Mechanisms of Tumorigenesis in Pancreatic Epithelial Cells

描述(申请人提供)：胰腺癌是美国成人癌症死亡的第四大原因。五年存活率继续保持在1-3%。在诊断时，大多数胰腺癌患者都有晚期和转移性疾病。虽然胰腺癌的基因图谱正在形成，但这些基因改变如何导致胰腺癌独特的表型和临床病程仍不清楚。胰腺上皮细胞致癌、侵袭和转移的潜在机制尚不清楚。由于缺乏正常的人胰腺导管上皮细胞来分析基因改变在肿瘤发生和转移中的作用，以及缺乏概括胰腺癌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了解。因此，这项研究的长期目标是使用E6E7永生化的人胰腺导管上皮细胞(HPDE/E6E7)来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这些基因变化导致的表型。最近的研究结果表明：(1)突变的K-ras4B(G12V)转化HPDE/E6E7并诱导小鼠原位成瘤；(2)鉴定了K-ras下游的靶基因；(3)突变的IkappaBalpha(S32，36A)介导的结构性抑制NF-kappaB激活抑制了胰腺癌细胞在裸鼠原位模型中的肝转移；(4)Smad4的过表达抑制了胰腺癌细胞系的成瘤。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的人胰腺导管上皮细胞中的表达而诱导肿瘤或转移表型的假说将得到验证。其具体目的是：(1)研究K-ras诱导HPDE/E6E7细胞致癌转化的机制；(2)确定组成性激活的核因子-kappaB在诱导转移中的作用。(3)确定Smad4在HPDE/E6E7细胞致瘤和转移表型启动中的作用。携带胰腺癌标志性突变的各种细胞系的产生应该能够识别诱导这种疾病的肿瘤形成和转移表型所需的基因改变。此外，肿瘤进展的机制，包括与这些胰腺癌标志性突变相关的基因组不稳定性和信号级联变化，可以在相关的体内和体外环境下使用分子和生化方法进行分析。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的治疗策略提供基础。

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.