Role of Annexin-II in growth factor/co-carcinogenic effects of progastrin

在过去的资助期间，我们证实了我们的主要假设，即孕激素多肽(PG)在体外和体内对肠/结肠上皮细胞株具有增殖/抗凋亡/共致癌作用，并增加了偶氮甲烷(AOM)导致结肠癌的风险。此外，我们还发现，Annexin II(ANXII)是PG的高亲和力受体，是介导PG对靶细胞的生长因子效应所必需的。β-连环蛋白、c-Src、PI3K/Akt、MAPK/ERK和NFkB在PG对正常和肿瘤肠上皮细胞的生长因子效应中起重要作用。基于这些发现，我们目前的资助方案的主要假设是，ANXII促进了一个或多个上述激酶/转录因子的激活，以响应PG。目标1和目标2中的实验将检验这一假设。在目标1中，将使用ANX-II表达改变的PG反应细胞系作为体外研究模型。对于体内研究，我们将使用过表达PG(Fabp-PG)的转基因小鼠，在ANXII表达调控之前或之后。我们将使用ANX-II基因敲除的小鼠，或者使用特定的siRNA来下调ANX-II的表达。在初步研究中，观察到PG与ANXII在原位、在过度表达自分泌PG或对外源PG有反应的细胞中的共定位。因此，在目标2中，将研究介导ANXII/PG细胞内转位的途径，以响应PG与细胞外ANXII的结合(及其功能意义)；将使用基于荧光的检测技术和生化分析。在目标3中，我们的发现的临床相关性将通过一项先导性的相关性研究来检验，这些样本是在结肠镜检查或结肠肿瘤手术切除时从结肠癌发生的不同阶段的患者获得的。我们的初步研究强烈表明PG多肽与结肠癌疾病的进展有关。因此，了解ANX-II和各种信号分子在介导PG的作用中的作用是临床上重要的目标。从上述研究中获得的数据将有助于制定更有效的战略，以针对PG的行动进行预防和治疗。

In the past funding period we confirmed our major hypothesis that progastrin peptides (PG) exert proliferative/anti-apoptotic/co-carcinogenic effects on intestinal/colonic epithelial cell lines, in vitro and in vivo, and increase the risk of colon carcinogenesis in response to azoxymethane (AOM). Additionally, we made the novel discovery that Annexin II (ANXII) functions as a high affinity receptor for PG and is required for mediating growth factor effects of PG on target cells. An important role of β-catenin, c-Src, PI3K/Akt, MAPK/ERK and NFkB in mediating growth factor effects of PG on normal and neoplastic intestinal epithelial cells was also identified. Based on these findings, the major hypothesis of our current grant proposal is that ANXII facilitates the activation of one or more of the above indicated kinases/transcription factors in response to PG. Experiments in Aims 1 and 2 will examine this hypothesis. In Aim 1, PG responsive cell lines, altered for ANX-II expression will be used, as an in vitro model of investigation. For the in vivo studies, we will either use transgenic mice over-expressing PG (Fabp-PG), before or after modulation of ANXII expression. We will either use ANX-II knock out mice, or use specific siRNA for down regulating ANXII expression. In preliminary studies co-localization of PG with ANXII in situ, in cells over-expressing autocrine PG or responsive to exogenous PG was observed. Therefore in Aim 2, pathways mediating intracellular translocation of ANXII/PG, in response to binding of PG to extracellular ANXII (and its functional significance) will be examined; fluorescence based detection techniques and biochemical analysis will be used. In Aim 3 the clinical relevance of our findings will be examined in a pilot correlative study with samples obtained from patients, consented at the time of colonoscopy or at the time of surgical resection of colonic tumors, at different stages of colon carcinogenesis. Our initial studies strongly implicate PG peptides in the progression of the colon cancer disease. Therefore, understanding the role of ANX-II and the various signaling molecules in mediating the actions of PG are clinically important goals. Data obtained from the above studies will facilitate the development of more effective strategies for targeting the actions of PG for preventative and treatment purposes.