Mechanism of Tumorigenesis in Pancreatic Epithelial Cell

描述（由申请人提供）：胰腺癌是美国成人癌症死亡率的第四大原因。5年生存率仍保持在1- 3%。在诊断时，大多数胰腺癌患者表现为晚期和转移性疾病。虽然胰腺癌的遗传特征正在出现，但这些遗传改变如何引起胰腺癌的独特表型和临床过程仍然是未知的。胰腺上皮细胞致瘤、侵袭和转移的潜在机制仍有待阐明。胰腺癌的分子基础的理解的进展受到阻碍，部分是由于缺乏正常的人胰腺导管上皮细胞的肿瘤发生和转移的遗传改变的作用进行分析，和实验动物模型，概括了这种疾病的分子发病机制和肿瘤生物学。因此，拟议研究的长期目标是使用携带这种疾病中的标志性遗传改变的E6 E7永生化人胰腺导管上皮（HPDE/E6 E7）细胞研究胰腺肿瘤发生和转移的分子基础，并使用原位小鼠模型确定由这些遗传改变诱导的表型。最近的调查结果表明：（1）突变型K-ras 4 B（G12 V）转化HPDE/E6 E7并在原位小鼠模型中诱导弱致瘤性;（2）鉴定K-ras下游靶基因;（3）突变型IkappaB α（G12 V）转化HPDE/E6 E7并在原位小鼠模型中诱导弱致瘤性。（S32，36 A）介导的组成型NF-κ B活化的抑制在原位裸鼠模型中抑制胰腺癌细胞的肝转移;（4）Smad 4过表达抑制胰腺癌细胞系的成瘤性。将对K-ras和NF-κ B的激活或Smad 4的失活通过改变其下游靶基因在永生化人胰腺导管上皮细胞中的表达来诱导致瘤性或转移性表型的假设进行检验。具体目标是：（1）研究K-ras基因诱导HPDE/E6 E7细胞恶性转化的机制。（2）研究组成性激活的NF-κ B在诱导肿瘤转移中的作用。(3)确定Smad 4在HPDE/E6 E7细胞中启动肿瘤发生和转移表型中的作用。产生的各种细胞系，携带的签名突变的胰腺癌应允许鉴定所需的遗传改变，在演唱会上，以诱导肿瘤和转移表型的这种疾病。此外，可以使用分子和生物化学方法在相关的体内和体外背景下分析肿瘤进展的潜在机制，包括与这些胰腺癌特征突变相关的基因组不稳定性和信号级联的改变。更好地了解基因改变诱导肿瘤发生和转移表型的机制将为胰腺癌的早期发现和有效治疗策略提供基础。

DESCRIPTION (provided by applicant): Pancreatic adenocarcinoma is the fourth leading cause of adult cancer mortality in the United States. The five-year survival rate continues at 1-3%. At the time of diagnosis, most pancreatic cancer patients present with advanced and metastatic disease. Although a genetic profile for pancreatic caner is emerging, it is still unknown how these genetic alterations elicit the unique phenotypes and clinical course of pancreatic cancer. The underlying mechanisms, by which pancreatic epithelial cells become tumorigenic, invasive and metastatic, remain to be elucidated. The advance in understanding of the molecular basis of pancreatic cancer is hindered in part due to the lack of normal human pancreatic ductal epithelial cells for analyzing the role of genetic alterations in tumorigenesis and metastasis, and experimental animal models that recapitulate the molecular pathogenesis and tumor biology of this disease. Therefore, the long-term objective of the proposed research is study the molecular basis of pancreatic tumorigenesis and metastasis using an E6E7-immortalized human pancreatic ductal epithelial (HPDE/E6E7) cells that carry the signature genetic alterations in this disease, and determine the phenotypes induced by these genetic alterations using an orthotopic mouse model. The recent findings show that: (1) mutated K-ras4B (G12V) transformed HPDE/E6E7 and induced weak tumorigenicity in orthotopic mouse model; (2) the K-ras downstream target genes were identified; (3) the mutant IkappaBalpha (S32, 36A) mediated inhibition of constitutive NF-kappaB activation suppressed liver metastasis of pancreas cancer cells in an orthotopic nude mouse model; (4) overexpression of Smad4 inhibits tumorigenesis of pancreatic cancer cell lines. The hypothesis, activation of K-ras and NF-kappaB, or inactivation of Smad4 induces tumorigenic or metastatic phenotype by altering the expression of their downstream target genes in immortalized human pancreatic ductal epithelial cells, will be tested. The specific aims are: (1) Study the mechanism of K-ras induced oncogenic transformation of HPDE/E6E7 cells (2) Determine the function of constitutively activated NF-kappaB in induction of metastasis. (3) Determine the role of Smad4 in initiating tumorigenic and metastatic phenotypes in HPDE/E6E7 cells. Generation of various cell lines that carry the signature mutation of pancreatic cancer should permit the identification of the genetic alterations required in concert to induce tumorigenic and metastatic phenotype of this disease. Furthermore, mechanisms underlying tumor progression, including genomic instability and alterations in signal cascades associated with these pancreatic cancer signature mutations can be analyzed in a relevant in vivo and in vitro context using molecular and biochemical methods. A better understanding of the mechanisms of genetic alterations in induction of tumorigenic and metastatic phenotypes will provide a basis for developing early detection and effective treatment strategies for pancreatic cancer.