Genetic expression in pancreatic adenocarcinoma cells predicts metastatic potential and response to oxidative stress

Abstract

Of all patients receiving a potentially curative pancreaticoduodenectomy for pancreatic adenocarcinoma, 70% will go on to develop liver metastases as their primary site of recurrence. All of these patients will then die a cancer-related death. Hematogenous pancreatic tumor metastasis to the liver is a highly selective process that occurs through non-random mechanistic steps including vascular invasion, embolization, sinusoidal arrest, extravasation into the parenchyma, and proliferation. Multiple mechanisms lead to the destruction of tumor cells during hematogenous metastasis. These mechanisms include mechanical shear stress after entrapment in the sinusoid and immunologic mechanisms such as the acute inflammatory response of the sinusoidal endothelial cells (SEC). SEC produce reactive oxygen species (ROS) in response to antigen stimulation causing oxidative stress to the tumor cells, leading to cell death. Tumor cells with inherent abilities to resist oxidative stress should produce metastatic disease more effectively. In our laboratory, multiple pancreatic tumor cell lines, including MiaPaCa-2 (Mia) and Panc-1, are utilized to produce orthotopic pancreatic adenocarcinoma xenografts in mice. Orthotopic Mia tumors produce metastatic disease in more than 80% of mice. In contrast, Panc-1 rarely produces metastases (less than 20%). We hypothesize the phenotypic differences noted between Panc-1 and Mia tumor metastases will be recapitulated through high-density genetic expression array analysis and through in vitro studies of these cells when exposed to oxidative stress. Evaluation of Mia and Panc-1 tumor cells using high-density gene expression array analysis revealed significant differences. Mia has a more than 5-fold higher expression of the free-radical scavenger Glutathione (GSH), which has been linked by prior experiments to resistance to oxidative stress. There is also a 3.6-fold higher expression of superoxide dismutase (SO), an enzyme that reacts with superoxide radicals and converts them into less noxious molecules. To evaluate the functional nature of these differences, Panc-1 and Mia cells were subjected to oxidative stress in vitro using hydrogen peroxide (H 2O 2). Using an MTT assay, cell proliferation and survival was compared between Mia and Panc-1 and non-stressed controls. Mia had only a 20% decrease in cell number when compared to control. Interestingly, Panc-1 cells in the same H 2O 2 concentration had a more than 70% reduction in the number of cells. In conclusion, the release of reactive oxygen species from sinusoidal endothelial cells plays an important role in the prevention of hepatic tumor metastasis formation. Characteristics at the cellular level imparting inherent resistance to oxidative stress lead to significant differences in metastatic potential. Manipulation of this system might therefore lead to novel methods of treatment for an otherwise recalcitrant disease.