In vitro Modeling of Human Pancreatic Duct Epithelial Cell Transformation Defines Gene Expression Changes Induced by K-ras Oncogenic Activation in Pancreatic Carcinogenesis

Abstract

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16INK4A , Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanINcarcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras G12V oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogenactivated protein kinase, AKT, and nuclear factor-KB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin B3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis. (Cancer Res 2005; 65(12): 5045-53)

K-Ras Promotes Growth Transformation and Invasion of Immortalized Human Pancreatic Cells by Raf and Phosphatidylinositol 3-Kinase Signaling

“…2B). Our results differ with a recent study that found that activated K-Ras did not alter the anchorage-dependent growth properties of immortalized pancreatic ductal epithelial cells (26).…”

“…To date, several pancreatic cell systems have been described to study Ras oncogenesis (21)(22)(23)(24)(25)(26). Although Lohr et al (22) generated immortalized cells expressing SV40 large T antigen and mutant K-Ras that formed contact-independent colonies in vitro and orthotopic tumors in mice, these were of bovine and not human origin.…”

“…Although Lohr et al (22) generated immortalized cells expressing SV40 large T antigen and mutant K-Ras that formed contact-independent colonies in vitro and orthotopic tumors in mice, these were of bovine and not human origin. Tsao et al (26) investigated human pancreatic ductal epithelial (HPDE) cells immortalized with human papillomavirus (HPV) 16 E6 and E7 oncogenes to inactivate p53 and Rb, respectively, and further transformed these cells with mutant K-Ras. The K-Ras-transformed HPDE cells formed tumors in nude mice but, interestingly, showed no indication of in vitro transformation as indicated by proliferation or contact-independent growth.…”

Keratinocyte Growth Factor/Fibroblast Growth Factor-7-regulated Cell Migration and Invasion through Activation of NF-κB Transcription Factors

“…The interplay is based on three basic processes: production of autocrine and paracrine factors, cell-matrix contact, and signaling by direct cell-cell interactions (39,40). To establish cell culture models for the study of the tumorigenesis of human pancreatic cancer in vitro, we have developed immortalized cultures of HPDE cells derived from normal epithelium (41). The aim of these studies was to investigate the immortalization-transformation sequence of these cells to understand better the implication of various mutations, including the role of paracrine factors such as KGF/FGF-7, a potent mitogenic paracrine mediator of epithelial cells, in the development of pancreatic cancer.…”

Establishment of three-dimensional cultures of human pancreatic duct epithelial cells

“…In particular, they have limited genetic aberrations, grow in an anchorage-dependent manner, and are non-tumorigenic in mice [8]. Further, stable expression of oncogenic Ki-Ras G12V caused transformation of HPDEE6E7 cells and activated signaling molecules implicated in human pancreatic cancer development such as the PI3K protein kinase mediator Akt [9].…”