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-neoplastic causative events in the normal breast. We need to understand the cancer-related genetic and physiological changes associated with breast development, aging, pregnancy, and the influence of lifestyle and dietary factors. Breast cancer is a complex disease, and the underlying genetics of disease heterogeneity seen in the clinic need clarification at the basic science level. We need more relevant cell and pre-clinical animal models of breast cancer. The key genetic and molecular signatures of the disease may provide useful biomarkers for better diagnosis and prognosis, so treatments can be individualized and women spared the use of ineffective drugs. The underlying cellular signaling pathways for growth control, cell death, DNA repair, and cell migration/metastasis require exploration to develop into new targets for therapy and prevention.

Two of CBCRP's research topics are presented in this section.

Biology of the Normal Breast: The Starting Point

Biology of the Normal Breast Funding Data:

Funding Data:
 

Proportion of Total

Biology of the Normal Breast grants awarded in 2004:

8

11%

Funded amount:

$1,605,672

19%

Biology of the Normal Breast Portfolio Summary:

The job of the breast appears to be quite straightforward, to produce milk for babies. However, in actuality the process requires a complicated structure responding to an intricate combination of cell signals and hormones. The breast (or mammary gland in animals) is composed of a collection of different types of cells, each of which must function properly for milk to be produced. The gland cells respond to the myriad hormonal signals during the menstrual cycle and throughout life by growing, maturing, and dying off. Researchers have had difficulty identifying the key changes that occur during tumor development because of the complex interactions that already exist in the normal mammary gland. The CBCRP applications funded in the Biology of the Normal Breast topic seek to understand the behavior of normal cells in hopes of eventually identifying the critical changes during tumor development.

The mammary gland is composed of branching ducts made of epithelial cells which are embedded in stromal cells and a network of extracellular matrix consisting of proteins. The epithelial cells are responsible for producing milk and delivering it to the nipple. They also are the origins of 98 percent of breast tumors. The stromal cells, which include fat cells and blood vessel cells, provide nutrients to the epithelial cells. The extracellular matrix provides a structure onto which cells attach and move. Research has shown that the interactions of these components are complex, and a change in one affects the behavior of the others.

The communication between cell types begins at the earliest stages of mammary gland development. Lindsay Hinck of the University of California, Santa Cruz, will undertake a three-year study to determine whether the Slit/Rob growth factor system, which is involved in attracting or repelling axons in nerves, is guiding the branching structure in the normal breast. This study could help us understand the factors affecting the movement of breast cells as they develop. Jacqueline Veltmaat from the Children's Hospital Los Angeles/Saban Research Institute will be studying the genetic control of mammary cells as they form the earliest vestiges of the mammary gland. She will investigate the role of Gli3 in early breast development by creating mice with mutant Gli3 and looking for effects on cell division, breast size, shape and production of breast-specific proteins.

The CBCRP funded two projects to study the different cell types and extracellular matrix interactions in the mammary gland. The manner in which cells connect to each other and the extracellular matrix can determine how they function. John Muschler of the California Pacific Medical Center Research Institute will explore the possibility that there are as yet undiscovered methods for cells to adhere to the basement membrane. He will generate cells lacking the major adherence proteins (dystroglycan and integrin beta) in transgenic mice and test whether there are any basement membrane receptors still functioning in the normal gland. Nancy Boudreau from the University of California, San Francisco, will test whether the loss of breast tissue organization acts as a trigger for the development of new blood vessels. These findings could help us to understand the tissue-related control points for tumor progression and metastasis.

Mammary gland growth and development is also controlled by hormones and growth factors. Hormones have often been found to have different forms, some of which encourage the mammary cells to proliferate and others of which signal them to mature or die. Situations that cause cells to produce or react to the maturation versions of the hormones may also be the ones that cause the breast to be resistant to tumor development. Two CBCRP-funded studies will investigate the protective role of hormones in the mammary gland. Postdoctoral fellow Leslie Hodges of the University of California, San Francisco, hypothesizes that ERβ (estrogen receptor beta) protects the mammary gland from developing tumors, because it is lost in the majority of breast tumors. She will use high throughput genetic screens to determine the molecular pathways that are modulated by ERβ and then test the physiological effects of the loss of ERβ in the mouse. Ameae Walker from the University of California, Riverside, is funded to investigate the potentially protective role of prolactin. Prolactin is the growth hormone responsible for causing breasts to grow, mature during pregnancy and produce milk. A growth inhibitory version of prolactin is found at elevated levels in breast milk, but the significance of its presence has not been explained. Dr. Walker will investigate the role of inhibitory prolactin on the milk side of the breast duct. This investigation could determine whether it contributes to the effect of early pregnancy on lowering a woman's subsequent risk for breast cancer.

There are thousands of genes that are being activated and inactivated inside the mammary cells in response to cell-cell interactions, hormones or growth factors. Two CBCRP investigators will look at the methods of gene regulation and their implications for breast cell behavior. Hosein Kouros-Mehr of the University of California, San Francisco, will examine the gene activation in different cell types of the developing mammary gland using a combination of histochemical techniques (for identifying the different cell types) and microarray (for studying the profiles of many genes at the same time). David Liston from The Salk Institute for Biological Studies will pursue a postdoctoral fellowship to examine the normal pattern of p16 (a gene involved in cell aging) inactivation through a chemical process called DNA methylation. These studies could lead to a better understanding of which genes are crucial for tumor development.

Biology of the Normal Breast Grants Funded in 2004:

Epithelial Polarity, Organization and the Angiogenic Switch
Nancy Boudreau, Ph.D.
University of California, San Francisco
Award type: IDEA
Duration: 1.5 years
$75,000

Axon Guidance Proteins in Mammary Gland Development
Lindsay Hinck, Ph.D.
University of California, Santa Cruz
Award type: RFA
Duration: 3 years
$449,228

Protective Role of Estrogen Receptor Beta in the Mammary Gland
Leslie Hodges, Ph.D.
University of California, San Francisco
Award type: Postdoctoral fellowship
Duration: 2 years
$90,000

Gene Expression Profiling in the Developing Mammary Gland
Hosein Kouros-Mehr
University of California, San Francisco
Award type: Dissertation
Duration: 2 years
$60,000

Targeting of DNA Methylation in Mammary Epithelial Cells
David Liston, Ph.D.
Salk Institute
Award type: Postdoctoral fellowship
Duration: 2 years
$90,000

Discovering Novel Cell-ECM Interactions in Breast Cells
John Muschler, Ph.D.
California Pacific Medical Center Research Institute
Award type: IDEA
Duration: 1.5 years
$160,000

The Role of Gli3 in Mouse Embryonic Mammary Gland Formation
Jacqueline Veltmaat, Ph.D.
Childrens Hospital, Los Angeles
Award type: Postdoctoral fellowship
Duration: 2 years
$90,000

Normal Mammary Biology of Phosphorylated Prolactin
Ameae Walker, Ph.D.
University of California, Riverside
Award type: RFA
Duration: 3 years
$541,444

Pathogenesis: Understanding the Disease

Basic science research, while often appearing to be unrelated to clinical problems and practical application, is the entry point for expertise from other research disciplines. Arthur Kornberg, the 1959 Nobel Laureate in Medicine, had these enduring thoughts, (1) “No matter how counter-intuitive it may seem, basic research has proven over and over to be the lifeline of practical advances in medicine,” and (2) “The pursuit of curiosity about the basic facts of nature has proven, with few exceptions throughout the history of medical science, to be the route by which the successful drugs and devices of modern medicine were discovered. Though it seemed unreasonable and impractical, counter-intuitive even to scientists, to solve an urgent problem of disease by exploring apparently unrelated questions in biology, chemistry and physics, these basic studies proved time and again to be utterly practical and cost-effective.” As novel paradigms and technologies in cell and molecular biology are advanced, we provide innovative project funding to explore their relevance to breast cancer.

The CBCRP encourages innovative and cross-disciplinary research on breast cancer tumor and stromal biology, including: (1) studies of relevant proteins and genes with an emphasis on their relationship to the actual disease and (2) elucidating key cell signaling, growth control, cell cycle, and apoptosis pathways. We especially encourage new research on the process of metastasis and the development of tools and models to better understand the key metastatic events that impact patient survival.

Funding Data:
 

Proportion of Total

Pathogenesis grants awarded in 2004:

10

24%

Funded amount:

$3,919,643

27%

Pathogenesis Portfolio Summary:

The predominant basic science topic funded by the CBCRP in 2004 was cancer invasion and metastasis, but the underlying approaches are quite varied. Two full-scale collaboration projects will link basic scientists in different disciplines and clinicians to tackle key research issues. Brunhilde Felding- Habermann and John Yates from the Scripps Research Institute are teaming with Evan Snyder at The Burnham Institute to explore the emerging theory that a small population of stem cells in breast tumors can seed the growth of new cancers. The stem cells make up a tiny fraction of the tumor and have properties similar to those of other pluripotent embryonic and organ stem cells. The CBCRP-funded project to Drs. Felding-Habermann, Yates, and Evans will use state-of-the-art proteomic, genetic, and immunochemical tests to characterize breast cancer stem cells, and determine whether these cells actually “seed” metastases to distant organs. Dr. Evans brings his expertise in neuronal stem cells as the “synergistic component” to this type of funding. If more effective ways of detecting and killing breast cancer stem cells can be devised, then disease recurrence might be greatly diminished. Benjamin Cravatt, a chemist-cell biologist at the Scripps Research Institute, is collaborating with Stefanie Jeffrey, a surgeon-cancer geneticist from Stanford University, to detect cell invasion-specific proteases using a new “functional proteomics” assay. In previous funding from the CBCRP, Dr. Cravatt has shown the ability of this new assay to detect and measure the activity of proteases in breast cells and animal tumor models. In the current project, they hope to translate these findings closer to a clinical application using primary tumor samples. Prior work from Dr. Jeffrey and colleagues at Stanford has shown that breast cancers can be genetically classified into five specific sub-types, so the addition of a proteomicsbased assay will serve to develop new information to make individualized metastasis-based prognosis closer to reality.

It has been known for decades that many cancer patients have tumor cells that circulate in the blood, but the clinical and prognostic significance remains uncertain. The CBCRP is funding two innovative projects to study circulating tumor cells (CTCs). Kristen Kulp, a basic scientist at Lawrence Livermore National Laboratory, will use imaging mass spectrometry (TOF-SIMS). The proof-of-principle for this approach will be to detect the protein fingerprints that distinguish metastatic and non-metastatic breast cancer cells spiked into whole blood samples as an initial in vitro model for CTCs. If successful, Dr. Kulp would extend these studies to tumors grown in animals, and eventually to detect CTCs from blood samples from human patients. Robert Carlson, an oncologist at Stanford University, plans to use advanced fluorescence-activated cell sorting (HiD-FACS) to simultaneously detect up to 12 biomarkers of interest. He will be comparing the CTCs biomarker profile to tumor cells obtained from patient bone marrow aspirates, a common metastatic site. Dr. Carlson is interested in refining a panel of biomarkers and ultimately developing a blood test that would be informative as to whether breast cancer might be recurring in patients several years following initial diagnosis. Novel paradigms are represented in the final two metastasis projects funded by the CBCRP. We awarded a fellowship grant to Lucy East from University of California, San Francisco, to determine the role of Hox genes in breast tumor angiogenesis. Dr. East is studying the normal endothelial cells that are induced by the tumor to form new blood vessels. Two master gene regulatory proteins, called HOX D3 and HOX D10, might become specific endothelial targets to modulate angiogenesis in breast tumors. Jeffrey Smith from The Burnham Institute will explore a link between the cell's protein-degrading machinery (the proteosome) and a mammary serine protease inhibitor, called “maspin”. The new paradigm to be tested is that maspin serves to alter the protein turnover in cancer cells by “tagging” of cell proteins by ubiquitin. These studies ultimately will address the effect of maspin's tumor suppressor activity in preventing cancer cell metastasis.

Even though the estrogen receptor (ER) is the most successful molecular target for treating breast cancer, it remains of high interest in basic research, especially the application of new technologies. We need new ER-targeted therapeutics, since SERMs and other estrogen antagonists fail to help many patients, and even those patients that benefit will often develop drug resistance. The CBCRP funded two grants that focus on ER biology. Alex So from University of California, San Francisco, will look at an alternate way of modulating the ER. Cells are believed to have mechanisms of maintaining the ER in an inactive state in the absence of estrogen. Working in laboratory of Dr. Keith Yamamoto, Mr. So will use a novel assay to determine whether a protein, called Hsp90, serves in this ER inactivation capacity. Understanding the “inactivation mechanisms” of the ER could provide alternate strategies for therapy. Next, the ER has long been thought to be a target for breast cancer-causing environmental agents, but many compounds in food or other sources might exert subtle influences on the ER that are difficult to detect. Bradford Gibson and Christopher Benz at Buck Institute for Age Research in Novato are funded to use new, state-of-the-art mass spectrometry to study the ER at the level of individual amino acids. They will test whether quinone oxidants, common in the human diet and endogenously produced by estrogen catabolism, serve to alter the arylatation and phosphorylation pattern on the ER. Their initial aim is to correlate the pattern of ER structural changes with known oxidant stress. Their ultimate goal is to better associate the environmental, dietary, and lifestyle effects for new model of breast cancer based on structural modifications of the ER.

Cancer progression is the topic of two other newly funded grants. First, Jason Bush at The Burnham Institute is using protein-based “proteomics” technology to study the transformation of epithelial cells into mesemcymal cells, the so-called EMT transformation that is an early physiological-morphological “switch” in cancer initiation. An adhesive receptor integrin, called α6β4, is a receptor for basement membrane components and is the interest for Dr. Bush's study of EMT. Using new inhibitory RNA technology (iRNA) and special protein “affinity tags”, he will be able to assess the role of this receptor in critical breast epithelial adhesion processes. Finally, apoptosis (programmed cell death) is involved in many aspects of cancer progression and failures of therapy. Beatrice Bailly-Maitre also from The Burnham Institute will study a novel pathway in breast cancer cell apoptosis. An anti-apoptotic protein, called BI-1 (Bax Inhibitor-1), appears to regulate a cell death pathway linked to stress in the endoplasmic reticulum. Working in Dr. John Reed's laboratory, Dr. Bailly-Maitre will study Bl-1 in animal models and tumor samples. By using BI-1 as a window into this poorly understood endoplasmic reticulumapoptosis pathway, she eventually hopes to devise strategies for bypassing the roadblocks to cell death that commonly arise as cancer progresses.

Pathogenesis Grants Funded in 2004:

Role of Bl-1 Protein in Breast Cancer Apoptosis
Beatrice Bailly-Maitre, Ph.D.
The Burnham Institute
Award type: Postdoctoral fellowship
Duration: 2 years
$90,000

Oxidative Stress and Estrogen Receptor Structural Changes
Christopher Benz, M.D. and Bradford Gibson, Ph.D.
Buck Institute for Age Research
Award type: SPRC Full
Duration: 3 years
$1,122,520

Proteomic Profiling of Adhesive Structures in Breast Cancer
Jason Bush, Ph.D.
The Burnham Institute
Award type: Postdoctoral fellowship
Duration: 2 years
$90,000

Characterizing Breast Cancer Cells in Blood and Bone Marrow
Robert Carlson, M.D. Stanford University
Award type: IDEA
Duration: 1 year
$156,108

Profiling Enzyme Activities in Human Breast Cancer
1Benjamin Cravatt, Ph.D.; and 2Stefanie Jeffrey, M.D.
1Scripps Research Institute and 2Stanford University
Duration: 2 years
Award type: TRC Full
1$469,250 and 2$400,000

Hox Transcriptional Regulation of Breast Tumor Angiogenesis
Lucy East, Ph.D.
University of California, San Francisco
Award type: Postdoctoral fellowship
Duration: 2 years
$90,000

Stem Cells in Breast Cancer Metastasis
1Brunhilde Felding-Habermann, Ph.D.; 1John Yates, M.D., Ph.D.; and 2Evan Snyder, M.D., Ph.D.
1Scripps Research Institute and 2The Burnham Institute
Award type: SPRC full
Duration: 2 years
$906,990

Identifying Metastatic Breast Cells from Peripheral Blood
Kristen Kulp, Ph.D.
Lawrence Livermore National Laboratory
Award type: IDEA
Duration: 1 year
$210,159

Maspin: Breast Cancer Suppression through Enzyme Inhibition?
Jeffrey Smith, Ph.D.
The Burnham Institute
Award type: STEP
Duration: 1 year
$285,266

A Novel Approach to Inactivate the Estrogen Receptor
Alex So
University of California, San Francisco
Award type: Dissertation
Duration: 2 years
$60,000