Bombesin-Activated Carcinoma Associated Fibroblasts in Colorectal Cancer

描述(申请人提供)：结直肠癌异常表达胃泌素释放肽(GRP)激素及其同源受体(GRPR)。大量的体内和体外实验表明，GRP或蛙皮素(BN)，GRP的药理同系物，促进结直肠癌的生长和进展。以前的研究主要集中在癌细胞上，然而，我们提供了令人信服的数据，在被检查的新鲜收获的人类结直肠癌中，95%的功能性GRPR由“癌相关成纤维细胞”(CAF)表达，而不是恶性上皮细胞。BN处理这些CAF可刺激其增殖，激活NF(B)，并以MEK/ERK通路依赖的方式诱导环氧合酶(COX2)。BN诱导CAF释放PGE2增加，PGE2刺激结肠上皮细胞迁移。这一建议的新假设是，BN刺激结直肠癌的发生，不是通过直接激活结肠上皮细胞，而是间接通过激活CAF。为了验证这一假设，我们将解决以下两个目标：目标1：确定BN激活大肠CAF的机制。我们将检测BN介导的CAF的增殖、MEK/ERK通路的激活、COX-2启动子活性的调节以及肝细胞生长因子在CAF中的上调。目的2：探讨BN激活的CAF在结直肠癌肿瘤进展和转移中的作用。我们将比较正常成纤维细胞、BN激活的CAF和它们的shRNA-GRPR敲除对应细胞与人结直肠癌细胞在小鼠异种移植和肝转移模型中的差异。这项建议的短期目标是确定CAF调节肿瘤上皮细胞生物学的机制。这些目标的成功完成将推进确定Gl肽激素通过调节间质/上皮相互作用来调节肿瘤进展的长期目标。由于只有8%的结直肠癌实际上存在伴随的APC、K-ras和p-53基因突变，其他表观遗传学事件，如CAF中GRPR的过度表达，可能是结直肠癌进展和转移的致病改变中的一个额外考虑因素。靶向CAF上的这些细胞表面受体，结合抗血管生成和细胞毒药物，可能会提供另一种合理的治疗选择。最后，由于我们已经证明GRPR是结肠癌CAF中COX2的上游激活剂，因此对GRPR信号机制的研究可能会阐明副作用较少的结肠癌预防方法。 相关：结肠癌异常表达胃泌素释放肽激素(GRP)及其受体(GRPR)。这种受体的激活控制着基因，这些基因可以增加肿瘤的生长、运动和允许癌细胞扩散的酶的分泌。我们发现，95%的结肠肿瘤在被称为成纤维细胞的支持细胞中表达GRPR，成纤维细胞与结肠癌细胞相互作用。通过对相关机制的研究，我们可以更好地设计针对这些支持细胞中GRPR的药物，从而提高患者的生存率。

DESCRIPTION (provided by applicant): Colorectal cancers aberrantly express gastrin-releasing peptide (GRP) hormone and its cognate receptor (GRPR). Ample in vivo and in vitro experiments suggest that GRP, or bombesin (BN), the pharmacological homologue of GRP, promotes colorectal cancer growth and progression. Previous studies have focused on the cancer cells, whereas, we provide compelling data, that in 95% of freshly harvested human colorectal cancers examined, functional GRPR is expressed by "carcinoma-associated fibroblasts" (CAFs), not the malignant epithelial cells. BN treatment of these CAFs stimulates their proliferation, activates NF(B, and induces cyclooxygenase (COX2) in an MEK/ERK pathway-dependent manner. BN induces increased PGE2 release from CAFs and PGE2 stimulates the migration of colonic epithelial cells in vitro. The novel hypothesis of this proposal is that BN stimulates colorectal carcinogenesis, not by directly activating the colonic epithelial cell, but rather, indirectly, by activating the CAFs. To test this hypothesis, we will address the following two aims: Aim 1: To determine the mechanisms by which BN activates colorectal CAFs. We will examine BN-mediated CAF proliferation, activation of the MEK/ERK pathway, regulation of COX-2 promoter activity and upregulation of Hepatocyte Growth Factor in CAFs. Aim 2: To determine how BN-activated CAFs contribute to neoplastic progression and metastasis in colorectal cancer. We will compare the differences between normal fibroblasts, BN-activated CAFs, and their shRNA-GRPR knockdown counterparts when co-implanted with human colorectal cancer cells in murine xenograft and liver metastasis models. The short-term goal of this proposal is to identify mechanisms by which CAFs modulate the biology of tumor epithelial cells. Successful completion of these aims will advance the long-term goal of defining the role of Gl peptide hormones in regulating tumor progression by mediating stromal/epithelial interactions. Since only 8% of colorectal cancers examined actually harbor concomitant mutations of APC, K-ras, and p-53, additional epigenetic events, such as the overexpression of GRPR, in CAFs may be an additional consideration in the pathogenic alterations of colorectal cancer progression and metastasis. Targeting these cell surface receptors on CAFs, combined with anti-angiogenic and cytotoxic drugs, may provide an additional, rational therapeutic option. Finally, because we have shown that GRPR is an upstream activator of COX2 in CAFs in colon cancer, the study of signaling mechanisms by GRPR may elucidate methods of colon cancer prevention with fewer side effects. RELEVANCE: Colon cancers aberrantly express gastrin-releasing peptide hormone (GRP) and its receptor (GRPR). Activation of this receptor controls genes which can increase tumor growth, motility, and secretion of enzymes that allow cancer cells to spread. We show that 95% of colon tumors express GRPR in supporting cells called fibroblasts, which interact with the colon cancer cells. By studying the relevant mechanisms, we may better design drugs to target GRPR in these supporting cells, and thus improve patient survival.