Molecular Genetics
Research Project Awards
- A Candidate Breast Tumor Suppressor Gene on Chromosome 13 – David R. Schott, Ph.D. California Pacific Medical Center
- Genes Involved in Multistep Mammary Tumorigenesis – Gregory M. Shackleford, Ph.D. Childrens Hospital, Los Angeles
- Genes Which Cause Increased Cellular DNA in Breast Cancer – Michael F. Press, M.D., Ph.D. University of Southern California
- Metabolism of Environmental Chemicals as Breast Cancer Risk – Regine Goth-Goldstein, Ph.D. Lawrence Berkeley National Laboratory
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 are tumor suppressor genes (TSG) that normally function 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 primary breast tumors and about half of 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
Gregory M. Shackleford, Ph.D.
Children's 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 transgenes) 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.
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. 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 tetraploidy. 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 (Broek et. al. Nature 349:388-393, 1991). 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.
Metabolism of Environmental Chemicals as Breast Cancer Risk
Regine Goth-Goldstein, Ph.D.
Lawerence Berkeley National Laboratory
Analysis of mutations in breast tumors suggests that environmental chemicals such as polycyclic aromatic hydrocarbons (PAHs) might be involved in the development of breast cancer. PAHs are ubiquitous in the environment, and are ingested and inhaled continuously. PAHs act as carcinogens only after being activated in the body. The balance of PAH-activating and detoxifying enzymes determines the amount of binding of PAHs to DNA, which in turn determines the risk of cancer initiation. The activity of these enzymes is determined by genetic and environmental factors. The synthesis of activating enzymes can be induced by PAHs themselves and other environmental toxins, such as dioxin. The interindivi-dual variations in activating and detoxifying enzymes contribute to individual susceptibility to cancer of the lung, colon and bladder. It is proposed to determine if the amount of PAH-activating and detoxifying enzymes in breast tissue represents a risk factor for breast cancer. If this is the case, women with increased susceptibility could be identified and certain preventive measures could be taken, such as more frequent examination, changes in lifestyle to reduce PAH exposure, and possibly changes in diet to alter the activity of PAH-metabolizing enzymes.
These hypotheses will be tested by examining the two major enzymes involved in PAH metabolism: the activating enzyme, cytochrome P4501A1, encoded by the gene CYPlAl, and the detoxifying enzyme glutathione S-transferase, encoded by the gene GSTM1. Variability in the activity of these enzymes is due in part to genetic variation, and in part to modification of gene expression by environmental agents. The pres-ence or absence of the GSTM1 gene, and expression of CYP1A1 will be determined in breast tissue. A collection of about one hundred microscopically nor-mal breast tissue specimens from mastectomy pa-tients or from reduction mammoplasties will be analyzed. CYP1A1 expression (determined by quanti-tative reverse transcription polymerase chain reaction) and GSTM1 genotype (determined by PCR) will be compared in individuals with breast cancer and healthy individuals. In addition, the correlation of PAH-DNA adduct formation and PAH-metabolizing enzymes will be examined in vitro by treating cells from specimens with different enzyme activity with a model compound and then measuring DNA adducts. These studies should indicate if there is a trend of higher CYP1A1 expression and/or absence of GSTM1 in breast cancer patients.
Innovative Developmental and Exploratory Awards
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 information 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 and is related to a poorer prognosis.
New Investigator Awards
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.
Postdoctoral Fellowship Award
Cripto: A Breast Cancer Marker?
Christina C. Niemeyer, Ph.D.
La Jolla Cancer Research Foundation
The purpose of this project is to study cripto, a protein produced at elevated levels in a majority of human breast cancer cells but not in normal breast cells. Cripto is a growth factor for breast cells and can cause morphological changes. Thus, it is a good candidate for a breast cancer marker and could be used in the earlier detection of the threat of cancer. However, very little is known about its role in mammary gland development and during the various stages of breast carcinogenesis. Before any marker for mammary carcinogenesis can be used in early detection strategies, prior knowledge of its mode of activation is essential. Also important is determining how it can be detected. The long term objectives of this project are to determine the role cripto plays in mammary gland carcinogenesis and to ascertain its use as a diagnostic marker for breast cancer. Knowledge gained from this study of cripto will also open an avenue for prevention strategies.
Questions, hypotheses, and methods: 1) Is the over- production of cripto an initiation event or are the elevated levels observed later in tumorigenesis? The time course during various stages of tumor formation and progression will be studied in mice that produce mammary tumors caused by the over-production of oncogenes. Cripto activation possibly precedes the onset of cancer and therefore can be used as a marker of tumor-precursor cells. 2) Can chemical agents modulate cripto production and thus the transforming ability of cripto over- production? Cripto production is decreased by retinoic acid in certain cells. The effect of clinical treatment agents such as retinoids, tamoxifen, and selenium will be assayed. 3) Does over-production of cripto in mammary cells lead to abnormal mammary growth and does it act with other known oncogenes? Cripto will be over- and under- produced in a mammary cell culture to test the effect of aberrant cripto production on cultured mammary cells' potential to become tumorigenic or grow abnormally. Cells over-producing cripto will be transferred into normal mouse mammary fat pads and the cells allowed to develop into gland. The effect of cripto on mammary development and the effect that components of the normal mammary gland have on cripto over-producing cells will be tested. The question of whether cripto acts synergistically with other known oncogenes will also be studied.
