Endocrinology

Research Project Awards


Breast Cancer Chemoprevention by Vitamin D Plus Retinoids

Marcia I. Dawson, Ph.D.
Stanford Research Institute International

Vitamin D3 and the retinoic acid family (oxidized forms of vitamin A) inhibit the proliferation and growth of human breast cancer cells, including the vascularization process in tumors, which is necessary for the tumor to maintain its growth. Therefore, these compounds have outstanding potential as chemopreventative agents to inhibit the recurrence and spread of breast cancer, provided that their toxic side effects can be reduced. Preliminary breast cancer cell growth inhibition experiments indicate that combinations of these substances will permit lower effective doses than would be required if each agent were used alone. Therefore, side effects should be reduced. We will investigate the interactive molecular mechanisms of action of vitamin D3 and several different retinoids in inhibiting breast cancer cell growth and proliferation.

Retinoids show a range of activity profiles that include such diverse effects on cancer cells as selectively inducing the synthesis of proteins involved in normal growth processes, inhibiting the synthesis of other proteins responsible for cell proliferation, and initiating the sequence of events that lead to tumor cell death. We propose to evaluate the effects of these retinoids alone and in combination with vitamin D3 on breast cancer cell growth under conditions in which cells become attached to surfaces (indicative of less likelihood of invading other tissues) and under conditions in which cells grow without the need for attachment (indicative of more invasive cancers). Our preliminary results indicate that both agents that mimic retinoid action and agents that block retinoid action inhibit the growth of cells under conditions requiring attachment, but that only agents that mimic retinoid action inhibit the growth of cells under conditions not requiring attachment. The growth of cells under these latter conditions correlates with their ability to grow as tumors and metastasize. We will also evaluate the effects of retinoids and vitamin D3 on breast cancer cell migration and invasion.

To assess the effects at the molecular level, we will evaluate these retinoids alone and with vitamin D3 on the levels of two markers of cell proliferation, epidermal growth factor and the gene c-myc, which is over-active in cancer cells. Our ultimate goal is to devise new strategies for the chemoprevention of breast cancer, including the prevention of either its recurrence or its further progression.

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Control of Estrogen Production in Breast Cancer

Shiuan Chen, Ph.D.
City of Hope National Medical Center

Estrogens have a major effect on the development of breast cancer. About 60% of premenopausal and 75% of postmenopausal breast cancer patients have estrogen-dependent tumors. In estrogen-dependent breast tumors, estrogens induce the production of peptide growth factors which are responsible for the proliferative response of cancer cells. Aromatase is the enzyme that synthesizes estrogens, so an ab-normality in the function of aromatase in breast cancer tumors must have a significant influence on tumor growth in breast cancer patients.

During this grant period, we will design and perform essential experiments to examine the function of aromatase in breast cancer and in surrounding fat cells and develop methods to regulate the function of aromatase in breast cancer patients, including evalu- ation of the amount of aromatase in breast cancer tumors and in the surrounding fat cells. In addition, experiments are being performed to determine the structural characteristics of aromatase and to provide insight into the interaction between aromatase and its inhibitors.

We anticipate that this research will have a direct impact on understanding the cause of estrogen-dependent breast cancer and on the prevention of further progression of cancer by controlling the func-tion of aromatase with aromatase inhibitors. This research is relevant to two of the BCRP priority issues for the grant cycle, i.e., Pathogenesis and Prevention of Breast Cancer.

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New Endocrine Strategy to Prevent Breast Cancer Progression

Richard J. Pietras, M.D., Ph.D.
University of California, Los Angeles

Endocrine therapy is commonly used to prevent breast cancer progression, but most patients eventually develop hormone-resistant disease. This failure of hormone therapy often associates with gene abnormalities found in some breast cancers. Tbe growth of breast cells is regulated by hormones and growth factors which react with specific receptor molecules. Alterations in specific genes in breast cancers lead to the production of multiple copies of growth factor receptors in two-thirds of breast cancers. These alterations play an important role in the development and progression of cancer and appear to predict a poor response to endocrine therapy. This project will assess mechanisms by which altered genes affect the pathogenesis and hormonal sensitivity of breast cancers, and the results may lead to improved prevention strategies. Aims of this project are:

  1. To evaluate the potential interaction of altered growth factor receptors with receptors for estrogen hormones in the development of hormone resistance in human breast cancer. Using preclinical models, cells with low levels of growth factor receptor and matched cells with high levels of growth factor receptor will be tested for their differential sensitivity to estrogen and to antiestrogen drugs which block estrogen effects, such as tamoxifen. Experiments will be done in vitro and in vivo, utilizing a mouse model. In addition, we will assess the advantage of antiestrogen therapy in combination with protein antibodies which counteract the effects of specific growth factor receptors, including the HER-2 growth factor receptor.
  2. To investigate effects of high levels of growth factor receptors on the production and biologic action of receptors for estrogen in human breast cancer cells. Effects of the growth factor, heregulin, on interaction of estrogen receptor with DNA-binding sites in the nucleus of breast cancer cells will also be investigated.
  3. To determine effects of estrogen and antiestrogens on the production of growth factor receptors and growth factors, such as heregulin, in human breast cancer cells. Heregulins may be induced by estrogens in breast cancer cells. This hypothesis will be tested directly in breast cells. Regulation of the production and activity of growth factor receptors, in turn, by estrogens and antiestrogens will be further evaluated.
  4. To use clinical specimens to evaluate the relationship of heregulin and growth factor receptors to estrogen receptor and development of the estrogen-independent state in human breast cancer. Data on levels of growth factor receptors (HER-1, HER-2, HER-3, HER-4 receptors), heregulin growth factor and estrogen receptors will be correlated with independent data on the growth of actual tumors and the clinical course of patients in the clinic.

These studies may help to guide patient management decisions to prevent disease progression and, possibly, lead to alternate targeted strategies in breast cancers with overexpression of growth factor receptors.

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Role of Estrogen in the Origin of Breast Cancer

Peter J. Kushner, Ph.D.
University of California, San Francisco

A fundamental feature of breast cancer is that it occurs far more frequently in women than in men, although men have breast tissue and sometimes get breast cancer. This sex difference appears to be due to the presence in women of high levels of the ovarian steroid hormone estrogen, which acts on the breast to make it susceptible to tumor formation. Our studies will investigate the mechanisms whereby estrogen affects the growth of mammary and uterine tissue and contributes to the development of breast cancer. The understanding that comes from this work will be directly relevant to the design of better antiestrogens for breast cancer prevention that would lack the dangerous side effects on the uterus of current antiestrogens (i.e., tamoxifen).

To carry out these studies, we will employ genetic means to alter the estrogen receptor, the protein which binds to estrogen, in mice. We will then examine whether the altered estrogen receptors can contribute to the growth of the breast and of the uterus and to estrogen- induced breast cancer in these animals. We hope to learn which of several pathways of estrogen receptor action lead to these effects and to identify targets for breast cancer prevention.

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Innovative, Developmental and Exploratory Awards (IDEAs)


Progesterone Action in Human Breast Cancer

G. Shyamala, Ph.D.
Lawrence Berkeley National Laboratory

Epidemiological studies have clearly established that, excluding the genetic background, reproductive history is an important and consistent "natural" risk factor associated with breast cancer. In particular, both early menarche (beginning of menstruation) and late age onset of menopause have been shown to be associated with an increased breast cancer risk. Thus, it appears that it is the total length of time between menarche and menopause which is associated with the risk factor.

During each menstrual cycle, the ovaries synthesize and secrete the female sex steroids, estrogen and progesterone. Accordingly, the total length of time between menarche and menopause can be translated as the total years to which the normal breast is exposed to estrogen and progesterone. This, together with the fact that these ovarian steroids have been implicated in breast cancer for more than half a century, argues the need to target estrogen and progesterone as key factors responsible for the observed relationships between the reproductive history and breast cancer risk.

The normal breast is composed of several cell types and among these, it is the epithelial cells which are the ones most likely to give rise to cancers. In a normal breast, the epithelial cells divide during the progesterone dominant phase of the menstrual cycle, and these cells have a finite life span, such that there is a balance between this cell division and cell death. This balance is uncoupled in tumors accounting for their unrestricted growth. Therefore, by understanding how progesterone controls the division of epithelial cells in the normal breast, we can identify the potential steps which can become deranged and give rise to tumors.

The action of progesterone is controlled by progesterone receptors which are synthesized in response to estrogen. Therefore, to understand the role of estrogen and progesterone in breast cancer risk, it is particularly critical to understand the role of progesterone receptors on the normal breast. For this, we need to culture human breast epithelial cells containing progesterone receptors. At present, such a culture system does not exist. Although breast tumor cells containing progesterone receptors are available, the use of tumor cells (cells which have already undergone a derangement) is counterproductive for understanding the very basis for such derangement.

Thus, the goal of our proposal is to create normal human breast epithelial cell lines containing progesterone receptors and examine these for their responses to progesterone so that we may understand the role of progesterone in the breast cancer risk associated with the reproductive history of the female.

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New Investigator Awards


Understanding Tamoxifen- A Drug for Breast Cancer

Paul Webb, Ph.D.
University of California, San Francisco

Tamoxifen is a widely used drug that is known to prevent breast cancer growth. When breast cancer first arises the cancer cells often continue to require estrogen to grow, just as their normal counterparts do. The requirement for estrogen provides an unparalleled opportunity to control the growth of the cancer. Tamoxifen acts by opposing the action of estrogens, and is therefore termed an antiestrogen. This property means that tamoxifen will arrest estrogen-dependent breast cancer growth while other treatments, such as surgery and chemotherapy, can be tried. Tamoxifen treatment for breast cancer has proven very successful. It is even presently proposed that tamoxifen should be given to healthy women to prevent formation of breast cancer. Unfortunately, this is not without risks. Instead of opposing estrogen action, tamoxifen can sometimes act just like estrogen. In wo-men who take tamoxifen, this can result in stimulation of the growth of later stage breast tumors and increases in the risk of cancer of the uterus. Our goal is to understand how tamoxifen causes these estrogen-like effects and to use this knowledge to design better cancer blocking drugs. Estrogen acts by binding to a specific protein, called the estrogen receptor that, in turn, stimulates cell growth. Tamoxifen binds to the estrogen receptor protein, and dislodges estrogen from the estrogen binding pocket on the receptor. We recently showed that tamoxifen, when attached to the estrogen receptor protein, stimulates the activity of certain proteins (AP-1) that are normally involved in cell growth and may be important in cancer development.

In this project we will study tamoxifen activity on AP-1 in cell culture models that are easy to manipulate. We will then use genetic and biochemical approaches to understand how tamoxifen stimulates AP-1 activity and how the estrogen-like effects of tamoxifen on cancer growth might arise. We hope to use this work to develop screening systems that will tell us whether new breast cancer drugs might have similar harmful effects, and to identify new strategies for preventive drug design. New antiestrogens with more desirable properties have the potential to reduce the human and economic costs of breast cancer in California.

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Xenoestrogens and Genetic Damage in Breast Cancer

Moire Robertson Creek, Ph.D., M.P.H.
Stanford Research Institute International

Estrogens are a class of hormones critical to female sexual development and functioning, but, paradoxically, strong evidence links them with the induction of breast cancer. Precisely which estrogen is responsible is unknown, but high levels of 16a-hydroxyes- trone (16a-OHE), a metabolite of the estrogen estradiol, are strongly correlated with the susceptibility to and presence of breast cancer in human and animals. Additional evidence suggests that certain dietary and environmental factors may alter estrogen production or metabolism and thereby function as "xenoestrogens." Xenoestrogens are broadly defined as substances that alter normal estrogen homeostasis (regulation) and function. For example, dietary substances found in cruciferous vegetables, e.g., broccoli or cauliflower, lower the effective levels of 16a-OHE and decrease mammary tumor incidence by inducing competing metabolic pathways. On the other hand, ethanol is thought to increase tumor formation by enhancing metabolic pathways that form 16a-OHE. Definitive studies showing the relationship between 16a-OHE levels, enzyme induction or inhibition, and xenoestrogen exposure have not been conducted and are the subject of this proposal. These studies will define the relative ability of estradiol and its metabolites (e.g., 16a-OHE) to alter genetic material and will examine how xenoestrogens influence the formation of these genotoxic compounds by altering the metabolic pathways leading to their formation. This knowledge may ultimately lead to effective methods for identifying susceptible populations, detecting early stages of breast cancer, reducing preventable causes of breast cancer, and designing chemicals that will help prevent development of breast cancer. We will address two priority issues of the Breast Cancer Research Program: the role of genotoxic agents in the origin of breast cancer, and early detection of breast cancer.

We hypothesize that 16a-OHE may initiate tumor formation by interacting directly with proteins and DNA, thereby leading to chromosomal breakage or loss. Furthermore, since the metabolism of estradiol to 16a-OHE is dependent upon xenoestrogen-inducible metabolic enzyme systems, we also hypothesize that the formation of this metabolite may be affected by xenoestrogens. Specifically, we will study the chromosome-damaging effects of estradiol and its metabolites, investigate covalent binding of estradiol and its metabolites to large molecules both in cell cultures and in breast tissue of animals with varying susceptibilities to breast cancer, study the effect of selected xenoestrogens on the profile of covalent binding, and examine how xenoestrogens alter estradiol metabolism and estradiol-induced genetic damage.

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Postdoctoral Fellowship Awards


Isolation of Estrogen Receptor Cofactors from Breast Tumors

Thorsten Heinzel, Ph.D.
University of California, San Diego


The estrogen receptor is a protein which, in the presence of estradiol, binds to DNA and activates genes involved in cell growth. Estrogen receptor is present in approximately two-thirds of all tumors of postmenopausal breast cancer patients. The growth of these tumors is stimulated by estrogen through the action of estrogen receptor. However, the precise mechanism by which estrogen receptor regulates cell growth is still unknown. Antiestrogens, in particular tamoxifen, are widely used as drugs in the treatment of breast cancer because they can inactivate the estrogen receptor. Recently, proteins have been identified which bind to the estrogen receptor in the presence of estradiol but not in the presence of antiestrogens. Experimental data suggest that these proteins play an important role in the activation of genes by the estrogen receptor. They are therefore referred to as coactivators. Mutations affecting these cofactors could potentially cause breast cancer or could be responsible for the development of drug resistance during tamoxifen treatment of breast cancer tumors. The identification and characterization of coactivator proteins that interact with estrogen receptor specifically in breast tumor tissue or cell lines, as proposed for this project, will improve our understanding of the causes of breast cancer. Ultimately a biochemical assay could be used to screen new antiestrogens for maximum effects on estrogen receptor-coactivator interaction. This assay would facilitate the development of new drugs for breast cancer endocrine therapy. Additional drugs would be extremely valuable for this type of therapy because most breast cancer tumors eventually develop resistance after initially responding to tamoxifen.

In this project the approach for the discovery of novel coactivators will be based on a biochemical assay which analyzes protein-protein interactions of the estrogen receptor bound to DNA. Proteins from breast cancer cells or breast tumor tissue will be identified that can bind to the receptor in the presence of estrogen. Following the initial characterization of these potential coactivators the proteins will be isolated in quantities allowing the determination of partial amino acid sequences. DNA amplification techniques can then be used to clone the coactivator genes. The effects of coactivators on target gene regulation by estrogen receptor will be evaluated by introducing these coactivators into cell lines that do not normally produce them. A gene deletion approach can be used to test the relevance of the coactivators for cell growth in cell culture and mutations that either affect coactivator genes directly or alter their regulation can be evaluated for their role in breast cancer.

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Prevention of Breast Cancer Tumor Growth by Retinoic Acid

Yi Liu, Ph.D.
La Jolla Cancer Research Foundation

A substantial body of data has demonstrated that retinoids, the natural and synthetic vitamin A derivatives, are effective in inhibiting the proliferation of breast cancer cells. Currently, retinoids are being used in clinical trials against breast cancer. However, the anti-proliferative effect of retinoids is most frequently observed in hormone-dependent human breast cancer cells, and this effect appears to diminish in hormone-independent cells during the progression of breast cancer. The molecular mechanism of growth inhibition by retinoids is currently unclear. In the proposed project, the molecular mechanism of the retinoid growth inhibitory effect will be studied. Results from this study may provide a molecular basis for developing more effective and potent retinoids in the prevention of breast cancer, and may also lead to early detection and valuable diagnostic markers for breast cancer.

The effect of retinoids is controlled by nuclear retinoid receptors (proteins which bind to retinoids and change the production of other proteins). Recently we have found that retinoic acid receptor b, a nuclear retinoid receptor, is the only retinoid receptor whose expression is induced by retinoic acid in hormone-dependent, but not in hormone-independent cells, which correlates well with the observed retinoic acid growth inhibitory effect. Preliminary studies have further shown that the introduction of retinoic acid receptor b into a hormone-independent breast cancer cell line that lacks the production of retinoic acid receptor b restores the retinoic acid growth inhibitory effect. Based on these preliminary results, the proposed project will study the necessity of retinoic acid receptor b for retinoic acid growth inhibition in breast cancer cells. Efforts will be undertaken to study the retinoic acid receptor b production and the recovery of retinoic acid growth inhibition in different hormone-independent cells. Furthermore, retinoic acid receptor b selective retinoids will be used to specifically activate and counteract retinoic acid receptor b activity in both hormone-dependent cells and retinoic acid receptor b producing hormone-independent cells. Retinoic acid receptor b will also be eliminated from hormone-dependent cells using molecular biology techniques. Our preliminary studies have also demonstrated that cancer cell death can be induced by retinoic acid only in hormone-dependent cells. Therefore, the molecular mechanism by which retinoic acid receptor b induces cancer cell death and how the process contributes to the anti-cancer effect of retinoic acid will be studied.

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