AnnexinA2/progastrin and stem cells: dietary cancer prevention

描述（由申请人提供）：累积证据强烈表明前体肽如原胃泌素（PG）对靶细胞发挥有丝分裂/共致癌作用，并增加肿瘤发生的风险。许多肿瘤表达PG，下调胃泌素基因/PG表达可减弱PG依赖性癌细胞的生长。我们最近发现胞外膜相关膜联蛋白A2 （ANXA2）是一种新的PG受体。同时，据报道，ANXA2的表达对于维持上皮癌细胞的生长至关重要。最近，我们发现HEK-293细胞中PG的过表达显著增加了DCAMKL+1/Lgr5/CD44阳性的干细胞群，并且令人惊讶地导致细胞的致瘤性转化。在一项重要的相关研究中，我们报道了PG通过抑制关键信号通路对癌细胞刺激生长的有效抑制。我们的初步研究表明，姜黄素可显著降低ANXA2和干细胞标志物的表达；有趣的是，姜黄素对转化上皮细胞和非转化上皮细胞中DCAMKL+1表达的相反作用被测量出来。基于这些新发现，我们将检验“化疗/饮食预防药物在非致瘤性上皮细胞和致瘤性上皮细胞中对生长因子介导的ANXA2和干细胞标记物的表达进行差异调节”的假设。为了实现这一目标，我们将重点研究姜黄素的作用。在Aim 1中，姜黄素对未转化/转化上皮细胞（富含ANXA2/干细胞标记物）生长的相对抑制效果将通过体外和体内研究模型进行检验。姜黄素对非致瘤性/致瘤性细胞中ANXA2/干细胞标记物表达的调节作用将与姜黄素的生长抑制作用有关。针对干细胞标记物的靶向siRNA/shRNA将用于增强姜黄素的抑制作用。这些研究的结果可能具有治疗意义。在Aim 2中，姜黄素减少PG对ANXA2/干细胞标记物表达的刺激作用的机制将在转化/非转化细胞系的蛋白质和RNA水平上进行研究；在转录水平上的抑制作用将在启动子报告子试验中进行检验。这些实验将扩大我们对生长因子、姜黄素和干细胞标记物之间相互作用的理解。在Aim 3中，我们将研究姜黄素对过表达PG的转基因小鼠结肠癌起始、促进和进展阶段的剂量依赖性作用，以及对ANXA2/干细胞标记物的影响。分析肿瘤前和肿瘤生长及周围正常粘膜的ANXA2/干细胞标记物的相对表达与2- catenin/NF:Bp65和其他转录因子的激活。这些研究的结果有望帮助我们开发基于机制的策略，用于预防和/或治疗循环PG（生长因子）和/或ANXA2表达肿瘤阳性患者的癌症。

DESCRIPTION (provided by applicant): Cumulative evidence strongly suggests that precursor peptides such as progastrins (PG) exert mitogenic/co-carcinogenic effects on target cells and increase the risk of tumorigenesis. Many tumors express PG, and down-regulation of gastrin gene/PG expression attenuates growth of PG-dependent cancer cells. We recently discovered that extra-cellular membrane-associated annexin A2 (ANXA2) represents a novel receptor for PG. At the same time, ANXA2 expression has been reported to be critical for maintaining the growth of epithelial cancer cells. More recently, we discovered that over-expression of PG in HEK-293 cells significantly increased stem cell populations, positive for DCAMKL+1/Lgr5/CD44, and surprisingly resulted in tumorigenic transformation of the cells. In an important related study we reported potent inhibition of PG stimulated growth on cancer cells via the inhibition of critical signaling pathways. Our preliminary studies suggest that curcumin significantly reduces expression of ANXA2 and stem cell markers; intriguingly opposite effects of curcumin were measured on DCAMKL+1 expression in transformed versus non-transformed epithelial cells, Based on these novel findings we will test the hypothesis that 'chemo/dietary preventive agents differentially modulate growth factor-mediated expression of ANXA2 and stem cell markers in non-tumorigenic versus tumorigenic epithelial cells'. Towards this goal, we will focus on examining the effects of curcumin. In Aim 1, relative inhibitory efficacy of curcumin on growth of non-transformed/transformed epithelial cells, enriched for ANXA2/stem cell markers will be examined using in vitro and in vivo models of investigation. Regulatory effects of curcumin on expression of ANXA2/stem cell markers in non-tumorigenic/tumorigenic cells will be examined in relation to growth inhibitory effects of curcumin. Target-specific siRNA/shRNA against stem cell markers will be used to augment inhibitory effects of curcumin. Results of these studies will likely have therapeutic implications. In Aim 2 mechanisms by which curcumin reduces stimulatory effects of PG on the expression of ANXA2/stem cell markers will be examined in transformed/non-transformed cell lines at protein and RNA levels; inhibitory effects at the transcriptional level will be examined in promoter-reporter assays. These experiments will expand our understanding of the interplay between growth factors, curcumin and stem cell markers. In Aim 3, dose-dependent effects of dietary curcumin against initiation, promotion, and progression phases of colon carcinogenesis in transgenic mice over-expressing PG, in relation to efects on ANXA2/stem cell markers will be examined. Pre-neoplastic and neoplastic growths and surrounding normal mucosa wil be analyzed for relative expression of ANXA2/stem cell markers in relation to activation of 2- catenin/NF:Bp65 and other transcriptional factors. Results of these studies are expected to help us develop mechanism-based strategies for prevention and/or treatment of cancers in patients, positive for circulating PG (growth factors) and/or ANXA2 expressing tumors. PUBLIC HEALTH RELEVANCE: In this grant application it is proposed to examine if a combination of dietary supplements and non- toxic small inhibitory molecules can prevent or treat the growth of solid tumors and eradicate the seed cancer cells so that the threat of recurrence is significantly reduced. We will also examine the mechanisms by which specific molecules, discovered in the past few years by our laboratory, increase the risk of developing cancerous growths, and the mechanisms by which dietary agents reduce this risk. The results of these studies are expected to help us develop more effective strategies for preventing and treating solid tumors in organs such as the colon, using less toxic methods.