Role of Alcohol as a risk factor in the induction of Pancreatic Carcinogenesis

胰腺癌是人类最致命和最具破坏性的恶性肿瘤之一，5年生存率为 约6%。由于缺乏可靠的诊断方法，胰腺癌（CaP）的早期诊断具有挑战性。 标志物和对病因的有限理解。大量饮酒构成了 这是一个严重的社会问题，并已被确定为胰腺癌的危险因素。酒精使用障碍 退伍军人和平民经常同时发生;因此，这些人群容易面临 不成比例的负担增加了患胰腺癌的风险。该应用程序将解决评估 酒精增加胰腺癌风险的细胞和分子机制。它将高度 这对于理解这种复杂疾病关联的潜在机制是重要和有益的。 因此，本申请的首要目标是检查酒精毒性在以下方面的生物学作用： 诱导胰腺癌和鉴定介导酒精诱导的细胞转化的基因， 胰腺癌的进展。胰腺癌的生长和持续依赖于癌症干细胞 肿瘤干细胞（CSC）也诱导复发并抵抗当前的化疗和放疗。我们的初步数据 表明乙醇诱导正常胰腺导管上皮细胞的细胞转化。慢性 乙醇暴露通过引起炎症诱导HPNE细胞恶性转化（上调IL-6， IL-8、TNFα和IL-1β基因抑制谷胱甘肽S-转移酶（GST）和谷胱甘肽过氧化物酶（GPx）， 和诱导的干细胞标志物）。有趣的是，我们发现Engrailed-2（EN 2）的表达模式， 在乙醇转化的HPNE细胞中，含有同源框的转录因子显著改变， EN 2的诱导与转化率相关。然而，生物学功能和分子 EN 2在胰腺癌中的作用机制尚未研究。我们证明， 人胰腺正常导管上皮细胞中的EN 2通过产生CSC诱导恶性转化， 提示EN 2的表达与胰腺癌的发生有关。 此外，我们已经表明，EN 2调节多能性维持因子的转录，这是必需的。 维持干细胞自我更新的能力，并且EN 2的表达受到严格调控， 癌EN 2在胰腺CSC和癌细胞系中高度表达，但在正常胰腺细胞中不表达， 正常胰腺组织。此外，我们已经表明抑制EN 2减弱了CSC特征， Notch途径和小鼠肿瘤生长。基于我们的初步研究，我们假设酒精 在转化过程中诱导炎性环境，导致诱导EN 2，其充当关键 多能性和自我更新的调节剂，并且在乙醇转化的细胞中抑制EN 2会减弱其 致瘤潜力研究酒精在胰腺癌中的作用，并解决上述假设 我们建议整合功能、分子、生物化学和组织学方法。EN 2的收益和损失 将在体外和体内进行功能研究。EN 2表达将被CRISPR敲低- Cas9体外技术。将生成新型三重转基因小鼠（LSL-KrasG 12 D; LSL-EN 2 +/+; Pdx-Cre） 并将在存在或不存在EN 2的情况下比较酒精对胰腺癌发生的影响 （KrasG 12 D/EN 2 +/+和KrasG 12 D/EN 2-/-小鼠）。我们将研究Notch-Hes 1通路参与 评估由EN 2介导的酒精的生物效应。本申请中提出的研究将提高 我们对乙醇诱导的正常胰腺导管细胞转化的生物学理解 上皮细胞，这对于胰腺癌的管理将是重要的。

Pancreatic cancer is one of the most lethal and devastating human malignancies, with a 5-year survival rate of approximately 6%. Early diagnosis of pancreatic cancer (CaP) is challenging due to lack of reliable diagnostic markers and limited understanding of the etiological factors. Heavy alcohol consumption constitutes a significant social problem and has been identified as a risk factor for pancreatic cancer. Alcohol use disorders frequently co-occur in military veterans and civilians; thus, these populations are vulnerable to face a disproportionate burden of increased risk to develop pancreatic cancer. This application will address evaluating the cellular and molecular mechanism by which alcohol increases pancreatic cancer risk. It will be highly significant and beneficial to understand the underlying mechanism of this complex disease association. Therefore, the overarching goal of this application is to examine the biological role of alcohol toxicity in the induction of pancreatic cancer and to identify genes mediating alcohol induced cellular transformation and progression of pancreatic cancer. Pancreatic cancer growth and persistence depend on the cancer stem cells (CSCs) that also induce recurrence and resist current chemo- and radiotherapies. Our preliminary data demonstrate that ethanol induced cellular transformation of normal pancreatic ductal epithelial cells. Chronic ethanol exposure induced malignant transformation of HPNE cells by causing inflammation (upregulated IL-6, IL-8, TNFα, and IL-1β genes, inhibited glutathione S-transferases (GSTs) and glutathione peroxidases (GPx), and induced stem cell markers). Interestingly, we identified that the expression pattern of Engrailed-2 (EN2), a homeobox-containing transcription factor was significantly altered in ethanol-transformed HPNE cells, and the induction of EN2 correlated with the rate of transformation. However, the biological function and molecular mechanism of EN2 in pancreatic cancer have never been examined. We demonstrate that overexpression of EN2 in human pancreatic normal ductal epithelial cells induces malignant transformation by generating CSCs, and regulating Notch-Hes1 pathway; suggesting that the expression of EN2 is oncogenic for pancreatic cancer. Further, we have shown that EN2 regulates transcription of pluripotency maintaining factors, that are required for sustaining the stem cells capacity for self-renewal and that the expression of EN2 is tightly regulated in cancer. EN2 is highly expressed in pancreatic CSCs and cancer cell lines, but not in normal pancreatic cells or normal pancreatic tissues. Further, we have shown that inhibition of EN2 attenuated CSC characteristics, Notch pathway, and tumor growth in mice. Based on our preliminary studies, we hypothesize that alcohol induces inflammatory environment during transformation resulting in induction of EN2, which acts as a key regulator of pluripotency and self-renewal and inhibition of EN2 in ethanol-transformed cells attenuates their tumorigenic potential. To examine the role of alcohol in pancreatic cancer and address the above hypothesis we propose to integrate functional, molecular, biochemical and histological approaches. Gain and loss of EN2 functional studies will be performed in vitro and in vivo. EN2 expression will be knocked down by CRISPR- Cas9 technology in vitro. Novel triple transgenic mice (LSL-KrasG12D; LSL-EN2+/+; Pdx-Cre) will be generated and effects of alcohol on pancreatic carcinogenesis will be compared in the presence or absence of EN2 (KrasG12D/EN2+/+, and KrasG12D/EN2-/- mice). We will examine the involvement of Notch-Hes1 pathway to assess the biological effects of alcohol mediated by EN2. The studies proposed in this application will enhance our understanding of the biology of ethanol-induced cellular transformation of normal pancreatic ductal epithelial cells, which will be significant for the management of pancreatic cancer.