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年存活率为 大约6%。由于缺乏可靠的诊断，胰腺癌的早期诊断具有挑战性。 标记物和对致病因素的认识有限。大量饮酒构成一种 这是一个重大的社会问题，已被确定为胰腺癌的危险因素。酒精使用障碍 通常在退伍军人和平民中共同发生；因此，这些人口很容易面临 患胰腺癌风险增加的不成比例的负担。这个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。这将是非常重要的 这对理解这种复杂疾病关联的潜在机制具有重要意义。 因此，这项应用的首要目标是检查酒精毒性在人体内的生物学作用。 诱导胰腺癌并鉴定介导酒精诱导细胞转化的基因和 胰腺癌的进展。胰腺癌的生长和生存依赖于肿瘤干细胞 (CSCs)也会导致复发并抵抗目前的化疗和放射治疗。我们的初步数据 证明乙醇可诱导正常胰腺导管上皮细胞转化。慢性 酒精暴露通过引起炎症(上调IL-6， IL-8、肿瘤坏死因子α和IL-1β基因可抑制谷胱甘肽S转移酶和谷胱甘肽过氧化物酶； 和诱导的干细胞标志物)。有趣的是，我们发现Engrailed-2(En2)，a的表达模式 在乙醇转化的HPNE细胞中，含有同源异型盒的转录因子显著改变，并且 EN2的诱导与转化率相关。然而，生物功能和分子水平 EN2在胰腺癌中的作用机制从未被研究过。我们证明了过度表达 人胰腺正常导管上皮细胞中的EN2通过产生CSCs诱导恶性转化， 调控Notch-Hes1通路，提示EN2的表达在胰腺癌中起致癌作用。 此外，我们还发现en2调节多能性维持因子的转录，这是必需的。 以维持干细胞自我更新的能力，并且EN2的表达在 癌症。EN2在胰腺CSCs和癌细胞中高表达，但在正常胰腺细胞或 正常的胰腺组织。此外，我们已经证明，抑制EN2可以减弱CSC的特征， Notch途径与小鼠肿瘤生长的关系。根据我们的初步研究，我们假设酒精 在转化过程中诱导炎症环境，导致EN2的诱导，而EN2是一个关键 乙醇转化细胞的多能性和自我更新调节因子EN2的抑制作用 致癌潜力。研究酒精在胰腺癌中的作用并解决上述假说 我们建议将功能、分子、生化和组织学方法结合起来。EN2的得失 功能研究将在体外和体内进行。EN2的表达将被CRISPR- Cas9技术在体外。将产生新的三重转基因小鼠(LSL-KrasG12D；LSL-EN2+/+；PDX-Cre 酒精对胰腺癌发生的影响将在有无EN2的情况下进行比较。 (KrasG12D/EN2+/+和KrasG12D/EN2-/-小鼠)。我们将研究Notch-Hes1通路是否参与 评估EN2介导的酒精的生物学效应。这项申请中提出的研究将增强 乙醇诱导正常胰腺导管细胞转化生物学的认识 上皮细胞，这将对胰腺癌的治疗具有重要意义。

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.