Molecular & Cell Biology
Research Project Awards
- Mechanisms of Aberrant Cell Growth in Breast Cancer – Daniel J. Donoghue, Ph.D. University of California, San Diego
- Regaining Control over Breast Cancer Cells – Michael Karin, Ph.D. University of California, San Diego
- Telomerase: A Factor for Early Detection of Breast Cancer – Nam Woo Kim, Ph.D. Geron Corporation
- The Role of MAR-Binding Protein in Breast Cancer – Terumi Kohwi-Shigematsu,
Ph.D. La Jolla Cancer Research Foundation
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 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 importance 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.
Regaining Control over Breast Cancer Cells
Michael Karin, Ph.D.
University of California, San Diego
Early in life most cells divide to increase the mass of the organism; at that point they are "undifferentiated," because they do not express specific traits. In response to specific signals, cells stop dividing and start expressing specific traits, thus they become differentiated. In an adult breast, most of the cells are differentiated as either fat cells or supporting tissue. A few undifferentiated epithelial cells divide during each menstrual cycle; some of the new cells differentiate as milk producing cell precursors. Without pregnancy hormones, the differentiated cells die. By contrast, breast cancer cells continue to divide and none "differentiate" to milk producing cells. Normal cells divide in response to "growth factors" which are "messenger" molecules from other parts of the body. Breast cancer cells also produce their own growth factors.
Undifferentiated cells recognize and respond to the growth factors through proteins called receptors embedded in their cell membrane. Cancer cells also produce excessive amounts of receptor proteins. One such protein that is overproduced in 25% of cell human breast cancer is called erbB2.
Recently a small protein that binds erbB2, called heregulin, was found; intriguingly, the effect of heregulin is inconsistent and difficult to predict. When applied to certain breast cancer cell lines, proliferation stops and differentiation is induced. However, when applied to other cells, proliferation is stimulated. Thus, heregulin acts as a growth factor. We propose to study the molecular basis for this variability. This may allow the design of strategies for preventing disease progression by allowing heregulin to induce differentiation in a highly consistent and efficient manner.
The chemical signals generated by erbB2 once it is bound to heregulin are transmitted to the cell's genetic machinery located in its nucleus through specialized enzymes called Mitogen Activated Protein Kinases (MAPKs). These enzymes transmit different signals that tell the cell to either divide, differentiate or commit suicide and die. This plethora of responses is made possible because there are three different types of MAPKs called ERKS, JNKs and p38/Mpk2. The exact biological functions of these enzymes are not well established. Therefore, we propose to examine the effects of heregulin on the three different enzymes in different types of breast cancer cells where it can induce either proliferation or differentiation. We propose that the exact cellular response to heregulin is determined by a "code" of MPK activation. We will try to "break" and interfere with this "code" through the selective inhibition of the enzymes described above. This, we hope, will allow different types of breast cancer cells to mount a consistent and predictable differentiation response to heregulin, thus stopping tumor growth.
Telomerase: A Factor for Early Detection of Breast Cancer
Nam Woo Kim, Ph.D.
Geron Corporation
According to recent research, normal cells have a predetermined lifespan controlled by the telomere. The telomere is a length of protective DNA that exists at the end of each chromosome and serves as a clocking mechanism that determines how many times a cell can divide and grow. In normal cells, the telomere shortens with each cell division, and when the telomere reaches a critically short length, the cell becomes "old" and stops dividing. Even in the presence of abnormal growth controls, breast cancer cells will have a finite lifespan due to the shortening of telomeres. For a breast cancer to continue to grow, the cancer cells must prevent critical telomere loss and become immortal. The cancer cells accomplish this by producing telomerase, an enzyme which prevents telomere shortening.
We will evaluate telomerase expression using Geron's highly sensitive proprietary assay to test blood, lymph node biopsies, and tumor biopsies from breast cancer patients. The data gathered from these studies will help correlate telomerase activity with the presence and stage of cancer. We also expect to develop a second generation telomerase assay that will be easier and more cost-effective to perform, and can be used to detect telomerase-positive cancer cells via microscopic examination of tissues. It is anticipated that our findings will result in more accurate and early detection of breast cancer, and a potential to predict which patients will experience more aggressive forms of the disease. This information can be used to reduce morbidity and mortality in breast cancer patients in California.
The Role of MAR-Binding Protein in Breast Cancer
Terumi Kohwi-Shigematsu, Ph.D.
La Jolla Cancer Research Foundation
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 of protein and resembles a mesh in design. Some segments of the DNA, called matrix-attachment 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.
Innovative Developmental and Exploratory Awards (IDEAs)
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 tumorigenic factors which induce its abnormal production, and further identify the assay's potential to detect very early breast cancers.
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.
New Investigator Awards
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.
Loss of p53 Tumor Suppressor Function in Breast Cancer
Jamil A. 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 misloca-tion in breast cancers. One protein previously dem-onstrated 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.
Postdoctoral Fellowship Awards
- EGFR Structure for Drug Design Targeted to Breast Cancer – Chung-leung Chan, Ph.D. University of California, San Diego
- Estrogen Receptor Regulation in Breast Cancer – Lisa A. McPherson, Ph.D. Stanford University
- Vitamin D and Breast Cancer Prevention: Cell Death vs Growth – Valerie M. Weaver, Ph.D. Lawrence Berkeley National Laboratory
EGFR Structure for Drug Design Targeted to Breast Cancer
Chung-leung Chan, Ph.D.
University of California, San Diego
Protein tyrosine kinases are a family of biomolecules that play crucial roles in maintaining a normal balance in cell growth and development. Two members of this family, EGFR and erbB-2, are particularly important in breast cancer because increased production frequently occurs in breast cancer. Tumor aggressiveness and patient outcomes can be directly correlated to their production levels. Drugs which can control their activity are able to stop the development of breast tumors and restore normal breast cell growth. However, the selectivity of this family of drugs is quite limited and often leads to serious side effects. Research proposed in this postdoctoral fellowship application addresses the fundamental issue of how to design specific drugs to prevent the progression of breast cancer. This proposal outlines several strategies that will allow us to examine the structure of EGFR in detail. The structural features that determine drug specificity will be identified. As the factors which control this specificity in EGFR begin to unfold, we will be able to design drugs with selective inhibitory effects on EGFR. Such progress may lead to specific drugs for controlling the growth of breast cancer cells.
Estrogen Receptor Regulation in Breast Cancer
Lisa A. McPherson, Ph.D.
Stanford University
In breast cancer, the majority of tumors found in postmenopausal women contain estrogen receptor while tumors in younger women often lack this protein. Estrogen receptor is a protein normally found in various reproduction-related tissues such as the breast and uterus. When estrogen receptor binds estrogen, it becomes activated and can interact with the genes of the cell, resulting in the activation of selected sets of responsive genes. This results in changes in the synthesis of specific RNA's and proteins involved in the regulation of cell proliferation, differentiation and physiologic function. Although normal breast tissue also makes estrogen receptor, the amount of this protein produced in positive breast carcinomas is significantly higher. This may account for some of the differences seen in the abnormal growth of various tumors and tumor cell lines when compared to normal breast tissue development. Breast carcinomas that do not make estrogen receptor tend to recur earlier than estrogen receptor-positive tumors and are also more aggressive, independent of any effect of hormonal therapy. Elucidating the mechanism by which the production of estrogen receptor is controlled in these tumors will lead to strategies by which the growth of aggressive estrogen receptor-negative tumors could be controlled.
Recent experiments have indicated the existence of a protein, Estrogen Receptor Factor-1 (ERF-1), which is involved in the regulation of estrogen receptor production and which is only made in estrogen receptor-positive breast and endometrial carcinomas. In addition to regulating estrogen receptor, it is possible that ERF-1 also regulates other cellular genes critical to breast cancer. We hypothesize that ERF-1 is involved in maintaining the characteristics associated with estrogen receptor production in breast carcinoma. This study seeks to determine the protein sequence of ERF-1 and to study the mechanism by which it regulates estrogen receptor production in breast cancer cell lines. The identification of ERF-1 could aid in the development of gene therapy protocols to stop the progression of tumors lacking both ERF-1 and estrogen receptor.
Vitamin D and Breast Cancer Prevention: Cell Death vs Growth
Valerie M. Weaver, Ph.D.
Lawrence Berkeley National Laboratory
Breast cancer occurs as a result of a transition of normal breast epithelial cell behavior to that of uncontrolled cell growth. A major factor in this transition is the gradual loss of the normal cellular balance between cell division and cell death. An important function of the genes in a normal breast cell is to control the appropriate time for the cell to die. Such cell death balances the growth which occurs due to normal cell division. In all non-menopausal women there is a sequential burst of cell division followed by a tightly controlled round of cell death with each menstrual cycle. We have found that the extracellular matrix (ECM, an interwoven network of proteins which is in contact with these breast epithelial cells and gives them strength and support) gives signals which provide information to these cells. Thus the ability of breast cells to respond correctly to these messages is critical for maintaining the normal cell balance. In breast cancer the genes of the breast epithelial cell are altered such that these cells are now unable to respond appropriately to breast tissue ECM-derived signals. There is reason to believe that the active compound of vitamin D may act on breast cells to regulate the genes required for this responsiveness. We hypothesize that vitamin D may re-establish the normal cell growth and death cycle and that these effects may be mediated through critical cell-ECM signals. Regardless of the mode of action, however, vitamin D may be effective in assisting with the prevention of both primary breast cancer and its recurrence following therapy.
We have access to a unique human breast cell line which mimics breast cancer progression as it happens in the body. In addition we have the expertise with a novel ECM assay which allows the reconstruction of the normal breast cell environment in culture. Using this system I will examine the ability of breast epithelial cells to respond appropriately to signals from the ECM as they undergo transition from normal to cancerous behavior. Emphasis will be placed on their ability to undergo appropriate cell death. After establishing when the critical changes occur in this transition, I will study the ability of vitamin D to rescue this aberrant behavior. Finally I will begin studies to determine what crucial factors are important for this altered responsiveness by isolating vitamin D-responsive genes. The validity of these results will be determined by using clinical samples of normal and cancerous human breast tissue. These results will have important consequences for understanding the development of both primary and recurrent breast tumors. They should also provide information for the development of better diagnostic techniques.
