Pathogenesis: Understanding the Disease
Innovative, Exploratory and Developmental Awards –
Type I
Innovative, Exploratory and Developmental Awards –
Type II
New Investigator Awards
Postdoctoral Fellowship Awards
The 17 new research grants awarded by the BCRP that address the priority issue of Pathogenesis examine the basic science-oriented processes in the initial development and progression of breast cancer. Some of the topics represented include: (i) cancer cell spread (metastasis) and blood vessel growth (angiogenesis) into tumors, (ii) enhanced growth of breast cancer through growth factor receptors (e.g., Her-2), (iii) the immunology of breast cancer, (iv) genes and mechanisms of gene regulation, and (v) intracellular signaling pathways, especially related to programmed cell death (apoptosis). The research methods employ the technologies of molecular biology, genetically altered (transgenic) animals, biochemistry, and immunology. Pathogenesis grants are mainly represented by three types of funding, (i) new investigator awards (ii) postdoctoral fellowships, and (iii) the Innovative, Developmental and Exploratory Awards (IDEAs). Respectively, this funding trains new researchers to tackle the breast cancer issues of tomorrow, and allows established researchers to test new approaches and ideas to break through current barriers in our understanding. The efforts to translate basic research into clinical application is seen in another BCRP priority issue: Innovative Treatment Modalities, discussed in the previous section.
The spread of breast cancer in the body is the least understood and most poorly treated aspect of the disease. Recent discoveries in angiogenesis offer new hope that effective therapy will be possible. To facilitate the development of these new findings, there is the need for model systems more closely representative of human disease to understand how breast cancer spreads. One project (Sonoko Narisawa) examines how breast cancer spreads to the bone, the most common site of metastasis. Pragada Sriramarao investigates the properties of newly formed tumor blood vessels with respect to binding circulating lymphocytes. The lack of an effective immune response sets the stage for breast cancer metastasis. However, some breast cancers, called ‘medullary’, have significant lymphocyte infiltration. Henrik Ditzel is looking for the breast cancer protein that stimulates this ‘medullary breast tumor’ T-lymphocyte infiltration, and could serve as the future basis for a vaccine. Earl Sawai is linking the processes of uncontrolled breast cancer cell growth to metastasis through a signaling molecule, phosphotidylinositol 3-kinase. Closely related to metastasis is the process of cell-cell adhesion that links the normal breast epithelial cells together and limits their growth. Karin Zeh is a postdoctoral fellow working with her mentor, Helene Baribault, to produce transgenic mice to study cell surface adhesion receptor-associated intracellular proteins. They plan to make whole animal mutations in these cytoskeletal proteins, called g-catenin and plakoglobin, to study their role as regulators of mammary development and cancer. Concurrent with spread to other sites, breast cancer cells lose contact with the local extracellular matrix. G. Shyamala is determining the relationship of the progesterone receptor with the activity of matrix metalloproteinases, presence of a matrix protein- laminin, and a cell surface receptor called E-cadherin.
A rapidly expanding topic in breast cancer research is the mechanism of intracelluar signaling and apoptosis. Normal cells will respond to either genetic damage, attack by the immune system, or chemotherapeutic treatment by initiating intracellular protein pathways that lead to cell death. In contrast, breast cancer cells show defects in these signaling functions and apoptosis, which allows genetic abnormalities to accumulate and makes the cells resistant to chemotherapy. Glenn Rosen is studying a compound derived from a traditional Chinese herb that sensitizes breast cancer cells to apoptosis. The protein signaling pathways inside breast cells that regulate apoptosis include the Bcl-2 (anti-apoptotic) family of proteins and the Bax (pro-apoptotic) family of proteins. Shu-ichi Matsuzawa is examining a family of p53 target proteins, called Siah, as downstream mediators of tumor suppressor function and apoptosis. p53 mutations are common in breast cancer, and some of these mutations could serve to disrupt other pathways inside the cell. Koji Itahana is looking at mutant p53s for their possible role in promoting breast cancer, beyond the loss of their normal function as tumor suppressors. The BRCA-1 gene product appears to act as a tumor suppressor and is normally located in the nucleus. Heinz Ruffner is examining the modification (phosphorylation) of BRCA-1 by looking for specific intracellular kinases. Another tumor suppressor protein, retinoblastoma, will be investigated for its interaction with associated proteins using x-ray crystallography (Kathryn Ely).
The search continues for the critical genes associated with either breast cancer or breast cell function. Breast cancer cells escape the normal limitations to growth and become immortal. Martha Stampfer is using a novel molecular approach to look for genes showing loss-of-function (tumor suppressor genes) and also permissive for immortalization. Fumiichiro Yamamoto is examining a novel set of genes that are believed to be associated with breast cancer development because they have a different number of methyl groups attached when compared to normal genes. Another aspect of chromosomal assembly is being investigated by Paul Kaufman, who will study Chromatin Assembly Factor (CAF)-I in breast cell senescence (aging). Kunxin Luo is working on a portion of a normal growth factor in mammary cells that could have a tumor suppressor function. Finally, Cary Lai is studying a possible regulatory binding protein, related to neuregulin, which could activate the ‘erb family’ of breast cancer growth receptors.
To summarize, the newly funded grants address a variety of topics at the forefront of research on breast cancer. Although these projects do not immediately impact breast cancer treatment, they provide new targets for future translation efforts. And, as we know more about the biochemical, molecular, and genetic differences between breast cancer and normal cells, drugs can be evaluated for their ability to target these critical elements to more effectively combat the disease.
Innovative, Exploratory and Developmental Awards – Type I
The Role of a Newly Discovered Neuregulin in Breast Cancer
Cary H.C. Lai, Ph.D.
The Scripps Research Institute
A number of protein factors and hormones affect the growth, survival and progression of breast cells from a normal to a malignant state. Our work focuses on finding molecules related to the molecule, neuregulin, which is known to activate the ErbB2 receptor that is expressed in roughly one-fourth of all human breast tumors. We believe that it is important to identify these molecules as their activities could affect the development of breast cancer and influence the growth rate of malignant cells.
We have identified a candidate novel neuregulin, AA238077, through a search of the databases that contain the DNA sequences of many genes of unknown function. When the sequence of AA238077 is compared with collections of known genes, it is most closely related to two of the previously identified distinct neuregulin genes, neuregulin-1 and neuregulin-3. While this similarity suggests that it too is a neuregulin, we must test its ability to activate ErbB2 and the related receptors ErbB3 and ErbB4 before its identity as a neuregulin-like molecule can be confirmed. We will accomplish this by first preparing the AA238077 protein in the laboratory and testing the ability of this protein to stimulate the activity of these receptors. Many breast cancer cells express one or more of the four ErbB receptors and we will use a set of tumor cell lines to assess the activity of AA238077.
The ability of neuregulin-1 to bind to breast cancer cells is currently being exploited as a way to target the specific destruction of ErbB2-expressing tumors. The general strategy is to couple a toxic substance to the neuregulin-1 molecule so that the breast tumor cells will be preferentially destroyed because they express more ErbB2 and other ErbB receptors than normal cells. Our previous identification and characterization of neuregulin-2 suggests that it binds to and activates certain breast cancer cells better than neuregulin-l. This suggests that each of the neuregulins, and perhaps AA238077, could be separately used to target different tumor populations. In addition, as it has been observed that neuregulin-1 can inhibit the ability of some breast cancer cells to divide, the distinct signaling properties of each of the neuregulins holds the promise that they may be useful to control the growth of different sets of breast tumors. Lastly, on a very practical level, it is important to know if any of the neuregulins is expressed (detectable) in the normal or cancerous breast as therapeutic strategies designed involving the use of these molecules could be affected by the presence of an as yet unidentified neuregulin-like molecule.
In order to best design therapies directed against breast cancer, it is important to identify as many of the molecular players involved. Although a complete understanding will certainly take many more years, we are in a position to make significant progress in finding the molecules that activate ErbB2 and the related receptors ErbB3 and ErbB4.
Intracellular Signaling Pathways in Metastasis
Earl Sawai, Ph.D.
University of California, Davis- School of Medicine
The spread of breast cancer to distant sites in the body is the most deadly of the many events in tumor progression. Much is known about specific surface receptors and adhesion events in metastasis, and our understanding of angiogenesis is creating clinical opportunities. However, there is a lack of specific information about the intracellular signaling events that potentially regulate metastasis. Signaling proteins inside the cell are the links between the genetic abnormalities (i.e., expression of oncogenes or loss of tumor suppressor genes) that characterize breast cancer and behavior (phenotype) of cancer cells (i.e., whether a certain tumor metastasizes). It may not be possible to correct specific genetic defects through gene therapy or other emerging technologies, but the metastatic phenotype could still be altered by targeting the regulatory factors operating within the cell.
Our focus is on a specific signaling molecule, called phosphatidylinositol 3-kinase (PI3K), a cell membrane-associated protein which is closely linked to critical growth factor receptors (e.g., Her-2). Our hypothesis is that PI3K connects uncontrolled tumor growth to the cell processes necessary for metastasis. We have developed a unique model system that allows us to study breast cancer metastasis. Using genetically altered breast cancer cell lines (called Met-1/Db-7) that vary in their metastatic potential when implanted into mice, we will determine whether activation of PI3K is critical for metastasis. This project will use a combined genetic, biochemical, and cell biology approach that will allow us to dissect the role of PI3K in metastasis. Our specific goals are to (i) determine whether the difference in metastatic potential of Met-1 and Db-7 is due to alterations in PI3K activation, (ii) determine whether disruption of the PI3K activation pathway affects metastatic and non-metastatic phenotypes in cell culture using either inactive or active mutants of PI3K, and (iii) analyze the metastatic potential of the tumor lines that inducibly express either the active or inactive mutants of PI3K in animals. The payoff is that we can compare the effects of different PI3K mutants on the ability of cancer cells to spread in the body.
We believe that further study of the pathways that control metastatic progression should facilitate both the identification of diagnostic markers and the development of novel therapeutic compounds that inhibit tumor dissemination.
Innovative, Exploratory and Developmental Awards – Type II
Role of g-Catenin in a Breast Cancer Mouse Model
Helene Baribault, Ph.D.
The Burnham Institute
Breast cancer is associated with numerous genetic alterations that lead to disregulated cellular functions. Our current interest is in the mechanism of defective cell adhesion signaling in breast cancer cells. Specifically, there are surface proteins called cadherins, which are located in cell-cell interaction sites. In normal epithelium, the interaction of cadherin proteins on adjacent cells signals the cells to stop dividing and form a normally functioning epithelial layer. This signaling becomes defective in cancer cells. There can be both a reduction in the amounts of the cadherins present, and in the intracellular signaling mechanism that arrest cell growth. The key signaling molecules linked to cadherins are called catenins. We are interested in two catenins, b-catenin and g-catenin (also called plakoglobin). Our hypothesis is that g-catenin acts like a tumor suppressor. Thus, we would expect that mutations that result in loss of g-catenin expression would be associated with the tumor phenotype, and overexpression or restoration of g-catenin would suppress tumor function. As supporting evidence for our hypothesis, it has been reported that chromosomal losses occur in breast cancer for the region where g-catenin is found and possible g-catenin loss-of-function mutations occur in breast cancer patient samples.
Our approach is to generate mice that overexpress g-catenin. For these experiments the gene for g-catenin is linked to a mammary virus gene regulation system and is introduced into mouse eggs. The resulting animals will overexpress g-catenin in the mammary glands. We will use molecular and biochemical methods to confirm the expression of our gene in the mammary gland of the mice. In addition, we will examine the structure of the mammary gland cells. One specific area of interest is the structure of the cell-cell epithelial junctions (desmosomes), which are formed through actions of the cell surface of cadherin receptors. There is evidence the g-catenin regulates the function of this key receptor in maintaining normal mammary epithelial structure. An additional experimental focus will be to look at the effects of the carcinogenic drug, ENU, in inducing cancer in the g-catenin overexpressing mice and whether any observed effects are associated with changes in the cellular mechanisms for programmed cell death (apoptosis).
Our study is designed to generate key information on how the mammary epithelial layer is organized, and what key genes are involved in sensitizing the cells to carcinogenic agents and causing cell death. These types of studies can only be performed in whole animals where the genes of interest can be manipulated in a known manner. This information would provide a rationale for further study of g-catenin in patients and strategies for breast cancer prevention and treatment.
Targets of B Cell Infiltrate in Medullary Breast Cancer
Henrik J. Ditzel, Ph.D.
The Scripps Research Institute
Medullary breast cancer, a subtype of human breast cancers that is characterized by white blood cell infiltrates in the tumor periphery, has been found to have a favorable prognosis as compared to other types of breast cancers at a similar disease stage. The presence of these white blood cells in the tumor has been suggested as a reason for the better prognosis. It is thus of great interest to identify the target of these white cells since this may have important implications for detection and treatment, not only for medullary breast cancers, but breast cancers in general. Previous studies indicate that the T white blood cells are not driving the immune response. The essence of the project is to evaluate the role of the B white blood cell infiltrates in medullary breast cancer. The proposal takes advantage of recently developed molecular techniques for the production of human monoclonal antibodies, which are secreted by the white cells, and identification of the target of these antibodies (the antigens). Preliminary results indicate that a few antibodies dominate the tumor-infiltrating B white blood cell response.
There are several goals for this study. The first is to identify human antibodies in the tumor-infiltrate of medullary breast cancer capable of binding to the cancer cells. The second is to characterize these antibodies by different techniques. The third is to identify the breast cancer component that is the target of these antibodies (by cDNA cloning) and determine where it is located in the cell and throughout the body. It is hoped that the results from this study will influence how breast cancer tumor vaccines are designed.
Role of Rb Protein and Cell Cycle Defects in Breast Cancer
Kathryn Ely, Ph.D.
The Burnham Institute
Cancer arises from an accumulation of multiple genetic mutations. Some key types of genes associated with breast cancer include oncogenes (e.g., Her-2), tumor suppressor genes (e.g., p53), and DNA repair genes. Our interest is in a well-characterized tumor suppressor, the retinoblastoma (Rb) protein. The role of this molecule is to regulate the cell cycle and proliferation of breast cancer cells. In this project, Rb and a newly discovered Rb-binding protein (RIZ) will be studied by structural methods. Our goal is to understand the three-dimensional structure of the individual proteins and fragments of these two proteins that involved in their direct binding activity.
We will use x-ray crystallography to study the interactions between the Rb and RIZ proteins to identify the molecular features of the interactions. Fragments of these molecules that encompass the interacting regions have been cloned and produced in sufficient quantities as recombinant proteins. For structural studies, these proteins will be crystallized alone and in combination. Production of these crystals will be a true breakthrough on the way to determining the structures of the proteins and the critical Rb/RIZ complex.
Ultimately, atomic models generated from these crystallographic studies will provide a critical insight into the structural and functional roles of these proteins in breast cancer. One possible outcome is to determine whether the PR domain element may represent a new class of tumor suppressors. It has been reported that some breast cancer cell lines that express mutant RIZ proteins lack the PR domain. In addition, RIZ is located on chromosome 1 in a region where deletion (loss of heterozygosity) has been reported for breast carcinoma. Our studies are performed in association with collaborators looking at biological and genetic assays focused on the role of RIZ in cell cycle arrest and apoptosis (programmed cell death). The wide application of purification, cloning, and functional studies on breast cancer genes/proteins provides insights into structure/function. However, the ability to see 3-dimensional picture of these interactions expands our understanding into their dynamic properties.
Chromatin Regulation of Breast Cancer Cell Senescence
Paul Kaufman, Ph.D.
Lawrence Berkeley National Laboratory- Division of Life Sciences
Normal mammalian cells can undergo only a fixed number of cell divisions. This intrinsic end to cell’s capacity to divide is termed cellular senescence. Tumor cells have mechanisms to evade these limitations, which contributes to cancer’s growth potential. Furthermore, the largest risk factor for the development of most types of cancer, including breast cancer, is age. This is thought to be due to the continuous accumulation of damaging mutations in critical genes. These mutations are rare random events that represent changes in the genetic instructions in a cell's chromosomes, and they can disrupt a cell's ability to undergo senescence. It is therefore of great interest to cancer researchers to understand the various ways that cells naturally try to protect themselves from mutational damage, and also to better understand the many uncharacterized aspects of cellular senescence. Every cell's chromosomes contain not only the genetic instructions (the DNA), but also proteins bundled with the DNA to form chromatin. Recent discoveries in yeast have shown that some chromatin proteins protect chromosomes from damage that accumulates as cells age.
This proposal seeks to extend these recent discoveries, explore how damage occurs to chromosomes in human breast cells during cellular aging, and how chromatin proteins might act to delay this process. We propose to test the relationship between chromatin proteins and cellular lifespan. To do this, we will determine the effects of disrupting the function of a protein complex called chromatin assembly factor (CAF)-I, which is responsible for forming the basic structural unit of chromosomes, the ‘nucleosome’. Our project involves both the introduction of CAF-I mutant subunits into breast cancer cells lines and the overexpression of normal CAF-I in other experiments. We will examine the effects of CAF-I on (i) chromosomal structure, (ii) the ability of cells to repair DNA damage induced by UV radiation, and (iii) cellular senescence measurements. Based on the previous data, we expect that these studies will clarify the relationship of CAF-I in regulating the lifespan and growth control of breast cancer cells.
Although chromosomal structure has been implicated in the regulation of cellular aging, and aging is correlated with increased cancer risk, very little direct experimentation has been done to associate these three topics with regard to human breast cell biology. Our results may lead to new approaches for the prevention or treatment of breast cancer that target specific chromatin proteins in order to block the ability of breast cells to proliferate.
A Novel Drug Induces Apoptosis in Breast Cancer Cells
Glenn Rosen, M.D.
Stanford University- School of Medicine
Despite the development of new chemotherapeutic agents and aggressive treatment regimens, improvements in the long-term survival for breast cancer patients has been disappointing. This is often due to genetic mutations in the cancer cells that make them resistant to otherwise effective therapy. Also, there are inherent limitations in the drugs used to treat breast cancer. For example, some problems with chemotherapy include toxicity, the development of tumors that are resistant to chemotherapy, and the ability of chemotherapy to kill only rapidly dividing cells. A critical genetic defect in breast cancer is found in the p53 tumor suppressor gene, which makes p53 non-functional. Normally, p53 sensitizes the tumor cell to chemotherapy, which will induce programmed cell death (apoptosis). Our efforts are focused on new approaches to induce apoptosis in breast cancer cells, even if the cells lack functional p53 or are resistant to chemotherapy.
Tumor necrosis factor (TNF), which regulates inflammatory responses and combats tumor growth, can induce cell death in tumor cells regardless of their p53 status and resistance to chemotherapy. TNF family members, however, also activate pathways in breast cancer cells that protect the cell from the cytotoxic action of the TNF. Recently, we have observed that a compound called PG490 that contains triptolide, a chemical derived from a traditional Chinese herb will (i) induce apoptosis in breast cancer cells, (ii) enhance chemotherapy-induced cytotoxicity, and (iii) induce apoptosis in multidrug-resistant breast cancer cells. PG490 also sensitizes breast cancer cells to apoptosis by members of the TNF family by blocking an inhibitor of apoptosis. Our project aims to investigate novel approaches to kill tumor cells both with PG490 alone and in combination with members of the TNF family. Most critical, we will study how to enhance cytotoxicity for breast tumors that have mutations in the p53 gene and where drug resistance is present. PG490 and members of the TNF family, unlike chemotherapy, also kill slowly dividing or quiescent cells. Most cells within a tumor at any one time are not dividing or are dividing slowly, so our approach has the advantage of targeting cells that evade the effects of chemotherapy and other common therapies aimed at dividing cells.
These studies aim to provide a framework for future efforts that will examine the efficacy and safety of PG490 and members of the TNF family in animal breast cancer models. Our eventual goal is to pursue these studies in patients with breast cancer. As new technologies to detect breast cancer in younger women are developed, we must focus our attention on the therapeutic issues to ensure their long-term survival.
Progesterone Receptor and Remodeling of Basement Membrane
G. Shyamala, Ph.D.
Lawrence Berkeley National Laboratory- Division of Life Sciences
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 accordance with this, experimental models have clearly established that the female sex hormones, estrogen and progesterone, are essential for the development of both the normal breast and the induction of breast cancer. It is also well established that cancers most often arise from the undifferentiated (immature) structures present most frequently in the breast of females who have never given birth. The breast is composed of many cell types including the epithelial cells, which are the targets of normal growth and are the ones most likely to give rise to cancers. In a normal breast, the epithelial cells grow in response to progesterone, e.g. during the progesterone dominant phase of the menstrual cycle and pregnancy. More importantly, during pregnancy, the undifferentiated structures of the breast are converted to their differentiated (mature) counterparts. Using genetically engineered mice, our laboratory has direct proof that (a) this conversion of undifferentiated structures to differentiated structures requires progesterone and progesterone receptors, the protein through which progesterone action is mediated, and (b) when there is an alteration in the expression of progesterone receptors and progesterone responsiveness, there is abnormal development of mammary glands.
Our recent studies also suggest that progesterone and progesterone receptors, in addition to regulating the growth and differentiation of epithelial cells, may also be involved in maintaining the integrity of the basement membrane present at the base of epithelial cells. Loss of basement membrane integrity can lead to migration of epithelial cells into adjacent tissue compartments and, in fact, contributes to the metastatic potential of tumors. Also, tumors with invasive properties are often hormone independent. Therefore, the goal of this project is to identify the potential involvement of progesterone and progesterone receptors in maintaining the integrity of the basement membrane and its link to progesterone and progesterone receptor mediated growth and differentiation of the breast. The results from these studies will provide crucial information with regard to whether a derangement in female sex steroid hormone action can trigger carcinogenesis due to inappropriate regulation and/or interactions with the basement membrane. Also, because of the potential utility of anti-progestins in the treatment of metastatic breast cancer, the results from these studies may also help in the selection of tumors most likely to respond to such treatments.
Leukocyte Recruitment, Angiogenesis, and Breast Cancer
Pragada Sriramarao, Ph.D.
La Jolla Institute for Experimental Medicine
In spite of advances in our current understanding and treatment of breast cancer, approximately 30% of breast cancer patients develop advanced, usually incurable metastatic disease. Unlike solid tumors, the metastatic micro-tumors originating from the breast are resistant to various therapies. Our interest is to study the immunopathology of metastatic breast cancer with the objective of enhancing or modulating the patient's own immune response to combat the disease. Recent studies with immune regulatory factors, such as cytokines and chemokines, have demonstrated their potential to eradicate experimentally induced tumors in animals. The inhibition of tumor growth in these studies was associated with enhanced infiltration of tumor killing lymphocytes. Thus, a better understanding of how immune cells interact with tumor microvessels in breast carcinomas will facilitate the development of strategies to target breast cancer.
Our project will investigate the properties of metastatic micro-tumors, which stimulate local formation of blood vessels through a process called angiogenesis. Our experimental technique is called 'intravital microscopy', which can directly visualize the interaction of immune cells with the tumor microcirculation. For this, we implant a transparent window chamber in the dorsum of a female mouse. Tiny tumor spheroids of human breast carcinoma (e.g., MCF-7 cells) are introduced into the chamber, and this stimulates production of a localized network of blood vessels within 10-14 days. This now becomes our model system where we can introduce immune cells, inhibitory molecules to prevent their attachment to the vessels, and immune-stimulatory compounds, such as cytokines. Our first aim is to test whether leukocyte/T-cell attachment is prevented by (i) inducing the expression of endothelial cell adhesion molecules by stimulation with inflammatory cytokines and/or (ii) blockade of angiogenic factors released by tumors. Next, we will inject mouse T-cells (and CD8+ cells) that have the ability to kill tumor cells, and determine whether vessel stimulation with specific cytokines and chemokines will help in T-cell recruitment into the tumor vessels. Finally, one key step in immune recruitment is the ability of leukocytes to leave the tumor vessels and move into the space where the tumors reside. Using our model of tumor angiogenesis, it will be possible to expose the cytokine-activated tumor vessels to various compounds and examine if the leukocytes or T-cells move from the blood into sites of tumor growth.
In cancer patients, immune cells continuously circulate in close proximity to cancer cells that spread and grow in different parts of the body. However, the ability of immune cells to stem the growth of these tumors appears to be compromised. It is hoped that new information will help in developing strategies to improve immune surveillance as a mechanism to effectively treat metastatic breast cancer.
Genes Involved in the Immortalization of Human Mammary Cells
Martha R. Stampfer, Ph.D.
Lawrence Berkeley National Laboratory- Life Sciences Division
Normal human mammary cells have a limited lifespan. After a certain number of cell divisions, they are no longer capable of further proliferation. Most scientists now believe that this limited lifespan, referred to as cellular senescence, arose as a cancer prevention mechanism in long-lived organisms such as humans. This hypothesis is based on two lines of evidence. First, invasive human breast carcinomas (and most adult cancers) are thought to result from accumulation of multiple genetic errors - around 9 errors have been estimated for breast cancer development. The limited proliferative capacity of normal breast cells makes it highly unlikely that so many errors could accumulate in the absence of some means of overcoming cellular senescence. Second, the vast majority of human breast cancers (and most human cancers) express an enzymatic activity, called telomerase, which is associated with an unlimited proliferative capacity - or immortality. This activity is not found in non-tumor breast tissues. Cells derived from normal human breast tissues exhibit cellular senescence when cultured under laboratory conditions, whereas some cells from tumor tissues can express an unlimited proliferative potential in culture.
Since it is thought that nearly all breast cancers have undergone changes that allow them to overcome normal cellular senescence, uncovering the underlying mechanisms that allow these cells to gain an extended or unlimited potential may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention in breast carcinogenesis. This proposal is concerned with uncovering those mechanisms involved in loss of normal cellular senescence and thus potentially responsible for the progression of early stages of breast cancer.
The vast majority of breast cancers originate from a type of cell called an epithelial cell. Our laboratory has pioneered the development and use of cultured human mammary epithelial cells (HMEC) in order to study the changes in growth control processes that occur during breast cancer progression. Using chemical carcinogens, we have been able to transform normal finite lifespan HMEC to immortality. However, we still do not know what molecular changes have permitted these transformed cells to overcome cellular senescence. The goal of this proposal is to identify the individual genes that normally prevent immortalization, based on the assumption that these genes are mutated in immortally transformed cells.
To identify such genes, we will use a relatively new methodology that employs gene fragments that may act as genetic suppressor elements. Finite lifespan HMEC which are likely to need just one more mutation to become immortal will be exposed to gene fragments (packaged in viral vectors) that may be able to disrupt the normal functions of the immortalization suppressor genes. Since immortally transformed cells will maintain growth after the finite lifespan cells have stopped growing, immortalized cells can be easily detected. The virally inserted gene fragments can then be identified by molecular markers attached to the viral vectors. The significance of the isolated and sequenced gene fragments will then be gauged by comparing their expression in normal vs. transformed HMEC, as well as by functional tests in which the full-length gene is introduced into immortal HMEC. The discovery of genes that normally suppress immortalization may aid in the design of drugs that specifically target the immortalization growth defect, provide diagnostic information, and greatly facilitate further studies on HMEC immortalization.
Alteration of Developmental Genes in Breast Cancer
Fumiichiro Yamamoto, Ph.D.
The Burnham Institute
Cancer is the result of genetic accidents that disrupt the normal regulation of cell proliferation, differentiation and survival. Such a disruption may result from permanent changes (mutations) in genes, but there are other more transitory ways that the DNA can be altered, also resulting in a disruption of these functions. A process called DNA methylation is the best understood of these types of mechanisms. In this process, genes undergo a chemical modification where a methyl group is added to one of their nucleotides. Thus, some spots in genes may be "methylated" or "unmethylated". Usually, a gene that has many methyl groups added to it (hypermethylated) no longer makes its protein. Methylation can therefore be a method to alter the function of the genes involved in developing cancer.
There are few techniques that will efficiently scan and identify changes in gene methylation. We have developed a method called Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP). This innovative technique allows the identification and isolation of DNA fragments showing methylation changes by looking for patterns called "DNA fingerprints". MS-AFLP pilot experiments using matched normal/tumor DNA of breast, prostate, and colon showed reproducible changes in gene methylation status, some of which were specifically associated with the tumors. In these pilot experiments we unexpectedly found that several of these fingerprint bands contained sequences derived from genes that are believed to be involved in developmental regulation called homeotic genes.
We believe that our observation of alterations in methylation in several homeotic genes by MS-AFLP fingerprinting may be the tip of the iceberg, and that DNA methylation changes in homeotic genes are very frequent in carcinogenesis in general and in breast cancer in particular. This hypothesis is consistent with the observation that cancer cells often possess characteristics specific to fetal cells. Because homeotic genes direct animal and cell maturation, inhibition of homeotic genes could result in a less mature cell. Thus, hypermethylation of homeotic genes may play a role in breast cancer by contributing to the escape of the cells from their normal constraints of cell maturation. The role of hypermethylation of some homeotic genes in the pathogenesis of breast cancer will be directly tested by introducing unmethylated forms of the homeotic genes into breast cancer cells in culture and monitoring their effects. This approach may open potential therapeutic avenues aiming at the reactivation of those genes.
New Investigator Awards
TGF-ß Receptor Signaling and Breast Cancer
Kunxin Luo, Ph.D.
Lawrence Berkeley National Laboratory- Life Sciences Division
Breast cancer cells differ from normal breast cells in two important aspects: 1) the growth of normal breast cells is tightly regulated, while breast cancer cells can grow in an uncontrolled manner; 2) in response to environmental stimuli, normal breast cells can stop growing and perform special functions such as producing milk, a process called differentiation. Cancer cells, in contrast, no longer respond to environmental signals and fail to perform breast-specific functions. The growth and differentiation of normal breast cells are two intimately related processes that are regulated by growth factors. Disruption of this regulatory program will result in formation of breast cancer. Therefore, in order to understand how breast cancer develops, we need to examine how growth and differentiation of normal breast cells are regulated, and how this regulation is disrupted in breast cancer cells.
One of the growth factors that play an important role in the regulation of breast cell differentiation is transforming growth factor-ß or TGFß. TGFß can inhibit the growth of normal breast cells and early stage breast cancer cells. It also triggers differentiation of normal breast cells. TGFß exerts these effects by binding to the surface of cells and by attaching to the TGFß receptor molecules. Activated TGFß receptors then send signals into the cells to inhibit growth and trigger differentiation. It is yet not clear how these signals are transmitted inside the cells. The goal of this proposal is therefore to study how TGFß regulates normal breast cell differentiation, and to investigate the role of TGFß receptor molecules in this process. We will use a cell culture system that mimics the three-dimensional breast environment inside our body. In this system, normal breast cells can be manipulated to differentiate to various degrees, while breast cancer cells fail to differentiate. Thus, this system can recapitulate the major difference between normal and cancerous breast cells. The effects of TGFß on normal breast cell differentiation will be examined in this culture system.
The role of TGFß receptors in mediating TGFß generated signals during differentiation of normal breast cells will also be investigated. These receptors will also be introduced into malignant breast cancer cells to examine whether they can restore the ability of these cancer cells to be growth inhibited by TGFß. If successful, this should represent a new strategy for breast cancer therapy. Taken together, these studies should allow a better understanding of the pathogenesis of breast cancer and may contribute to better diagnosis and treatment of breast cancer.
Understanding Breast Cancer Metastasis to Bone
Sonoko Narisawa, Ph.D.
The Burnham Institute
Breast cancer commonly spreads to bone at a frequency of approximately 70% in patients having distant metastasis. However, the mechanism of bone metastasis is not well understood. One possibility is that the environment within bone marrow, highly rich in growth factors and cytokines, is suitable for the proliferation of breast cancer cells. A more likely explanation is that specific mechanisms of adhesion occur between the bone endothelial (blood vessel lining) cells and breast cancer cells that favors spread to this site. We hypothesize that breast cancer cells released into the circulation adhere to the endothelial cells of bone marrow through a specific receptor interaction, and we will employ a novel molecular technology to study this process.
In our initial experiments we will use a massive collection ('library') of short, random protein sequences that we fix to the surface of virus-like particles called phage. We can then incubate these phage peptide libraries with cultured bone marrow endothelial cells. The small fraction of phage that specifically bind to endothelial cells can be recovered, re-grown in large numbers, and the process repeated to select out a tiny fraction for further study. Thus, the protein sequence on these phage could represent the same recognition site present on breast cancer cells that targets them to bone. Some of these could be already identified genes and proteins, and others should be previously unknown. Next, we will inject the purified phage directly into the circulation of mice- allowing them to briefly circulate, sacrificing the animals, and determining which organ binds the phage most selectively.
Naturally, our aim is to find the phage and its protein sequence that targets to bone, thus mimicking the situation for breast cancer. In advanced experiments we can incorporate these bone-targeting phage protein sequences directly into a membrane protein that we place on the surface of cultured mammalian cells. These cells (i.e., surrogate breast cancer cells) will be injected into the circulation of mice to provide a more ‘real world’ test of adhesive function. In final experiments, our studies will involve actual breast cancer cells with different metastatic potential. We would expect that a bone-homing peptide would confer bone localization when placed on the surface of a previously non-metastatic breast cancer cell line.
In addition to identifying novel proteins and mechanisms of breast cancer spread to bone, we will also determine the precise binding site that regulates this process. This puts us much closer to finding a therapeutic inhibitor and working in the direction of blocking this most lethal phase of breast cancer.
Postdoctoral Fellowship Awards
Novel Binding Functions of Mutant p53 in Breast Cancer Cells
Koji Itahana, Ph.D.
Lawrence Berkeley National Laboratory- Division of Life Sciences
The development of human breast cancer is a multistage process that is initiated by birth of multiple new cells from a single cell that has gained a selective growth advantage. The first step (gaining a selective growth advantage) may be either a genetic "mistake" or caused by factors from outside the cell (e.g., the immediate microenvironment). During tumor progression, further genetic alterations accumulate in the cells to increase the malignant potential. DNA and chromosomal studies of breast cancer indicate the presence of multiple genetic alterations. Often these mutations occur in oncogenes (genes that promote tumors and are "turned off" or inactivated in normal cells), such as Her-2 and tumor suppressor genes (genes that suppress tumors and are "turned on" in normal cells), such as p53. The incidence of tumor suppressor gene mutations is especially high in breast cancer cells, suggesting that their inactivation plays a central role in genetic defect progression.
p53 is a key tumor suppressor gene, which serves to protect the cell from damage and will trigger programmed cell death (apoptosis) and other processes if the cell’s DNA is damaged by radiation or chemical agents. Mutations in p53 that inactivate it, therefore, will lead to the extensive genetic changes characteristic of cancer. Although many different p53 mutations have been reported, this project focuses on mutant p53 forms frequently observed in breast cancer. Our central hypothesis is that many p53 mutations do not completely abolish activity, but rather serve to reprogram p53 to bind to novel intracellular ‘partners’ to alter their normal function. We plan to detect these novel p53-binding partners by producing mutant p53 proteins and using them in a procedure called yeast 2-hybrid screening. Then, we will test these mutant p53 ‘partner proteins’ for their relevance to breast cancer. For this we will clone them and introduce them back into cells to see how they function in the presence of either normal or mutant forms of p53. Finally, we plan to develop both antibodies and inhibitory RNAs (antisense) to the novel ‘partners’ of p53 to test their ability to inhibit cancer processes.
The genetic alterations of cancer are often more subtle than a complete loss or gain of a particular gene or protein. Our interest is in the processes leading to slow accumulation of genetic changes in breast cancer cells through minor alterations in p53 and the potential reprogramming of its normal function. Understanding these from a molecular standpoint will lead to new approaches to slow down cancer development, make present treatments more effective by restoring normal cell functions, and perhaps in the area of cancer prevention.
Siah-Family Genes: Effectors of p53 in Breast Cancer
Shu-ichi Matsuzawa, Ph.D.
The Burnham Institute
Normal cells have protective mechanisms to detect damage, especially to the cell’s DNA. Defects in these protective genes, called tumor suppressor genes, will permit damage to DNA to persist undetected and result in the accumulation of mutations leading to cancer development and progression. Importantly, mutations that inactivate the tumor suppressor protein, p53, occur commonly in breast cancers and are associated with aggressive clinical behavior of tumors and shorter patient survival. p53 has multiple functions in cells, including an ability to stop cell division and to trigger programmed cell death (called apoptosis) in some cell types. Simple model organisms offer an excellent opportunity to unravel the interaction between p53 and its intracellular signaling processes. Our approach was to first compare the p53-like proteins and pathways in the fruit fly, Drosophila. We noted that a Drosophila gene called "seven in absentia" or 'sina' was required for photoreceptor cell formation during eye development. Recently, a mammalian sina homologue we call 'Siah' was reported to be a p53-inducible gene. Using this information, we are in a position to examine the p53-associated events related to Siah function with specific reference to breast cancer.
In our preliminary experiments we were able to introduce the Siah genes into human tumor cell lines and show evidence of their critical link to the arrest of cell division and apoptosis in a p53-dependent manner. In the present project, we will test the hypothesis that the Siah family proteins play an important role in the pathways by which p53 controls cell proliferation and death in human breast cancer. First, we will examine which of the three known Siah-family proteins, Siah-1A, Siah-1B, or Siah-2 are expressed in breast cancers following cell injury to induce p53. Next, we will introduce Siah genes into breast cancer cells and examine the effects on cell proliferation and apoptosis. We will use mutant forms that produce active and inactive Siah proteins to confirm the cellular effect. Finally, the molecular targets of Siah family proteins will be sought in order to place them in the context of what is known about intracellular signaling within breast cancer cells.
Taken together, our overall goal is to develop novel ways to restore growth control or induce apoptosis in breast cancers that have lost p53 tumor suppressor activity. This would serve to limit the genetic changes in these tumors that contribute to aggressive growth and progression.
Biochemical and Functional Characterization of BRCA1
Heinz Ruffner, Ph.D.
The Salk Institute for Biological Studies
About half of the patients from families with a predisposition to breast cancer, and even more patients from families in which both breast and ovarian cancer occur, harbor a genetic change (mutation) within the BRCA1 gene (breast cancer gene 1). This gene encodes a large protein, called the BRCA1 protein that acts by an unknown mechanism to impede breast and ovarian cancer. It has been proposed that BRCA1 regulates growth, differentiation and death of cells. And, it may play a role both in maintaining the cell's genetic material and by regulating other genes.
In our previous work, we found that BRCA1 was mainly present within the nucleus of breast cancer and other cells. Moreover, we could demonstrate that BRCA1 undergoes changes during cell growth and as a result of damage to the genetic material. Specifically this involved phosphorylation (addition of phosphorus to the protein) of the BRCA1 protein by enzymes named protein kinases. This was followed by reversal of phosphorylation by other enzymes. It is believed that these chemical modifications cause a change in BRCA1 protein activity. To begin understanding the exact function of BRCA1, it is important to identify the specific protein kinase(s) that act on it. As a first step, I have identified possible sites on the BRCA1 protein where these modifications occur. Next, I am going to identify the individual targeted amino acids that may give a clue about the type of kinase responsible. These results will serve as a basis for isolating the kinase(s) by conventional biochemical approaches and associated gene cloning techniques. We believe that identifying the kinase(s) will provide insights into the regulation of normal BRCA1 function and elucidate how cancer occurs when BRCA1 is present in a mutant form. Since BRCA1 has been suggested to control the activity of other genes, I also intend to search for these genes. For these studies we have generated several human cell cultures in which we can produce BRCA1 protein at high levels in a regulated fashion. These cells will allow investigation of the effects of BRCA1 on cell growth and on the genes that it regulates.
Identification of BRCA1 in families having a high incidence of breast cancer is only the first step to understanding its complex role in normal cells. Although mutations in BRCA1 do not appear to be associated directly with the more common causes of sporadic breast cancer, the further study of BRCA1 still offers the opportunity to identify novel genes that may be critical for the progression of breast cancer and to elucidate events that lead to breast cancer in general.
Cell Adhesion Signaling Defects in Breast Cancer
Karin Zeh, Ph.D.
The Burnham Institute
In normal organs, including the breast, one way for cells to communicate with each other is through specific molecules on the cell surface, called adhesion molecules. Inside the cell, other proteins, named catenins, serve as a switchboard and organize a response to the incoming messages. This communication system allows the cells to grow, divide and attach in organized groups of cells. In breast cancer, cells have lost the ability to communicate with their neighbors properly. This results in an unregulated division and/or escape from the cell attachment which causes spreading (metastasis) of the cancer in the body. Since the catenins play an important role in the communication of an outside message into an inside response of the cell, we decided to investigate their role in the development of breast cancer. There are two sister catenins, g-catenin (also called plakoglobin) and g-catenin. It has been found that either too much or too little catenin in the cell causes unregulated cell division. For example, g-catenin causes colon cancer when present in elevated amounts. In contrast, plakoglobin is thought to have the opposite effect, such that reduced amounts cause cancer. When plakoglobin is reintroduced into kidney cancer cells, they revert to a normal state. More significantly, plakoglobin mutations have been found in breast cancer cells in women.
To study if and how these mutations contribute to the development of breast cancer, we propose to generate mice without plakoglobin in the mammary gland. This will be done by using a novel technology, called the Cre-loxP system. This method allows us to produce mice without the protein of interest in the mammary gland itself. We will also investigate whether the lack of plakoglobin can cause breast cancer or whether it can aggravate or suppress an existing breast cancer induced by a carcinogenic drug. In further studies, we propose to investigate a possible relationship between plakoglobin and an oncogene directly associated with breast cancer. This oncogene, named c-erb/neu, is found in highly elevated amounts in breast cancers in many women. These studies in mice will be complemented by analyzing cells that have been isolated from the breast. We will test them for the ability to crawl onto and penetrate an extracellular matrix, which are key properties of metastatic cancer cells.
In conclusion, the experiments proposed here will serve to elucidate how the loss of plakoglobin disturbs the communication between mammary glands cells thereby leading to breast cancer. It will also potentially provide new molecular targets to design novel drugs to treat and cure this disease.
