Role of IL-6/STAT3 Signaling in the Neoplastic Progression of Barrett's Esophagus

描述（由申请人提供）： 胃食管反流病和Barrett食管是退伍军人患者的常见疾病，是食管腺癌的主要危险因素，食管腺癌是一种致命的肿瘤，在过去的几十年中其发生率增加了六倍以上。由于对Barrett化生的肿瘤进展的分子事件的了解有限，阻碍了预防或治愈这种致命肿瘤的治疗方法的发展。 在巴雷特食管的癌变过程中，遗传改变积累，使细胞能够提供自己的生长信号，避免生长抑制信号，抵抗细胞凋亡，无限制复制，合成新血管，侵入邻近器官和转移。Hanahan和温伯格在2000年描述了这些癌症的生理“标志”，正常细胞可以通过破坏少数几个关键的生长调节途径来获得这些标志。最近，癌症相关炎症已被提出作为癌症的另一个生理标志，其可以通过两种途径建立：1）外在途径，其中诸如反流性食管炎的疾病引起有助于致癌的组织炎症，和2）内在途径，其中癌前细胞获得产生炎性肿瘤微环境的遗传异常。这两种途径都可以集中在某些关键的下游靶点上，例如IL-6，一种已知可促进细胞生长的强效炎症细胞因子，以及STAT 3，一种在IL-6信号转导中起关键作用的转录因子。反流性食管炎引起的慢性食管炎症被广泛认为是促进Barrett食管癌变的外在途径，但很少有人关注Barrett食管化生恶性进展中的内在炎症途径和IL-6/STAT 3信号传导的作用。 由于缺乏合适的模型系统，对巴雷特化生转化为腺癌的早期分子事件的研究受到阻碍。在我们的Merit Review资助的最后一个周期中，我们通过建立非肿瘤性巴雷特上皮（BAR-T）细胞的培养物来解决这一缺陷，我们通过端粒酶的强制表达使这些细胞永生化。使用p53敲低和致癌H-RasG 12 V的强制表达的组合，我们已经诱导了那些BAR-T细胞的恶性转化，并且在该过程中，建立了一系列具有明确定义的遗传改变的巴雷特上皮细胞系，其代表了从良性巴雷特化生到癌症的转变中的各个阶段。我们的初步数据表明，转化的巴雷特细胞表达IL-6 mRNA，并表现出IL-6蛋白分泌的显着增加。此外，我们发现IL-6信号传导导致转化细胞中磷酸化STAT 3水平的增加，并且IL-6/STAT 3途径的激活赋予细胞抵抗凋亡和分泌血管生成促进因子的能力。我们建议使用我们独特的Barrett上皮细胞系系列来探索IL-6/STAT 3通路在驱动Barrett食管恶性转化中的作用。 我们假设IL-6/STAT 3信号通路的激活在驱动Barrett化生的肿瘤转化中起关键作用。基于我们的初步数据，我们研究的目的是描述p53失活和Ras活化导致Barrett上皮细胞中IL-6/STAT 3信号通路活化的机制，以确定IL-6/STAT 3通路的活化和失活对Barrett细胞系中肿瘤转化水平的影响，并使用来自患有非发育异常的巴雷特食管、发育异常的巴雷特食管（低级和高级）和巴雷特腺癌的患者的活检标本，在体内巴雷特食管的肿瘤进展期间将p53失活和Ras途径活化与IL-6/STAT 3途径活化相关联。 公共卫生相关性： 对退伍军人医疗保健的潜在影响是，我们的研究将确定关键的分子途径，可以有针对性地预防癌症在我们许多患有巴雷特食管的退伍军人患者中发展，并治疗那些已经患有巴雷特食管癌症的患者。

DESCRIPTION (provided by applicant): Project Summary/Abstract Gastroesophageal reflux disease and Barrett's esophagus, common disorders in Veteran patients, are the major risk factors for esophageal adenocarcinoma, a lethal tumor whose frequency has increased more than six-fold in the past few decades. The development of therapies to prevent or cure this lethal tumor has been hampered by limited understanding of the molecular events underlying the neoplastic progression of Barrett's metaplasia. During carcinogenesis in Barrett's esophagus, genetic alterations accumulate that enable the cells to provide their own growth signals, avoid growth inhibitory signals, resist apoptosis, replicate without limit, synthesize new blood vessels, invade adjacent organs and metastasize. These physiologic "hallmarks" of cancer, which were described by Hanahan and Weinberg in 2000, can be acquired by normal cells through disruptions in surprisingly few key growth regulatory pathways. Recently, cancer-related inflammation has been proposed as another physiologic hallmark of cancer that can be established through two pathways: 1) an extrinsic pathway in which disorders such as reflux esophagitis cause tissue inflammation that contributes to carcinogenesis, and 2) an intrinsic pathway in which the precancerous cells acquire genetic abnormalities that produce an inflammatory tumor microenvironment. Both pathways can converge on certain key downstream targets such as IL-6, a potent inflammatory cytokine that is known to promote cellular growth, and STAT3, a transcription factor that has a key role in IL-6 signal transduction. Chronic esophageal inflammation due to reflux esophagitis is widely regarded as the extrinsic pathway for promoting carcinogenesis in Barrett's esophagus, but little attention has been directed at the intrinsic inflammatory pathway and the role of IL-6/STAT3 signaling in the malignant progression of Barrett's metaplasia. Studies on the early molecular events that transform Barrett's metaplasia into adenocarcinoma have been hampered by the lack of appropriate model systems. During our last cycle of Merit Review funding, we addressed this deficiency by establishing cultures of non- neoplastic Barrett's epithelial (BAR-T) cells that we immortalized through the forced expression of telomerase. Using a combination of p53 knockdown and forced expression of oncogenic H- RasG12V, we have induced the malignant transformation of those BAR-T cells and, in the process, established a series of Barrett's epithelial cell lines with well defined genetic alterations representing various stages in the transition from benign Barrett's metaplasia to cancer. Our preliminary data demonstrate that the transformed Barrett's cells express IL-6 mRNA and exhibit a significant increase in IL-6 protein secretion. Moreover, we have found that IL-6 signaling causes an increase in phospho-STAT3 levels in the transformed cells, and that activation of the IL-6/STAT3 pathway endows the cells with the ability to resist apoptosis and to secrete angiogenesis-promoting factors. We propose to use our unique series of Barrett's epithelial cell lines to explore the role of the IL-6/STAT3 pathway in driving the malignant transformation of Barrett's esophagus. We hypothesize that activation of the IL-6/STAT3 signaling pathway plays a key role in driving the neoplastic transformation of Barrett's metaplasia. Based on our preliminary data, the objectives of our study are to delineate the mechanism(s) whereby p53 inactivation and Ras activation result in activation of the IL-6/STAT3 signaling pathway in Barrett's epithelial cells, to determine the effects of activation and inactivation of the IL-6/STAT3 pathway on the level of neoplastic transformation in Barrett's cell lines, and to correlate p53 inactivation and Ras pathway activation with the activation of the IL-6/STAT3 pathway during the neoplastic progression of Barrett's esophagus in vivo using biopsy specimens from patients with non- dysplastic Barrett's esophagus, dysplastic Barrett's esophagus (low-grade and high-grade), and Barrett's adenocarcinoma. PUBLIC HEALTH RELEVANCE: Project Narrative The Potential Impact on Veterans Health Care is that our studies will identify key molecular pathways that can be targeted to prevent cancer from developing in our many Veteran patients who have Barrett's esophagus, and to treat those who already have cancers in Barrett's esophagus.