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.