosed with PC. PUBLIC HEALTH RELEVANCE: This project is focused on elucidating the mechanism of Epithelial-to-Mesenchymal Transition (EMT) in gemcitabine-resistant (GR) pancreatic cancer (PC) cells related to the regulation of miR-21 and its targets, and further investigate the mechanism(s) by which "natural agents" such as curcumin and our novel synthetic analog (CDF) of curcumin could effectively kill EMT-type cells alone or in combination with conventional chemotherapeutic agents. We will examine the molecular mechanism in vitro and will also test our hypothesis in vivo using xenograft and K-ras transgenic animal model by assessing whether CDF or curcumin alone or in combination with conventional therapeutics could be useful for the prevention of tumor progression and/or treatment. Our proposed investigation is highly relevant to the mission of NCI, NIH and is likely to have a significant impact toward saving lives of patients diagnosed with PC in the immediate future.