Biology of the Normal Breast:
The Starting Point

The Biology of the Normal Breast is a greatly understudied area. The breast is a complex structure composed of several cell types. We know that the milkforming epithelial cells are most associated with tumors, but there are many questions remaining. How do the different types of cells interact in the breast under normal conditions? What normal changes are necessary for the breast to function properly? Without knowing the answers to these questions, it requires a leap of faith to be able to identify the abnormal changes associated with cancer.

What we do know about the breast is that it is an organ in constant flux. Researchers are finding that how the breast remodels itself under the influence of internal and external factors dictates how it functions. The production of milk depends on the maturity (differentiation) of the breast cells, which in turn is controlled by hormones and growth factors and the immediate environment of the cells, as well as the internal and external physical structure of the cells.

Research Conclusions

Eight CBCRP grants studying the Biology of the Normal Breast were completed in 2003.

The Role of N-CoR During Normal Mammary Gland Development. Tumors that are estrogen receptor positive (ER+) depend on estrogen for their growth. However, precisely how estrogen regulates these processes is still unclear. Sung Hee Baek, Ph.D., of the University of California, San Diego, studied the role of a protein, N-CoR, in the growth and development of the normal mammary gland. N-CoR is a molecule inside the nucleus that is involved in estrogen regulation of cell growth. Using a test called immunohistochemistry, which allows researchers to detect the presence of specific proteins in cells or tissues, Dr. Baek found that some protein signals could lead to a progressive decrease of N-CoR. Dr. Baek also found that when N-CoR is not present, tamoxifen, a drug used to treat breast cancer tumors that are (ER+), stimulates the estrogen receptor. These findings have the potential to impact the diagnosis and treatment of human breast cancer, and have been published in Cell 111 (2002):673-685 and Cell 110 (2002):55-67.

Coactivators in Mammary Gland Development and Tumorigenesis.
Estrogen and progesterone, two hormones that are produced by the ovaries, control the growth of the epithelial cells in the breast by attaching to the estrogen and progesterone receptors. Once these receptors are turned on, they can turn on genes that play a role in both normal breast development and breast cancer. Two genes that facilitate the function of the p160 gene, p/CIP and SRC1, increase the activity of the estrogen and progesterone receptors. Also, high levels of the p/CIP gene and its proteins are found in human breast and ovarian cancers. Zhiyong Wang, Ph.D., at the Salk Institute for Biological Studies in La Jolla, looked at whether p/CIP and SRC1 could cause cancer to develop in mice. His team found that when just one gene was not working, no problems developed. But when both genes did not work, the mice developed a defect in their mammary gland after puberty. This suggests that p/CIP and SRC1 are required for normal development of the mouse mammary gland. Dr. Wang’s team also developed a mouse that makes too much p/CIP in the mammary gland, and they found that these mice develop tumors as they get older. Dr. Wang published results of his study in Cancer Cell 4 (2003):499-515.

Method for Measuring Breast Epithelial Turnover in Humans.
Each time a normal epithelial cell divides, the chance of a genetic mutation increases. The accumulation of genetic mutations is a hallmark of cancer. Thus, a reliable way to measure the division rate of cells in the breast has the potential to increase understanding of how cancer develops and provide a way to test cancer prevention treatments. Marc Hellerstein, M.D., Ph.D., at the University of California, Berkeley, investigated a technique developed in his laboratory to measure cell division rates directly, without using radioactivity or toxic substances. His team found that this technique could successfully measure a woman’s cell division rate by using breast tissue from core biopsies. They have also found that genistein, a substance found in soybeans, decreases the epithelial cell division rate in rats. Dr. Hellerstein’s team is now conducting a study that aims to establish normal rates of epithelial breast cell division and to determine factors that might be associated with this turnover rate in women, such as age, weight, ethnicity, and diet. His team also intends to look at whether their technique can predict who is most at risk for developing breast cancer. Dr. Hellerstein presented an abstract of his research in FASEB Journal 14(4):A786.

Telomere Dynamics During Breast Development.
Unlike most other cells, breast cells undergo changes during breast development at puberty and throughout most of adulthood, especially during pregnancy and lactation. Sahn-Ho Kim, Ph.D., at the Lawrence Berkeley National Laboratory, studied how telomeres and telomere-associated proteins influence the development of breast cells. Telomeres are the protective caps that are on each end of a chromosome’s four arms (a chromosome looks like an X), and keep the chromosome in working order. TIN2 is a human telomere-associated protein that plays an important role in the cell. Dr. Kim found that the TIN2 proteins regulate telomere length. He also found that removing the TIN2 proteins from cells causes the cell to die, which suggests that TIN2, along with telomeres, are necessary for a cell to survive. In addition, Dr. Kim found that an abnormal form of TIN2, TIN2-13, increases telomere length. This abnormal TIN2-13 also disrupts the development of breast cells in cell culture. Results of this study were published in Oncogene 21 (2002):503-11 and the Journal of Biological Chemistry 277 (2002):28609-17.

Cell Growth Control of Breast Epithelial Cells.
Intracellular Rho GTPases, including Rac, Cdc42 and Rho, provide an important regulatory mechanism to connect cell-surface generated signals with the nucleus. Ulla Knaus, Ph.D., at The Scripps Research Institute, La Jolla, investigated two proteins that appear to be involved in the growth of normal breast cells. The two proteins are called Rac3 and Rac1. The expression of the proteins is turned on by hormones and by proteins called growth factors that come from outside the cell. Rac3 is consistently expressed in tumors, while Rac1 is not. This might mean that Rac3 is tricking cells into growing at inappropriate times. Rac3 does not have mutations, so Dr. Knaus investigated where inside breast cells this protein is attached. Her team introduced fluorescent copies of both proteins into normal human breast epithelial cells. They found that Rac1 distributed itself throughout the cell, but Rac3 attached to membranes inside the cell. They also found that several growth-stimulating substances that activate Rac1 don’t activate Rac3. This information provides clues about the basic biology of the breast that may prove useful in the development of new treatments for breast cancer.

A Vascular Restriction of Mammary Tumor Progression.
A number of growth factors are associated with the tumor and normal cell’s blood supply. VEGF (vascular-endothelial growth factor) is a significant part of angiogenesis (blood vessel growth). When a person is injured, VEGF is helpful —it aids healing by stimulating the growth of blood vessels. When cancer is present, VEGF is not helpful—it allows tumors to grow new blood vessels, which enables them to grow and spread. Robert Oshima, Ph.D., at The Burnham Institute, La Jolla, developed a genetically engineered mouse that was prone to have both breast cancer and a higher level of VEGF. Dr. Oshima compared tumor growth in the mice he developed with tumor development in normal mice. He found that the mice with the higher levels of VEGF began to develop tumors immediately after the mammary gland began to develop and that these tumors not only grew dramatically faster but also quickly produced new tumor blood vessels. Based on these findings, Dr. Oshima concluded that the increased blood supply that VEGF provides is important for large tumors and appears to play a critical role in the transformation of fast-growing cells into small tumors. Dr. Oshima’s results were recently published in Cancer Research 64 (2004):169-79.

Defining a Role for Endothelial Precursor Cells in Breast Cancer.
Blood vessels are lined with endothelial cells; new endothelial cells can come from existing blood vessels or from endothelial precursor cells that originate in the bone marrow and circulate in the blood. Longchuan Chen, Sc.D., at the La Jolla Institute for Molecular Medicine, investigated the role endothelial precursor cells play in both normal breast development and in breast tumors. Dr. Chen’s team used mice with breast tumors to track the movement of the endothelial precursor cells from the marrow. They found groups of these cells in the tumor blood vessels, but they were only a small percentage of the total number of endothelial cells. Dr. Chen is going to continue to study the role that the endothelial precursor cells that come from bone marrow play when cancer begins to develop. He is also going to continue to study an antibody his team found that could stop the endothelial precursor cells from forming new blood vessels. This antibody could become a breast cancer treatment.

Research in Progress

A number of ongoing CBCRP grants in the topic of Biology of the Normal Breast reported substantial progress in 2003.

Role of IKKa in Mammary Gland Development.
In the tiny space between the cell’s membrane (where information is received) and the cell’s nucleus (where genes are regulated), there exist a multitude of signaling pathways. Collectively the study of “signal transduction” involves all areas of cancer biology. A particularly interesting pathway is called NF-?B, which plays an important role in the regulation of the immune response, cell death, inflammation, cell cycle progression, and cancer. Activation of NF-?B is thought to be part of a stress response induced by growth factors, cytokines, UV, and pharmacological agents. Michael Karin, Ph.D., at the University of California, San Diego, is studying an NF-?B regulatory element, called IKK?. Dr. Karin has shown that blocking this enzyme decreases or delays the development of breast cancer in mice. Dr. Karin is now investigating how IKK??causes tumors to grow. Blocking this enzyme doesn’t affect other tissues or organs, so it could eventually be a target for a treatment that would have few side effects. Dr. Karin and his colleagues have published numerous articles on this topic, which they recently reviewed in Seminars in Cancer Biology 13 (2003):107-114 and Nature Reviews Cancer 2002 Apr; 2(4):301-10.

Steroid Receptor Coactivators in Mammary Gland Development.
Cells in normal breast tissue and in estrogen receptor (ER)-positive breast cancers need estrogen to grow. Breast cells that do not get estrogen stop proliferating and die. This is why ER-positive breast cancers are treated with drugs that block estrogen activity. Shi Huang, Ph.D., at The Burnham Institute, La Jolla, and colleagues discovered a new tumor suppressor gene, RIZ1, and they are investigating this new gene and the protein it produces. They have found laboratory evidence that suggests that the breast needs RIZ1 to respond to the hormones estrogen and progesterone, and they are now exploring the relationship between RIZ1 and the estrogen receptor. This research could lead to the development of new drugs for the prevention and treatment of breast cancer.

Genetic Aspects of Physiological Response During Lactation.
When a clump of tumor cells grows too large, the level of oxygen in the tissue decreases (i.e., hypoxia). In response, a protein, HIF-1, increases and activates genes that control new blood vessel growth. Recent studies have shown that high levels of HIF-1 have been found in a variety of tumors, including breast tumors. Randall S. Johnson, Ph.D., at the University of California, San Diego, is investigating whether the HIF-1 response to lowered oxygen levels contributes to mammary gland development and the production of milk in mice. These findings could lead to new ways to block blood vessel growth in breast tumors and to new breast cancer treatments. Results from this research were published in Development (2003) 130:1713-1724.

Statistical Techniques for Breast Biology and Cancer Research.
New technologies allow scientists to rapidly and simultaneously measure thousands of genes, proteins, and other molecules within cells, and much of this information is in publicly-accessible databases; however, statistical techniques to identify important and useful patterns in the data are not available. Saira Mian, Ph.D., at the Lawrence Berkeley National Laboratory, is developing a variety of cutting-edge statistical techniques that can identify these patterns. This approach could make it easier to diagnose and treat breast cancer and may help explain why cancer treatments work on some people, but fail for others. Results from Dr. Mian’s research were published in Journal of Biological Chemistry 278 (2003):3882-3890, Signal Processing 83 (2003):729-743, Lancet 362 (2003):440-445, and Mechanisms of Aging and Development 124 (2003):109-114.

Effect of Breast Cell Environment on Repair of DNA Damage.
Breast cells become cancerous when they no longer respond to signals that control their growth. Signals regulating cell growth come from both within and outside the cells. Within the breast tissue, cells contact other cells as well as the scaffold material, called the extracellular matrix (ECM) that surrounds them. Aylin Rizki, Ph.D., at the Lawrence Berkeley National Laboratory, is investigating how communication between cells and the ECM affect a cell’s ability to repair damage to its DNA. When the DNA is not repaired properly, it can accumulate genetic changes, which sets the stage for cancer to occur. She is also continuing to explore the finding that restoring proper communication between the cells and the ECM can turn cancer cells back into normal cells. Results from this research were published in Differentiation 70 (2003):537-46 and Signal Processing 83 (2003):729-743.

Rac/STAT5 Signaling.
Normal breast function depends on proper interactions between breast epithelial cells and the cells that surround them. These interactions regulate the responses of epithelial cells to hormones, like prolactin and estrogen, and allow them to grow normally. Disruption of these interactions can result in breast cancer. Hee Kwang Choi, Ph.D., at The Burnham Institute, La Jolla, is investigating two molecules, called Rac and STAT5, and the theory that they function as a master switch that not only controls how the genes in the breast respond to prolactin and estrogen but also aids in the development of breast cancer and tumors that are drug resistant. His research on the role Rac and STAT5 play in both normal and breast cancer cells will provide important information about normal breast biology and cancer progression.

Role of Chromatin Regulator in Breast Cell Growth.
To grow, both normal cells and cancer cells need the information carried on their DNA to synthesize a large number of different proteins. Chromatin is the substance that forms chromosomes. It contains DNA, RNA, and various proteins. Hongwu Chen, Ph.D., of the University of California, Davis, has found that one of the proteins that regulates chromatin is also present in elevated amounts in breast tumors. Dr. Chen is investigating how this protein controls normal breast cells and spurs breast tumor cell growth. This research has allowed Dr. Chen to develop a new model for why tumors stop responding to the hormonal treatment tamoxifen. It also has the potential to lead to new breast cancer treatments.

The Importance of Growth Inhibitory Signals in Normal Breast Cells.
HER-2 is a protein found in larger than normal amounts in about 30 percent of breast cancer cases. Scientists do not yet understand how having too much HER-2 promotes breast cancer. Cindy Wilson, Ph.D., at the University of California, Los Angeles, is testing the hypothesis that HER-2 promotes breast cancer by inhibiting the action of proteins in the breast that are the body’s first line of defense against breast cancer. Dr. Wilson and her colleagues are also continuing to explore their finding that higher than normal levels of HER-2 can make cells less sensitive to a protein called transforming growth factor beta, which may control the growth of breast epithelial cells. This research could lead to the development of new treatments for women with HER2-positive breast cancer.

Telomere Clustering is Lost in Mammary Epithelial Tumors.
Paul Kaminker, Ph.D., of Lawrence Berkeley National Laboratory, is investigating a part of the nucleus associated with the telomeric protein, TIN2, called a “TIN2 cluster.” The absence of these clusters has been shown previously to be an indicator that a tumor is malignant. Dr. Kaminker’s research will provide more information about what types of proteins comprise these clusters, how likely it is that cells that do not have these clusters will become cancerous, and whether keeping the clusters together will affect whether tumors are formed. Dr. Kaminker and his mentor, Dr. Judith Campisi, recently reviewed their progress on this topic in Oncogene 21 (2002):503-11.

Understanding Aging Effects in the Breast.
As the body ages or senesces, the supporting stromal cells become less functional and this affects the overall biology of the breast. Ana Krtolica, Ph.D., at the Lawrence Berkeley National Laboratory, is investigating a type of breast cell called a fibroblast. Fibroblasts do not usually become cancerous, but they are part of the structure that supports the cells that do. Dr. Krtolica is studying which mutations in breast cells make them sensitive to the senescent fibroblasts around them. What she learns about how cells in the breast age and how their aging affects nearby cells could lead to new advances in breast cancer prevention and treatment. Results from this research were published in International Journal of Biochemistry and Cell Biology 31 (2002):1401-1414 and Cytometry 49 (2002):73-82.

Genetic Alterations in MRI Screen-Detected Breast Lesions.
James Ford, M.D., and Sylvia Plevritis, Ph.D., at Stanford University, are using Magnetic Resonance Imaging (MRI) screening in women with inherited BRCA1 and BRCA2 mutations who are at high risk for developing breast cancer. The tissue from breast lumps found through MRI will be analyzed for genetic changes that may be able to predict whether a benign lump later becomes cancerous. This research could lead to more extensive use of MRI for breast cancer screening and to new genetic tests for predicting who is at risk for breast cancer. Drs. Ford and Plevritis recently published their results in Cancer 100 (2004):479-89.

Understanding Telomere Dynamics in the Breast.
Telomeres, which cap the ends of chromosomes, shorten as we age, and when they get too short, a cell can no longer divide. Cancer cells learn how to keep the telomeres from getting too short, which allows them to divide indefinitely. Steven Artandi, Ph.D., at Stanford University, is studying how normal breast cells respond to telomere shortening as they age. He is also investigating how telomeres are reactivated. This research on how breast cancer evolves could lead to new methods of prevention and treatment.

Research Initiated in 2003

The CBCRP funded eight new individual grants in 2003 to pursue studies on Biology of the Normal Breast. The earliest stage of embryonic breast development involves the migration of the breast epithelial cells to the location on the body where the breast will eventually form. Saverio Bellusci, Ph.D., of the Children’s Hospital Los Angeles, received a three-year grant to investigate this process. He will study how the interactions between the growth factor FGF10 and WNT gene family direct breast epithelial cell migration. Ultimately this research may give us insights into the metastatic process of breast tumors.

Three investigators were awarded grants to study the regulation of gene activation and inactivation in the normal breast cell. John Conboy, Ph.D., of the Lawrence Berkeley National Laboratory, will investigate the changes in cell behavior due to “alternate splicing”—when one gene produces different proteins from the same code. Dr. Conboy will study the mechanism for the determining which protein is produced under different cellular conditions. Peter Kaiser, Ph.D., of the University of California, Irvine, will also use an IDEA to study the genetic regulation of the breast cancer susceptibility gene BRCA1 through a process of protein degradation, called ubiquitylation. Yuehai Ke, Ph.D., at The Burnham Institute in La Jolla, was awarded a postdoctoral fellowship to determine the role of a set of proteins called tyrosine phosphatases, which are known to regulate the activation of other proteins, in the development and normal functioning of the mammary gland.

The aging of breast cells and supporting stroma result in changes that may contribute to breast cancer. Two newly-funded CBCRP grants will investigate the role of DNA integrity in the function of normal breast cells. Kimberly McDermott, Ph.D., of the University of California, San Francisco, will undertake a postdoctoral fellowship to investigate the how the cellular structures that regulate DNA replication (centrosomes) function to protect it from mutations and chromosomal abnormalities. Paul Yaswen, Ph.D., of the Lawrence Berkeley National Laboratory, will investigate a newly discovered gene, called BORIS, for its role in controlling the integrity of DNA and determine whether it plays a role in the early transformation of breast cells.

The early changes in the transition from a normal breast cell to a breast tumor cell are subtle, but it is the goal of two studies funded this year to define the key genetic changes in this transition. Thea Tlsty, Ph.D., of the University of California, San Francisco, developed an early precursor model of breast cancer, called the variant Human Mammary Epithelial Cell (vHMEC) that has specific genetic characteristics. Dr. Tlsty will determine whether these same characteristics can be found in early pre-cancer breast lesions, and therefore used to distinguish them from normal cells. Finally, the information age has opened new avenues for characterizing and understanding important changes to tissues at the protein level. One new technology is called proteomics, which is the simultaneous comparison of proteins in tissues under different physiological conditions. Drs. Dave Hoon, Armando Giuliano, and Lori Wilson (co-PIs) at the John Wayne Cancer Institute in Santa Monica will apply this new technology to the breast. The two major goals of the project are to: (1) determine whether it is possible to develop a proteomic profile signature of normal breast tissue during different stages of physiology, and (2) determine if proteomic profile signatures of various types of benign breast disease can be used for diagnosing early stages of pre-cancerous breast disease.