Our research hopes to develop appropriate treatments or preventive strategies that reduce Breast Cancer (BC) by characterizing the mechanisms contributing to malignant transformation, tumorigenesis, cancer development and progression.
Recent evidence demonstrates the role of inflammation and inflammatory mediators in malignant transformation and BC, and inflammatory mediators are effective predictors of poor clinical outcome in BC of diverse origin. Inflammation and inflammatory mediators contribute to malignant transformation and BC by still poorly defined means that may include promoting carcinogenesis, altering mammary epithelial cell differentiation, and/or stimulating the epithelial-mesenchymal transition.
Our laboratory recently observed an unexpected contribution to mammary gland differentiation by the ROS generating enzyme XOR that, coupled with recent clinical and genetic observations, may be of particular significance to BC and the role of inflammation and inflammatory mediators.
While present data support a critical role for sustained expression of XOR in development and mammary epithelial cell differentiation, clinical data on the other hand suggest that down regulation of XOR may be functionally linked to aggressive breast cancer.
Recent data also demonstrate XOR to be an upstream regulator of both COX-2 expression and p38 activation. COX-2 has been directly linked to carcinogenesis and BC, and overexpression of COX-2 in mammary epithelial cells promotes tumorigenesis, while conditional knockout of COX-2 in the mammary epithelium reduces tumorigenesis as does pharmacological inhibition. These data identify a critical link between XOR and COX-2 in mammary epithelial cells. XOR regulates COX-2 expression and COX-2 and XOR exhibit opposite expression in aggressive BC. XOR is also an important mediator of the acute phase inflammatory response in mammary epithelial cells and this may underlie its role in the mammary gland. Both XOR and COX-2 gene expression are regulated by physiological stress, growth factors, and inflammation in mammary epithelial cells.
Our hypothesis is that XOR provides a critical function in mammary epithelial cell differentiation that serves to inhibit transformation and tumorigenesis in part through redox regulation of COX-2 and p38 MAPK and that inflammation and inflammatory mediators perturb intracellular redox signaling thus altering differentiation and contributing to malignant transformation and tumorigenesis.
It is well recognized that intracellular ROS stimulate cell proliferation at low or moderate levels, but as intracellular ROS concentration rises may induce apoptosis. While conventional antioxidant therapy has sought to dampen proliferation through the use of ROS scavengers, we imagine that over-expressing ROS generators like XOR in mammary epithelial tumors may offer a novel therapeutic possibility for inducing apoptosis specifically in tumor cells, and this is the long term objective and translational value of the work proposed. However, since many unpredictable consequences may arise from manipulation of intracellular redox status, our present experiments have been designed to first characterize the role of XOR regulation of COX-2 and p38 MAPK in epithelial cell differentiation and inflammatory response. As proof of concept, we will determine the effect of manipulating XOR in vitro on an in vivo xenograft model of BC using well established human mammary carcinoma cells.
Significance and Potential Outcome of our Research
The development of new chemopreventive or chemotherapeutic strategies appropriate to BC requires a more profound understanding of the mechanisms of epithelial tumorigenesis. The contribution of inflammation to the pathogenesis of BC has been recognized and is well documented.
However, the realization that leukocytes (MNP) and/or fibroblasts are required for the normal cycle of proliferation, differentiation, and involution has suggested a critical new view of the potential for these cells and their secreted cytokines to promote BC.
As obligatory components of the developing and differentiating mammary gland, MNP exert essential influences on the course of differentiation. Furthermore, inflammatory cytokines in the epithelial cell microenvironment may perturb the normal program of mammary gland differentiation leading to transformation, proliferation, and BC. Our laboratory observed an unexpected contribution to mammary gland development by the purine catabolic enzyme, xanthine oxidoreductase (XOR), whose function has since been found indispensable for both neonatal development and for mammary gland development in particular.
XOR regulation by inflammation and inflammatory cytokines may significantly perturb development and differentiation in unknown ways. Significantly, XOR is an upstream regulator of both p38 MAPK and COX-2, whose expression is regulated by inflammation and inflammatory cytokines and has been positively linked to BC.
Aggressive BC is directly associated with high COX-2 expression and loss of XOR expression. As a source of intracellular ROS, XOR effectively links inflammation to epithelial differentiation and tumorigenesis, and our data have suggested a potential novel therapeutic use of XOR as an endogenous source of ROS whose overexpression is anticipated to induce epithelial cell death.
The development of new transgenic models of BC as initiated by our experiments will identify the contribution of inflammatory regulation of XOR to expression of p38 MAPK and COX-2 and to mammary epithelial cell proliferation, differentiation, and apoptosis in a well established mouse model of mammary cancer, in vivo.
We will independently test the role of XOR in vitro in mouse mammary epithelial cells and in human mammary carcinoma cells. Subsequently, we will examine the effect of XOR overexpression or specific knockdown on a xenograft model of BC using these cells. We anticipate that the information obtained from these studies will lead to a more rational, mechanism based, approach to BC therapy. Mechanism based targeting of XOR or ROS signaling pathways that control differentiation and proliferation of epithelial cells represents a novel approach to BC management that has great potential to impact BC management. Our experiments will develop a novel strategy for directing ROS overexpression in mammary tumor cells with the goal of inducing directed tumor cell death.
Inflammation and Innate Immunity Interest Statement
Our second project is focused on the family of mammalian molybdenum iron-sulfur flavoproteins that are also sources of intracellular metabolic oxygen derived free radicals. This family of genes encodes the enzymes aldehyde oxidase, sulfite oxidase, and xanthine oxidodreductase (xanthine oxidase/dehydrogenase). We have cloned, mapped, and determined the structure of several members of this family and have uncovered dramatic similarities in the sequences and structures of the genes for all these enzymes. Working with colleagues from several different institutions we have developed a picture of the organization of the human molybdenum hydroxylase genes on chromosome 2. These observations have linked the molybdenum iron-sulfur flavoproteins genes to hereditary xanthinuria and the aldehyde oxidase group to amyotrophic lateral sclerosis (ALS). Previous studies suggested a role for xanthine oxidoreductase in oxidative stress induced injury in ischemia/reperfusion and in inflammatory disorders. However, current analyses have revealed the far more subtle role in redox signaling. Knockout genetics of xanthine oxidoreductase demonstrated its physical and regulatory interaction with cyclooxygenase-2 and have confirmed the role of xanthine oxidoreductase in intracellular free radical signaling in inflammation and in the mammary gland. Current analyses are focused on the mechanisms by which molybdenum iron-sulfur flavoproteins mediate intracellular free radical signaling. Two models are presently in use. In one, the contribution of intracellular free radical signaling to lung inflammation is being studied in the mononuclear phagocyte cell population. In the other, conditional cell specific knockout of the XOR gene is being developed for the purpose of examining its role in specifically in the innate immune system with possible application to breast cancer or other innate inflammatory disorders..
Our future plans will continue to develop an understanding of the mechanism by which oxygen regulates expression of mitochondrial electron transport genes, and we will expand our understanding of free radical based intracellular signaling by the molybdenum iron-sulfur flavoproteins in breast and lung cancer where these enzymes may be playing significant roles. Furthermore, it has become abundantly clear that the iron-sulfur flavoproteins are important enzymes in a network of intracellular free radical signaling linked to cyclooxygenase and nitric oxide synthase, and this has extended the scope of our goals to include inflammation mediated carcinogenesis. Thus, one of our immediate goals will be to delve into these mechanisms both for mammary and lung carcinogenesis. We will develop new models of inflammation induced cancer in mouse lung and mammary glands where transgenic models are presently limited.