Executive Summary

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 coordinate migration, proliferation, and apoptosis (cell death) over space and time. In cancer progression these processes become deregulated, initially at the genetic level that leads to the physiological changes associated with malignancy. An inability to recognize and properly repair damage to DNA that occurs in normal cell physiology and enhanced by environmental factors is recognized as driving force of cancer progression. An emerging paradigm identifies progenitor 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 of breast cells in response to hormones and pregnancy. If this paradigm proves correct, then only a small fraction (1-2%) of cells in a tumor mass retain stem/progenitor cell properties, and these “cancer stem cells” must be selectively targeted to achieve an effective eradication of the disease. Important basic science topics represented in CBCRP’s portfolio include: exploring the role of stem cells in normal and tumor breast; cell proliferation control mechanisms through the estrogen receptor and growth factor receptors (e.g., Her-2); alterations in DNA repair processes that permit genetic damage to accumulate in cancer cells; cell cycle changes that permit division under conditions where normal cells would undergo programmed cell death (apoptosis); novel biomarkers to distinguish pre-cancerous and cancerous cells from normal breast epithelium and their validation as potential new detection and therapy targets; and developing methods for accounting for the complexity of the interplay of all of these factors in breast cancer.

Two of the CBCRP’s research areas are presented in this section.

Research Concluded in 2009

Regulation of Mammary Epithelial Invasion by MMPs and FGFs
The mature mammary gland does not develop until the end of puberty, and its structure changes extensively during the hormonal cycles that accompany pregnancy, lactation, and involution. Andrew Ewald, Ph.D., at the University of California, San Francisco, and colleagues studied the underlying cellular mechanisms of normal mammary tissue invasion in animal cells in order to learn how the epithelial cells turn into invasive cancer cells. Using advanced electron microscopy techniques, they identified a new mechanism of cellular growth and invasion in breast tissue. They also showed that this process was markedly similar to that seen in human cancer cells. Dr. Ewald used this study to establish his own laboratory at Johns Hopkins Medical School, where he now using these techniques to study how the stromal cells in connective tissue and the proteins in the extracellular matrix (the environment that surrounds a cell) regulate breast tumor invasion. This work has the potential to lead to new breast cancer therapies. Findings from this research were published in: Developmental Biology 306(2007)193; Nature Reviews, Molecular Cellular Biology 8(2007)221; Molecular Biology of the Cell 18(2007)1693; Cancer Cell 13(2008)141; Developmental Cell 14(2008)570; Current Biology 18(2008)507; Developmental Biology 321(2008)77; Disease Models and Mechanisms 1(2008)155.

Cytoskeletal Regulation of Invading Breast Cells
In many early stage breast cancers, the cells that are in the acini, the milk producing glands of the breast, not only look unusual but have started to spread into the surrounding tissue. These changes are precursors to metastatic breast cancer. Catherine Jacobson, Ph.D., at the University of California, San Francisco, used a mouse mammary epithelial cell line called EpH4 to investigate and identify precisely how breast cells begin to migrate away from the acini and invade the surrounding tissue. This work will contribute to our understanding of the how cancer cells metastasize.

Telomerase, Mammary Stem Cells, and Breast Cancer
Telomeres are the special caps that protect each end of the four arms of a chromosome. The telomeres get shorter each time the cell divides. When they get too short to do their work, they send the cell a message telling it to stop dividing. Telomerase is an enzyme that can add more DNA to the telomeres. In cancer cells, telomerase keeps the telomeres from becoming shorter, enabling these cells to reproduce endlessly. Previous work by Steven Artandi, M.D., Ph.D., at Stanford University, in Palo Alto, and colleagues suggested that telomerase might also play a role in breast cancer development by stimulating tissue stem cells. Their new studies found that telomerase is a cofactor in the Wnt pathway, which is one of the most important circuits in cancers and stem cells. It was already known that Wnt signaling was important in both breast development and breast cancer, but these findings provide the first evidence that Wnt and telomerase are intimately linked. These studies could lead to the development of new breast cancer treatments that work by inhibiting telomerase. Findings from this research were published in Nature 240(2009)66.

Competition for ADA2 and 3 to Inhibit p53 in Breast Cancer
Breast cancers are characterized by the abnormal behavior of proteins, called transcription factors, which determine which genes are used in a particular cell. A tumor suppressor gene called p53 is a transcription factor that is inactivated in about one-fourth of breast cancer cases. However, it’s not clear how p53 is inactivated in breast cancers that do not have a p53 genetic mutation. To investigate this question, Min Yang, M.D, M.S., at the University of California, Irvine, and colleagues used molecular biology techniques to study interactions that occur between the proteins beta-catenin and p53 in breast cancer cell lines. Their studies showed that two other proteins, called ADA2a and ADA3, are required for these proteins to be fully active. This information could lead to new breast cancer treatments that simultaneously target different gene regulation pathways. Findings from this research were published in Cancer Biology and Therapy 7(2008)120.

Targeting Tissue Factor in Breast Cancer
Tissue factor (TF) works along with factor VII to initiate the blood clotting that is necessary to prevent excessive bleeding and initiate wound healing. TF is also expressed on tumor cells, and studies have found that it is associated with more aggressive cancers. Florence Shaffner, Ph.D., at the Scripps Research Institute, in La Jolla, used two different antibodies that block different components of TF activity in mouse models to learn more about its different biological functions. Dr. Shaffner and her team showed that blocking TF signaling reduced tumor growth and spontaneous metastasis and resulted in tumors with fewer blood vessels. This suggests that TF signaling plays an important role in breast cancer development by regulating how a tumor develops blood vessels and gains the ability to metastasize. Dr. Shaffner and her team are continuing to study TF signaling, and are now trying to determine if it influences other breast cancer pathways. These studies could lead to the development of new breast cancer therapies that target and block TF signaling. Findings from these studies were published in Arteriosclerosis, Thrombosis, and Vascular Biology Aug. 6, 2009 Epub and Cancer Research 68(2009)7219.

Breast Tumor Responses to Novel TGF-beta Inhibitors
There is strong evidence that increased TGF-β signaling can contribute to tumor progression. Furthermore, anti-TGF-ß therapy has been shown to reduce both the size and aggressiveness of breast tumors. However, there is concern that anti-TGF-β therapy may have adverse effects in some patient populations. Kelly Harradine, Ph.D., at the University of California, San Francisco, and colleagues investigated how different types of breast tumors respond to TGF-β inhibition. Her team’s preliminary findings suggest that breast tumor subtypes have different TGF-β dependence, which, in turn, causes differences in response to anti-TGF-β therapy. These findings could help investigators predict which patients are most likely to benefit from anti-TGFß therapy, and spare patients whose tumors will not respond well to treatment.

Trask, a Candidate Breast Cancer Metastasis Protein
Trask is a protein that is active in normal cells only when the cell is dividing. However, in cancer cells, Trask is active all of the time. This suggests that Trask may play a role in tumor metastasis. Ching Hang Wong, Ph.D., at the University of California, San Francisco, and colleagues previously showed that when the amount of Trask that is present in breast cancer cells increases, the cells detach and separate. This is similar to what happens in metastasis. Dr. Wong and his team are developing breast cancer cell lines that can be used to study the role Trask plays in cancer progression. They are also using a cell culture model to study the interaction between Trask and beta-catenin, a protein that regulates cell-cell adhesion. This work could lead to the development of new breast cancer treatments that target Trask. Findings from these studies appeared in Clinical Cancer Research15(2009) 2311.

Determination of Stromal Gene Expression in Breast Cancer
Cancer cells are surrounded by a complex mixture of blood vessels, inflammatory cells, and different types of connective tissue (stromal) cells. These stromal cells are not cancerous, but they have been shown to play a crucial role in cancer development and progression. Robert West, M.D., Ph.D., at the Palo Alto Institute for Research & Education, and colleagues are investigating whether it is possible to develop cancer therapies that target these stromal cells by studying low-grade soft tissue tumors. Dr. West received two additional years of CBCRP support to continue this project, which will involve developing clinically useful biomarkers of stromal expression patterns in invasive cancer and identifying stromal response patterns associated with pre-invasive breast cancer. This work could lead to new ways of treating breast cancer. Findings from this research appeared in Laboratory Investigations 88(2008)591 and Clinical Cancer Research 15(2009)778.

Profiling Drug Metabolism (P450) Proteins in Breast Cancer
The cytochrome P450 family plays a role in normal breast cell regulation. One P450 enzymes, called aromatase, is necessary for the body to make estrogen, and is the target of a class of anti-estrogen breast cancer drugs called aromatase inhibitors. Aaron Wright, Ph.D., at the Scripps Research Institute in La Jolla, and colleagues developed a new chemical probe that can analyze human P450 activity. They then used it to evaluate how two anti-estrogen breast cancer therapies impact P450 activity. These probes provide a new way to analyze the effect of new breast cancer drugs on P450 activities, and could lead to the development of new breast cancer treatments that inhibit and regulate the P450 enzyme. Findings from this research were published in Chemistry and Biology 14(2007)1043 and the Annual Review of Biochemistry 22(2008)383.

The Role of Chk1 in Breast Cancer DNA Damage Repair
Cells duplicate their DNA during every cell cycle. Chk1 and Claspin are two genes that work at the DNA damage checkpoints that operate during cell division. Their job is to prevent errors from being passed on when the cell divides. Jennifer Scorah, Ph.D., at the Scripps Research Institute, in La Jolla, and colleagues used DNA fiber technology, which can analyze DNA replication at the level of individual molecules, rather than the whole genome, to learn more about these two genes. Their findings provided the first detailed analysis of these genes’ replication functions and suggested that although Claspin is required to activate Chk1 at the cell cycle checkpoint, its role in replication is actually independent of Chk1. This work could lead to the development of new breast cancer drugs that target Chk1 or Claspin. Findings from this research were published in the Journal of Biological Chemistry 283(2008)17250.

Inflammation Alters Transcription by ER in Breast Cancer
Estrogen acts through the estrogen receptor (ER), a powerful regulator of cell behavior that can switch specific genes on or off. Most research on ER function has focused on its ability to activate genes. Eliot Bourk, B.A., at the University of California, San Diego, and colleagues used gene expression profiling experiments and genome wide location analyses to investigate which genes are shut off by ER in response to estrogen, and how some of these genes are then reactivated by inflammation. Their work showed that in the presence of inflammation, repression of certain genes by estrogen could be reversed in ER-alpha expressing cells but not in ER-beta expressing cells. This was a previously unknown difference between these two estrogen receptors. Mr. Bourk intends to continue to conduct studies on the impact that inflammation has on genes shut off by ER. These findings could advance our understanding of the estrogen receptor and its effects on gene expression.

A New Mouse Model of PI3-Kinase Induced Breast Cancer
Some breast cancers are caused by genetic mutation. One gene, called PIK3CA, is mutated in about 30 percent of breast cancer patients. Jun Zhang, Ph.D., M.D., at the University of California, San Francisco, and colleagues are trying to develop a mouse model with a PIK3CA mutation that could be conditionally activated in animal tissue to model breast cancer in humans. This research may lead to the development of a mouse model that could be used to develop new breast cancer therapies that target PIK3CA.

Lipid Raft Composition in Deregulated ERBB2 Signaling
About 25 to 30 percent of all breast cancer cells have extra ERBB2/HER2 receptors. The ways in which these receptors interact with the cells in the microenvironment that surround them are not well known. Ralf Landgraf, Ph.D., at the University of California, Los Angeles, and colleagues investigated whether changes that occur in the microenvironment that surrounds the ERBB2/HER2 receptor might help to explain why some HER2-positive cancers stop responding to the drug Herceptin. Their studies indicated that both HER2/ERBB2 and ERBB3 localize preferentially to certain lipid rafts in the cell’s membrane. (Lipid rafts are an area of the cell membrane that creates a favorable environment for saturated fatty acids and other proteins.) Dr. Landgraf and his team are continuing to investigate whether changes in the ratio of saturated and unsaturated fatty acids affects these rafts in ways that, in turn, impacts ERBB2 recruitment. This work could lead to new treatments for HER2-positive tumors.

MicroRNA Expression in Breast Cancer Stem Cells
Current evidence suggests that breast cancer stem cells are more resistant to standard therapies than other breast cancer cells. MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression and control a variety of cell functions, including cell proliferation and stem cell maintenance. Abnormal expression of certain miRNAs in human cancers is associated with cancer progression and a patient’s prognosis. Yohei Shimono, M.D., Ph.D., at Stanford University, in Palo Alto, and colleagues investigated whether miRNAs are important regulators of breast cancer stem cells. They identified 37 miRNAs that were expressed at different levels in human breast cancer stem cells and normal breast stem cells. They found that one of the down-regulated miRNAs, called miR-200c, controlled expression of BMI1, a known regulator of stem cell self-renewal. It also suppressed the ability of human breast cancer stem cells to form tumors in vivo and the ability of normal breast stem cells to form breast ducts. These findings provide evidence that cancer stem cells and normal stem cells share molecular mechanisms that regulate cell growth, and may help explain, in part, how cancer stem cells encourage breast cancer growth. These findings could lead to new breast cancer treatments that target breast cancer stem cells. Findings from this research were published in the Annual Review of Cell and Developmental Biology 23(2007)675 and Cell 138(2009)592.

The Relationship of BRCA1 and HMGA2 in Breast Cancer
Mutations in a gene called BRCA1 account for 50 percent of all hereditary breast cancer cases. BRCA1 is known to play an essential role in maintaining genomic integrity by repairing damaged DNA and monitoring cell growth. However, it’s not precisely clear why a BRCA1 mutation increases breast cancer risk. Connie Tsai, B.S., at the University of California, Irvine, and colleagues used an array of laboratory techniques to study the relationship between BRCA1 and a gene called HMGA2 that promotes cell proliferation. Their findings suggest that the BRCAl/CtIP/ZBRK1 repressor complex mediates HMGA2. However, they were not able to establish a specific relationship between BRCA1 and HMGA2. This led them to conclude that HMGA2 is regulated by ZBRK1, independent of BRCA1. These findings add to our understanding of how BRCA1 genetic mutations increase breast cancer risk.

Research Initiated in 2009

Breast Cancer Tumor-Stroma Interactions in an In Vivo Model
Per Borgstrom
Vaccine Research Institute of San Diego

A Molecular Strategy to Inhibit Breast Cancer Metastasis
Frances Brodsky
University of California, San Francisco

Podocalyxin as a Basal-like Breast Cancer Stem Cell Marker
Graham Casey
University of Southern California

The Role of Estrogen Receptor in Endocrine Resistance
Hei Chan
Beckman Research Institute of the City of Hope

Understanding the Role of GATA3 in Breast Cancer
Jonathan Chou
University of California, San Francisco

Finding BRCA1 Ubiquitinated Substrates in Breast Cancer
Sonia del Rincon
The Burnham Institute for Medical Research

Substrate Profiling of Breast Cancer Related Proteases
Melissa Dix
Scripps Research Institute

A Genetic System for Identification of Mammary Stem Cells
Dannielle Engle
Salk Institute for Biological Studies

The Regulation of SATB1 in Metastatic Breast Cancer
Laurie Friesenhahn
Lawrence Berkeley National Laboratory

Novel Tumor Suppressors in Breast Development and Cancer
Margaret Fuller
Stanford University

Targeting MYC in Human Breast Cancer
Dai Horiuchi
University of California, San Francisco

Role of Circadian Rhythm Gene Homolog PER3 in Breast Cancer
Kuang-Yu Jen
University of California, San Francisco

Control of BRCA2-mediated Homologous Recombination
Damon Meyer
University of California, Davis

Discovery of Fusion Genes in Breast Cancer
Jonathan Pollack
Stanford University

Proline Metabolism in Metastatic Breast Cancer
Adam Richardson
The Burnham Institute for Medical Research

P32: New Functional Target in Breast Cancer Brain Metastasis
Karin Staflin
Scripps Research Institute

Role of p68 in Breast Cancer
Daojing Wang
Lawrence Berkeley National Laboratory

Novel Akt Regulatory Factor PHLPP in Breast Cancer
Noel Warfel
University of California, San Diego

Stroma Expression Patterns in Breast Cancer
Robert West
Palo Alto Institute for Research & Education

The Role of EGF Variant mLEEK and Grp78 in Breast Cancer
Albert Wong
Stanford University

Research in Progress

Breast Cancer Studies in a 3-D Cell Culture System
Robert Abraham
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

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

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

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

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

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

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

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

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

Chemokine Receptor Signaling in Breast Cancer
Morgan O’Hayre
University of California, San Diego

Dietary Metabolite Inhibition of Breast Cancer Cell Survival
Holly Hantz
University of California, Berkeley

Dissecting the Role of Twist in Breast Cancer Metastasis
Janine Low-Marchelli
University of California, San Diego

Global Analysis of Protein Ubiquitination in Breast Cancer
Stefan Grotegut
Sidney Kimmel Cancer Center

Maternal Embryonic Leucine Zipper Kinase in Mammary Tumors
Robert Oshima
The Burnham Institute for Medical Research

Nanolipoproteins to Study Breast Cancer Growth Receptors
Paul Henderson
University of California, Davis

Regulation of Breast Stem-Progenitor Cell Chromatin by Pygo2
Bingnan Gu
University of California, Irvine

Role of Estrogen-modulated Protein AGR2 in Breast Cancer
Mikhail Geyfman
University of California, Irvine

Tumor Suppressor 14-3-3sigma in Breast Cancer Progression
Aaron Boudreau
Lawrence Berkeley National Laboratory