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The Stroma as a Gatekeeper of Tumor Development
Background: Twenty years ago, E. M. Rivera showed that rat mammary gland (MG) tumor fragments implanted into “cleared” (epithelium-free) fat pads (CFPs) of mature syngeneic rats generated normal ducts in addition to tumors. Intrigued by these findings, we performed a pilot experiment and observed that the CFPs from 150 day-old (mature) and multiparous rats prevented neoplastic development and encouraged normal ductal growth of grafted epithelial cancer cells (ECCs) isolated from nitrosomethylurea (NMU)-induced tumors. In contrast, when ECCs from the same tumors were injected into CFPs from 50 day-old (young) rats, they grew as tumors. Most remarkably, these data suggest a parallel to the phenomenon of age-dependent susceptibility to chemical carcinogens; susceptibility is maximal around 50 days of age and decreases significantly once the MG completes adult development. Pregnancy further reduces susceptibility.
Objectives/Hypotheses: i) To discern whether treatment with estrogen (E) and progesterone (P) (to mimic pregnancy) confers to the stroma the ability to mediate normalization of ECCs and ii) to identify the molecular components mediating this phenomenon.
Specific Aims: 1) To assess the role of the ovarian hormones E and P, on the normalizing properties of MG stroma of parous rats. 2) To identify putative mediators of the normalizing effect of parous and E+P-treated MG stroma by DNA microarray analysis.
Study design: Aim 1) ECCs isolated from NMU-induced mammary tumors will be injected into the CFPs of i) 50 day-old virgin rats, ii) parous 100 day-old rats, iii) 100 day-old virgin rats, and iv) 100 day-old virgin rats treated with 20 ug E and 20 mg P for 21 days to mimic pregnancy. The presence of normal ducts and neoplastic lesions in these grafts will be assessed in whole-mounts and in tissue sections. We expect that CFPs of groups ii) and iv) will normalize the growth of ECCs. Aim 2) RNA will be extracted from CFPs of young, E+P-treated, mature and parous rats for DNA microarray analysis. The expression of candidate RNAs will be ascertained by quantitative RT-PCR and northern analysis, and that of candidate proteins by immunohistochemistry.
Potential outcomes and benefits of the research: The data collected should provide a better understanding of both susceptibility and normalization of the cancer phenotype. The gene expression analysis will identify candidate genes that may mediate these processes. As a consequence, a new set of preventive, prognostic and therapeutic points of intervention will become available.
Background: The majority of human cancers are of epithelial origin (carcinomas). Alterations in the stroma (the connective tissue) have been thought to occur as a consequence of the malignant transformation of epithelial cells. However, using a tissue recombination rat mammary gland model, we recently observed that epithelial cells are transformed into malignant cells when only the stroma was exposed to the chemical carcinogen nitrosomethylurea (NMU); epithelial tumors appeared regardless of whether or not the epithelial cells were exposed to the carcinogen. These data suggest that the stroma, rather than the epithelium, is the crucial target of NMU.
In a separate experiment, we surgically removed the epithelium from the mammary glands leaving a cleared mammary stroma or cleared fat pad (CFP), and injected epithelial cancer cells (ECCs) isolated from NMU-induced rat mammary tumors. Those ECCs formed tumors when injected in CFPs of young hosts. In contrast, tumor formation was substantially decreased or absent when tumor cells were injected into CFPs of mature hosts or CFPs of hosts that have had multiple pregnancies. Instead normal ducts formed.
Objective/hypothesis: To further investigate the role of pregnancy on the normalization of ECCs. We aim i) to discern whether treatment with ovarian hormones, estrogen (E) and progesterone (P), at concentrations found during pregnancy allows the stroma to normalize the ECCs and ii) to identify the molecular components mediating this phenomenon.
Study design: We will compare the ability of CFPs to normalize ECCs and to inhibit tumor formation by injecting ECCs into CFPs of 1) young virgin animals (high tumor incidence expected), 2) animals that have undergone pregnancy (lowest tumor incidence expected), 3) virgin animals of similar age to those that have undergone pregnancy (intermediate incidence expected), and 4) virgin animals treated with E+P at concentrations found during pregnancy (incidence expected to be similar to Group 2). The pattern of gene expression in the CFPs of high and low susceptibility groups will be compared to identify candidate genes expressed in the stroma that may mediate the normalization of cancer cells.
Potential outcomes and benefits of the research: We anticipate that these experiments will result in a better understanding of both cancer susceptibility and normalization. Consequently, we expect that a new set of preventive, prognostic and therapeutic points of intervention will become available, allowing an alternative approach to the use of unspecific chemotherapeutic agents.