The relationship between reproductive history and breast cancer is complex. A full term pregnancy in younger women is protective against breast cancer later in life, while a first full term pregnancy after 40 years of age is associated with higher cancer occurrence. In addition, there is an increase in breast cancer within the first two years of pregnancy, regardless of age. Pregnancy associated breast cancer also presents at a later clinical stage and is more invasive than non-pregnancy associated breast cancers. We hypothesize that the cancer promotional effect of pregnancy is due to physiologic remodeling of the mammary stroma that occurs after pregnancy. During parturition or weaning-induced mammary gland involution, mammary stroma is degraded and stroma levels of matrix metalloproteinases (MMP) and pro-inflammatory mediators are elevated. We propose that these involution-associated changes in mammary stroma promote an epithelial to mesenchymal transition (EMT) in pre-existing neoplastic cells. The resulting activated tumor cells, coupled with the weakened stromal barrier, increases the likelihood of tumor cell extravasation. Since the likelihood of having occult tumor cells increases with age, pregnancy associated gland involution is more likely to effect EMT and metastases in older women.
In Aim 1. the hypothesis that tumor cell metastatic potential is modulated by reproductive status will be tested in vivo using xenograft models. Human mammary tumor cells will be injected directly into teats or mammary fat pads of immunodeficient mice at different reproductive stages (i.e. nulliparous, 'pregnancy-associated', and parous). The ability of tumor cells to exit the gland is predicted to be dependent on the degree of tissue remodeling at the time of injection: low in nulliparous and parous, and high in regressing glands. Primary tumors will be evaluated histologically and metastases to lung, liver, brain and long bone of the leg will be measured by semi-quantitative PCR using a unique human sequence, the CCR5 gene.
In Aim 2, the hypothesis that endocrine-induced changes in stroma composition can either promote or suppress EMT will be evaluated in vitro using reconstitution models. Human mammary epithelial cells, which differ in metastatic potential from non-tumorigenic to metastatic, will be combined with mammary stroma isolated from rats with different reproductive states. It is anticipated that quiescent mammary stroma (nulliparous and parous) will inhibit, while involuting matrix will promote EMT in highly metastatic cells. Functional evidence for EMT will be obtained by measuring stroma-dependent changes in cell adhesion, motility and invasiveness using modified Boyden chambers assays. Morphological and biochemical evidence for EMT will be evaluated in a 3-dimensional model of tumor formation. The effects of mammary matrix on key markers of EMT, actin fiber organization, and expression of E-cadherin, vimentin, gelsolin and Fspl proteins, will be determined biochemically.
In Aim 3, the hypothesis that involuting mammary matrix induces, while nulliparous and parous mammary matrices suppress expression of MMP in tumor cells will be evaluated by reporter gene assays. Luciferase-based reporter constructs containing either the stromelysin (MMP-3), gelatinase A (MMP-2), or gelatinase B (MMP-9) promoters will be tested for activity in tumor cells plated on different mammary matrices. The results of these studies will contribute to our understanding of the effects pregnancy-associated changes in mammary stroma have on breast cancer progression, and should provide insights into epidemiological data describing the dual effect of pregnancy on breast cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA085944-03
Application #
6633698
Study Section
Metabolic Pathology Study Section (MEP)
Program Officer
Mohla, Suresh
Project Start
2001-06-04
Project End
2005-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
3
Fiscal Year
2003
Total Cost
$266,175
Indirect Cost
Name
Amc Cancer Research Center
Department
Type
DUNS #
016207271
City
Denver
State
CO
Country
United States
Zip Code
80214
Fornetti, Jaime; Martinson, Holly A; Betts, Courtney B et al. (2014) Mammary gland involution as an immunotherapeutic target for postpartum breast cancer. J Mammary Gland Biol Neoplasia 19:213-28
Martinson, Holly A; Lyons, Traci R; Giles, Erin D et al. (2013) Developmental windows of breast cancer risk provide opportunities for targeted chemoprevention. Exp Cell Res 319:1671-8
Ramirez, Robert A; Lee, Amy; Schedin, Pepper et al. (2012) Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling. Dev Dyn 241:890-900
Billups, Stephen C; Neville, Margaret C; Rudolph, Michael et al. (2009) Identifying significant temporal variation in time course microarray data without replicates. BMC Bioinformatics 10:96
Laffin, Brian; Wellberg, Elizabeth; Kwak, Hyeong-Il et al. (2008) Loss of singleminded-2s in the mouse mammary gland induces an epithelial-mesenchymal transition associated with up-regulation of slug and matrix metalloprotease 2. Mol Cell Biol 28:1936-46
Kwak, Hyeong-Il; Gustafson, Tanya; Metz, Richard P et al. (2007) Inhibition of breast cancer growth and invasion by single-minded 2s. Carcinogenesis 28:259-66
Schedin, Pepper; O'Brien, Jenean; Rudolph, Michael et al. (2007) Microenvironment of the involuting mammary gland mediates mammary cancer progression. J Mammary Gland Biol Neoplasia 12:71-82
McDaniel, Shauntae M; Rumer, Kristen K; Biroc, Sandra L et al. (2006) Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis. Am J Pathol 168:608-20
Schedin, Pepper J; Eckel-Mahan, Kristin L; McDaniel, Shauntae M et al. (2004) ESX induces transformation and functional epithelial to mesenchymal transition in MCF-12A mammary epithelial cells. Oncogene 23:1766-79
Schedin, Pepper; Elias, Anthony (2004) Multistep tumorigenesis and the microenvironment. Breast Cancer Res 6:93-101