Masako Nakanishi, Ph.D. - US grants

Project Summary Activation of the COX-2/mPGES-1/PGE2 signaling axis is a hallmark of many cancers, including colorectal (CRC), leading to the implementation of therapeutic strategies for targeting COX-2 activity. Despite their demonstrated chemopreventive efficacy, long-term treatment with COX inhibitors poses significant health risks as a result of the global suppression of physiological prostanoids. We have shown that targeting the downstream terminal PGE2 synthase, mPGES-1 (Ptges), specifically reduces inducible PGE2 formation and confers protection against colon carcinogenesis in several mouse colon cancer models. In our lipidomic analysis to define the redistribution of the COX products, we found significant metabolic shunting towards PGD2 and thromboxane B2 (TXB2) in the colon tumors of mPGES-1-deficient Apc-mutant mice. These metabolites are primarily produced by mast cells, a cell type that has been frequently detected within the tumor microenvironment. Prostanoids have been shown to directly influence the functional activity of mast cells, and a large body of evidence suggests that mast cell-derived mediators including PGD2 contribute to cancer pathogenesis. These observations serve as a basis for our hypothesis that mPGES-1 targeting causes metabolite shifts in the mast cells, which alters tumor microenvironment towards less growth-promoting. We propose experiments to develop a better understanding of functional roles of mast cells on colon tumor development, and how genetic targeting of mPGES-1 will affect its actions. Aim 1 will extend our pilot lipidomic analysis of colon tumors to confirm the extent of prostanoid shunting and also to identify metabolically distinct prostanoid profiles that are associated with both size and macroscopic features of Apc-driven tumors formed in the presence or absence of mPGES-1. Aim 2 will isolate tumor-associated mast cells from the colon of Apc mice and examine the impact of mPGES-1 blockade on mast cell activation. We will measure the release and production of mediators including histamine, proteases and a panel of prostanoids and cytokines, which directly impact the tumor microenvironment. We will also explore how mast cell responds to PGE2, which is often found in high concentrations within the tumor microenvironment. Aim 3 will generate tumor organoids from Apc mice with or without mPGES-1, and test the direct effects of mast cell-derived mediators (Aim 2) on cancer stem cell homeostasis. Results from these studies will shed light on the roles of mPGES-1 activity in mast cells, and also provide a novel paradigm for the tumor protective mechanism by PGE2 inhibition.

Project Summary The development of genetically engineered mouse models (GEMMs) together with the advent of Cre- LoxP technologies and tissue-specific promoters has become excellent tools to assess the functional roles of cancer-associated genes. In recent years, several promoters have been successfully adapted to drive Cre expression specifically within the intestinal epithelium. These promoters have proven useful for manipulating gene expression in normal cells throughout intestinal tract; however, a genetic system that will specifically target intestinal tumors for transgene expression has not yet been developed. Therefore, this exploratory proposal aims to establish a novel mouse model in which the forced expression of a given transgene can be re-introduced through tamoxifen-controlled Cre expression in intestinal tumors. To achieve tumor-specific expression, we will use stearoyl-CoA desaturase (Scd)-3 gene locus as a promoter. We have recently found that Scd3 was consistently up-regulated in intestinal tumors, but not in normal mucosa, suggesting that it may serve as a specific intestinal tumor marker in mice. In Aim 1, we propose to generate Scd3-CreER+/- mice, and Cre activity is validated using a reporter mice. Optimal timing and duration of Cre activity and the efficiency of Cre-Lox recombination will be determined to establish a viable mouse model. In Aim 2, we will then characterize the efficiency of Scd3-CreER system in Apc?14/+ tumor model. Finally, in Aim 3, we will extend our analysis to chemically-induced colon cancer model to test the applicability of our Scd3-CreER+/- mice to additional cancer etiologies. We believe that the successful generation of this experimental mouse system will provide a way to test the impact of reactivation of suppressed tumor-associated genes directly within neoplastic tissue, and also enable us to examine the therapeutic effects associated with the functional restoration of the lost genes.