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 precancerous, 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 that 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 how extracellular and breast/tumor stromal factors contribute to tumor progression. 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%) 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.

The CBCRP funded 13 new grants in 2007 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 Normal Breast Cell Portfolio Summary:

Three newly funded grants study the biology of the normal breast. Throughout a woman’s life, the breast undergoes a series of hormonally-driven developmental changes that involve signaling pathways to prevent abnormal expansion of mammary cells. The retinoblastoma protein (pRb) is a tumor suppressor protein that becomes dysfunctional in many types of cancer. A major function of pRb is to prevent the cell from dividing or progressing through the cell cycle. Thus, when pRb is ineffective at this role, mutated cells can continue to divide and may become cancerous. Deborah Burkhart from Stanford University was funded through a dissertation award to study the function of the Rb-related proteins, p107 and p130, which need to be inactivated, in addition to pRb, before cancer can occur. She will focus on the regulation and function of p107 in the mammary gland of mice, with the specific goal of understanding how p107 can block cancer in pRb-deficient breast cells. Next, telomerase has been proposed as a key to cellular immortality, a so-called “fountain of youth.” In both cancer and normal stem cells the presence of telomerase allows them to divide repeatedly. Telomerase is “turned on” in 90% of human breast cancers and DCIS, making its re-activation one of the most common changes in the disease. Steven Artandi also at Stanford University will examine an alternate function of telomerase (i.e., the Telomerase Reverse Transcriptase, or the TERT protein) to promote the proliferation and expansion of mammary stem/progenitor cells. In this IDEA renewal grant Dr. Artandi will further develop the hypothesis that conditional activation of TERT in adult mouse epithelium leads breast cancer by promoting an expansion of mammary stem/progenitor cell populations. Finally, many types of early breast cancer lesions are characterized by a loss of normal breast’s acinar organization (i.e., the berry-shaped terminal regions of mammary ducts), including a loss of cell-cell adhesion and polarity, increased proliferation, and cellular invasion into the surrounding tissue. These changes are precursors to invasive, metastatic breast cancer. Catherine Jacobson from the University of California, San Francisco, will study the function of the microtubule-organizing center (MTOC) and the nuclear orientation of mammary epithelial cells along the direction of migration. She will test the function of proteins, such as Rho GTPase and Cdc42, for their ability to shift the migration of normal mouse epithelial cells grown in three-dimensional culture from an acinar to a migratory phenotype.

Three newly funded projects focus on processes central to breast tumor cell invasion and metastasis. Transforming growth factor-beta (TGFβ) is a secreted protein that is generated in abundance from tumors cells, and there is a strong correlation between high levels of tumorderived TGFβ and poor clinical prognosis. In cell culture and animal models, TGFβ has been shown to promote processes that stimulate metastasis. Various anti-TGFβ therapeutics are currently entering clinical trials for oncology applications, but despite the potent anti-metastatic action of TGFβ inhibitors for certain tumors, it has been known for a decade that TGFβ can have opposite, tumor-promoting actions in others. Kelly Harradine from the University of California, San Francisco plans to profile the gene expression patterns of breast tumors to study gene signatures associated with TGFβ. The goal is to develop useful information for clinicians that will predict the breast tumor’s response to novel small molecule inhibitors of TGFβ signaling, ultimately facilitating patient selection for treatment. Next, Src is a tyrosine kinase oncogene and is found to be “activated” in most breast cancers. Src sits at the center of a complex web of cellular communication, taking messages from a variety of cell-surface receptors and passing them on to proteins that control cell differentiation and proliferation. Trask is a new Src target protein that, when phosphorylated (phospho-Trask) in breast cancer cells, causes the cells to detach and separate, similar to metastasis. Ching Hang Wong also at the University of California, San Francisco plans to test the hypothesis that that phospho-Trask brings Src kinases in proximity to the E-cadherin/β-catenin complex, where Src can phosphorylate β-catenin more readily. This would provide a novel mechanism of breast tumor cell metastasis. Finally, tissue factor (TF) is the primary initiator of the blood coagulation cascade, which is physiologically important to prevent excessive bleeding and initiate wound healing. In addition to its crucial role in blood clotting, TF is expressed by tumor cells and this correlates with a poor prognosis. Florence Schaffner from the Scripps Research Institute will study how blocking the function of TF in both coagulation and in cell signaling may influence breast cancer growth and metastasis. She will treat established tumors in mice with TF-specific antibodies to establish the differential effects of inhibiting either TF signaling or coagulation.

Three grants study processes central to breast cancer cell growth control. Elevated amounts of the ErbB2 (Her-2) oncogene growth receptor are clinically important in 25-30% of breast cancers. Yet, despite the availability of drugs (such as Herceptin®) that specifically target ErbB2, the therapy may fail because we still do not fully understand the cellular components that control ErbB2 activity. Ralf Landgraf from the University of California, Los Angeles, received IDEA funding to study the role cell membrane lipid domains, called “lipid rafts”, play in modulating ErbB2 signaling, dimerization with other ErbB family members, and cell resistance to Herceptin®. It is possible that saturated fatty acids and gangliosides mediate this interplay between lipids and ERBB2, and this ErbB2-lipid raft interplay may provide new insights into the regulation of cell growth, resistance to therapy, and how environmental/dietary factors may influence the course of the breast cancer. Next, the dietary indole, indole-3-carbinol (I3C) found in cruciferous vegetables, has been shown to decrease estrogen receptor-α (ERα) levels, but the mechanism underlying this effect has not been determined. Crystal Marconett at the University of California, Berkeley, will determine which portions of the ERα gene promoter are sensitive to I3C. This new information could potentially connect critical breast cancer cell pathways known to be affected by I3C, of find novel I3C-regulated ERα pathways. Finally, phopshoinositide (PI) 3-kinases have been linked to a diverse group of cellular functions, including growth, proliferation, differentiation, motility, survival and intracellular protein trafficking. Jun Zhang at the University of California, San Francisco, aims to generate the first mouse model for examining the role of “activating mutations” in PI 3-kinase α in breast cancer. Once the mouse is generated, Dr. Zhang will examine the interplay PI 3-kinase α mutations with other common breast tumor genetic defects, such as p53 and ARF (GTP-binding proteins of the Ras superfamily). Lastly, this project involves using the mice to test the effectiveness of a new generation of PI 3-kinase family inhibitors for the treatment of breast cancer.

Two newly funded grants focus on gene regulatory and epigenetic events in breast cancer. Epigenetics includes processes that alter gene activity without changing the DNA sequence. DNA methylation, a chemical modification involving addition or removal of methyl groups from DNA, is a common type of epigenetic mechanism that can “silence” genes, especially tumor suppressors and apoptosis genes that would otherwise block disease progression. Global changes in DNA methylation are known to occur during progression of breast cancer. A loss of the normal cell turnover mechanisms (apoptosis) that keep cell numbers in check is a common feature of breast cancer. Lorena Puto from The Burnham Institute of Medical Research will study the Daxx adapter protein (death-associated protein 6) that suppresses RelB, a DNAbinding protein that controls the activity of several anti-apoptotic genes. She will focus on the role that DNA methylation plays in the process. Very few examples exist in literature that would explain how specific genes are silenced by DNA methylation in cancer, and none exists in breast cancer. Next, 25% of breast cancers have inactivating mutations in the p53 gene. In the remainder of breast cancers, the p53 gene and protein is normal. Thus, it is unclear how p53 functions in most cancer cells are regulated. Min Yang at University of California, Irvine, will examine how two histone acetyltransferase (HAT) “docking factors”, called ADA2 and ADA3, become altered in breast cancer to reduce p53 gene transcription. Dr. Yang’s hypothesis is that transcription factors overexpressed in breast cancers, such as the estrogen receptor and β- catenin, not only turn on genes that promote growth, but also indirectly inhibit tumor suppressors, in particular p53. The mechanism being tested is that the estrogen receptor and beta-catenin recruit ADA2 and ADA3 away from p53.

Finally, two new projects seek to understand events involved in tumor progression. Cancer cells are surrounded by a complex mixture of blood vessels, inflammatory cells, and different types of connective tissue cells. These stromal cells are themselves not cancerous, but have been shown to play a crucial role in cancer development and progression. An alternative avenue of therapy focuses on targeting various non-neoplastic cells that are associated with the tumor microenvironment. Robert West from the Palo Alto Institute for Research & Education will study the gene expression of soft tissue tumors (STT, including the malignant variants called sarcomas) to dissect and understand the gene expression of normal connective tissue cells, using these SSTs as surrogates for normal connective tissue cells. Dr. West’s initial studies show that genes differentially expressed in STTs vary among groups of breast cancers. They plan to confirm and extend these observations to find breast cancer “stromal reaction patterns” as a novel tumor classification scheme. In addition to their use as prognostic markers, potential therapeutic targets may also be discovered in the group of SST genes, if confirmed in breast cancers. Lastly, BRCA1 gene mutations account for 50% of hereditary breast cancers. In sporadic breast cancers, although the BRCA1 gene is intact, its protein expression is often reduced. BRCA1 is known to play an essential role in maintaining genomic integrity mainly through direct involvement in repairing damaged DNA and monitoring cell proliferation. However, these functions of BRCA1 do not adequately explain how its deficiency accelerates breast cancer progression. Connie Tsai at the University of California, Irvine, recently demonstrated that BRCA1 has a direct role in regulating the expression of an array of genes involved in tumor progression, one of which is HMGA2 (i.e., one of the high mobility group chromosomal proteins with an AT-hook DNA-binding motif that is frequently associated with benign and malignant tumors). Her dissertation award project will study the regulation of HMGA2 expression by BRCA1 and the effects of altering HMGA2 amounts in breast cancer cells and in mouse tumor models.

Biology of the Breast Cell Grants Funded in 2007:

Telomerase, Mammary Stem Cells, and Breast Cancer
Steven Artandi, M.D., Ph.D.
Stanford University
Award type: IDEA-competitive renewal
$419,400

Novel Regulation of the Rb Pathway in Breast Epithelium
Deborah Burkhart
Stanford University
Award type: Dissertation
$76,000

Breast Tumor Responses to Novel TGF-beta Inhibitors
Kelly Harradine, Ph.D.
University of California, San Francisco
Award type: Postdoctoral Fellowship
$90,000

Cytoskeletal Regulation of Invading Breast Cells
Catherine Jacobson, Ph.D
University of California, San Francisco
Award type: Postdoctoral Fellowship
$90,000

Lipid Raft Composition in Deregulated ERBB2 Signaling
Ralf Landgraf , Ph.D.
University of California, Los Angeles
Award type: IDEA
$100,000

Indole (I3C) Control of Breast Cancer by ER Downregulation
Crystal Marconett
University of California, Berkeley
Award type: Dissertation
$76,000

Mechanisms of Daxx-Mediated Apoptosis in Breast Cancer
Lorena Puto
The Burnham Institute of Medical Research
Award type: Dissertation
$76,000

Targeting Tissue Factor in Breast Cancer
Florence Schaffner, Ph.D.
Scripps Research Institute
Award type: Postdoctoral Fellowship
$90,000

The Relationship of BRCA1 and HMGA2 in Breast Cancer
Connie Tsai
University of California, Irvine
Award type: Dissertation
$76,000

Determination of Stromal Gene Expression in Breast Cancer
Robert West, M.D., Ph.D.
Palo Alto Institute for Research & Education
Award type: IDEA
$139,441

Trask, a Candidate Breast Cancer Metastasis Protein
Ching Hang Wong, Ph.D.
University of California, San Francisco
Award type: Postdoctoral Fellowship
$90,000

Competition for ADA2 and 3 to Inhibit p53 in Breast Cancer
Min Yang, M.D.
University of California, Irvine
Award type: Dissertation
$76,000

A New Mouse Model of PI3-Kinase Induced Breast Cancer
Jun Zhang, Ph.D.
University of California, San Francisco
Award type: Postdoctoral Fellowship
$90,000