Biology of the Breast Cell

To understand the origin of breast cancers, more research is needed on the pre-cancerous, causative events in the normal breast. In breast development, cell populations must co-ordinate migration, proliferation, and apoptosis (cell death) over space and time. In cancer progression, these same processes become dysregulated, initially at the genetic level, it leads to the physiological changes associated with malignancy. To better mimic breast and tumor architecture, 3-D cell culture models provide a means to explore potential underlying mechanisms and show the structure of the breast and interaction of its different cell types lead to the development of a tumor. An emerging paradigm identifies “stem cells” as the key to the origin of tumors. Stem cell populations reside in body organs to provide the raw material for tissue regeneration, repair, and for the cyclic proliferation responses to hormones and pregnancy in the breast. If this theory proves correct, then only a small fraction (1- 2 percent) of cells in a tumor mass retain stem cell properties, and these “cancer stem cells” must be selectively targeted to achieve an effective eradication of the disease.

Tumor biology, which the CBCRP refers to as pathogenesis, typically involves basic science cell-based studies. In the past, researchers approached tumor biology from the reductionist level (i.e., studying the contributions of individual genes and proteins to the development of disease). However, over the past decade researchers have realized that the underlying mechanistic driving forces of tumor biology operate though complex, concurrent genetic changes in numerous molecular pathways. Still, it remains the metastatic process that presents the greatest hurdle in our efforts to contain and destroy cancer as it too often presents itself at the time of diagnosis. Breast cancer can spread to almost any region of the body, although metastases are most common to the bone, lung and liver. Understanding the gene and physiological regulatory mechanisms for this cancer cell diaspora is crucial for the design of therapeutic strategies. Other important basic science topics represented in CBCRP’s portfolio include: (1) cell proliferation control mechanisms through the estrogen receptor and growth factor receptors (e.g., Her-2), (2) alterations in DNA repair process that permit genetic damage to accumulate in cancer cells, (3) cell cycle changes that permit division under conditions where normal cells would undergo programmed cell death (apoptosis), and (4) novel biomarkers to distinguish pre-cancerous and cancerous cells from normal breast epithelium and their validation as potential new detection and therapy targets.

Two research topics are presented in this section.

Research Conclusions

Discovering Novel Cell-ECM Interactions in Breast Cells
Breast cancer begins in the epithelial cells that line the breast duct. Normal epithelial cells are in contact with a complex mixture of proteins released from the basement membrane (BM) of the extracellular matrix, a framework that surrounds and supports these cells. Normal epithelial cells use proteins, known as receptors, to communicate with the BM. This communication organizes the cells into tissues and prevents uncontrolled cell growth. In cancer cells this communication process has stopped functioning properly, which allows the cells to break through the BM and invade surrounding tissue. Two types of proteins, called integrins and dystroglycan receptors, are known to contribute to cancer progression. John Muschler, Ph.D., at the California Pacific Medical Center Research Institute, San Francisco, established six new human breast epithelial cell lines that lack ß1 integrin and dystroglycan receptors. By looking at how cancer develops in cells that lack these receptors, Muschler and his colleagues hope to find and identify currently unknown receptors and signaling pathways involved in breast cell-BM communication.

The Role of Gli3 in Mouse Embryonic Mammary Gland Formation
Scientists recognize that tumors arise when the genes that play a role in normal development stop functioning properly. Mutant mice in which the genes Gli3, Fgf10, or Fgfr2b do not properly function have similar defects in breast development. Jacqueline Veltmaat, Ph.D., at Childrens Hospital, Los Angeles, used these mutant mice to study the role that these three genes play in the developing breast. Normal mice form five pairs of breasts. Dr. Veltmaat and her team found that in the Gli3 and Fgf10 mutant mice, the fourth breast pair developed, but the third pair did not. In addition, in the Gli3 mutants, the second breast pair developed abnormally. The team also found that when Gli3 was absent, the production level of Fgf10 remained too low to induce signaling between Fdf10 and another molecule called Wnt. This finding suggested that there is an optimal level of signaling between Fgf10 and Wnt that needs to occur to maintain a breast cell's identity. It also suggested that Gli3 might start to function as a breast cancer gene when its levels get too high. The team is now investigating how to return Gli3 functioning that is too high to normal levels.

Epithelial Polarity, Organization, and the Angiogenic Switch
For a tumor to become invasive, it first must develop the new blood vessels that will allow it to grow and spread, a process known as angiogenesis. Nancy Boudreau, Ph.D., at the University of California, San Francisco, explored whether there is a process that occurs in early tumor development that triggers breast epithelial cells (the cells in which breast cancer begins) to start producing the special molecules, called angiogenic factors, that a cell needs to create new blood vessels. Dr. Boudreau and her team found that normal cells, which appear organized (or polarized) inside, suppressed expression of angiogenic factors whereas disorganized cells and breast tumors cells had higher levels of these factors. They also found that by restoring expression of a gene, called HoxD10, which is missing in aggressive tumors, they could revert tumor cells back to an organized, polarized state. The team is now exploring the role that a receptor called ß4 integrin, which interacts with HoxD10, plays in this process. They are also investigating whether cells need a protein called ß-catenin to grow new blood vessels. This work could lead to the development of new breast cancer treatments that stop tumor cells from
growing by inhibiting angiogenesis.

Role of Telomerase in Mammary Stem Cell Function
Telomeres are the protective caps on each end of a chromosome’s four arms. Each time a chromosome splits during cell division, the telomeres get shorter; when they get too short, the cell dies. Telomerase is an enzyme that tells the telomeres to grow. It is expressed in stem cells and in cancer cells, but not in the vast majority of normal cells. Steven Artandi, M.D., Ph.D., at Stanford University, Palo Alto, and colleagues discovered that telomerase could activate inactive tissue stem cells, such as those found in the mammary gland. To further explore this finding, the team created a genetically engineered mouse (GEM) in which a protein subunit of telomerase, called TERT, could be switched on and off. They found that when TERT was on, it led to excessive cell growth and early breast cancers. Dr. Artandi and his team also showed that mice that don’t have telomerase exhibit telomere shortening that impairs mammary gland development during pregnancy; that they could reconstitute the mammary gland when stem cells from one GEM mouse were transplanted into a normal mouse; and that the function of mammary stem cells is impaired when the telomeres in mice without TERT become very short. These studies provide important insights into the role telomerase plays in maintaining telomeres and preserving mammary stem cell function when cancer starts to develop.

Identification of BRCA1 Ubiquitylation Targets
The tumor suppressor gene BRCA1 is mutated in 50-90 percent of hereditary breast and ovarian cancers. Although how BRCA1 suppresses tumor growth is not fully understood, ubiquitinligases, which help attach the small protein ubiquitin to other proteins, are believed to play a role. Peter Kaiser, Ph.D., at the University of California, Irvine, and colleagues developed a procedure to identify which target proteins in BRCA1 are attached to ubiquitin. By comparing cells with BRCA mutations to normal cells, they were able to identify many ubiquination sites and to measure quantitative changes in ubiquination profiles in response to DNA damage. This work could provide a way to identify the meaningful mutations in BRCA1. This could, in turn, lead to more reliable genetic counseling of individuals with an extensive family history of breast cancer. Findings from this research were published in Molecular and Cellular Proteomics 2(2005)366 and 5(2006)737, Genome Biology 6(2005)233, EMBO Journal 8(2007)817, and Biochemistry 11(2007)3553. Dr. Kaiser recently received funding from the National Institutes of Health to purchase an instrument, called a mass spectrometer, which will permit his team to better identify BRCA1 ubiquitination targets.

Modulation of TGF-beta Signaling in Mammary Epithelial Cells
Transforming Growth Factor-beta (TGFß) strongly inhibits the growth of mammary epithelial cells. Although it has the ability to suppress tumor growth, TGFß also can promote invasion and metastases of tumor cells as breast cancer progresses. To develop effective TGFß-based treatments, it is necessary to understand how TGFß converts from a tumor suppressor to a tumor promoter. Xiaoman Xu, B.S., at the University of California, Irvine, and colleagues investigated whether a gene regulatory protein, called LMO4, which is found at high levels during mammary gland development and in over half of all breast cancer cases, transforms TGFß in breast cancer. The team showed that LMO4 affects cell growth by helping TGFß put the brakes on cell proliferation, and that increasing or removing LMO4 enhances TGFß-stimulated transcription. This suggests that LMO4 regulates the transcriptional response to TGFß in two different ways. Mr. Xu and his team also showed that LMO4 associates with a gene promoter, called PAI-1, in a way that could mediate the effects of LMO4 on TGFß signaling. And they found that a protein that is a member of the TGF-ß superfamily of proteins, called BMP7, is a direct target of LMO4. This work, which shows that LMO4 has a role in TGFß signaling, has the potential to advance our understanding of how breast cancer progresses and could lead to the development of new breast cancer treatments. Findings from this research appeared in Oncogene 25(2006)2920 and 26(2007)6431.

Isolation of Cancer Precursors from Normal Human Breasts
Cancer researchers are trying to identify biomarkers that can be used for early detection, prognosis, and prevention. Bob Liu, Ph.D., at the University of California, San Francisco, and colleagues in the lab of Dr. Thea Tlsty are using a cell culture model system to grow normal human mammary epithelial cells (HMEC) that allows them to study the earliest events in breast cancer development. The research team has identified a population of cells that have abnormal growth control and malignant characteristics, which they call “variant” HMEC (vHMEC). These cells do not express an important tumor suppressor gene, called p16INK4a. Dr. Liu and his team developed cell surface markers, CD73+ and CD90-, that allowed them to identify vHMEC in women without breast cancer who have pre-malignant characteristics. They then characterized the cell populations that were identified by the CD73 and CD90 markers by whether or not they had malignant characteristics. The team found that there were sub-populations of breast cells that appeared to be able to silence tumor suppressors genes. They also found that CD73 “high” and CD90 “low” cells appear to overlap with basal-like breast cancers, which, due to their aggressive nature, have a poor prognosis. If future research confirms that CD73 and CD90 are good biomarkers, this work could lead to new ways of identifying and treating basal-like breast cancer.

Stem Cells in Breast Cancer Metastasis
Many scientists believe that stem cells may play a role in breast cancer. John Yates, M.D., Ph.D., and Brunhilde Felding-Habermann, Ph.D., at the Scripps Research Institute, La Jolla, and Evan Snyder, M.D., Ph.D., at The Burnham Institute for Medical Research, La Jolla, tried to identify a population of aberrant stem-like cells within breast tumors that might play a critical role in the initiation of metastatic disease. They found that the majority of cells from patients with metastatic breast cancer display several of the properties associated with small subpopulations of cells found in primary tumors. Using a mouse model, the team showed that these tumor cells have multiple ways of homing in on different organs. The mouse model also revealed that breast cancer cells that have the ability to spread to the brain, which has a unique microenvironment, derive their energy predominantly from glucose oxidation, which is a hallmark of brain cell metabolism. These brain-homing breast cancer cells also are able to activate pathways that enhance their ability to survive and grow in the brain. The team is now studying whether normal human brain stem cells could be used to deliver treatments for metastatic brain lesions. This work could lead to the development of new approaches to prevent and treat metastatic breast cancer. Findings from this research were published in Molecular and Cellular Proteomics 5(2006)53, Clinical Cancer Research 13(2007)1656, and Cancer Research 67(2007)1472.

Histone Methylation as a Marker of Breast Cancer Progression
Histone methylation is a normal cell event that is often altered during breast cancer progression. Histones are proteins that organize the DNA in our cells. When they undergo a chemical change called methylation it results in gene misregulation, DNA damage, cell cycle defects, and genomic instability – all of which are hallmarks of cancer. Judd Rice, Ph.D., at the University of Southern California, Los Angeles, and colleagues investigated whether histone methylation could be a breast cancer biomarker. They began by identifying locations on the genome of breast cancer cells where changes in histone methylation had occurred. They then looked at these locations on normal cells. However, no significant differences were present. The team then conducted the study again, using a panel of histone methylation-specific antibodies. This time they found that specific methylated forms of the histones were dramatically altered on the cancer cells, which suggested that the antibodies themselves could be used as a biomarker. To explore this further, Dr. Rice has begun looking for correlations between cancer progression and degrees of histone methylation. This work could lead to new methods of breast cancer detection, assessment, and treatment.

Apaf-1 is a Transcriptional Target for the ZNF217 Oncogene
An oncogene is a gene that has the ability to transform normal cells into cancer cells. In 20-30 percent of early stage breast tumors, an oncogene called ZNF217 has too many copies (amplification) of the gene and too many proteins (overexpression) on its surface. This amplification and overexpression appears to play a key role during the early transformation of normal breast epithelial cells into cancer cells. Studies have shown that putting ZNF217 into normal breast epithelial cells not only allows them to reproduce indefinitely but it protects breast tumor cells from the chemotherapy drug doxorubicin. Sheryl Krig, Ph.D., at the University of California, Davis, identified target genes for ZNF217 in three different cell lines and showed that ZNF217 targets genes in a cell-type specific manner. These findings led Dr. Krig and her team to hypothesize that the normal function of ZNF217 may be to help keep cells in a proliferative state. Findings from this research were published in Genome Research 16(2006)890 and Journal of Biological Chemistry 282(2007)9703.

Integrated Proteomic and Metabolic Analysis of Breast Cancer
Proteins, such as proteases, play a central role in promoting the aggressive properties of metastatic breast tumors. To better understand how proteases and related enzymes impact breast cancer, Kyle Chiang, B.S., at the Scripps Research Institute, La Jolla, and colleagues developed a new way to measure proteins called activity-based protein profiling (ABPP) that can analyze changes in activity in large enzyme families. Using this technique, they identified a new enzyme, called KIAA1363, which appears at increased levels in aggressive breast cancer cells. The team also identified a KIAA1363 inhibitor, but it only inactivated KIAA1363 in lab studies and not in living organisms. The team is now trying to develop a better KIAA1363 inhibitor. Using breast and ovarian cancer cell lines with reductions in KIAA1363 expression, Mr. Chiang and his team showed that by interfering with KIAA1363 they could significantly reduce tumor growth rates in mouse models. The team is now investigating models of human breast cancer development and metastasis in KIAA1363 (-/-) mice. Findings from this research were published in Chemistry and Biology 12(2006)1041.

Novel Approach to Analyze Estrogen Action in Breast Cancer
Estrogen promotes breast cancer by inducing cell proliferation through estrogen receptors (ERs) and their associated signaling pathways. Anti-estrogen therapy is widely used to treat ERpositive breast tumors and is assumed to work by blocking estrogen-induced cancer cell division. Brian Eliceiri, Ph.D., at the La Jolla Institute for Molecular Medicine, and colleagues explored whether estrogen also influences host tissues, such as blood vessels, to promote tumor metastasis independent of its effects on the tumor cells themselves. They began by identifying a breast cancer cell line that does not respond to estrogen. They then injected these cells into mice with very low estrogen levels and mice with normal levels of estrogen. (They control the estrogen levels in the mice by removing the ovaries, the organs that produce the most estrogen; they then implant slow-release estrogen pellets in half the mice.) They found that the estrogen promoted metastasis to the lungs, a finding that could have significant implications for the treatment of ER-negative and ER-positive breast cancers. Findings from this research were published in Cancer Research 66(2006)3667.

Survivin: Target for Breast Cancer Brain Metastases
Metastatic breast cancer to the brain, which affects about 10-15 percent of patients with advanced breast cancer, has a poor prognosis. Part of the problem is that the chemotherapy agents that are used to treat breast cancer are unable to penetrate the blood-brain barrier. Survivin is a protein that is found in tumor blood vessels. It is also present at high levels in tumorassociated brain endothelial cells (TuBEC), where it makes these cells drug resistant. Studies have shown that blocking survivin production can induce cell death. Florence Hofman, Ph.D., at the University of Southern California, Los Angeles is exploring whether reducing survivin in tumor-associated blood vessel cells will disrupt the blood-brain barrier thereby allowing chemotherapy to kill the tumor cells. Dr. Hofman and her team have shown that reducing survivin levels in TuBEC increases their sensitivity to a cancer treatment called temozolomide. Now, they are using a mouse model of human breast cancer to determine how human tumorassociated blood vessel cells with reduced survivin support human breast cancer growth. These studies will show whether survivin can be implanted into the rodent brain. This research could open the possibility for using a wide range of chemotherapy agents for brain metastases. In addition, if anti-survivin therapy is found to be effective in stopping the formation of new blood vessels, it could lead to the development of new drug combinations for blocking tumor growth as well as treating brain metastases. Findings from this research were published in Neurosurgery Focus 20(2006)E22.

The Role of B-Myb in Human Breast Cancer Progression
Myb-related proteins play a role in various aspects of normal chromosome biology. Clinical studies have shown that B-Myb is one of a small number of genes that can predict disease recurrence in breast cancer patients who are lymph node negative and treated with the antiestrogen drug tamoxifen. Joseph Lipsick, M.D., Ph.D., at Stanford University, Palo Alto, and colleagues investigated whether high levels of B-Myb are predictive of recurrence because of the role it plays in aneuploidy—the additions or deletions of chromosomes —in breast cancer cells. The team created two versions of the human B-Myb gene. One version produced a full-length BMyb protein; the other produced a truncated Myb protein that contained only its DNA-binding domain. The team found that high levels of B-Myb caused cell death (apoptosis) in a MCF-7 human breast cancer cell line, whereas moderate levels caused more rapid entry into the cell cycle and increased invasion into the extracellular matrix, the framework that surrounds cells. They also found that inhibition of B-Myb caused an increase in aneuploidy without apoptosis. Dr. Lipsick and his colleagues are continuing to explore the role of B-Myb. They also are currently testing rabbit antibodies they prepared against human B-Myb as potential diagnostic agents. This work could lead to new ways of diagnosing and treating breast cancer.

Defining Mammary Cancer Origins in a Mouse Model of DCIS
The more scientists understand about early events in breast cancer progression, the easier it will be for them to develop new prevention therapies and strategies. Alexander Borowsky, M.D., at the University of California, Davis, and colleagues are using mouse models of mammary cancer that progress from precancerous ductal carcinoma in situ (DCIS) to invasive cancer to explore whether genetic changes that commit a mammary cell to become a cancer cell occur before an actual lesion is formed. They are also investigating whether these changes commit the cell to become a cancer cell with specific behavioral properties. This research confirmed the stability of their mouse model system. It also indicated that there were significant differences in the ratio of "stem"- like cells in their pre-cancer mouse model and tumor tissues, and that both ratios differed from normal mammary gland tissue. Dr. Borowsky is now collaborating with the Kent Erickson laboratory at UC Davis in an effort to identify new stem cell markers. He is also collaborating with several other laboratories to use the mouse model in a variety of contexts. This work could lead to a better understanding of how breast cancer develops. Findings from this research appeared in BMC Cancer 6(2006)275 and Clinical Cancer Research 12(2006)2613.

Role of Oxidative DNA Damage to Breast Tumor Progression
For decades, scientists have believed that cancer is caused by environmental factors that result in mutations in the DNA code. A more contemporary theory suggests that oxygen metabolism produces free radicals, such as hydrogen peroxide, that produce a type of DNA damage, called oxidation, that can result in cancer. In some breast cancers, increased levels of oxidative DNA damage have been associated with tumor progression. However, it has been hard to measure this damage accurately, as the most commonly used marker of DNA oxidation, 8-oxoG, is chemically unstable. Paul Henderson, Ph.D., at Lawrence Livermore National Laboratory previously found that the secondary products that 8-oxoG produces when it is oxidized are both chemically stable and easily result in mutations. This project explored the role these products might play in the development of breast cancer. Dr. Henderson and his team found, in part, that exposure to the hormone estradiol (E2), which is present in breast tumors, damages 8-oxodG in the DNA. They also found that a key repair enzyme, called MTH1, which targets 8-oxodG metabolites in cells, increases in concentration with increasing E2 concentration. This work could lead to the identification of molecular markers that could be used to diagnose breast cancer or monitor treatment response. Findings from this research were published in Chemical Research in Toxicology 18(2005)12, Bioorganic Medical Chemistry 15(2005)3627-31 and Proceedings of the National Academy of Sciences of the United States of America 104(2007)11203.

A Role for p53 and Splicing Factor SAP145 in Breast Cancer
Two of the proteins known to play a role in breast cancer are p53, which helps suppress tumor growth, and Cyclin E, which helps regulate the cell cycle. Both are found at higher than normal levels in aggressive human breast cancers. Lan Truong, B.S., at the University of California, Irvine, and colleagues investigated how these two proteins work together to modify, or splice, the genes involved in the initiation and progression of breast cancer through a splicing factor called SAP145. Their initial findings suggested that another cell cycle regulatory protein, p21, may also be involved in this process. This led them to examine how SAP145, p53, p21 and Cyclin E interact. The research team found that SAP145 only interacts with p53 and p21 under conditions of no or low stress-induced cell death. They also found that following high stress or damage, activated p53 decreases SAP145 protein levels, an effect that cannot be rescued by Cyclin E. In addition, they showed that the loss of SAP145 is p53-dependent following conditions of high stress and that it results in apoptosis, or programmed cell death. These findings, which suggest that p53’s work as a tumor suppressor protein may also include mediating apoptosis, could lead to new approaches for treating breast cancer.

Breast Cancer Studies in a 3-D Cell Culture System
Breast tumors exist in a complex environment where cells are growing, dividing, and invading other tissues. As a result of these changes, cancer cells are subjected to stresses, such as limiting amounts of oxygen and nutrients. These so-called metabolic stresses affect how the cells communicate with each other, how they respond to signals from the environment, and how they respond to breast cancer treatment. Kristiina Vuori, M.D.,Ph.D., at The Burnham Institute of Medical Research, La Jolla, and colleagues developed a three-dimensional (3-D) culture system that captures the metabolic stresses seen in living tissue better than the single-layer cell dishes currently used to study tumor growth. After investigating a number of cell lines, Dr. Vuori’s team found that the human breast cell line T47D worked best in this 3-D model, and they are now using this cell line in their model to explore how breast cancer cells respond to radiation and chemotherapy and whether cell death is more likely to occur in nutrient- and oxygen-stressed cells.

Evaluating the Role of the RIN1 Gene in Breast Cancer
Ras proteins help regulate the pathways that control cell growth, differentiation, and cell death. These proteins alternate between inactive (GDP-bound) and active (GTP-bound) states. In about 30 percent of tumors RAS becomes “activated.” Marc Milstein, B.S., at the University of California, Los Angeles, and colleagues are studying what occurs downstream in the RAS pathway. Their lab previously identified an unknown breast tumor suppressor protein, called RIN1. This protein is a Ras “effector” that regulates epithelial cell functions. The team has determined that RIN1 expression is frequently blocked in breast cancer cell lines and human breast tumors, and they have characterized two mechanisms that silence RIN1. The team also found that restoration of RIN1 inhibits the growth of tumor cells in cell culture and in mice. In addition, they have shown that the RIN1 gene is tightly clustered with two other tumor suppressor genes, BRMS1 and B3GNT1, and that the three genes display coordinated silencing in multiple breast tumor cell lines and a tissue sample. Additional studies showed that treatment with the protein TGFß caused a reduction in B3GNT1, BRMS1 and RIN1 expression in normal mouse epithelial cells and tumor cells, and that B3GNT1, RIN1, and BRMS1 each independently acted as negative regulators of cell migration. The discovery of this tumor suppressor gene cluster could lead to the development of new breast cancer treatments.

Oxidative Stress and Estrogen Receptor Structural Changes
There is extensive evidence showing that the estrogen receptor alpha (ER, alpha isoform) plays a critical role in driving both the initiation and promotion of most human breast cancers. Oxidative stress induces aging and age-related diseases, and there is biological and clinical evidence to suggest that oxidative stress changes ER structure and function in ways that could help promote the development of breast cancer. Bradford Gibson, Ph.D., and Christopher Benz, M.D., at the Buck Institute for Age Research, Novato, used an analytical technique called mass spectrometry to monitor the effect of oxidative stress chemical changes on the ER structure. Their research identified several structural changes in ER that had previously been suspected but had never before been detected. They also showed that two of these newly detected structural changes in ER could be translated into a potential new method of diagnosing ER-positive breast cancers. This work has the potential to advance our understanding of how ER-positive breast cancer develops and to reveal environmental exposures that contribute to the development and progression of the disease. Findings from this research were published in American Association for Cancer Research 45(2005)a560, Drug Metabolism Reviews 38(2006)601, and Analytical Chemistry 79(2007)3083.

Profiling Enzyme Activities in Human Breast Cancer
Developing new ways to diagnose and treat breast cancer relies heavily on the discovery of new protein biomarkers and therapeutic targets. To streamline analyses of human breast tumors and cells, Benjamin Cravatt, Ph.D., and Stefanie Jeffrey, M.D., at the Scripps Research Institute, La Jolla, combined a chemical methodology called activity-based protein profiling (ABPP), which was developed in their laboratory to identify enzyme activities, with a multidimensional protein identification technology (MudPIT). Using this new methodology, Drs. Cravatt and Jeffrey and their team identified more than 50 enzyme activities in human breast tumors, nearly a third of which represented previously uncharacterized proteins. They also embarked on a project to disrupt the function of these enzymes in breast cancer models. These studies led to the discovery that the enzyme KIAA1363 regulates an ether lipid signaling network in human breast and ovarian cancer cells. Using a mouse model, Drs. Cravatt and Jeffrey showed that disruption of the KIAA1363-ether lipid network reduced breast and ovarian tumor growth, suggesting that this enzyme could be a therapeutic target. This is one example of how this new technology could advance our ability to diagnose and treat breast cancer. Findings from this research were published in Journal American Chemical Society 126(2004)15640, Proceedings of the National Academy of Sciences 101(2004)13756, Nature Method 2(2005)691, and Chemical Biology
13(2006)1041.

Defining Mutagenesis Pathways in Breast Cancer Evolution
Breast cancer is a genetic disease that begins with the mutation of DNA. It has long been believed that these mutations occur due to failure of the DNA replication and repair systems. More recently, researchers have come to believe that the cell itself must also influence the proteins that help induce mutations. Ewa Lis, B.A., at the Scripps Research Institute, La Jolla, and colleagues studied mutation processes in yeast, which is an excellent model organism for studying cell cycle and DNA repair pathways, and then translating these finding to human breast cancer. The team screened 4,847 yeast gene deletion strains to identify 10 genes involved in the mutation of a gene called CAN1. The team was able to identify new pathways that appear to play a role in inducing genetic mutations. They also showed that they could use a small molecule to inhibit a specific genetic mutation. This suggests that if imilar pathways exist in human cells, intervention in some forms of mutation may be possible.

Reactivation of the Inactive X Chromosome and Breast Cancer
In females, one of the two X chromosomes is inactivated to equalize X-linked gene dosage with XY males. Specific types of human breast cancer, including basal-like cancer, have acquired X chromosomal abnormalities such as the loss of the inactive X (Xi) and/or a gain of active X (Xa) chromosomes. These abnormalities are associated with an increased expression of at least 30 Xlinked genes, including some that have previously been shown to be involved in breast cancer. These observations suggest that deregulation of X inactivation may play a role in breast cancer. Angela Andersen, Ph.D., at the University of California, San Francisco, and colleagues analyzed X inactivation in different mouse models of breast cancer. Xist is an X-linked gene expressed exclusively from the Xi; this non-coding RNA coats the Xi and plays a role in maintaining the silent state. Loss of Xist RNA enrichment from the Xi correlates with human basal-like cancer. Dr. Anderson and her team found that most cells from each mouse model had Xist RNA coating a single Xi, and that genes normally subject to X inactivation were expressed exclusively from the single Xa. This work could lead to new screening techniques and new treatment strategies that utilize assays for the presence of multiple Xa chromosomes.

Structural Analysis of Cancer-Relevant BRCA2 Mutations
Inherited mutations in BRCA1 and BRCA2 are responsible for about 5-10 percent of all breast cancer cases and about one-half of all familial cases of breast and ovarian cancer. While evidence for a role of BRCA2 in the recombinational repair of DNA damage is mounting, the precise molecular functions of this protein and its biochemical properties remain unknown. Henning Stahlberg, Ph.D., at the University of California, Davis, and colleagues developed a three-dimensional structure to test the hypothesis that a subgroup of mutations results in a folded BRCA2 protein that has a reduced ability to bind to Rad51, the central protein in recombinational repair. This, in turn, could elevate cancer risk. Dr. Stahlberg has successfully developed a new sample preparation method that will enable his team to work with the very fragile BRCA2 protein. This new preparation method has the potential to advance other breast cancer protein research projects. It also could lead to the identification of mutant BRCA2 proteins and, in turn, new drug treatments.

Grants in Progress: 2007

Analysis of MicroRNA Expression in Breast Cancer Stem Cells
Yohei Shimono
Stanford University

Axon Guidance Proteins in Mammary Gland Development
Lindsay Hinck
University of California, Santa Cruz

Breast Cancer Studies in a 3-D Cell Culture System
Robert Abraham
The Burnham Institute of Medical Research

A Candidate Marker of Mammary Tumor Initiating Cells
Alexey Terskikh
The Burnham Institute of Medical Research

Defining Mammary Cancer Origins in a Mouse Model of DCIS
Alexander Borowski
University of California, Davis

Functional Analysis of BORIS, A Novel DNA-binding Protein
Paul Yaswen
Lawrence Berkeley National Laboratory

Identification of Metastasis Competent Breast Cancer Cells
Barbara Mueller
La Jolla Institute for Molecular Medicine

Identifying Metastatic Breast Cells from Peripheral Blood
Kristin Kulp
Lawrence Livermore National Laboratory

Imaging RhoC-induced Breast Cancer Invasion and Angiogenesis
Konstantin Stoletov
University of California, San Diego

Inflammation Alters Transcription by ER in Breast Cancer
Eliot Bourk
University of California, San Diego

Modeling, Targeting Acetyl-CoA Metabolism in Breast Cancer
Chen Yang
The Burnham Institute of Medical Research

MYC and CSN5 in the Breast Cancer "Wound Signature" Profile
Adam Adler
Stanford University

A New Marker for Mammary Epithelial Stem Cells?
Robert Oshima
The Burnham Institute of Medical Research

Normal Mammary Biology of Phosphorylated Prolactin
Ameae Walker
University of California, Riverside

Novel Approach to Analyze Estrogen Action in Breast Cancer
Brian Elicieri
La Jolla Institute for Molecular Medicine

A Novel Epithelial-Stromal Model of Metastatic Breast Cancer
Richard Neve
Lawrence Berkeley National Laboratory

Profiling Drug Metabolism (P450) Proteins in Breast Cancer
Aaron Wright
Scripps Research Institute

Reactivation of the Inactive X Chromosome and Breast Cancer
Angela Anderson
University of California, San Francisco

Regulation of Mammary Epithelial Invasion by MMPs and FGFs
Andrew Ewald
University of California, San Francisco

Role of Cell Division Asymmetry in Breast Cancer Stem Cells
Claudia Petritsch
University of California, San Francisco

The Role Chk1 in Breast Cancer DNA Damage Repair
Jennifer Scorah
Scripps Research Institute

The Role of the ECM in Breast Cancer DNA Damage Repair
Albert Davalos
Lawrence Berkeley National Laboratory

The Role of Estrogen-Related Receptors in Breast Cancer
Anastasia Kralli
Scripps Research Institute

Role of Integrins in Lymphangiogenesis During Breast Cancer
Barbara Susini
University of California, San Diego

The Role of LMO4 in Breast Cancer
Zhengquan Yu
University of California, Irvine

The Role of Podosomes in Breast Cancer Metastasis
Barbara Blouw
The Burnham Institute of Medical Research

The Role of Serine and Metallo-Hydrolase's in Breast Cancer
Sherry Niessen
Scripps Research Institute

Stem Cells in Breast Cancer Metastasis
Brunhilde Felding-Habermann, John Yates & Evan Snyder
Scripps Research Institute and The Burnham Institute of Medical Research

Stem Cells of Molecularly Diverse ER Negative Breast Cancers
Stefanie Jeffrey
Stanford University

Structural Analysis of Cancer-Relevant BCRA2 Mutations
Henning Stahlberg
University of California, Davis

Twist Activation in Breast Cancer Metastasis
Jing Yang
University of California, San Diego

Research Initiated in 2007

Breast Tumor Responses to Novel TGF-beta Inhibitors
Kelly Harradine
University of California, San Francisco

Competition for ADA2 and 3 to Inhibit p53 in Breast Cancer
Min Yang
University of California, Irvine

Cytoskeletal Regulation of Invading Breast Cells
Catherine Jacobson
University of California, San Francisco

Determination of Stromal Gene Expression in Breast Cancer
Robert West
Palo Alto Institute for Research & Education

Indole (I3C) Control of Breast Cancer by ER Downregulation
Crystal Marconett
University of California, Berkeley

Lipid Raft Composition in Deregulated ERBB2 Signaling
Ralf Landgraf
University of California, Los Angeles

Mechanisms of Daxx-Mediated Apoptosis in Breast Cancer
Lorena Puto
The Burnham Institute of Medical Research

A New Mouse Model of PI3-Kinase Induced Breast Cancer
Jun Zhang, Ph.D.
University of California, San Francisco

Novel Regulation of the Rb Pathway in Breast Epithelium
Deborah Burkhart
Stanford University

The Relationship of BRCA1 and HMGA2 in Breast Cancer
Connie Tsai
University of California, Irvine

Targeting Tissue Factor in Breast Cancer
Florence Schaffner
Scripps Research Institute

Telomerase, Mammary Stem Cells, and Breast Cancer
Steven Artandi
Stanford University

Trask, a Candidate Breast Cancer Metastasis Protein
Ching Hang Wong
University of California, San Francisco