Pathogenesis

Understanding the origin and metastatic spread of breast cancer is essential to the development of more effective prevention and treatment methods. For this reason, pathogenesis (the structural and functional changes in cells and tissues which lead to disease) was chosen by the Council as a priority area.

The Breast Cancer Research Program funded 25 grants in the area of pathogenesis to provide new information on the mechanisms by which breast cancer originates and spreads to distant body sites. The funded studies fall into three major categories.

The first category is the identification of genetic markers that indicate early events in breast cells which are associated with the development of breast cancer or the capacity of cancer cells to spread to distant sites. Specifically, the role of proteins such as BAX, ErbB2, Neu, p53, and p114 in cellular growth control and proliferation are being assessed. These investigations will also test whether alterations in the expression and/or function of specific gene products can serve as predictors of clinical outcome.

The second category includes investigations of the fundamental mechanisms of growth control in normal and cancerous breast cells and some important events that lead to the metastatic spread of breast cancer. These research projects aim to identify novel signaling pathways, new tumor suppressor genes, and new oncogenes that contribute to the development of tumors and tumor spread. A new method is being developed to compare the differential expression of multiple genes in normal and cancerous cells. Two proposals were funded to examine whether alterations in chromosome structure or chromosome number contribute to breast cancer progression.

The third catagory of studies addresses how changes in intracellular molecular signaling processes result in deregulation or loss of growth control. Funding was also provided to study molecules involved in extracellular cell-cell interactions that are important in the spread of breast cancer. In particular, the role of L-plastin, cell adhesion proteins, and metalloproteinases in promoting invasiveness of tumors is being assessed.

ABSTRACTS

The Invasive Nature of Epithelial Breast Cancer Cells

Pierre-Yves Desprez, Ph.D.
Lawrence Berkeley National Laboratory

Growth and differentiation (transition from one function to another) are intimately connected and controlled in normal cells. Cancer cells differ from normal in at least three fundamental ways. First, cancer cells grow inappropriately. Second, they become aberrantly differentiated. These two features are fundamental to virtually all cancers, and are inseparable. Cancer cells may also leave the body site from which they originated, invade the surrounding tissue and grow in otherwise foreign sites. This research will provide critical insights into how cancer cells lose proper control over growth and differentiation.

We developed a cell culture system of mammary epithelial cells, the cells from which 90% of all breast cancers arise. We found that an extracellular matrix (ECM, an interwoven network of proteins that give strength and support to cells) in contact with the cells is critical for inducing normal growth cessation and differentiation.

We also found that an intracellular protein known as Id-1 is critical for coordinating the changes in growth and differentiation induced by the ECM. In the mammary gland and in culture, Id-1 is produced when cells are growing, but not when they are growth arrested. We created breast epithelial cells in which Id-1 is always present. When contacted by ECM, these perpetual Id-1-expressing cells failed to undergo the change in differentiation, and migrated from their original site, grew and invaded the surrounding ECM.

How does Id-1 do this, and can we reverse the effect of Id-1 and induce abnormal cells to behave more normally? We know that Id proteins act by binding and inactivating another class of proteins called bHLH transcription factors. We hypothesize that normal breast epithelial cells produce bHLH proteins, which are essential for the cells to arrest growth, differentiate, and remain non-invasive. We will introduce the identified bHLH genes into undifferentiated breast cancer cells and ask whether the cells can now be induced to cease growth and differentiate. We will introduce these genes into metastatic cells and ask whether the cells lose the ability to invade surrounding tissue and colonize foreign sites. We will also test the effects of anticancer agents such as vitamin D3 and ask whether they act by reducing Id-1 expression. We propose that master genes like those encoding bHLH or Id proteins can alter the pattern of genes expressed by breast cancer cells, and thus alter their growth, differentiation and invasiveness.

BAX Gene Expression in Breast Cancer

John Reed, Ph.D.
La Jolla Cancer Research Foundation

Essentially all chemotherapeutic drugs commonly used in the treatment of cancer, as well as radiation, have been shown to ultimately kill cancer cells by triggering a form of cell death called “apoptosis.” Consequently, alterations in the regulation of cellular genes that control the apoptotic cell death process can play an important role in determining the relative chemosensitivity and resistance of tumors. Recently we obtained evidence that the pro-apoptotic gene BAX is normally turned-on (“expressed”) in breast tissue, but becomes inactive in approximately one-third of invasive breast cancers. In a study of 119 women with metastatic breast cancer, we found that patients whose tumors had lost BAX activity had poorer responses to combination chemotherapy, faster time to tumor progression, and shorter overall survival.

We now propose to extend these studies to larger groups of women to determine the prognostic significance of BAX as a predictor of responses to chemotherapy and clinical outcome for women with metastatic breast cancer. We also propose to study the molecular mechanisms that regulate the activity of the BAX gene in breast cancers, so that we might one day be able to improve tumor responses to currently available anticancer drugs. In this regard, it has been shown that radiation and DNA-damaging anticancer drugs can induce expression of BAX, suggesting that turning on this pro-apoptotic gene may be important for chemoresponses. We have recently delineated one of the factors that can regulate BAX gene activity. This regulator of BAX is called p53, and is a recognized tumor suppressor gene.

The p53 tumor suppressor gene becomes inactivated in over half of all human cancers, including many carcinomas of the breast. Loss of p53 has been associated with poor responses to chemotherapy and radiotherapy in several types of cancer, including adenocarcinoma of the breast. This tumor suppressor controls cellular growth through two mechanisms: (a) inhibiting cell division and (b) triggering of apoptosis. Though considerable advances have been made towards understanding the mechanisms by which p53 inhibits cell division, little is known about how p53 stimulates apoptosis. Recently, however, we showed that p53 directly turns on the BAX gene. This observation suggests for the first time a mechanism by which p53 might induce apoptosis. A further goal of this proposal therefore is to delineate the molecular mechanisms by which p53 regulates the expression of the BAX gene, and to determine whether increases in BAX gene expression are required for apoptosis induction by p53, as well as by radiation and anticancer drugs.

Taken together, these investigations will determine the clinical relevance of decreases in BAX gene activity for women with breast cancer, and will begin to provide insights into some of the molecular mechanisms that account for loss of BAX gene expression in metastatic breast cancers.

Growth Factor Control of Breast Tumor Cell Proliferation

Richard C. Kurten, Ph.D.
University of California, San Diego

The normal cyclic renewal of breast tissues is accompanied by variations in epithelial cell proliferation that are regulated by steroid hormones and peptide growth factors which are produced elsewhere in the body. I recently discovered two new human genes involved in the action of epidermal growth factor (EGF), a regulator of cell proliferation in breast tissues. I propose to evaluate their potential utility as targets for controlling the growth of breast tumor cells in tissue culture. This research is intended to provide a new foundation on which to design strategies for preventing disease progression and for treating advanced breast cancer in women. The potential impact of the planned research would be to provide another line of defense against breast cancer.

Epidermal growth factor (EGF) regulates cell proliferation by binding its receptor, a large protein spanning the thickness of the cell membrane. The receptor protein has several sections, each of which performs a different, but related function. On the outside of the cell is a section that binds to EGF. When that binding occurs, a section on the inside of the cell becomes active as an enzyme to catalyze reactions. This section of the receptor will be studied in this project because it regulates some protein interactions that preceed cell division. The genes I isolated code for two proteins, corkii-1 and corkii-2, that bind the enzymatic section of the molecule and regulate its level of activity. In so doing, corkii-1 and corkii-2 appear capable of controlling the ability of the EGF receptor to transmit information deeper into the cell. Most importantly, by controlling catalytic efficiency, these molecules set the sensitivity of a cell to EGF and determine the rate at which the cell divides; cells that divide too rapidly form tumors.

I plan to measure the levels of corkii-1 and corkii-2 in normal and malignant cultured cells derived from human breast tissue to establish the relationship between their levels and cellular proliferation rates. In a second line of investigation, molecular approaches employing antisense DNA technology and transfer of synthetic genes will be used to change the level of corkii-1 and corkii-2 in cultured breast tumor cells. The effects of these manipulations on proliferation of cultured breast tumor cells will serve as indicators of the potential utility of DNA-based drugs in slowing or eliminating tumor growth.

ErbB2 and Control of Growth in Breast Cancer Cells

Deborah L. Cadena, Ph.D.
University of California, San Diego

In order to address the important health issues surrounding the detection, diagnosis and prevention of breast cancer, it is important to understand the fundamental mechanisms of growth control in normal and malignant breast tissue. In approximately 30% of human breast cancers, a protein called ErbB2 is present at levels significantly above those found in normal cells. Breast cancer tumors which involve these larger amounts of ErbB2 protein are correlated with very aggressive cancer. The experiments proposed in this grant are intended to understand how the ErbB2 protein carries out its job in normal and malignant growth of breast cells. Ultimately, these results could lead to novel preventative approaches which could decrease the morbidity and mortality of breast cancer.

The ErbB2 protein is a member of a very important group of proteins called receptor tyrosine kinases. These receptors sit on the outer surface of cells where they wait to receive messages that are transported through the blood stream from other cells in the body. Such messages include hormones, such as insulin, and growth factors. When these receptors receive the appropriate message, they pass information to the inside of the cell by using an enzyme activity called tyrosine kinase. The tyrosine kinase family of enzymes serve extremely important functions by passing information inside cells, termed signal transduction, and are key regulators of cell growth. Because of this important function in regulating when cells grow, these receptor tyrosine kinases can contribute to cancer if their activity is abnormal.

In order to understand the relationship of ErbB2 to breast cancer, it is important to understand the fundamental mechanisms underlying signal transduction by the receptor. It is proposed that proteins that directly interact with ErbB2 will function in important signaling mechanisms. Using molecular biology techniques, molecules will be identified that interact with specific parts of ErbB2. The importance of these molecules will be determined by studying a human mammary cell line. Certain cell lines have characteristics of cancer cells. The newly identified molecules will be tested to see if parts of the molecule can actually alter these breast cancer cells. This result would strongly indicate that the molecule performs an important signaling function in breast cells. By analyzing the way that ErbB2 carries out its function in breast cells, it is possible that new strategies of preventing the progression of certain breast cancers may be developed.

Mechanisms of Aberrant Cell Growth in Breast Cancer

Daniel J. Donoghue, Ph.D.
University of California, San Diego

A number of different genetic alterations have been correlated with breast cancer, including frequent aberrations in genes that are important for regulation of cell division. Increased production of the protein coded for by one of these genes, named Neu, occurs in 20-30% of primary human breast tumors. Despite little understanding of the mechanistic details, it seems clear that aberrant activation of Neu plays a significant role in breast cancer by contributing to the proliferation of tumor cells. Therefore, understanding the role Neu plays in breast cancer may allow the design of interventions to prevent progression of the disease.

The Neu gene codes for a receptor protein which is normally functional only in response to the presence of a specific molecule, called a ligand, which has not yet been identified. When the ligand is present, two molecules of receptor come together and dimerize, leading to activation of the receptors, which in turn causes cell proliferation. The Neu receptor protein is embedded in the cellular membrane and, structurally, consists of a membrane- spanning domain which connects an ‘extra’-cellular domain to an ‘intra’-cellular domain. Upon dimerization, there is an activation of processes inside the cell, whereas, without dimerization, there is no signal to trigger cell growth. Although the Neu receptor protein is present in normal cells, breast cancer cells frequently exhibit increased dimerization and activation of Neu receptor molecules.

Neu has specialized functions in developmental and growth regulation of normal cells. Dimerization, activation, and the stimulation of cell proliferation are all normal processes of Neu; however, in oncogenic forms of Neu, these processes are unregulated and out of control, contributing to the characteristic growth of cancer cells. Previous work on the oncogenic activation of Neu has clearly established the importance of its transmembrane domain, which plays a central role in controlling the transmission of extracellular signals to the intracellular environment. The transmembrane domain is clearly involved in the aberrant dimerization and activation of oncogenic forms of Neu, which occurs in the absence of the normal protein that binds to Neu. The fundamental goal of this proposal is to increase our biochemical understanding of the im-portance of the transmembrane domain in the dimerization of Neu in breast cancer. This work will increase our understanding of the molecular and cell biology of breast cancer cells, and may allow the development of strategies for inhibiting the proliferation of breast cancer cells.

Regulatory Mechanisms for Growth & Motility in Breast Cancer

Ulla G. Knaus, Ph.D.
The Scripps Research Institute

The development and progression of breast cancer is not well understood, but appears to involve changes in normal cellular regulatory mechanisms for growth and motility. Important parts of this regulation involve proteins known as kinases. Kinases activate or inhibit other proteins by attaching a chemical phosphate group. The formation of cancers has been shown to depend on at least one of these kinases, known as MAP kinase. Recent findings suggest additional kinase pathways may also play critical roles in the developing tumors and in their ability to spread to other parts of the body. Our studies will investigate a possible novel pathway through which breast cancer and other tumors might develop. Knowledge of this new mechanism will lead to greater understanding of the pathogenesis of breast cancer and will enable us to identify new biological markers able to predict the course of the cancer (prognostic indicators). Information obtained on regulation of the motile responses of breast cancers will allow us to develop means to reduce or prevent such spreading. This will be a tremendous benefit, since cancers of the breast can be readily accessed prior to their spread into additional organs.

Previous studies have shown that activity of the normal MAP kinase pathway is controlled by Ras protein and that Ras directly induces the formation of tumors in this way. Ras is one of a family of proteins which bind a signaling molecule known as GTP. Other GTP-binding proteins of the Ras family, termed Rac and CDC42, may control other equally important regulatory kinases. We have identified such kinases, which are called p21-activated kinases or Paks. Based on a variety of data, we hypothesize that the Rac GTP-binding protein, via its ability to modulate the activity of Pak, regulates another set of mammalian MAP kinases. These new MAP kinases are likely to be important for controlling breast cancer development and spreading (metastasis). Using molecular biological and biochemical approaches, we will first determine whether Pak controls one of these novel MAP kinase pathways. We will then proceed to identify the actual sequence of proteins that may be intermediate between Pak(s) and the novel MAP kinases. These findings will be evaluated directly in breast-cancer derived cells, assessing the effects of the novel pathway(s) on growth control and motility. The studies described here form the required initial steps to understand the molecular basis for breast cancer, ultimately and directly leading to a greater ability to intervene effectively in this disease.

The Role of MAR-Binding Protein in Breast Cancer

Terumi Kohwi-Shigematsu, Ph.D.
La Jolla Cancer Research Center

The nucleus of a cell contains the genetic information of each individual, such as DNA. This DNA material is organized upon a framework within the nucleus called the nuclear matrix, which is made out of protein and resembles a mesh in design. Some segments of the DNA, called matrix-attach-ment regions (MARs), anchor themselves onto the nuclear matrix. Our laboratory has isolated a protein called p114 that binds to MARs. The MAR binding activity of this protein is found only in malignant human breast tumor specimens, not in normal breast tissues or benign breast disease tissues. This fact indicates that p114 might be an excellent diagnostic tool for the detection of cancerous cells. In addition, strong p114 MAR- binding activity has been detected in aggressive tumors that are more fully developed, while significantly weaker p114 activity has been observed in less aggressive tumors.

In this project, we will clone the gene for p114 and develop antibodies specific for p114 to evaluate the use of p114 as a diagnostic/prognostic marker for breast cancer. The information generated from this study may help us to understand how normal cells become cancerous and to develop better tools for the early detection and prevention of breast cancer.

Loss of P53 Tumor Suppressor Function in Breast Cancer

Jamil Momand, Ph.D.
City of Hope National Medical Center

The p53 tumor suppressor protein is largely responsible for protecting cells from cancer- causing DNA-damaging agents. In breast cancer, the p53 protein can be inactivated by four different mechanisms. First, a mutation within the gene can result in a p53 protein that is not functional. Second, a mutation within the gene can result in no expression of the p53 protein. Third, the p53 protein can be expressed in a normal fashion, but a second protein binds to the p53 protein and inactivates it. Fourth, the p53 protein can be expressed in a normal fashion but is located in the wrong place within the cell. This proposal sets out to identify proteins that bind and inactivate p53 and to uncover the mechanism responsible for p53 mislocation in breast cancers. One protein previously demonstrated to be a natural inhibitor of p53 is mdm-2. Mdm-2 binds normal p53 and inactivates the tumor suppressor activity of p53. While recent evidence indicates that some breast cancer cells express significant levels of mdm-2 it is not known if mdm-2 actually binds p53 and inhibits p53 activity. We propose to measure the relative levels of free p53 and p53 bound to mdm-2 in breast cancer cells and determine if p53 is functional. This will test the hypothesis that cells expressing a high proportion of p53 bound to mdm-2 exhibit the lowest p53 tumor suppressor activity. P53 may be inactivated by its inability to travel to the nucleus of the cell. In order for p53 to protect cells from DNA damage it must bind DNA in the nucleus. Studies have shown that several breast cancers contain normal p53, but that it is located outside the nucleus. It is unclear what prevents p53 from entering the nucleus but preliminary work has shown that a short-lived protein is required. We propose to purify this short-lived protein and identify it by protein sequence analysis. Identification of these alternate mechanisms of p53 inactivation has immediate relevance to increasing our understanding of breast cancer pathogenesis for the following four reasons: 1. Since the p53 gene can be the first gene mutated in breast cancer, identification of the proteins responsible for p53 inactivation will directly contribute to our understanding of breast cancer initiation at the molecular level; 2. If mutations in the genes coding these putative proteins are inherited, genetic screening of breast cancer families may provide a useful risk factor; 3. These proteins may be suitable targets for the design of more effective or less invasive cancer therapies; 4. The clinical outcome of breast cancer patients may correlate with the abnormal regulation of these proteins and, therefore, their identification may be used as a guideline for future therapy modalities.

Biology of Telomere Length Conservation in Breast Cancer

Darryl K. Shibata, M.D.
University of Southern California

Breast cancer is likely a heterogeneous disease which occurs after the accumulation of many different genetic alterations. This heterogeneity complicates diagnostic and prevention advances since multiple approaches are necessary. However, all dividing cells suffer from the shortening of their chromosomes since normal cells cannot copy chromosomal ends (telomeres). The progressive loss of telomeres prevents normal cells from dividing forever. In contrast, breast cancer cells can divide forever and kill because they abnormally express an enzyme (telomerase) which repairs telomeres. The transition between telomerase negative and telomerase positive breast cells marks a critical conversion between cells which cannot divide forever and cells which are immortal. Therefore, a single telomerase assay can potentially identify all “immortal” or tumor breast cells from any patient. Combined with the high specificity of telomerase activity for cancer, this highly sensitive assay may facilitate the molecular detection of breast cancer.

It is unknown when telomerase activity is expressed in breast cancer. Since many breast cancers contain both early and late tumor regions, microdissection of these regions followed by telomerase detection may identify whether it is produced very early or late in breast cancer development. A newly developed assay based on the polymerase chain reaction allows the sensitive detection of telomerase activity from as few as one cancer cell. Characterization of when telomerase is expressed during breast cancer progression should help identify tumor-igenic factors which induce its abnormal production, and further identify the assay's potential to detect very early breast cancers.

New Method for Measuring Breast Cancer Gene Expression

Daniel Pinkel, Ph.D.
Lawrence Berkeley National Laboratory

This IDEA grant provides the opportunity to perform proof of principle studies on a new concept for analysis of genetic function in breast cancer. If successful, this technique will provide new information on fundamental biological characteristics of breast tumors and their associated risk factors, and help contribute to the identification of new genes involved in breast cancer development. Such infomation is critical to the development of early detection and prevention stategies.

Malignant breast cancer cells contain abnormalities in the genetic code of their DNA which alter critical aspects of normal function. The first step in translation of the genetic code into function is the production of messenger RNA (mRNA) molecules, a different type for each gene. A mistake in the genetic code in a gene will result in an abnormal type of mRNA. In many cases this leads to an increase or decrease in the amount of that type of mRNA in the cell. Thus detection of abnormal mRNA levels provides one important view of the genetic events involved in the development of malignancy. Current techniques are only able to analyze mRNA produced by a few genes at a time, and these genes must have already been discovered. Since a large number of breast cancer genes are already known, and many remain to be discovered, more comprehensive analytical approaches are needed. We propose to develop a method that will: a) measure the expression of multiple genes in a single test; and b) contribute to discovery of new breast cancer genes.

The proposed method involves extraction of the mRNA produced by all of the genes in a breast tumor and labeling that mixture with a green fluorescent dye. Similarly all the mRNA is extracted from normal cells and labeled with a red dye. These are then combined and reacted with an array. Each element of the array contains many copies of the same DNA molecule, but the molecules differ among the elements. The mRNA produced from a gene binds specifically to the element that contains DNA with the genetic code from which it was originally made. Thus each element becomes stained with a mixture of green and red dyes, and the ratio of the two colors is proportional to the ratio of that mRNA in the tumor and normal cells. For example if a type of mRNA in a tumor becomes elevated its green to red ratio would be higher than the ratio for an mRNA that remains the same in tumor and normal cells, while if it became reduced in abundance its ratio would be lower. We will test this method by analyzing the expression of the gene cERBB2, which is known to be at elevated levels in some breast tumors, which is related to a poorer prognosis.

Genes Which Cause Increased Cellular DNA in Breast Cancer

Michael F. Press, M.D., Ph.D.
University of Southern California

The vast majority of human breast cancers have an increased, abnormal DNA content in each tumor cell; this condition is termed tetraploidy or aneuploidy. The normal amount of DNA, referred to as diploidy, consists of two copies of each gene on 23 pairs of chromosomes. Doubling of the normal amount of DNA in a cell is referred to as tetra-ploidy. Any other increase in DNA content is referred to as aneuploidy. The high frequency of increased ploidy in breast cancers suggests that events which increase the DNA content of cells (tetraploidy or aneuploidy) play a causative role in the genesis of some cancers. Our working hypothesis is that alterations in genes controlling DNA synthesis and cell division in the cell cycle are important for the development of human breast cancers.

To date no vertebrate gene has been identified as having the potential to disrupt the cell cycle causing an increase in ploidy. A sensitive assay has been developed in yeast to identify genes which increase the DNA content of cells. The yeast species used is one whose control of the cell cycle is remarkably similar to that of mammals. This species normally has one copy of each chromosome. In this assay, a single human gene is introduced into one yeast cell and each yeast cell is allowed to form a colony. Thus each colony will have one human gene added to its own genome. We propose to use this assay to screen for colonies, and thus genes from breast cancer which increase the amount of DNA in yeast cells. The genes associated with an increase in the yeast cell DNA will be identified and characterized. We will also determine whether the identified genes can induce a DNA ploidy increase when active in human breast epithelial cells. Further, we will determine whether the identified genes have oncogenic potential or whether they are mutated in human breast tumor DNAs.

The search for oncogenes and tumor suppressor genes has increased our understanding of breast cancer development. However, this understanding will continue to be incomplete until we understand the mechanism underlying the increased ploidy associated with most breast cancers. The lack of research concerning DNA ploidy increases has likely overlooked an important event(s) common to the genesis of the majority of breast cancers. The novel nature of this proposal and the lack of active research in this field, affecting the vast majority of breast cancers, provide an opportunity for significant advances in this important area of investigation. The lack of progress in this area of breast cancer pathogenesis is very likely due to the lack of a genetic assay for such genes in the past.

A Candidate Breast Tumor Suppressor Gene on Chromosome 13

David R. Schott, Ph.D.
California Pacific Medical Center

One of the fundamental goals in breast cancer research is to identify genes that are involved in the origin and progression of cancer. One group of cancer-related genes is the tumor suppressor genes (TSG) that normally functions to prevent or suppress tumors. When the TSG is mutated or lost and its function is lacking, tumors are then capable of unimpeded growth and proliferation. We have isolated a candidate gene (designated Brush-1) that is located on chromosome 13 in a region of frequent genetic instability in breast tumors. The Brush-1 gene's function is lost in more than one third of the primary breast tumors and in about half of the breast cancer cell lines tested. Furthermore, the loss of Brush-1 function occurs specifically in those breast tumors carrying the chromosome 13 genetic anomaly. This differential loss of Brush-1 function for both primary tumors and breast cancer cell lines is the expected pattern for a breast TSG. We will show a role for the normal Brush-1 gene in suppressing breast tumors by inserting a normal Brush-1 gene directly into breast cancer cells lacking Brush-1 function. We will then examine the effect of restoring Brush-1 function in these breast cancer cells. We will characterize these genetically modified cells for a number of cancer properties including high growth rate and motility and also the ability to invade other tissues and form tumors. Loss or reduction of tumor-related properties will support a key role for Brush-1 in suppressing breast cancer. To further characterize Brush-1, we will determine the localization and patterns of Brush-1 gene function in normal and tumor tissues. The proposed characterization of Brush-1 is a necessary first step toward an understanding of the processes involved in breast cancer. Besides serving as an important new genetic marker for breast cancer, the replacement of normal Brush-1 function may prove to be an important strategy in the prevention of a significant proportion of breast cancers.

Genes Involved in Multistep Mammary Tumorigenesis

Greg M. Shackleford, Ph.D.
Childrens Hospital, Los Angeles

It is generally accepted that breast cancer arises via a multistep process consisting of the accumulation of genetic lesions over time. The general objective of this research program is to gain a greater understanding of the genes involved in multistep mammary tumorigenesis. Toward this goal we are working to rapidly identify and isolate genes that contribute to the formation of mammary tumors. The identification of genes involved in the pathogenesis of breast cancer is a specific goal of the Breast Cancer Research Program. Defining the cumulative genetic events that lead to breast cancer and learning how the involved genes work together is ultimately necessary to fully understand the genetic and biochemical processes involved in the initiation and progression of this disease. This fundamental knowledge is important for the generation of directly pertinent genetic markers for breast cancer and for the design of targeted strategies for the prevention, earlier detection and treatment of breast cancer.

The foundation of our system for the identification of breast cancer genes (also called oncogenes) consists of transgenic mice. Transgenic mice are mice that have been genetically engineered to contain one or more additional genes (called trans-genes) in the cells of their tissues. Our system takes advantage of transgenic mice that already harbor two active breast cancer genes but require additional oncogene activations for a true tumor to arise. We have devised a powerful strategy that allows us to rapidly identify and isolate activated cellular oncogenes that cooperate with the oncogenes present in the transgenic mice. We have had excellent success using a similar strategy with transgenic mice carrying only one oncogenic transgene: four oncogenes have been identified to date that cooperate with the transgene in tumor formation. Using mice carrying two active oncogenes, we propose here to take the system a step further and identify genes at the third stage in multistep mammary tumorigenesis.

Malignant Transformation in Breast Epithelium

Heinz Furthmayr, M.D.
Stanford University

Several mechanisms are involved in the invasion of tissues by, and metastatic spread of, tumor cells. Among these are loss of differentiation, differences in communication between cells and with the extracellular environment, activation of proteolytic enzymes and changes in intracellular transfer of signals. The abnormal cell behaviour of invasive tumor cells is not the result of a single change but rather likely is caused by several interdependent events. The research during this sabbatical period will focus on mechanisms breast cancer cells use to communicate with each other and to sense their environment.

The extracellular matrix plays a critical role in differentiation in the breast gland and in growth regulation, motility and death of the cells. In response to signals from the extracellular matrix, cells change shape as a result of precisely regulated re-arrangements of proteins within the cell. These involve modifying enzymes, which act upon internal cytoskeletal proteins and regulate protein-protein interactions. It is believed that such signals are received by delicate and transiently formed extensions, called pseudopodia, which specifically recognize the extracellular matrix and transmit information for processing. These cellular structures are critical for cell movement and hence, for invasion of tissues and the ability of tumor cells to metastasize. Fluorescent derivatives of several relevant proteins will be prepared for introduction into mammary cells by microinjection and other methods. These cells will then be studied by time-lapse video and fluorescence microscopy.

The ultimate goal of this research program is the ability to interfere with the invasive property of breast cancer cells.

Role of L-Plastin in Breast Cancer

Ching-Shwun Lin, Ph.D.
University of California, San Francisco

L-plastin is a protein normally found in leukocytes (white blood cells) only. In these cells L-plastin regulates cell movement by interacting with actin, a major cellular protein that directly controls cell movement. Normal breast epithelial cells do not have L-plastin. However, we have discovered that all cultured breast carcinoma cells that were derived from breast tumors contain L-plastin. Furthermore, the amount of L-plastin contained in the cells correlates with the degree of invasiveness of these cells, as determined by matrigel invasion assay (where cell movement through a gel is measured). Therefore, it appears that L-plastin may be important for the motility of breast cancer cells. In this project we will (1) determine whether there is a correlation between the pattern of L-plastin synthesis and the degree of malignancy of clinically defined stages of breast tumors, (2) through the use of gene transfer determine whether overproduction of L-plastin will result in enhanced invasiveness, and (3) determine how the L-plastin gene is activated in breast cancer cells. For the first aim we will screen a panel of breast tumors that have been clinically graded at the UCSF Medical Center. We will use an anti-L-plas-tin antibody to detect the L-plastin protein or use an L-plastin DNA probe to detect the messenger RNA in these specimens. For the second aim we will introduce L-plastin, by a gene transfer technique called transfection, into normal breast epithelial cells. We will then perform matrigel invasion assay to see if increased L-plastin synthesis will result in enhanced invasiveness. Alternatively we will introduce a genetically engineered molecule called rybozyme into cultured breast cancer cells. We expect this molecule to specifically destroy the L-plastin messenger RNA, thereby preventing the synthesis of L-plastin. If so, the rybozyme-treated cells may lose the ability to invade. The third aim of this project is to elucidate the mechanisms by which the L-plastin gene gets turned on in breast cancer cells. We have isolated the entire L-plastin gene and identified a region that controls the synthesis of L-plastin messenger RNA. In this region, we have further recognized a few highly specialized DNA sequences that may be responsible for the activation of L-plastin gene in breast cancer cells. We will continue testing these DNA sequences by deleting or modifying the nucleotides to precisely determine how L-plastin gene is activated in breast cancer cells.

Cell Microenvironment and Progression of Breast Cancer

Andre Lochter, Ph.D.
Lawrence Berkeley National Laboratory

Progression of breast cancer from benign cells to a life-threatening disease is characterized by acquisition of migratory and invasive properties of tumor cells as they move through the breast tissue in search of blood vessels. It is this process and the subsequent infiltration of blood vessels and colonization of other organs which constitutes metastasis. From our own past and ongoing research, and from published reports, we have concluded that a major factor in tumor progression is the interaction of tumor cells with their surrounding microenvironment. This microenvironment consists of stroma, cells that create a supporting framework often composed of connective tissue for a gland or organ, and extracellular matrix (ECM), the interwoven network of proteins which provides additional support and vital information to surrounding cells.

It is known that normal functioning cells require the surrounding structure of the ECM. Although attempts have been made to understand the interaction of the cell with its microenvironment, the lack of suitable tissue culture models has hampered the precise interpretation of the data obtained and raised questions about their physiological relevance. We have now begun to reconstitute the cellular and ECM microenvironment of tumor cells in three-dimensions by taking into culture a cell line which recapitulates the different stages of tumor progression. With this designer microenvironment in hand, we are able to analyze the role of tumor cell-stroma interactions and molecules of the ECM in tumor progression and to test our hypotheses by experimental manipulation. In our investigation, we will focus on a class of matrix-degrading enzymes, metalloproteinases, which are frequently found in large amounts in breast tumors, and are believed to promote tumor cell invasion by breaking down the components of ECM.

The proposed study is expected to enable a better understanding of the development of breast cancer and to identify molecular changes associated with the early stages of tumor progression in the breast. Detection of these changes in tumor patients would thus provide a valuable tool for the diagnosis of breast cancer. Furthermore and foremost, we expect to identify those molecules which are indispensable for tumor migration and invasion. By interfering with their function we hope to block tumor progression. We will also search for signals inside the ECM which attenuate or abolish tumor progression. Upon identification of these inhibitory signals we will engineer small peptides (chains of amino acids) which can be administered to breast tissues and block tumor progression.

Breast Cancer Progression and the Extracellular Matrix

Francis S. Markland, Jr., Ph.D.
University of Southern California

The most common cause of death in breast cancer patients is the spread, or metastasis, of the cancer cells from the breast to the bones, lungs, liver and brain and the progressive growth of the cancer cells at these sites. Therefore, controlling breast cancer metastasis represents an effective method of preventing progression of the disease. We propose to use a multidisciplinary approach to not only study how the surrounding tissue effects the ability of breast cancer cells to spread, but also to evaluate a unique pharmacologic approach for combating breast cancer metastasis.

Research proposed in this application will lead to an enhanced understanding of the pathogenesis of breast cancer, one of the CBCRP's priority research issues. Recent studies suggest that cell adhesion proteins on breast cancer cells interact with the extracellular matrix (ECM), the tissue surrounding the tumor cells. This interaction induces increased production by the breast cancer cells of proteins that degrade the ECM. This degradation enables the tumor cells to invade the surrounding tissue and ultimately to enter the circulatory system. Once in the circulation, tumor cells travel to other organ sites where they progressively grow. Our investigations should lead to a better understanding of the role of cell adhesion proteins in breast cancer invasion and dissemination. A clearer understanding of this process will provide a basis for developing effective therapies for this most lethal aspect of breast cancer.

Our studies are also aimed at developing more effective interventions for preventing progression of breast cancer, another priority research issue of the CBCRP. Our approach will be aimed at blocking cell adhesion proteins on the breast cancer cells to prevent them from interacting with the ECM. This should also inhibit growth and dissemination of the breast cancer cells by: (1) preventing the growth of new blood vessels that provide nourishment for the cancer cells, a process called neovascularization; and (2) interfering with pathways leading to the production of proteins essential to the invasive properties of breast cancer cells.

Inhibition of cell adhesion can be achieved by treating breast cancer cells with proteins called disintegrins. The Principal Investigator has purified and characterized disintegrins and shown that they are well tolerated in animals. We will study the inhibitory effect of disintegrins on cancer growth, neovascularization, and metastasis in mammary fat pads. The results from this study will be directly applicable to over 90% of women with metastatic breast cancer in the state of California. Data gathered by our studies will support an important, new, and exciting approach for the prevention of breast cancer.

Model of Human Breast Cancer Development and Progression

Satyabrata Nandi, Ph.D.
University of California, Berkeley

The clinical course of human breast lesions and cancer is extremely variable. Our proposal is directly aimed at gaining insight into the variable clinical course of human breast cancer development and progression. Through a better understanding of these processes, new approaches will hopefully be identified for the development of both the prevention and treatment of human breast cancer. Perhaps the only definitive way to study human breast cancer development and progression consists of sampling the patient's breast lesion as a function of time until the very terminal stage. Such a longitudinal study, of course, cannot be done for ethical reasons, and surgical intervention is most often undertaken. The clinical course of the undisturbed lesion in vivo in a patient therefore can no longer be followed. However without such human experimentation, the biological course of various breast lesions and cancer can never be adequately analyzed.

The availability of a model system which could propagate and maintain human breast lesions, similar to those actually seen in surgical breast specimens, would greatly enhance our understanding of the human breast cancer development and progression. We propose in this application just such a model system, the “surrogate human breast” in mice. In this system the breast cells are isolated from human surgical breast specimens, embedded in collagen gel, and then transplanted subcutaneously in mice. Histological sections of recovered transplants show that various breast lesions and cancer similar to those actually seen in surgical breast specimens, as well as normal cells, can be maintained and propagated in this system. In essence, the breast lesion from a single patient has now been propagated into multiple “surrogate human breasts” in an animal for study. This model system thus provides an unique opportunity to study the development and progression of different breast lesions as well as various factors influencing their biological course. The influence of various hormonal environments and gene therapy on the development and progression of these lesions can be determined. Specifically, we propose to alter the hormonal environment of the host animals to determine the conditions affecting both positively and negatively the biological course of various breast lesions. On the analyses of genetic influence, we propose to utilize a highly efficient transfer of genes implicated to be of importance in human breast carcinogenesis. Collectively, these results using a physiologically relevant model system, the surrogate human breast in mice, will not only provide a better understanding of human breast cancer development and progression but may have far-reaching implications in both the prevention and treatment of human breast cancer.

HSP27 Regulation of Breast Tumor Blood Vessel Growth

Randolph S. Piotrowicz, Ph.D.
The Scripps Research Institute

The growth and spread of breast cancer is dependent on the establishment of a blood supply to the cancerous tumor. This is accomplished by the extension of existing blood vessels into the tumor mass by the migration and growth of the endothelial cells which line the intruding blood vessels. Highly vascularized tumors are generally larger and more likely to spread to other organs. The extent of tumor vascularization, therefore, is correlative with the risk of morbidity. Understanding how the generation of a tumor blood supply is regulated is essential to the understanding of the progression of the disease and may provide future avenues of prevention. Work in our laboratory has identified a small heat shock protein (HSP27) as a potential regulator of vessel growth. Increasing HSP27 levels by placing the human gene into bovine arterial endothelial cells results in enhanced growth and migration. HSP27 has been of interest to cancer researchers and oncologists because estrogen treatment of certain breast cancer cells, which causes accelerated growth of these cells, also causes increased HSP27 production. Since endothelial cell HSP27 is also responsive to estrogen, we propose that endothelial cell HSP27 regulates the vascularization of tumors which generate estrogen and/or grow in a milieu where estrogen is a key regulatory factor, eg. breast carcinoma. To test this hypothesis, the following is proposed. First, the effects of estrogen treatment or the co-culture of breast cancer cells on the growth and migration of endothelial cells producing elevated HSP27 levels or mutant HSP27 will be measured. In addition, the effect of lowering endothelial cell HSP27 levels (through the infection of adenovirus vectors carrying anti-sense DNA) on these processes will be tested. Second, culture conditions will be modified to support the formation of capillary-like structures. The effect of estrogen or co-cultured breast tumor cells on the generation of these structures will be measured. Finally, mice expressing human HSP27 in addition to mouse HSP27 will be used to test the effect of elevated endothelial cell HSP27 levels on the formation of new blood vessels. The effect of elevated circulating and local estrogen levels (the latter approximating the effect of tumor-generated estrogen) will also be assessed. These studies address the complex communications which occur between breast tumor cells and invading vascular cells and will delineate the role of a key player in breast tumor vascularization.

Changes in Transport into the Nucleus in Breast Cancer

Deborah J.Sweet, Ph.D.
The Scripps Research Institute

The aim of the research described in this proposal is to characterize the relationship between changes in movement of proteins between the nucleus and cytoplasm and the development of breast cancer. Exchange of proteins between the nucleus and cytoplasm occurs continuously and at a rapid rate, and is a fundamental aspect of normal cellular activity. It is also an important element of cell growth regulation, particularly for transmission of growth signals such as steroid hormones to the genome to produce changes in gene expression that alter the growth rate. Characterization of differences in the transport capacity of normal breast cells compared to those at different stages of conversion to a cancerous state will provide new insights into a previously unexplored aspect of the development of breast cancer. Improved understanding of these changes will provide additional information about the characteristics of breast tumors and could be used to enhance hormone based strategies.

Transport of proteins from the cytoplasm to the nucleus across the nuclear envelope, i.e. nuclear protein import, is a signal and energy-dependent process that requires the activity of both nuclear envelope associated and cytoplasmic factors. The nuclear import capacity of cells has been shown to vary depending on their growth state: rapidly growing cells have significantly higher nuclear import rates than cells that are not actively growing. The proposed research will characterize differences in the nuclear import capacity of human breast cell lines representing normal, proliferating and malignant tumor cells, with the aim of identifying the changes that occur during the development of cancer.

Mislocalization of steroid hormone receptors is a feature of some forms of breast cancer, so this study will also include a focus on the nuclear import of these receptors. It has recently become clear that steroid hormone receptors move continuously between the nucleus, where they affect gene expression, and the cytoplasm. Specific or general changes in the nuclear import pathway could therefore have significant effects on receptor localization within the cell and be a contributory factor in the development of cancer.

In the last few years, rapid progress has been made in understanding the molecular mechanism of nuclear protein import. Differences observed between the various cell types will therefore be investigated in more detail to determine whether they can be attributed to known transport factors, to better understand the molecular basis of breast cancer.

Control of Estrogen Production in Breast Cancer

Shiuan Chen, Ph.D.
Beckman Research Institute of the City of Hope

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 abnormality 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 evaluation 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 function of aromatase with aromatase inhibitors. This research is relevant to two of the Priority Breast Cancer Research Issues for the grant cycle. i.e., Pathogenesis and Prevention of Breast Cancer.

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 post-menopausal 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.

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.

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.

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. The 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 anti-estrogens 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 antiestro-gens 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.

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 counter-productive 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.

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 women 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 begin 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 cause cancer. 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.