Biology of the Normal Breast: The Starting Point

As any woman who performs breast self-exams knows, the normal breast is a constantly changing organ. The breast's normal changes can obscure the more ominous changes associated with cancer. Researchers have worked hard to determine what constitutes a cancerous change in the breast, but the lack of a thorough understanding of the normal breast makes this work more difficult.

Because a relatively small amount of research is being done in this area, the California Breast Cancer Research Program earmarks funds especially for it. We hope these studies will provide a strong foundation for distinguishing the difference between benign and malignant breast changes.

Research Conclusions

Breast Development

Hormonal Regulation of TGF-β-1 During Mammary Development.

The reproductive organs of female mice go through the estrus cycle, where fertile periods (when pregnancy is possible) alternate with infertile periods (when it isn't). Levels of the hormones estrogen and progesterone rise and fall in a pattern during the mouse estrus cycle, as they do during the human menstrual cycle. Mary Helen Barcellos-Hoff, Ph.D., at the Lawrence Berkeley National Laboratory, investigated how changes in hormone levels interact with a protein, transforming growth factor β-1 (TGF-β-1), found in some human breast (and mouse mammary) cells. There is evidence that TGF-β-1 can both promote and suppress human breast tumors and that it plays a role in the formation of new cells during breast development. TGF-β-1 is chemically locked up in cells, and has to be released in order to be active in the cell processes. The research team found that the hormones estrogen and progesterone play a role in activating TGF-β-1. They also found that TGF-β-1 is present in all cells that produce a protein called the estrogen receptor, which allows cells to combine with, and be affected by, the hormone estrogen, and that TGF-β-1 is also in most of the cells that produce the progesterone receptor. Depriving mice of TGF-β-1 makes these cells multiply faster than normal during puberty, pregnancy and estrus. TFG-β-1 prevents cells from multiplying in response to estrogen and progesterone. Based on this research, the team hypothesizes that disruption of the action of TGF-β-1 is involved in breast cancer in cells that produce the estrogen receptor, a finding that could lead to new treatments for this type of the disease.

Breast Function

Breast Cancer Chemoprevention by Retinoids.

Xiaokun Zhang, Ph.D., at The Burnham Institute La Jolla, investigated how Vitamin A inhibits breast cell growth. Vitamin A and its natural and synthetic derivatives, retinoids, are promising preventive agents for women at risk for breast cancer. Most of the retinoids investigated so far do not prevent the most malignant breast cancers, or they must be given at such high doses that they are toxic to patients. Dr. Zhang investigated the molecule-level interactions between retinoids and the retinoid receptor proteins that allow cells to take them in. The team found that one retinoid receptor protein inhibits two cancer genes and also causes breast cancer cell death. They identified two proteins that interfere with retinoid receptor proteins. Another protein, TR3, is necessary for a new group of retinoids to cause breast cancer cells to die. This research could contribute to the development of a more effective breast cancer-preventive drug based on Vitamin A. Results from this study have been published in Molecular and Cell Biology (2000;20(3):957-970) and Cancer Research (2000;60(12):3271-80).

Mechanisms of Fluid Transport in Human Mammary Epithelium.

Fluid accumulates in the breasts of many women who have not yet reached menopause. This condition causes breast tenderness and pain; it also leads to increased cancer risk. Sheldon Miller, Ph.D., at University of California, Berkeley, investigated proteins and chemical reactions involved in fluid moving through epithelial cells, the cells where most breast cancers arise. The research team identified several “transport proteins” involved in the movement of fluids in and out of mouse mammary epithelial cells (the mouse equivalent of human breast cells). The team also identified a number of chains of chemical reactions, known as signaling pathways, within these cells. These signaling pathways are involved in the movement of fluids and also of ions—substances that include sodium, potassium and calcium—into and out of these cells. The team plans to continue this research toward the goal of a medication to prevent abnormal fluid accumulation in the breast. Such a medication could reduce breast cancer scares and unnecessary tests caused by abnormal fluid buildup being misdiagnosed as possible breast cancer, and could also reduce breast cancer risk.

Method for Measuring Breast Epithelial Turnover in Humans.

Epithelial cells in the breast produce milk and deliver it to the nipple, and are also the source of most breast cancers. Breast cancer cells divide more rapidly than normal cells. Each time a normal cell divides, the chance of a genetic mutation goes up, and so does the risk that the mutation will lead to cancer. Therefore, it is important to have a reliable way to measure the division rate of cells in the breast. Marc Hellerstein, M.D., Ph.D., at the University of California, Berkeley, developed a method to measure cell division rates directly, without using radioactivity or toxic substances, using breast tissues from core biopsies. The research team found that the cell division rate varies in samples of tissue taken from different parts of a woman's breast. Postmenopausal women tend to have lower rates of cell division than women who are still menstruating. The research team also found that exposing rats to genistein, a substance found in soybeans, both before and after the rats are sexually mature, decreases their cell division rate by 28%.

Pregnancy and Breast Cancer: an Immunological Connection?

A full term pregnancy at an early age protects a woman against developing breast cancer. But what changes occur in the breast to explain it? Many theories concentrate on hormones. Michael Campbell, Ph.D., of the University of California, San Francisco, investigated whether the immune system plays a part. Dr. Campbell examined the sera (a part of blood) from women who had multiple pregnancies and compared it to the sera of women who had never had a baby. He looked for antibodies that recognize breast tumors. Using several increasingly sensitive screening methods, the research team at first found no antibodies, then found possible evidence of antibodies. The team plans to continue this research, using sera from women whose last pregnancy was one year earlier, compared to four years earlier for these experiments, because these antibody levels may drop over time. Success with this approach could provide the basis for vaccines to prevent breast cancer.

The Role of Nitric Oxide and Arginine in the Breast.

The breast cells responsible for milk production, the epithelial cells, are where most breast cancer starts. When these cells grow too much and develop the ability to invade other tissues, it leads to cancer. However, these cells also often grow too much in a way that does not lead to cancer. Carol MacLeod, Ph.D., at the University of California , San Diego, systematically investigated the biology and genetics of these cells, looking for ways to distinguish truly precancerous cells from harmless ones. The team made progress with genes, developing a method to assess the role of one gene in the progression from normal cells, to cells that grow too much, to cells that later become cancer. They also developed a method for determining biological properties of these cells. This method will be used in future research to assess the potential of these cells to turn cancerous in the presence of various substances that do, or can, circulate in the body. Funding from the CBCRP led to a grant from another funding agency to pursue this research. The team is currently testing a substance derived from soy beans for its ability to halt the early changes cells go through on the way to becoming cancerous. The CBCRP-funded research was published in Cancer Res. 2001 61:8298-305.

Genetic Repair of Oxidative Damage: Effect of Estrogen.

Nicholas J. Rampino, Ph.D., at The Burnham Institute, La Jolla, investigated how three breast cancer prevention medications work in cells on the biochemical level. The three medications are raloxifene, tamoxifen, and ICI 182,780. The team tested how well each worked at protecting genes from the kinds of mutation that lead to cancer. Raloxifene did the best at stimulating the normal DNA repair process in cells. This process repairs mutations in DNA that lead to cancer. Raloxifene was also most effective at stopping a number of other molecule-level gene-related processes in cells that can lead to cancer, and stimulating others that allow cells to overcome cancer. These differences help to explain, at least in part, raloxifene's superior ability to prevent breast cancer. This research helps explain how these medications prevent breast cancer and could, in addition, provide the basis for developing more potent medications of this type.

Role of the POP1 Gene in Breast Cancer Genomic Stability.

Peter K. Jackson, Ph.D., at Stanford University, investigated the human POP1 gene for its role in breast cancer, but found no clear evidence that it plays one. The team then shifted its focus to another gene, Fbx5. They found that the Fbx5 gene controls the entry of cells into the phase where they prepare to divide. This gene is more active in 30-40% of breast tumors, compared to normal breast cells. The gene is also overactive in several other types of tumors. This research was published in Nat Cell Biol. 4(5):E119-20.

Breast Aging

The Role of PAK2 in Breast Cancer Cell Death.

Normally, the body maintains a critical balance between the growth of new cells and the death of the old. When the balance shifts away from normal levels of cell death, then cells multiply abnormally and cancer develops. Gary M. Bokoch, Ph.D., at The Scripps Research Institute, La Jolla, investigated a protein, p21-activated kinase, or PAK, that plays a role in this balance. PAK interacts chemically with other proteins within cells, probably in the course of both normal and cancerous cell growth and death. The research team found that PAK was necessary but not sufficient for another protein, Jun kinase, to cause cells to die. Surprisingly, PAK could block the action of another cell death protein, called Bad. The team found that PAK was abnormally high in some kinds of breast cancer cells. PAK is also involved in the ability of cells to migrate in the course of normal development and in cancer cells spreading to other body parts. The team plans to pursue this research further to find out if PAK could be a target for a therapy to treat some types of breast cancer.

Cloning of Senescence Genes in Mammary Epithelial Cells.

Normal cells have only a limited capacity to grow in lab cultures. They soon stop growing, entering a state called replica-tive senescence. The inability of senescent cells to continue to divide in two and form new cells has led researchers to suggest that replicative senescence may be a tumor suppression mechanism that prevents normal cells from turning into cancer. There is evidence that gene activity determines senescence. Hong Zhang, Ph.D., at Stanford University, used techniques called random homozygous knock-out, or RHKO, and cDNA microarrays to investigate which genes are active in cells during senescence. Dr. Zhang compared the “genetic fingerprint” of senescent cells with cells in growth arrest. Many changes in the activity of genes previously thought to reflect senescence turned out to also reflect growth arrest. Dr. Zhang is currently investigating two genes that have abnormal activity only during senescence. Dr. Zhang also compared the activity of genes in senescent human breast epithelial cells (the cells where most cancers arise) with the activity of genes in the cells that make up connective tissue in the breast. The genes that were active were very different for the two types of cells, indicating that the process of senescence is different for different types of cells.

Research in Progress

Genetic Aspects of Physiological Response During Lactation.

Scientists believe that when tissue grows too large for its existing blood vessel network, the level of oxygen in the tissue drops. In response, a protein, HIF-1α, increases and activates genes that control new blood vessel growth. HIF-1α is present in larger than normal amounts in tumors that grow rapidly Randall S. Johnson, Ph.D., at the University of California, San Diego, is investigating the role of HIF-1α in the development and function of the normal breast. The research team has found that female mice bred with no HIF-1α don't develop enough milk-producing cells. These mice don't produce enough milk to feed their babies, and their babies are 33-50% underweight. Mice bred with too much HIF-1α have a greater than normal number of blood vessels in their milk-producing glands, but these glands don't fully develop, and the milk these glands produce has blood mixed with it. The goal of this research is to generate information that will lead to better therapies to block tumor growth and development of a blood supply.

Telomere Clustering is Lost in Mammary Epithelial Tumors.

Telomeres—structures of DNA and proteins that cap the end of chromosomes—play a role in normal breast development, aging, and cancer. Paul Kaminker, Ph.D., of Lawrence Berkeley National Laboratory, is investigating whether disrupting the structure of telomeres in cells leads to the formation of breast cell tumors. When a protein called Tin2 is present in larger than normal amounts, cells develop a structure similar to cancer cells and the cell DNA loses telomere clusters. The team has found that as normal cells mature, Tin2 detaches from telomeres, and this causes clusters to form. The team is trying to find out if Tin2 reattaches to telomeres later in the cell developmental process.

Genetic Changes in Normal Epithelium of the Cancerous Breast.

Shanaz Dairkee, Ph.D., of the California Pacific Medical Center Research Institute, San Francisco, is trying to identify genetic changes in normal-appearing breast cells that indicate a propensity to become breast cancer. Some genes, including ATM and the normal version of BRCA2, suppress breast cancer. In inherited cancer, these genes undergo mutations that make a woman more susceptible to breast cancer. In non-inherited cancer, these genes are frequently turned off because the cell loses this part of the DNA when it reproduces itself. This year the team investigated normal-appearing cell structures (called terminal ductal lobular units, TDLU) in breast tissue samples that also contained cancerous tumors. In four out of eleven tumor samples, the normal-looking cell structures had lost the ATM tumor suppressor gene. In two out of four, the normal-appearing cell structures had lost the BRCA2 gene. This strongly suggests that for a subset of breast tumors, loss of these genes are among the earliest signs that the disease is developing, detectable well before any tumor forms. The team also found that a woman who has a cancerous lump removed where the TDLU have lost DNA in the chromosome 3p24 region has a five times greater than average risk of recurrence. It takes longer than average for the cancer to recur, suggesting that the cancer is arising from normal cells that have lost part of their DNA, rather than from tumor cells that were not removed. This research led to a publication in Cancer Research 62:1000, 2002.

Research Initiated in 2002

Breast Development

Role of Epimorphin and Progesterone in Breast Development.

Jamie Bascom, at the Lawrence Berkeley National Laboratory, is investigating epimorphin, a protein that stimulates breast epithelial cells to become specialized milk producing cells. The research team will determine if there is a relationship between epimorphin and the hormone progesterone in this process.

Steroid Receptor Coactivators in Mammary Gland Development.

Shi Huang, Ph.D., at The Burnham Institute, La Jolla, is investigating the RIZ1 gene and the protein this gene produces, to see if it plays a role in the action of the hormone estrogen on normal breast development.

Breast Function

Defining a Role for Endothelial Precursor Cells in Breast.

New blood vessel growth is necessary for both normal tissue expansion and breast cancer. Blood vessels are lined with endothelial cells; new endothelial cells can come from existing blood vessels or from endothelial precursor cells that circulate in the blood. Longchuan Chen, Ph.D., at the La Jolla Institute for Molecular Medicine, is investigating the significance of endothelial precursor cells in both normal breast development and in breast tumors, and will try to design a way to block these cells to see if this stops tumor growth.

Rac/STAT5 Signaling.

Normal breast function depends on proper interactions of breast epithelial cells with the cells that surround them. These interactions regulate the responses of epithelial cells to hormones 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, to see if they function together as a master switch that allows breast cells to respond normally to hormones, and the molecules' possible role in breast cancer.

The Importance of Growth Inhibitory Signals in Normal Breast Cells.

HER-2 is a protein found in larger than normal amounts in about 30% of breast cancer cases. Scientists do not understand what role HER-2 plays in promoting 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 that are naturally present in the breast and that are the body's first line of defense against breast cancer.

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. However, statistical techniques to identify the significant patterns in this information are not available. Saira Mian, Ph.D., at the Lawrence Berkeley National Laboratory, will develop a variety of statistical techniques that could help provide more reliable diagnoses, uncover unrecognized categories of breast cancer, and explain why cancer treatments work on some people, but not on others.

Targeting Estrogen Receptors to Mammary Epithelial Cells.

The hormone estrogen causes normal breast growth and also the growth of tumors, but scientists don't know exactly how. Estrogen receptors are proteins that allow cells to take up estrogen. Richard H. Price, Jr., Ph.D., at the University of California, San Francisco, will engineer mice with more than the usual amount of estrogen receptors in the cells where mammary cancer (the mouse equivalent of breast cancer) usually arises. They will compare the growth of these cells with those in normal mice.

Effect of Breast Cell Environment on Repair of DNA Damage.

Aylin Rizki, Ph.D., at the Lawrence Berkeley National Laboratory, will investigate whether scaffolding cells that support milk-producing cells interact with the milk-producing cells in a way that protects the milk-producing cells from DNA damage.

Breast Aging

Understanding Telomere Dynamics in the Breast.

Telomeres are DNA sequences on the ends of all our chromosomes that cap and protect them from breaking down and joining with other chromosomes. Telomeres shorten in many of our organs as we age, limiting the organs' ability to replace worn-out cells. In the breast, shorter telomeres can also allow cells to become cancerous. Steven Artandi, Ph.D., at Stanford University, will study how normal breast cells react to telomere shortening as they age.

Understanding Aging Effects in the Breast.

Ana Krtolica, Ph.D., at the Lawrence Berkeley National Laboratory, is investigating a type of breast cell called fibroblasts. Fibroblasts do not usually become cancerous, but they are part of the structure that supports the cells that do. Dr. Krtolica is investigating whether old fibroblasts that have lost the ability to divide into new cells create an environment that allows nearby cells to become cancerous.

Separating Normal from Abnormal Breast Structures

Genetic Alterations in MRI Screen-Detected Breast Lesions.

James Ford, M.D., and Sylvia Plevritis, Ph.D., at Stanford University, will use Magnetic Resonance Imaging (MRI) screening to detect breast lumps, then see whether analyzing the genes in these tissues can detect genetic changes that could predict whether a benign lump may later become cancerous.