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The Tumor Suppressing Effect of Pregnancy: Can the Mammary Stroma do the Job?
Tumor Cell Biology IV
Background: Mechanisms underlying the protective effect of pregnancy against breast cancer remain unclear. Evidence indicates that: 1) administering pregnancy levels of estrogen (E) and progesterone (P) to carcinogen-exposed virgin animals reduces tumor incidence; 2) epithelial tumor cells transplanted into epithelium-free stroma of parous rats are normalized, forming normal ducts instead of tumors. However, this evidence comes from animal experiments where systemic effects cannot be separated from local effects. Hypothesis: Pregnancy’s protective effect is due to local changes occurring in the mammary stroma. We will test this using stroma-like 3 dimensional (3D) matrices derived from fibroblasts from: 1) parous rats and 2) virgin rats treated in vitro with pregnancy levels of E+P. Specific Aims: Aim 1) To characterize 3D matrices generated by fibroblasts obtained from mammary glands (MG) of parous rats and compare it against matrices generated by fibroblasts from age-matched virgin rats. Aim 2) To study the effects of pregnancy levels of E and P alone and in combination on the 3D matrices derived from fibroblasts obtained from the MG of virgin rats. Aim 3) To study the effect of parous and virgin fibroblast-derived 3D matrices on the behavior of normal and neoplastic mammary epithelial cells. Study Design: Aim 1: 3D matrices derived from parous or virgin fibroblasts from MG of Wistar-Furth rats will be assessed by: 1) Thickness : measured by immunostaining for fibronectin. 2) Composition : laminin, fibronectin, tenascin, type I collagen and clusterin will be assessed by immunostaining and Western blot. 3) Topography : using immunostaining we will assess the number and orientation of fibers. Aim 2: Fibroblasts will be isolated from virgin MG. E and P treatment (concentrations resembling those during pregnancy) will last 8 days. Topography and biochemical composition of the matrices will be analyzed as in Aim 1 and compared to the parous matrices. Aim 3: Non-tumorigenic FSK-3 and tumor epithelial PN8A cells will be separately plated on parous and virgin 3D matrices. Immunostaining for integrins a5, ß1 and 4, and MMPs 2 and 3 will show matrix adhesions. Cytoskeletal organization (actin, keratin and vimentin), cell proliferation, migration and invasion will also be tested . Potential outcomes and benefits: Using this model we will identify the stromal molecules involved in repressing tumor formation and open a new avenue for prevention therapy .
Epidemiological studies as well as animal research show that breast cancer incidence is significantly reduced in individuals that have experienced pregnancy. During intrauterine life, gland development is governed by interactions between the epithelium and the surrounding stroma. These physical and biochemical interactions continue during adult life and are key to maintaining a normal, healthy mammary gland (MG). Our experimental data indicates that the stroma of a parous rat (2 pregnancies, lactation and involution) has the ability to not only suppress tumor initiation but to instruct tumor cells to become normal ducts. To fully understand the protective effect of pregnancy and also explore the possibility of recreating a parous-like stroma in a nulliparous individual as a preventive therapy, we must identify if the pregnancy effect is local (takes place in the MG) or systemic (the effect occurs at the hormonal level). Common cell culture experiments are 2-dimensional (2D) lacking the 3D context of in vivo tissue and cannot be compared to the level of organization observed in the MG. We will use an in vivo -like 3D culture of mammary stromal-fibroblasts (a major cell type of the stroma involved in directing the stromal half of the dialogue with the epithelium to maintain tissue homeostasis). These cells form a 3D matrix resembling the tissue of origin. We will use fibroblasts from parous and virgin rats to compare the matrix spatial organization and composition. We will test whether treating virgin fibroblasts with pregnancy levels of estrogen and progesterone produces matrices similar to those derived from parous fibroblasts. Finally we will test the effects of parous and virgin fibroblast-derived matrices on the behavior of tumor and normal mammary epithelial cells. We will focus our attention on some of the most common tumor cell features: migration, invasion, proliferation and adherence to the matrix. We expect the biochemical and/or spatial organization of the parous and virgin fibroblast-derived 3D matrices to be different with regard to repressing the tumor behavior. The success of this project will allow us to test the identified putative “tumor repressor” molecules, through their signaling to the epithelium (soluble factors or physical forces). If we show that extracellular matrix composition and organization play important roles in supporting or repressing tumors, we may revise the current strategies used to prevent tumor formation.