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    The Role of the Stroma in Carcinogenesis

    Scientific Abstract:
    The Role of the Stroma in Carcinogenesis Background: Mammary gland (MG) tumor susceptibility to the carcinogen N-nitrosomethylurea (NMU) varies considerably among rat strains. Wistar-Furth (WF) rats are highly susceptible, whereas Copenhagen (COP) rats are resistant. Although the initial pre-neoplastic changes in the epithelium of both rat strains are similar, in COP rats the lesions disappear prematurely and do not complete the neoplastic transformation. This process of “redifferentiation/normalization” occurs within 2 months of NMU exposure. Meanwhile, the same pre-neoplastic lesions develop in the WF rats and progress into tumors. No differences were found in proliferation or apoptosis rates, incidence of Ha-ras gene mutations, etc. that would explain such a different response to the same injury. Recently, using WF rats, we showed that non-exposed mammary epithelial cells (MEC) developed neoplasias when placed into NMU-exposed mammary stroma. In contrast, recombinants of non-exposed stroma and NMU-exposed MEC developed phenotypically normal ducts, suggesting damage to the stroma initiates the neoplastic transformation of non-exposed MEC. Objective/Hypothesis: We hypothesize that the tumor-resistant phenotype in COP rats is due to the stroma’s ability to reverse the damage generated by the exposure to NMU through the “normalization” of stromal-epithelial interactions in their MGs. This hypothesis will be tested through two different approaches: 1) By recombining either the stroma or epithelium component of the tumor-resistant COP MG with either the epithelium or stroma of the tumor-susceptible WF rats. These recombinants will be introduced into an immunotolerant host such as the SCID mouse, and 2) By identifying which genes are differentially expressed in the MG stroma of either COP or WF rats exposed to NMU. Specific Aims: Aim 1) To determine whether the MG tumor-resistant phenotype seen in the COP rats stems from the stroma or the MEC; Aim 2) To assess whether the COP tumor-resistant phenotype is due to a different gene expression profile in the stroma of NMU-exposed COP, compared to NMU-exposed WF. Study Design: Aim 1: The experimental design has two distinct components: 1) in vitro: MEC (grown from young virgin rats, either WF or COP) and 2) in vivo: (a) the SCID mouse host and (b) donors of stroma and MEC (COP and WF rats). At 17 days of age, WF (Groups 1 and 4) and COP (Groups 2 and 3) 4th and 5th abdominal MGs will be cleared of epithelium (CFP) and transplanted into the abdominal wall of age-matched SCID mice. The host mice will be injected i.p. with 50 mg NMU/kg B/W at 50-55 days of age. Five days later, MEC from WF will be transplanted into CFPs of Groups 1 and 2 and MEC from COP will be transplanted into CFPs of Groups 3 and 4. Control groups: Group 1 is the tissue recombination positive control. Group 5: whole MG from 17 day-old WF rats will be transplanted into SCID mice; the mice will receive NMU at 50-55 days of age (positive control for tumor formation). Group 6: whole MG from 17 day-old COP rats will be transplanted into SCID mice; the mice will receive NMU as above (control for tumor resistance). Group 7: same as Group 5 but the mice will receive vehicle (negative control). Group 8: same as Group 6 but the mice will receive vehicle. In all groups, transplants will be harvested at 45, 90 and 150 days after MEC transplantation. We will score the appearance and/or disappearance of pre-neoplastic and neoplastic lesions by evaluating structural changes in MG whole mounts and histochemistry. The quality of the stroma (both cellular and extracellular components) will be assessed by immunohistochemistry and Western blot. Aim 2: 17 day-old, WF and COP MG will be cleared of epithelium (CFP). Both strains will receive either NMU or vehicle at 50-55 days of age; 5 days later the CFP will be harvested and processed for RNA isolation. The RNA samples will be analyzed by DNA microarray technology. The selected up- and down-regulated genes will be confirmed by quantitative RT-PCR and Western blot when appropriate. Potential outcomes and Benefits of the Research: These experiments will provide the phenomenological basis for exploring the molecular mechanisms responsible for tumor resistance. The realization of these aims will help in applying a more rational approach to breast cancer prevention and therapies.

    Lay Abstract:
    The Role of the Stroma in Carcinogenesis In 1971, President Nixon declared the "War on Cancer". After more than three decades, the question still remains: "Are we winning the war?" Cancer is the second leading cause of death after heart disease in the United States despite intensive efforts to reduce this threat. Breast cancer has been widely studied using both cell culture techniques and animal models. The most common animals used in breast cancer research are mice and rats, the latter being the one that most resembles the breast cancer in women. Attention has been mostly focused on generating tumors more efficiently, in the shortest period of time. Therefore, most studies have been performed using tumor-susceptible rat strains. However, there are strains of rats that are resistant to mammary tumor induction and very little is known about where this resistance originates. My project aims at learning from these animals that are resistant to mammary tumor development. In terms of tissue architecture, the mammary gland of a rat is comparable to that of women. It is formed by an epithelium that covers the ducts and alveoli and a stroma (the connective tissue scaffolding of this organ). These two compartments are in continuous interaction during embryonic development and throughout adulthood. The current theory of carcinogenesis, the Somatic Mutation Theory, is based on the assumption that a somatic cell (for instance the epithelial cells of the mammary gland) in the adult organism would undergo successive mutations that would accumulate in a mammary epithelial cell and, eventually, these mutations would be responsible for the cancer. However, this theory does not explain the many tumors where mutations are not found or, equally important, how some tumor cells reversed to their normal phenotype when placed into a normal stroma. The analysis of data collected led others and us to propose the alternative that cancer is a problem of developmental biology gone awry. In other words, disruption in the interaction between cells and tissues would induce a neoplasia. We called this theory the Tissue Organization Field Theory. Using a rat mammary gland tissue recombination model, we have shown that mammary epithelial cells that were not exposed to a carcinogen developed neoplasias when placed into a carcinogen-treated stroma. In contrast, carcinogen-treated epithelial cells developed normal-like mammary ducts when placed into a non-treated stroma. Because the stroma is a target of a carcinogen in tumor-susceptible rats, we now hypothesize that the stroma may also play a role in tumor resistance. We propose to investigate which compartment of the mammary gland in tumor-resistant rats is responsible for the resistant phenotype. We will expose the stroma of both susceptible and resistant rats and recombine them as follows: epithelial cells from susceptible strains with stroma from resistant strains and stroma from susceptible strains with epithelial cells from resistant strains. We aim to discern whether the resistance stems from the stroma or the epithelium of the mammary gland. We will analyze whether there is/are any difference/s in the expression of genes in the carcinogen-exposed stroma of these two strains of rats that will allow us to identify possible gene products associated with resistance. Eventually, a wider effort to identify which among the multiple components of the stroma play the prominent role in the disease will become necessary. We expect that our experiments will help us to better understand the biology of tumorigenesis and the mechanisms responsible for resistance. If the experiments proposed in this application show that the stroma also plays an important role in tumor-resistance, it will be important to focus our attention on this tissue compartment to study, how to prevent tumor formation as well as how metastatic growth is controlled.