Basic Breast Biology
One of the issues identified at the March, 1996 Advisory Meeting as an under-funded, but critical, area for breast cancer research was normal breast physiology and development. Research in this area could easily provide a basis for understanding the changes in the breast that lead to the development of cancer, ultimately fostering the development of more effective prevention therapies against breast cancer. In order to achieve this end, we issued a Request For Applications (RFA) targeting the Basic Breast Biology Relevant to the Development of Breast Cancer in Cycle III. This RFA was designed to elicit research that would lead to a better comprehension of the physiology, biochemistry and molecular biology of the normal breast.
Five grants were funded through this RFA. They examine the genetic and hormonal influences on the breast that could lead to breast cancer development. In one grant, a variety of hormones will be examined for their effect on human breast cells that are grown in mice (Jason Yang, PI). The advantage of this model is that hormones can be manipulated in mice more easily than in humans and the effect of hormones on the growth of human breast cells can be observed. In another grant the biological action of retinoids in the breast are examined (Xiao-Kun Zhang, PI). Some retinoids are known to be protective against the growth of breast cancer cells but can have undesirable side effects. The basic biological functions of retinoids in the breast uncovered by this study could provide the rationale for designing a better set of preventive retinoids. Estrogen is a known risk factor in the development of breast cancer, but not enough is known about its actions inside the cell. The third grant examines how estrogen regulates breast cell growth and asks whether the method of regulation could possibly select for mutant breast cells (Nicholas Rampino, PI). The fourth grant concentrates on the molecular signals that tell breast cells when it’s time to die and asks whether a breakdown in this regulation could lead to metastatic potential (Gary Bokoch, PI). The fifth grant examines the effect of the environment on the breast in the form of organochlorine insecticides (Essam Enan, PI). This grant will take a molecular and cell biology approach to examine the interaction of estrogen and cancer genes with organochlorine insecticides in the breast. Together, the scope of research in these grants spans a breadth of areas in breast physiology that will bring us closer to answering the critical question - what differentiates a normal breast cell from a breast cancer cell?
Request for Application (RFA) Awards
- The Role of PAK2 in Breast Cancer Cell Death – Gary M. Bokoch, Ph.D., The Scripps Research Institute
- Organochlorine Insecticides and Breast Cancer – Essam E. Enan, Ph.D., University of California, Davis
- Genetic Repair of Oxidative Damage: Effect of Estrogen – Nicholas J. Rampino, Ph.D., The Burnham Institute
- Hormonal Control of Normal Human Breast Tissue – Jason Yang, M.D., Ph.D., Cancer Research Fund of Contra Costa
- Breast Cancer Chemoprevention by Retinoids – Xiao-kun Zhang, Ph.D., The Burnham Institute
The Role of PAK2 in Breast Cancer Cell Death
Gary M. Bokoch, Ph.D.
The Scripps Research Institute
The breast cells of pregnant women undergo dramatic changes in structure during the process of milk production, which must then be reversed as the child is weaned. This normal reshaping process requires the now unwanted milk-producing cells to undergo a process known as programmed cell death. The breast cells respond to hormonal signals by undergoing biochemical changes that cause the cells to effectively kill themselves. Normally developing breast tissue is controlled by a balance between growing cells and dying cells. When this process becomes abnormal, so that the cells no longer die as they should, then the remaining cells can grow out of control and become breast cancers.
The mortality associated with breast cancer is highly correlated with its ability to spread from the original site in the breast to other areas of the body. An important observation has been made that the breast cancer cells most resistant to the process of cell death are also those that are most likely to effectively move into other tissues. Thus, there is a relationship between an abnormal resistance to cell death and increased spread of breast cancers. Up until now, the basis for this relationship has been unknown.
We have discovered a protein present in breast cancers that can regulate both cell death and cell movement. Because of its regulatory activities in both of these areas, this protein, called PAK2, is a likely candidate to explain the connection that has been noted between resistance to cell death and the ability of breast cancers to invade the body. PAK2 normally becomes active during cell death responses we have studied in other systems. We will determine whether PAK2 is also an important mediator of breast cancer cell death responses. It is possible that PAK2 does not get activated normally to induce cell death in the cells that develop into breast cancers, and we will examine this possibility in our studies. We will investigate whether PAK2 can then regulate the ability of the death-resistant breast cancer cells to move from other body sites. Our studies will provide new insights into the problem of how breast cancers might be more effectively induced to die by chemotherapeutic drugs, and will also suggest mechanisms that might allow us to inhibit the spread of breast cancers.
Organochlorine Insecticides and Breast Cancer
Essam E. Enan, Ph.D.
University of California, Davis
Recent epidemiological evidence suggests that there is a relationship between the incidence of breast cancer and the presence of organochlorine insecticides in the body. There are many study results indicating that environmental factors have strong influence on the occurrence of breast cancer and that estrogenic actions of various chemicals are one of the most strongly suspected contributing factors. The overall objective of this project is to understand the cellular mechanisms by which organochlorine pesticides act to cause cancer-like transformation of human breast cancer cells under laboratory (in vitro) conditions.
The role of environmental organochlorines in breast cancer is the subject of continuing debate and research. Epidemiological studies have reported numerous environmental chemical contaminants to be present in normal and cancerous breast tissue, including organochlorine insecticides and polychlorinated biphenyls. The proposed study will address the interaction between estrogen and insecticides in human breast cancer cells and normal cells. Specifically, we will study the role of growth factors and the process of transmitting the growth cues from outside of the cell to the cellular proteins (signal transduction) in the insecticide-induced foci formation (a biomarker for cancer promotion).
Our preliminary study identified two components involved in the signal transduction pathway in human breast cancer cells that are activated by organochlorine insecticide treatment. These two components are c-Neu kinase and c-src kinase. We believe that the interaction between these two enzymes is a key point at which organochlorine insecticides promote breast cancer. Our study will address whether the blocking of c-Neu and c-src kinases signaling using specific inhibitors for these two enzymes will prevent the induction of foci formation by organochlorine insecticides in cells. In addition, we will examine the interaction of DDT and Lindane with the estrogen response pathway in breast cancer cells. Furthermore, it is the goal of this proposal to identify and establish a biomarker for early detection of breast cancer promotion. We will compare the level of pesticide residues found in breast tissues of cancer patients with the levels that are required to affect breast cancer cells in the laboratory.
Why are these basic mechanistic studies on cell transformation necessary? The major benefits of these studies are to provide the logical basis for understanding the action of pesticides on breast tissue and to identify other environmental chemicals that may increase the risk of breast cancer in exposed women.
Genetic Repair of Oxidative Damage: Effect of Estrogen
Nicholas J. Rampino, Ph.D.
The Burnham Institute
Estrogen has been implicated in the pathogenesis of breast cancer. Since many tumors grow in response to estrogen, synthetic estrogen antagonists have been widely studied as potential therapeutic agents for hormone dependent cancers. Tamoxifen, a synthetic anti-estrogen, is among the most successful specific therapies for breast cancer. Tamoxifen, however, displays estrogen-like effects in the uterus, increasing the risk of uterine cancer. Our ultimate goal is the identification of specific anti-estrogens that can stop breast cancer without stimulating cancer in other tissues, particularly the uterus.
Estrogen strongly stimulates breast cell division. When a cell divides, it goes through a process where it makes copies of its DNA and then splits in two new cells, each of which contains a carbon copy of the original DNA. The cells pause in what is called a resting phase between each division. During the resting phase, the cells check their DNA for damage (mutations) and repair it. We hypothesize that at least one way that estrogen may drive cancer development is by causing the cells to divide quickly and rush through this resting phase, which would mean that they would not have time to repair all of the DNA damage. The damaged DNA would produce changes in the cells that eventually cause them to become cancers. We also hypothesize that anti-estrogens will keep the cells in resting phase, thus prolonging the amount of time the cell has to repair DNA damage.
To test this hypothesis, we will make use of human breast and uterine cells that we expose to an oxidative stress, which causes DNA damage, and estrogen or anti-estrogen. Identification of specific anti-estrogens that can decrease DNA damage in breast tissue without unwanted side effects in other tissues, particularly uterus, will provide highly effective anti-cancer therapies for the prevention and/or cure of breast cancer.
Hormonal Control of Normal Human Breast Tissue
Jason Yang, M.D., Ph.D.
Cancer Research Fund of Contra Costa
Hormones and reproductive factors, such as early menarche, late menopause, and late age at first full-term pregnancy, are associated with human breast cancer development and progression. It has been proposed that breast cancer risk may be greatly influenced by the total cumulative exposure of breast tissue to hormones and the associated cumulative cell proliferation. However, very little is known about the role of hormones in human breast development. Our proposal is directly aimed at gaining insight into the hormonal control of human breast cell growth. Perhaps the only definitive way to study human breast cell growth consists of sampling the patient’s breast in response to various hormonal manipulations and following the clinical course. Such a longitudinal study, of course, cannot be done, and the analyses of the hormonal stimulated breast cell growth in a patient can never be followed. Therefore, past studies have predominantly utilized mammary glands from mice and rats, as well as established cell lines from human breast cancer origin which have been propagated in culture dishes for an extended period of time. The generality of findings from these studies to breast cells in human females, unfortunately, cannot be assumed.
The availability of a model system that could propagate and maintain human breast cells similar to those actually seen in a patient would greatly enhance our understanding of the hormonal control of human breast cell growth. We plan to develop such a model system, the "reconstituted human breast" in mice. In essence, the breast cells from a single patient can be propagated into multiple "reconstituted human breasts" in an experimentally manipulatable animal system. This model system thus provides a unique opportunity to study normal human breast cell proliferation and estrogen/progesterone receptor expression, as well as the influence of various hormonal environments. Specifically, we plan to provide answers to 3 questions: (1) We will inquire whether estrogen alone, or estrogen plus progesterone combined is more growth promoting in vivo for normal human breast epithelial cells. (2) We will test whether an environment similar to human pregnancy can be mimicked in our model system. First-trimester pregnancy is a period in which the greatest extent of breast cell proliferation is seen in a woman’s life. (3) We will inquire which, among the many growth factors, is most stimulatory in vivo for proliferation of normal human breast epithelial cells. Collectively, these results using a physiologically relevant in vivo model system will not only provide a better understanding of normal human breast cell proliferation but may have far-reaching implications in both the prevention and intervention of human breast cancer. One direct translational potential of these studies may be to provide a framework on which to select the best formulation for oral contraceptive and/or hormone replacement therapy with minimal effect on breast cancer risk, similar to what has already been accomplished for endometrial cancer. The breast cancer risk may be greatly influenced by the total cumulative lifetime exposure to estrogen and the associated cumulative cell proliferation. Therefore, should progesterone augment the effect of estrogen in terms of cell proliferation, exogenous hormones consisting of both estrogen and progesterone may increase the risk for breast cancer. We believe that our model system, aside from human experimentation in clinical trials, may come as close as possible in determining the hormonal control on human breast development.
Breast Cancer Chemoprevention by Retinoids
Xiao-kun Zhang, Ph.D.
The Burnham Institute
Natural retinoids (vitamin A derivatives) and their synthetic analogs can prevent the development of breast cancer in animal models. Several retinoids are now being evaluated for their ability to prevent breast cancer recurrence in patients at risk. These retinoids can inhibit the growth of cancer cells or cause them to undergo a type of cell death (apoptosis) in which their genetic material is degraded. Although one of these retinoids showed promising activity against breast cancer development in pre-menopausal women in a recent clinical trial, most retinoids either do not prevent the appearance of more malignant breast cancers that have lost their dependence on estrogens for growth or must be given at such high doses to prevent new tumors that they are toxic to patients. The antiestrogen tamoxifen is in clinical use to prevent new tumors, but patients whose tumors are originally inhibited may only respond for 2 to 5 years, after which more aggressive tumors can appear. Therefore, more effective compounds for prevention of breast cancer are urgently needed.
Our proposed project will focus on identifying retinoids to prevent breast cancer development in both pre-menopausal and post-menopausal women. The research will be based on molecular and cellular mechanistic studies of how retinoid effects are mediated through the retinoid receptors (RAR a, b, and g and RXRa, b, and g) to develop retinoids that can inhibit the growth of both estrogen-dependent and -independent breast cancer cells. Our team of molecular and cancer biologists and chemists has exciting preliminary results on retinoids that we are investigating for prevention and treatment of breast cancer through their interactions with retinoid receptors in cells. We found that the natural retinoids can induce RARb and cell death in estrogen-dependent breast cancer cells that express high levels of RARa. Although they had little effect on the more malignant, estrogen-independent breast cancer cells due to low RARa levels in the cells, we discovered that several RXR-selective synthetic retinoids, when used together with RAR-selective retinoids, can effectively induce RARa and cell death of the cells, probably through a different retinoid signaling pathway. In addition, we have identified a number of other novel RAR selective retinoids that alone can effectively induce cell death in both breast cancer cell types. Very recently, we found that retinoid receptors can interact with Bag-l, a gene that prevents cell death, thereby providing an opportunity to develop retinoids that selectively promote receptor/Bag-1 interaction and induce apoptosis. We will build on these results to (1) gain a better understanding of how retinoids and their receptors prevent breast cancer development and (2) identify improved retinoids as breast cancer preventive agents.
We propose to identify how these retinoids exert their anti-breast cancer effects and then adapt these mechanisms to identify more effective retinoids. We will first study how retinoids, on binding to their receptors in both estrogen-dependent and -independent breast cancer cells, induce the synthesis of RARb, which is lost or inactivated when cells become cancerous. We will analyze the effects of these retinoids on RARb activities and induction of Bag-1 to clarify their mechanisms of action. These results will enable us to design and prepare more selective retinoids with enhanced cancer preventive activity, which will then be evaluated in molecular and cellular assays for breast cancer cell growth inhibition and cell death induction. The most effective and selective retinoid found in this assay, and two modified versions that permit it to concentrate in breast tissue, will be evaluated for breast cancer prevention in the rat. The outcome of our improved understanding of how retinoids prevent breast cancer development should lead to identifying more effective retinoids to prevent this devastating disease in California residents.
