Mechanisms of Tumorigenesis in Pancreatic Epithelial Cells

描述（由申请人提供）：胰腺腺癌是美国成人癌症死亡的第四大原因。5年生存率维持在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。虽然胰腺癌的遗传谱正在出现，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未知。胰腺上皮细胞成为致瘤性、侵袭性和转移性的潜在机制仍有待阐明。由于缺乏正常的人类胰腺导管上皮细胞来分析遗传改变在肿瘤发生和转移中的作用，以及缺乏概括这种疾病的分子发病机制和肿瘤生物学的实验动物模型，阻碍了对胰腺癌分子基础的理解的进展。因此，本研究的长期目标是利用携带该疾病标志性遗传改变的E6E7永生化人胰腺导管上皮细胞（HPDE/E6E7）研究胰腺肿瘤发生和转移的分子基础，并利用原位小鼠模型确定这些遗传改变诱导的表型。最近的研究结果表明：(1)突变的K-ras4B （G12V）在原位小鼠模型中转化HPDE/E6E7并诱导弱致瘤性；(2)鉴定出K-ras下游靶基因；(3)在原位裸鼠模型中，突变体IkappaBalpha （S32, 36A）介导的组成型NF-kappaB激活抑制了胰腺癌细胞的肝转移；(4) Smad4过表达抑制胰腺癌细胞系的肿瘤发生。K-ras和NF-kappaB的激活或Smad4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，这一假说将被验证。具体目的是：(1)研究K-ras诱导HPDE/E6E7细胞癌化转化的机制(2)确定组成型活化NF-kappaB在诱导转移中的作用。(3)确定Smad4在启动HPDE/E6E7细胞的致瘤和转移表型中的作用。携带胰腺癌特征突变的各种细胞系的产生应该允许鉴定诱导这种疾病的致瘤性和转移性表型所需的遗传改变。此外，肿瘤进展的潜在机制，包括基因组不稳定性和与这些胰腺癌特征突变相关的信号级联的改变，可以在相关的体内和体外环境中使用分子和生化方法进行分析。更好地了解遗传改变诱导致瘤性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.