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Study Of Mammary Stem Cells To Examine Mechanism of Parity-Induced Protection Against Breast Cancer
An early full term pregnancy is the best known factor to lower the lifetime risk of developing breast cancer. Although parity-induced protection against mammary tumorigenesis in humans and animal models has been known for almost half a century, nobody has so far investigated the changes that occur in the mammary stem cells which may mediate the lowering of breast cancer risk. I hypothesize that changes in the mammary stem cells exerted by pregnancy and involution mediate the protective effect against breast cancer development. The major goal of my proposal is to study the alterations in the mammary stem cells occurring due to pregnancy and involution that are responsible for the decreased risk of developing breast cancer over the lifetime. I propose a new paradigm that the altered protein expression profiles in the mammary stem cells mediates the decreased susceptibility to oncogene activity and carcinogenic challenges.
Preliminary data show that I can successfully isolate stem/progenitor cell fractions from mouse mammary glands using flow cytometry (FACS). The isolated stem cells grow as mammospheres in culture and can be re-injected into cleared mouse mammary fad pads to regenerate a fully functional mammary gland. Using quantitative real-time PCR (Q-RT-PCR) I have shown that the isolated stem cells differentially express known markers correlating the parity status of the donor mice, and thereby validating my isolation protocol.
Using isolated mammary stem cells I propose to determine if parity and involution protect against breast cancer development. I will investigate mRNA and protein expression patterns in greater detail using proteomic and microarray analysis to determine factors that are involved or responsible for altered malignancy initiation and cancer susceptibility. In particular, I will focus on genes involved in genomic/chromosomal instability and tumorigenesis. One candidate cancer susceptibility protein is separase, an endopeptidase that cleaves the cohesion of sister chromatids. Over-expression of separase leads to premature separation of sister chromatids and ultimately to aneuploidy, a hallmark of breast cancer. Our laboratory has shown that induction of separase causes aneuploidy and tumorigenesis in mouse mammary models (submitted). Separase expression is regulated by estrogen, progesterone and p53. I hypothesize that differences in the expression regulation of separase in the mammary gland, which varies with parity status, mediates at least in part the altered risk for breast cancer development. I will use both in vitro and in vivo assays using separase-transformed mammary stem cells in cleared fat pads to determine effect of parity/involution on separase-induced chromosomal instability in breast cancer development. Furthermore, I will re-inject untransfected stem/progenitor cells from the virgin and from parous/involuted animals and subject these mice to carcinogen treatment with the carcinogen 7,12-dimethylbenzanthracene (DMBA). I expect to see differences in the tumor development confirming the protective effect of parity against carcinogen challenge. I will analyze the stem cell fraction in the developing tumors from separase as well as DMBA treated animals to determine the alterations compared to the initially injected stem cells. I am confident our approach will lead to insights into the changes intrinsic to mammary stem cells following parity that prevent tumor formation, and hence help to find new breast cancer prevention strategies that can be used to improve treatment.
Specific Aims are as follows:
1) Characterize the differentially expressed proteins using gene-expression and proteomic analysis from stem/progenitor cells isolated from virgin and from parous/involuted mouse mammary glands. (Timeline: months 1-12).
2) Characterize alterations in signaling pathways and their role in mediating protective effects against breast cancer development. (Timeline: months 9-36).
3) Investigate the role of carcinogens and separase expression in parity-induced protection against aneuploidy development using a lentiviral delivery system and a DMBA model for breast cancer development. (Timeline: months 10- 36)
The knowledge gained by the proposed experiments will certainly not only help us to understand the protective changes that occur in the mammary stem cells during pregnancy and involution, but will also enable us to develop new strategies for breast cancer prevention. Furthermore, we will gain more insights into the initiation of cancer, which will enable us to find new targets for breast cancer therapy.
An early full term pregnancy (including delivery and nursing of the baby) is the best known factor to lower the overall lifetime risk of developing breast cancer in women. However, the mechanisms that mediate this protective effect remain elusive. It is widely believed that a tiny percentage of slowly dividing cells (called stem cells), rather than the majority of other cells in the mammary gland, give rise to tumors. Therefore, I hypothesize that: Pregnancy induces changes in the mammary stem cells that mediate protection against breast tumor development. These alterations not only lower the lifetime risk of developing breast cancer, but also protect normal mammary gland function i.e. for later pregnancies. I propose here to investigate in a mouse mammary stem cell model the changes in gene and protein expression that persist after a pregnancy compared to the virgin siblings of the same age. I propose that we will be able to achieve this protective effect in women, without going to an actual pregnancy early in life, as a prevention strategy to lower the lifetime risk of breast cancer in humans.
Furthermore, the knowledge of the protective genes might also help to interfere with existing cancers and offer alternative approaches for treatment. I therefore, propose to analyze expression of the messenger RNA as well as proteins in mammary stem cells before and after pregnancy. Combining both the gene and protein expression data will enhance the confidence of my analysis, while eliminating the false positive results. I therefore expect to find a number of changes in protein expressions that I will classify based on their involvement in different cellular pathways. I will primarily focus on genes involved in cell division and genome stability, as almost all of the breast tumors have uncontrolled cellular growth and aberrant chromosomal number. Chromosomal instability is caused at least in part by aberrant separation of chromosomes during mitosis. Therefore I will focus on changes in gene expressions related to chromosomal cohesion, separation, mitotic checkpoint and cell cycle regulation during mitosis. I will however publish all the results, so that other researches can investigate additional interesting alterations that might contribute to the pregnancy-induced protection against breast cancer.
To validate the candidate genes from our screen, I will inject the stem cells isolated from the virgin and post pregnancy glands into mice where the mammary gland has been surgically removed, only leaving the fat tissue behind. I can show that the stem cells from both groups grow out in the fat tissue to form a normal functional mammary gland. I will than treat these mice with carcinogen or over-express oncogens in the stem cells before the injection, to see if previous pregnancy protects these stem cells from tumor formation. I expect to see no or a lot less tumor development in the outgrowth from pregnancy stem cells. The arising tumors will than be analyzed and compared in gene expression to the initially injected stem cells. With this approach I can on one hand confirm the protective effect and on the other evaluate the alterations in the generated tumors to determine what caused the malignant transformation. These results will validate the gene expression profiles for breast cancer prevention strategies and give us more insights in pathways that are altered in the emerging tumors to generate new treatment approaches for existing breast cancers.