Surgical Studies of GI Peptides - Mechanisms of Action

描述（由申请人提供）：胃肠道（GI）癌症仍然是一个重要的和具有挑战性的临床问题。手术切除是治疗胃肠道恶性肿瘤的主要手段;然而，只有肿瘤局部化并且没有扩散到淋巴结和远处器官，才能实现治愈。对控制胃肠道癌细胞生长的分子机制的进一步了解是开发与手术切除联合使用的新型治疗策略所必需的。胃肠道肽激素可以刺激正常和肿瘤性肠道组织的生长。多年来，我们的研究一直集中在确定肠肽，胃泌素（G-17），及其同源受体，调节细胞生长的分子机制。最近，我们发现了一种新的CCK-B/胃泌素受体的剪接变体，称为CCK-BRi 4sv，它在结肠癌和胰腺癌中表达，但在正常组织中不表达。CCK-BRi 4sv与野生型CCK-BR（CCK-BRwt）相比，表现出明显不同的信号传导特性，包括G-17非依赖性的细胞生长刺激、细胞内Ca 2+的调节和亚细胞运输。此外，我们发现，丝裂原活化蛋白激酶（MAPKs）在激动剂刺激前后CCK-BR介导的信号传导中起关键作用。MAPK激酶（MEK）调节CCK-BRwt对G-17刺激的敏感性，并介导G-17刺激对下游效应物的影响。最后，我们发现CCK-BRi 4sv和CCK-BRwt介导G-17诱导考克斯-2基因表达。基于我们的研究结果，我们假设CCK-BR变体通过激动剂依赖性和非依赖性机制调节GI细胞生长，并且MAPK通过调节受体对激动剂刺激的敏感性和作为激动剂诱导的信号转导的下游效应物在受体介导的细胞生长调节中发挥核心作用。为了检验这些假设，我们计划了三个具体目标的实验。目的1：明确CCK-BRwt和CCK-BRi 4sv的内化和细胞内受体转运的机制。目的2：探讨MAPK在CCK-BRwt和CCK-BRi 4sv介导的细胞内信号转导中的作用。目的3：研究CCK-BRi 4sv和CCK-BRwt表达对基因表达的影响。

DESCRIPTION (provided by applicant): Gastrointestinal (GI) cancers continue to be a significant and challenging clinical problem. Surgical resection is the mainstay for cure of GI malignancies; however, cure can only be achieved if the tumors are localized and have not spread to lymph nodes and distant organs. Increased understanding of the molecular mechanisms controlling GI cancer cell growth is required for the development of novel thera-peutic strategies to be used in combination with surgical resection. GI peptide hormones can stimulate the growth of normal and neoplastic gut tissues. For years, our studies have focused on determining the molecular mechanisms by which the gut peptide, gastrin (G-17), and its cognate receptors, regulate cell growth. Recently, we have discovered a novel splice variant of the CCK-B/gastrin receptor called CCK-BRi4sv that is expressed in colonic and pancreatic cancers, but not the normal tissues. CCK-BRi4sv exhibit distinctly different signaling properties when compared to the previously characterized wild-type CCK-BR (CCK-BRwt) including G-17-independent stimulation of cell growth, regulation of intracellular Ca 2+and subcellular trafficking. Also, we found that mitogen-activated protein kinases (MAPKs) play a key role in CCK-BR-mediated signaling both before and after agonist stimulation. MAPK kinase (MEK) regulates CCK-BRwt sensitivity to G-17 stimulation and mediates the effects of G-17 stimulation on downstream effectors. Finally, we found that CCK-BRi4sv and CCK-BRwt mediate G-17-induction of COX-2 gene expression. Based on our findings, we hypothesize that CCK-BR variants regulate GI cell growth by both agonist-dependent and -independent mechanisms, and that MAPKs play a central role in receptor-mediated regulation of cell growth by modulating the sensitivity of the receptor to agonist stimulation and by acting as downstream effectors of agonist-induced signal transduction. To examine these hypotheses, we have planned experiments with three Specific Aims. Aim 1: To define the mechanisms of CCK-BRwt and CCK-BRi4sv internalization and intracellular receptor trafficking. Aim 2: To define the role of MAPKs in CCK-BRwt- and CCK-BRi4sv-mediated intracellular signal transduction. Aim 3: To determine the effects of CCK-BRi4sv and CCK-BRwt expression on gene expression.