Biology of the Breast Cell: The Basic Science of the Disease
Overview: To understand the origin of breast cancers, more research is needed on the pre-cancerous causative events in the normal breast. We need to understand the cancer-related genetic and physiological changes associated with breast development, aging, pregnancy, and consider the influence of lifestyle and environmental factors. Breast cancer is a complex disease, and the underlying genetics of the variability seen in the clinic need clarification at the basic science level. Basic scientists need to use more relevant cell and pre-clinical animal models of breast cancer. It is hoped that new genetic and molecular “cancer signatures” of cancer sub-types and stages of progression may provide useful biomarkers for better diagnosis and prognosis, so treatments can be individualized and women spared the use of ineffective drugs. More research on the underlying cellular signaling pathways for growth control, cell death, DNA repair, and cell migration/metastasis are needed to develop into new targets for therapy and prevention.
Some recent advances in basic science have altered our conceptual view of breast cancer and promise to have a significant impact over the next few years. First, researchers at the University of Michigan, including Dr. Michael Clarke and Dr. Max Wicha, demonstrated the existence of breast cancer stem cells. According to this paradigm of breast cancer origin and progression, a small population of pluripotent stem cells acquire mutations that lead to tumor formation, tumor spread to distant organs, and resistance to most current therapies. Only a small fraction (1-2 percent) of cells in a tumor mass retain stem cell properties, and these are the tumor component that must be targeted in any effective therapy. Over the past twenty years, we have seen the limit of cancer therapies that merely shrink tumors, but allow the cancer stem cell population to persist and lead to recurrence.
Second, cancer epigenetics research is gaining strength with diffusion of the technology and informatics spawned from the Human Genome Project. Epigenetic changes alter gene functions without modifying the genetic code and are essential to normal development. In terms of cancer, sometimes the epigenetic changes will disable tumor suppressor genes and DNA repair mechanisms. Studies have suggested that epigenetic effects may be as common in some tumor cells as actual genetic mutations. At least a dozen drugs that target epigenetic mechanisms, such as methylation, are in clinical trials and more are in development. One of these drugs is now used to treat a rare bone marrow disorder, called myelodysplastic syndrome.
Third, the use of RNA-interference (RNAi) technologies is becoming widespread. Discovered in plants, and then in nematode worms in 1998, RNAi is an elegant, endogenous mechanism of “gene silencing” with potential therapeutic utility. In the basic research setting, siRNA (small interfering RNA) is commonly being used to dissect signaling pathways and knock out the expression of single genes. Although promising in cell-based studies, it remains unclear if this approach can make the leap into the anti-cancer therapeutic arena.
A critical area where basic science could benefit from a new organizational approach is through the discipline of systems biology. Thus, the traditional way of doing science in single labs or small research groups might give way to a team-based, integrative style. Systems biology is the study of living organisms in terms of their underlying network structure rather than by dissecting their individual molecular components (i.e., reductionist logic). A system can be anything from a gene regulatory network to a cell, a tissue, or an entire organism. Because systems biology requires the consideration of all interacting components simultaneously, high-throughput, computational technologies are essential. Perhaps the most articulate proponent is Leroy Hood, M.D., Ph.D., from The Institute for Systems Biology, Seattle. As the complexity and heterogeneity of breast cancer becomes more obvious, then progress will demand that researchers adapt to new paradigms to effectively tackle the disease. The NIH has adopted this approach by incorporating “Research Teams of the Future” as a part of the new NIH Roadmap plan. Director Dr. Elias Zerhouni has written, “The scale and complexity of today's biomedical research problems increasingly demand that scientists move beyond the confines of their own discipline and explore new organizational models for team science.”
Two of the CBCRP’s research topics are presented in this section.
• Biology of the Normal Breast: The Starting Point
• Pathogenesis: Understanding the Disease
Biology of the Normal Breast Funding Data:
Funding Data: |
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Proportion of Total |
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Biology of the Normal Breast grants awarded in 2005: |
24 |
45% |
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Funded amount: |
$3,996,716 |
52% |
Biology of the Normal Breast Portfolio Summary:
Breast cancer begins with early pre-cancerous changes in individual cells, which could be breast stem cells or their immediate progenitors. As cancer progresses, the molecular events that regulate chromosomal surveillance, DNA repair, cell division and differentiation, movement, apoptosis (programmed cell death), and epithelial-stromal interactions become defective. However, the window of opportunity for prevention, early detection, and treatment is extensive, since the period from initiation to clinical diagnosis can span a decade or more.
Stem cells are the focus of three newly funded grants. Alexander Borowsky, M.D., from the University of California, Davis, will use pre-cancerous breast tissues from a genetically defined mouse cancer model to identify gene patterns important for progression and to find evidence for an early breast cancer stem cell in these tissues. Once isolated, Dr. Borowsky will be able to test his underlying hypothesis that the “multiple gene hits” commit a stem cell to begin the journey to cancer, and, once committed, cancer progression does not depend on additional genetic mutations.
Steven Artandi, Ph.D., at Stanford University will be studying the role of telomerase in the context of breast stem cells in mice. Telomerase maintains the ends of chromosomes and is “turned on” in 90 percent of human breast cancers, including DCIS. Dr. Artandi has postulated a novel function for telomerase in the proliferation of breast stem cells, and this function is thought to be critical for cancer initiation as well.
Stefanie Jeffrey, M.D., also from Stanford University, will attempt to isolate stem cells from the most aggressive type of breast cancer, estrogen receptor- and progesterone receptor-negative tumors that are either Her-2 positive or negative. Patients with these tumors have a similar genetic profile, and their tumors and metastatic sites are very resistant to current therapies. If the tumor-generating stem cell population can be isolated from these patients, then more information can be obtained on how to eradicate them. It is thought that current therapies often fail the patient because, although tumors will usually shrink, the stem cell population persists and becomes the source of disease recurrence.
Four newly funded grants examine breast cancer in the context of early disease progression. Albert Davalos, Ph.D., at Lawrence Berkeley National Laboratory is studying human mammary epithelial cells (HMECs) to examine the contribution of the microenvironment in regulating how cells respond to DNA damage. Dr. Davalos will focus on extracellular matrix (ECM)-driven signaling to see how breast cells that lack critical repair proteins, mainly BRCA1 and NBS1, are able to respond to DNA damage. If successful, this model system has the potential to identify novel markers for early detection and targeted therapies.
Andrew Ewald, Ph.D., from the University of California, San Francisco, will study invasiveness of breast cells, and how matrix metalloproteinase-2 and fibroblast growth factor receptor-2 influence cell movements. The invasive process is essential to forming the primary ductal network in breast development and might be a mechanism of epithelial invasion when aging makes our tissues more permissive to the cancer phenotype.
Transforming growth factor-beta (TGF-?? has inhibitory effects in normal breast cells, but it can promote invasion and metastasis later in cancer progression. Xiaoman Xu from the University of California, Irvine, was funded though a dissertation award to determine whether a gene-regulatory transcription factor, called LMO-4, is a modulator of TGF???This study will use genetically modified mice. Given that TGF?? has such diverse effects, this project could provide biomarkers to predict the response to therapy in patients.
Zhengquan Yu, Ph.D., also at the University of California, Irvine, will also study LMO-4 in the context of signaling through the Her-2 oncogene pathway. LMO-4 levels are increased in about 50 percent of breast cancers, and it may serve to increase cell proliferation and influence cell growth and death pathways.
Cancer-causing oncogenes and cancer-preventing tumor suppressor genes are the focus of a number of newly funded grants. Peter Kaiser, Ph.D., from the University of California, Irvine, is funded to continue his studies on the BRCA1 protein’s function to selectively promote the degradation of other cellular proteins. Proteins that are essential to critical processes, such as the cell cycle pathway, are frequently marked for destruction by the attachment of ubiquitin. Dr. Kaiser will compare BRCA positive and negative cells for differences in their cellular ubiquitination patterns. In addition to giving new insight into how this critical breast cancer hereditary gene works, it could provide new disease biomarkers and strategies to prevent cancer.
The other major breast cancer hereditary gene, BRCA2, is the focus of another newly funded innovative grant to Henning Stahlberg, Ph.D., at the University of California, Davis. BRCA2 functions in DNA repair and is associated with a protein called Rad51. The aims of Dr. Stahlberg’s project are to clone and express cancer-associated BRCA2 mutants, investigate the structure of these mutants, and study the interaction of normal or mutant forms of BRCA2 with Rad51.
Sheryl Krig, Ph.D., also from the University of California, Davis, received a postdoctoral fellowship to study the ZNF217 oncogene, which contributes to the early progression of breast cancer by promoting cell "immortality." Dr. Krig will analyze whether ZNF217 suppresses Apaf-1 (apoptotic protease activating factor-1), which is essential for caspase activation that triggers apoptosis (cell death).
Myb oncogenes were initially described in Drosophila (fruit flies), and they have a role in leukemia and lymphoma. Joseph Lipsick, M.D., Ph.D., at Stanford University will extend his studies on B-Myb into human breast cancer. Although B-Myb is included as one of the 21 genes in the Oncotype DXTM test to predict recurrence in tamoxifen-treated, node-negative breast cancer patients, its effects on chromosomal number (ploidy) in cancer progression are not well described.
Marc Milstein at the University of California, Los Angeles, will investigate RIN1, which regulates the Ras oncogene. Ras was one of the earliest oncogene families discovered. The Ras proteins deliver signals from cell surface receptors, such as growth factor receptors and G-protein coupled receptors, to ultimately regulate such functions as DNA synthesis and cytoskeletal organization. Mr. Milstein’s dissertation project aims are to determine whether RIN1 can act as a tumor suppressor by influencing Ras, and whether the loss of RIN1 function plays a role in breast cancer.
P53 is a well-studied tumor suppressor that, while not lost or mutated as frequently in breast cancer as some other cancers, still is a subject of detailed investigation. Lan Truong, also from the University of California, Irvine, will conduct her dissertation research on the binding of p53 to a pre-mRNA splicing factor, called SAP145. Ms. Lan is interested in how the binding of Cyclin E to this complex could have an effect on the pre-mRNA splicing. The breast cancer endpoints include the cell growth and death pathways.
Breast cancer cells, even at the early DCIS stages, show profound changes in chromosomal structure that include gene deletions, duplications, and rearrangements. It remains a mystery how the genetic sequence, which is so closely monitored and repaired in normal cells, can be so profoundly altered in cancer cells and still allow proliferation. Ewa Lis at The Scripps Research Institute will identify and study basic mechanisms of mutagenesis-promoting genes in yeast, then study the corresponding genes in human breast cancer cells. Model organisms, such as yeast, have been valuable tools to study evolutionarily conserved processes, such as DNA repair and the cell cycle. In addition to facilitating cancer progression, it is thought that mutagenesis-promoting genes might be a major cause of drug resistance.
Women have two X chromosomes, but it was long believed that one of them was inactivated early in life and remained so permanently. Now it’s thought that the “inactive X” might be re-activated in cancer, and this is the topic of the fellowship award to Angela Andersen, Ph.D., from the University of California, San Francisco. The activation of the X chromosome and the regulatory protein, called Xist, will be studied in a variety of mouse breast cells, stem cells, early tumor hyperplastic outgrowths, and transformed cells. The X chromosome contains at least 70 cancer-related genes, so the re-activation of these genes could be the equivalent to gene duplication in other chromosomes.
Continuing along with the theme of epigenetic changes, Judd Rice, Ph.D., from the University of Southern California, will study the patterns of a specific histone modification in a series of breast cancer cell lines with the goal of identifying markers associated with breast cancer progression. Dr. Rice will perform chromatin immunoprecipitation (ChIP) analysis to determine different degrees of methylation for histone 4/lysine20 on cell lines derived from normal breast epithelium, primary lesions, and metastatic sites. Histones are proteins that package chromosomal DNA. Although histone methylation patterns in cancer are not fully understood, one hypothesis is that they may play a role in silencing key tumor suppressor genes and open the door to cancer progression even in the absence of other genetic changes.
The final major topic of newly funded CBCRP tumor biology grants is metastasis and angiogenesis. Although angiogenesis research appeared to promise a real breakthrough in cancer treatment five to ten years ago, actual translation to the clinical setting has been painfully slow. Genentech’s introduction of Avastin in 2004 to treat colorectal cancer is the first of an anticipated new generation of molecularly-targeted therapeutics aimed at angiogenesis. Three newly funded grants focus on angiogenesis from unique perspectives.
Barbara Susini, Ph.D., at the University of California, San Diego, will study the role of lymphatic vessel growth (lymphangiogensis) and the altered integrin (i.e., cell surface adhesion receptor) profiles within tumor lymph-specific endothelial cells. It might seem strange that one of the body's major organs, the lymphatic system is so poorly understood. Lymphatic vessels collect fluid that has leaked into tissues from the bloodstream and return it to the blood through lymph nodes where key cells of the immune system are located. We know that tumor cells can pass through the lymphatic system, because lymph node biopsy has been a mainstay of tumor prognosis for decades. However, the mechanism of lymphatic vessel entry into tumors is an unexplored topic.
Konstantin Stoletov, Ph.D., from The Scripps Research Institute will use a unique animal model system, the zebrafish, to study angiogenesis. These fish are transparent, so it is easy to see organ and tissue morphology, especially in development. They have been widely used in genetics research, and the zebrafish genome has been entirely sequenced. Dr. Stoletov has shown that human breast cancer cells will grow progressively and induce angiogenesis in zebrafish, and he will focus on the RhoC gene. RhoC, whose full name is RhoC-GTPase, is a protein involved in changing the internal skeleton of a cell to allow a cell to polarize or move. It has been associated with inflammatory breast cancer, and might be a key factor in angiogenesis.
We need to know more about breast cancers that have spread to various organs, so that new treatments can target metastatic disease. Florence Hofman, Ph.D., at the University of Southern California is focusing on breast cancer metastasis to the brain, which occurs in 10-15 percent of patients, by seeking ways to kill the tumor-associated brain blood vessel cells. Dr. Hofman will determine whether survivin, a trigger for apoptosis (cell death), can be targeted by RNA-interference molecules that are delivered by lentiviral vectors. While not directly killing tumor cells, this approach would make the tumor vulnerable to other chemotherapeutic agents by disrupting the brain-blood barrier selectively at tumor sites.
In any diagnosis of cancer, the patient and clinician want to know whether metastasis is likely to have occurred. Most tumors constantly shed small numbers of cells into the blood, so these circulating tumor cells (CTCs) are a promising source of biomarkers for metastasis and prognosis. Kristen Kulp, Ph.D., at Lawrence Livermore National Laboratory is developing a new technology to detect CTCs through the statistical analysis of molecule-specific images derived from individual cells. This technology is based on “time of flight secondary ion mass spectrometry” (ToF-SIMS). Dr. Kulp achieved proof of principle in her previous CBCRP funding, so a two-year renewal grant will continue these studies in both animal and human settings. The initial phase of this study is to validate that known metastatic vs. non-metastatic cells derived from culture can be isolated and distinguished when spiked into blood or grown as tumors in animal models.
Richard Neve, Ph.D., from Lawrence Berkeley National Laboratory will study ephrins and their receptors, which are important regulators of tissue morphogenesis. He plans to develop a new model system to co-culture various breast cell lines with normal and cancer-associated fibroblasts. Then, Dr. Neve will use RNA-interference to knock down EphA2 or EphA1 and determine whether this affects tumor formation and metastasis.
Also utilizing a novel model system is Robert Abraham, Ph.D., at The Burnham Institute, who will grow tumor spheroids in a three-dimensional culture system. These conditions are thought to better duplicate the tumor setting compared to using tumor cells grown on plastic dishes. Dr. Abraham will use the new cell culture system to test inhibitors of the mTOR signaling pathway. The anti-fungal drug, rapamycin, has a mammalian target, called mTOR. The mTOR pathway is critical to signaling through the PI3K/Akt apoptosis pathways, so inhibiting mTOR may be useful in sensitizing tumor cells to existing therapeutics.
Brian Eliceiri, Ph.D., from the La Jolla Institute for Molecular Medicine, will test the novel hypothesis that estrogen promotes metastasis of breast cancer through actions on host tissues rather than on the cancer cells. He will use a breast cancer cell line that does not respond to estrogen and measure metastasis in mice that have their systemic estrogen levels modulated. Changes in the tumor vascular permeability and the extracellular matrix will be the focus of these studies.
Kyle Chiang from The Scripps Research Institute is funded for a dissertation project to continue research funded by the CBCRP previously to his mentor, Benjamin Cravatt, Ph.D. Mr. Chiang’s project will focus on a protease, called KIAA1363, and he will test its relevance to breast cancer metastasis in biochemical, cell and animal models. This protease appears to be increased in aggressive cancer, so the discovery of KIAA1363’s substrate and inhibitors would be major steps towards pre-clinical studies.
Biology of the Breast Cell Grants Funded in 2005:
Breast Cancer Studies in a 3-D Cell Culture System
Robert T. Abraham, Ph.D.
The Burnham Institute
Award Type: IDEA
$191,000
Reactivation of the Inactive X Chromosome and Breast Cancer
Angela Andersen, Ph.D.
University of California, San Francisco
Award Type: Postdoctoral fellowship
$90,000
Role of Telomerase in Mammary Stem Cell Function
Steven Artandi, Ph.D.
Stanford University
Award Type: IDEA
$236,519
Defining Mammary Cancer Origins in a Mouse Model of DCIS
Alexander Borowsky, M.D.
University of California, Davis
Award Type: IDEA
$150,000
Integrated Proteomic and Metabolic Analysis of Breast Cancer
Kyle P. Chiang
The Scripps Research Institute
Award Type: Dissertation
$76,000
The Role of the ECM in Breast Cancer DNA Damage Repair
Albert R. Davalos, Ph.D.
Lawrence Berkeley National Laboratory
Award Type: IDEA
$252,791
Novel Approach to Analyze Estrogen Action in Breast Cancer
Brian P. Eliceiri, Ph.D.
La Jolla Institute for Molecular Medicine
Award Type: IDEA
$310,950
Regulation of Mammary Epithelial Invasion by MMPs and FGFs
Andrew J. Ewald, Ph.D.
University of California, San Francisco
Award Type: Postdoctoral fellowship
$135,000
Survivin: Target for Breast Cancer Brain Metastases
Florence M. Hofman, Ph.D.
University of Southern California
Award Type: IDEA
$243,733
Stem Cells of Molecularly Diverse ER Negative Breast Cancers
Stephanie Jeffrey, M.D.
Stanford University
Award Type: IDEA
$234,165
Identification of BRCA1 Ubiquitylation Targets
Peter Kaiser, Ph.D.
University of California, Irvine
Award Type: IDEA, competitive renewal
$200,000
Apaf-1 is a Transcriptional Target for the ZNF217 Oncogene
Sheryl R. Krig, Ph.D.
University of California, Davis
Award Type: Postdoctoral fellowship
$53,649
Identifying Metastatic Breast Cells from Peripheral Blood
Kristen S. Kulp, Ph.D.
Lawrence Livermore National Laboratory
Award Type: IDEA, competitive renewal
$490,774
The Role of B-Myb in Human Breast Cancer Progression
Joseph Lipsick, M.D., Ph.D.
Stanford University
Award Type: IDEA
$156,106
Defining Mutagenesis Pathways in Breast Cancer Evolution
Ewa Lis
Scripps Research Institute
Award Type: Dissertation
$67,520
Evaluating the Role of RIN1 in Breast Cancer
Marc Milstein
University of California, Los Angeles
Award Type: Dissertation
$72,335
A Novel Epithelial-Stromal Model of Metastatic Breast Cancer
Richard M. Neve, Ph.D.
Lawrence Berkeley National Laboratory
Award Type: IDEA
$216,674
Histone Methylation as a Marker of Breast Cancer Progression
Judd C. Rice, Ph.D.
University of Southern California
Award Type: IDEA
$162,500
Structural Analysis of Cancer-Relevant BCRA2 Mutations
Henning Stahlberg, Ph.D.
University of California, Davis
Award Type: IDEA
$100,000
Imaging RhoC-induced Breast Cancer Invasion and Angiogenesis
Konstantin V. Stoletov, Ph.D.
The Scripps Research Institute
Award Type: Postdoctoral fellowship
$135,000
Role of Integrins in Lymphangiogenesis During Breast Cancer
Barbara Susini, Ph.D.
University of California, San Diego
Award Type: Postdoctoral fellowship
$135,000
A Role for p53 and Splicing Factor SAP145 in Breast Cancer
Lan N. Truong
University of California, Irvine
Award Type: Dissertation
$76,000
Modulation of TGF-beta Signaling in Mammary Epithelial Cells
Xiaoman Xu
University of California, Irvine
Award Type: Dissertation
$76,000
The Role of LMO4 in Breast Cancer
Zhengquan Yu, Ph.D.
University of California, Irvine
Award Type: Postdoctoral fellowship
$135,000
