Biology of the Breast Cell: The Basic Science of the Disease

Overview: To understand the origin of breast cancers, more research is needed on the pre-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.

The recent achievement of generating induced pluripotent stem cells (iPSCs) derived from differentiated adult cells by modulating four pivotal genes suggests that stem cell transformations related to cancer may involve a very limited number of key initiating events. Importantly, two recent publications supported by CBCRP funding have shed new light on the relationship of stem cells and breast cancer. First, Dr. Steven Artandi at Stanford University showed that telomerase affects a key Wnt/catenin signaling pathway in stem cell biology and differentiation that may explain a key process in tumor progression. Next, a major new research interest involves the discovery and study of microRNAs which regulate gene expression. CBCRP-funded postdoctoral researcher Dr. Yohei Shimano and his mentor, Dr. Michael Clarke and other colleagues, also from Stanford University, reported that certain microRNAs regulating a key self-renewal factor become decreased both in normal mammary epithelial stem cells and breast tumor-initiating cells. These findings suggest new ways of targeting cancer stem cells.

The CBCRP funded 20 new grants in 2009 to advance research knowledge in our Biology of the Breast Cell priority issue. Two of the CBCRP’s research topics are presented in this section.

Biology of the Breast Cell Portfolio
Two newly funded grants explore aspects of normal breast biology relevant to breast cancer.  Of course, knowing how cells function in their normal tissues and organs will prove crucial in explaining what goes wrong in cancer. In this respect, stem cells have gained increasing research attention. An entire functional mouse mammary gland can be regenerated using a single mammary stem cell. In fact, recently an entire mouse was generated from using iPSCs derived from the skin of adult animals. Despite these remarkable feats, it is still very difficult to isolate normal mammary or breast cancer stem cells for the mechanistic laboratory studies central to basic science. Dannielle Engle at the Salk Institute for Biological Studies is funded for a dissertation award to utilize special Wnt signaling reporter mice to define pathways during mammary gland development, which should provide a platform to characterize and isolate mammary stem cells. Under the direction of her mentor, Dr. Jeffrey Wahl, and working with other colleagues, Ms. Engle hopes to improve on the current level of mammary stem cell enrichment of only 1 in every 60 cells capable of re-populating a mouse mammary gland. Further, successful completion of this project will serve to identify the key pathways (i.e., definitive molecular markers) that regulate mammary stem cell self-renewal and reveal the actual location of the stem cells within the mammary gland.

Key biological mechanisms are often conserved in evolution, so there is great potential for extrapolating discoveries from lower organisms to humans. Thus, changes in the molecular switches that regulate stem cell proliferation versus differentiation may underlie the pre-neoplastic changes seen in cancer. Margaret Fuller from Stanford University is an accomplished Drosophila (fruit fly) researcher who will use RNA-interference strategies to test whether mammalian genetic counterparts of specific fruit fly tumor suppressorsare required for the switch from proliferation to differentiation in mammary gland stem cell lineages.

Six newly funded grants focus on processes related to breast cancer metastasis.

Cancer cells are surrounded by a complex mixture of blood vessels, inflammatory cells, and different types of connective tissue cells. This supporting stroma is not cancerous, but has been shown to play a crucial role in cancer development and progression. For example, as a person ages, the stromal component of the breast deteriorates and becomes more permissive for altered breast epithelial cells to progress into invasive cancers. In addition, it has been hypothesized that these stromal elements adjacent to tumors contain proteins and cellular genetic changes that could as biomarkers to predict cancer progression, classify tumors, and assess therapy outcome. Robert West at the Palo Alto Institute for Research and Education used previous CBCRP IDEA funding to develop a novel approach to discover unique types of “stromal reaction patterns” through gene expression profiling of soft tissue tumors (STT). In this approach, STTs are used as a discovery tool to classify various types of breast cancer stromal reaction patterns to the presence of tumors. Dr. West received two additional years of funding to screen 300 DCIS cases for validation that stromal reaction patterns predict disease progression. Thus, it may be possible to modulate stromal components to reduce the progression and invasion potential, especially for early stage cancers and DCIS.

During the metastatic spread of breast cancer, tumor cells must invade and grow in new organ sites that are receptive to metastatic spread. This concept was first put forth in Paget's 1889 proposal that metastasis depends on molecular cross-talk between selected cancer cells (the “seeds”) and specific organ microenvironments (the “soil”), which still holds forth today (refined most recently by Dr. Isaiah Fidler). Per Borgstrom from the Vaccine Research Institute of San Diego will use a combination of intravital video-microscopy (IVM) and gene profiling to better identify the stromal requirements for selective tumor growth in various organ sites. In these studies, Dr. Borgstrom and collaborators will co-implant mouse mammary tumor cells with different stroma, including mammary fat pad, liver, lung, and skin to study primary and metastatic breast tumor growth. Using IVM will allow continuous monitoring of tumor growth and the removal of tumor/stromal tissues at various points of disease progression to analyze the underlying genetic changes in “seed and soil.”

Brain metastases are the most feared complication in breast cancer, and occur in nearly 30% of patients. However, there are few animal models to allow researchers to study this process, so our basic science mechanistic knowledge for brain metastasis is minimal. Karin Staflin from the Scripps Research Institute will study the role of a unique protein, called p32, using unique human breast cancer cell lines that were established from patients with brain lesions. P32 is a multi-functional protein shown to localize to hypoxic/nutrient deprived areas within tumors. It is involved in the regulation of apoptosis (programmed cell death) and autophagy (degradation of a cell's own components), which are key stress responses in tumor cells. In this mouse model system, Dr. Staflin will be able to track brain metastasis by external bioluminescent imaging.

Frances Brodsky from the University of California, San Francisco is funded for an IDEA grant to study the role of a possible metastasis protein, called Hip-1 (Huntingtin Interacting Protein-1). Dr. Brodsy’s lab will validate the relationship of Hip-1 in breast cancer with studies on a panel of cell lines, use animal models to study Hip-1’s influence in forming tumors and metastasis, and screen a chemical library to isolate compounds interfering with the Hip-1/clathrin interaction that might prove useful for therapeutic investigation.

Serine proteases have been shown to be important regulators of breast cancer growth and metastasis and represent about 0.6% of the genes in the human genome. They catalyze one of the most pervasive post-translational regulatory processes in biology; cleavage of other proteins resulting in either activation or inactivation. Despite their fundamental importance, a complete endogenous substrate profile for most human proteases is lacking. An increase in the activity of certain proteases has been strongly linked to tumor spread. Melissa Dix from the Scripps Research Institute will focus her dissertation research on the urokinase-type
plasminogen activator (uPA), a protease which has proven to be a useful marker for the clinical diagnosis of breast cancer. Using a novel proteomics technology, called PROTOMAP (PROtein Topography and Migration Analysis Platform), developed in her mentor’s (Dr.Benjamin Cravatt) lab, she will search for novel biomarkers which could then be integrated into a screening platform that would allow early detection of breast cancer in terms of disease progression and metastasis.

It is well documented that breast cancer cells have altered metabolic programs, but the exact role of these metabolic shifts associated with tumor invasion and metastasis remains undefined. Recently it has been reported that the enzymes whose function is to oxidize and degrade proline (a cyclic structure, non-essential amino acid) are pro-apoptotic and can be activated by the tumor suppressor protein, p53. Adam Richardson from The Burnham Institute for Medical Research will create breast tumor lines with altered endogenous proline biosynthesis pathways to study this underlying proline connection. Dr. Richardson will confirm the alteration of key metabolic pathways, and then test the ability of increased proline biosynthesis to suppress apoptosis in both adherent and non-adherent cell settings. It is thought that metastatic cells are more resistant to the programmed cell death caused by increased cellular proline.

Four newly funded grants study various aspects of cell growth and growth factor signaling.

Most patients treated with selective endocrine receptor modulators (SERMs) (e.g., tamoxifen) or aromatase inhibitors (AI) will eventually develop resistance, but the underlying mechanisms are unclear. For his dissertation project, Hei Chan at the Beckman Research Institute of the City of Hope will utilize special tamoxifen and AI-resistant cell lines cell lines developed in the lab of his mentor, Dr. Shiuan Chen. Mr. Chan will survey estrogen receptor binding to DNA in these cell lines to detect candidate transcription factors across the entire human genome. Together, they will correlate ER binding sites with bioinformatics analysis using the consensus sequences of other DNA binding proteins along with microarray (gene profiling) data. A comparison of the various resistant cell lines will reveal the differences in estrogen receptor signaling and potentially identify new pathways for therapeutic intervention for drug resistant breast cancers.

MYC is a transcription factor that regulates expression of nearly one-third of the genes in the human genome. Abnormal MYC amplification has been found in approximately 50% of human breast cancers, including those that are hormone receptor-negative. MYC amplification has also been linked to resistance to existing therapies and a decrease in breast cancer patients’ survival. However, no targeted therapy currently exists to treat these difficult-to-treat breast cancers having high MYC levels. Dai Horiuchi from the University of California, San Francisco previously discovered that breast and other cell lines engineered to over-express MYC could be effectively killed by an inhibitor against a protein, called Cyclin-dependent kinase 1 (CDK1), a central regulator of cell division. Dr. Horiuchi, working in his mentor’s lab (Dr. Andrei Goga), will test a large collection of breast cell lines established from primary human breast tumors, instead of using man-made engineered cells. In follow-up experiments they plan to study associated signaling mechanisms and apoptosis, in order to develop a strategy to target interactions between MYC and CDK1.

Signaling pathways within a cell control the balance between death and survival. When these pathways become deregulated, cells can begin to divide and grow uncontrollably, leading to cancer. One of the most important and prevalent signaling pathways activated in breast cancer is the Akt pathway. Akt drives the formation of many types of cancer, including breast cancer unless its activity is tightly regulated. Noel Warfel at the University of California, San Diego will focus his dissertation research on the role of a novel family of phosphatases, called PHLPP (PH domain and Leucine-rich repeat Protein Phosphatases), which serve as a “brake” on Akt signaling. PHLPP phosphatases were recently discovered in 2005 in the lab of Dr. Alexandra Newton, Mr. Warfel’s mentor. The goal is to study the Akt regulatory loop that becomes defective in breast cancer.

Breast cancer cells are highly adapted to resist stress that result from a lack of nutrients, a reduced blood supply (hypoxia), or are caused by radiation and chemotherapy (DNA damage). A molecular chaperone protein, called Grp78, has been identified as a key factor helping breast cancer cells resist stress by mediating a cell pathway, called the unfolded protein response (UPR). Albert Wong at Stanford University has discovered an epidermal growth factor receptor variant that retains only the C-terminus of the receptor, called mLEEK. Dr. Wong is funded to validate the relationship between mLEEK and Grp78 using breast cancer tumor samples, experimentally alter mLEEK amounts in cell lines, and evaluate a possible mechanistic link to cell growth and apoptosis. If successful, this project could set the stage for future work to generate a monoclonal antibody as a mLEEK inhibitor.

Five newly funded grants involve studies of novel genes and DNA repair processes associated with breast cancer.

Women that inherit a mutated BRCA1 gene have a lifetime risk of 36–85% for developing breast cancer. The large, multi-functional BRCA1 protein has been most studied for its role in DNA double-strand break repair. However, another of BRCA1’s tasks is tagging proteins with a small protein called ubiquitin, which leads to protein turn-over inside cells. Interestingly, several inherited BRCA1 gene mutations prevent BRCA1 from ubiquitinating proteins, suggesting the importance of this task in protecting breast epithelial cells from becoming cancerous. To date, little is known regarding how loss of BRCA1’s ubiquitin ligase function contributes to breast cancer development, and this has primarily been due to an inability of researchers to identify the proteins tagged by BRCA1. Sonia del Rincon from The Burnham Institute for Medical Research will utilize a novel experimental method to identify proteins that become ubiquitinated by BRCA1. This involves probing protein microarrays consisting of glass slides that are spotted with more than 8,000 human protein samples. Any hits will be validated in cell systems, especially to determine whether cells having BRCA1 mutations exhibit shifts in key ubiquitination target proteins

In human cells, normal metabolic activities (oxidation) and environmental factors (UV light and radiation) result in DNA damage at an estimated level of 1 million individual events per cell per day. The BRCA2 tumor suppressor also functions in DNA repair, and the loss of its critical repair function leads to the genome instability that characterizes most cancers. Damon Meyer at the University of California, Davis will study the function of two BRCA2 accessory proteins, DSS1 and RAD54, using reconstituted test tube conditions. In this setting the purified proteins and portions of proteins are combined, and their ability to catalyze critical steps in DNA repair can be analyzed. This project is unique because so few researchers study BRCA biology using purified proteins.

A fusion gene is a hybrid formed from two previously separate genes. Fusion genes are found in hematological cancers, sarcomas and prostate cancer. These may result in the production of a novel fusion protein with cancer-causing activity. The prototypic example is the BCR-ABL gene fusion in chronic myelogenous leukemia. Importantly, this finding led to the development of the promising cancer drug, Gleevec. In 2005, a prostate cancer-specific TMPRSS2 and ETS fusion oncogene was discovered, which raised the possibility of comparable gene fusions in breast cancer. Jonathan Pollack from Stanford University is funded to apply a novel DNA microarray approach to discover fusion genes in breast cancer. The goal is to focus on estrogen-regulated and oncogenic fusion partners by profiling 50 breast cancer cell lines and 150 primary breast tumors. New fusion gene discoveries in breast cancer offer the dual appeal that those producing a functional protein could prove to be a unique target for therapy, while silent fusion genes still offer the potential for developing a useful diagnostic test.

Emerging evidence suggests that disruption of circadian rhythms (the 24-hour cycle in the biochemical, physiological or behavioral processes) and circadian rhythm genes may play a significant role in cancer. Epidemiologic studies demonstrate that women with disrupted sleep cycles are more likely to develop breast cancer. In fact, Period 3 (PER3), a mammalian counterpart of the Drosophila circadian rhythm gene period, contains a DNA sequence change that is associated with an increased risk of breast cancer in younger women. Kuang-Yu Jen from the University of California, San Francisco will study multiple aspects of PER3 in breast cancer, including a role in the DNA damage response in mice, as a prognosis factor in patient samples and for the ability to alter cell sensitivity to hormones or anti-hormone therapies.

Helicases are motor proteins that move along a nucleic acid backbone, separating two annealed nucleic acid strands. They are involved in many aspects of DNA and RNA metabolism, such as replication, recombination, repair, and transcription. Daojing Wang at Lawrence Berkeley National Laboratory is taking a “systems biology” approach to examine an RNA helicase, called p68, in order to gain a mechanistic understanding of its role in breast cancer, with particular emphases on cell invasion and drug resistance. P68 expression in breast cancer cells could alter their drug and biological responsiveness by reprogramming a variety of signaling/transcription networks.

Three newly funded grants explore various aspects of tumor progression.

Breast cancers are heterogeneous in their clinical course and response to therapy. This is largely due to differences in the underlying biology, with at least five different types (gene-expression profiles) of breast cancers being recognized. Of these, “basal-like” is one of the most aggressive forms (akin to “triple negative” tumors) of breast cancer and is associated a high risk of metastases. Graham Casey from the University of Southern California will study podocalyxin (PODXL), a cell surface glycoprotein that is expressed on the surface of a wide range of cells. PODXL is best characterized in the kidney, where it functions to maintain open filtration pathways between neighboring podocyte foot processes. Dr. Casey will determine whether PODXL and PODXL signaling are associated with BRCA1 and the development of a basal-like breast cancer stem cell phenotype.

GATA3 is a protein expressed in normal breast epithelial cells to maintain their differentiated state. In human breast tumors, GATA3 expression is lost in malignant cells, which serves as a negative prognostic indicator. Jonathan Chou from the University of California, San Francisco received a dissertation award to search for microRNA targets of GATA3 that may promote or suppress tumor metastasis. Mr. Chou, working with his mentor (Dr. Zena Werb), will profile the microRNA landscape in mammary epithelial cells, then test those that are lost as breast tumor cells progress to the point of metastasis.

Chromatin is the complex association of DNA, RNA, and protein that makes up chromosomes. SATB1 (Special AT Sequence Binding Protein-1) is a genome organizer that works by tethering chromatin elements together. SATB1 expression is absent in normal breast epithelium, but, surprisingly, it is detected specifically in a subset of breast cancers that are aggressive and metastatic. Laurie Friesenhahn from Lawrence Berkeley National Laboratory will use genomics technologies to discover candidate genes that activate or maintain SATB1 expression in cancer cells. In addition, she will compare this information to other cancer stem cell markers and use mouse models to determine whether SATB 1 amounts affect the underlying metastatic potential.

Biology of the Breast Cell Grants Listing
Breast Cancer Tumor-Stroma Interactions in an In Vivo Model
Borgstrom, Per, Ph.D.
Vaccine Research Institute of San Diego
Award type: IDEA
$284,250

A Molecular Strategy to Inhibit Breast Cancer Metastasis
Brodsky, Frances, D.Phil.
University of California, San Francisco
Award type: IDEA
$150,000

Podocalyxin as a Basal-like Breast Cancer Stem Cell Marker
Casey, Graham, Ph.D.
University of Southern California
Award type: IDEA
$243,676

The Role of Estrogen Receptor in Endocrine Resistance
Chan, Hei
Beckman Research Institute of the City of Hope
Award type: Dissertation
$76,000

Understanding the Role of GATA3 in Breast Cancer
Chou, Jonathan
University of California, San Francisco
Award type: Dissertation
$76,000

Finding BRCA1 Ubiquitinated Substrates in Breast Cancer
del Rincon, Sonia, Ph.D.
The Burnham Institute for Medical Research
Award Type: IDEA
$191,000

Substrate Profiling of Breast Cancer Related Proteases
Dix, Melissa
Scripps Research Institute
Award type: Dissertation
$76,000

A Genetic System for Identification of Mammary Stem Cells
Engle, Dannielle
Salk Institute for Biological Studies
Award type: Award type: Dissertation
$76,000

The Regulation of SATB1 in Metastatic Breast Cancer
Friesenhahn, Laurie, Ph.D.
Lawrence Berkeley National Laboratory
Award type: Postdoctoral fellowship
$90,000

Novel Tumor Suppressors in Breast Development and Cancer
Fuller, Margaret, Ph.D.
Stanford University
Award type: IDEA
$231,058

Targeting MYC in Human Breast Cancer
Horiuchi, Dai, Ph.D.
University of California, San Francisco
Award type: Postdoctoral fellowship
$90,000

Role of Circadian Rhythm Gene Homolog PER3 in Breast Cancer
Jen, Kuang-Yu, M.D., Ph.D.
University of California, San Francisco
Award type: Postdoctoral fellowship
$90,000

Control of BRCA2-mediated Homologous Recombination
Meyer, Damon, Ph.D.
University of California, Davis
Award type: Postdoctoral fellowship
$90,000

Discovery of Fusion Genes in Breast Cancer
Pollack, Jonathan, M.D., Ph.D.
Stanford University
Award type: IDEA
$160,000

Proline Metabolism in Metastatic Breast Cancer
Richardson, Adam, Ph.D.
The Burnham Institute for Medical Research
Award type: IDEA
$284,895

tax checkoffP32: New Functional Target in Breast Cancer Brain Metastasis
Staflin, Karin, Ph.D.
Scripps Research Institute
Award type: Postdoctoral fellowship
$90,000

Role of p68 in Breast Cancer
Wang, Daojing, Ph.D.
Lawrence Berkeley National Laboratory
Award type: IDEA
$165,339

Novel Akt Regulatory Factor PHLPP in Breast Cancer
Warfel, Noel
University of California, San Diego
Award type: Dissertation
$75,998

Stroma Expression Patterns in Breast Cancer
West, Robert, M.D., Ph.D.
Palo Alto Institute for Research & Education
Award type: IDEA renewal
$358,000

The Role of EGF Variant mLEEK and Grp78 in Breast Cancer
Wong, Albert, M.D.
Stanford University
Award type: IDEA
$241,380