The 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 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 2010

The Role of Podosomes in Breast Cancer Metastasis
Early detection has greatly reduced breast cancer mortality. However, once breast cancer has metastasized treatment options are limited. Studies suggest that cancer cells use specialized structures called podosomes to help them invade surrounding tissue. These podosomes, which are located on the front of the cellular membrane, are composed of a number of different proteins. One of these proteins is called Tks5, and studies have found that its presence in human breast cancer correlates with tumor progression. Barbara Blouw, Ph.D., at Sanford-Burnham Medical Research Institute in La Jolla used a mouse model of breast cancer to investigate whether Tks5 and podosomes play a role in breast cancer tumor growth and progression. Preliminary data suggest that when the levels of Tks5 are reduced, growth of the primary cancer is decreased. Dr. Blouw is conducting experiments to verify these findings and to further analyze the role Tks5 and podosomes may play in metastasis. This work could lead to new treatments for metastatic breast cancer. Findings from this research were published in the European Journal of Cell Biology 87(2008)555.

Novel Regulation of the Rb Pathway in Breast Epithelium
For a normal cell to transform into a breast cancer cell multiple mutations must occur in the cell’s genes. Some of the genes that are often lost in breast cancer are called tumor suppressors. Their job is to keep breast cells from abnormally multiplying. Deborah Burkhart and colleagues at Stanford University in Palo Alto are studying the pRB family of tumor suppressors, which is comprised of pRB (a retinoblastoma protein), and its associated proteins p107 and p130. Normally pRB prevents the cell from replicating damaged DNA by blocking its progression through the cell cycle. This project focused on learning more about p107, and how it can block cancer in pRB-deficient breast cells. The team successfully developed and published a novel p107-GFP reporter transgenic mouse line. Their studies using this mouse line showed that in wild-type animals p107 levels appear to decrease over the course of development, even during mid-pregnancy when the ducts are expanding. They also showed that in some cells in the mammary gland p107 expression could increase in the absence of Rb. This research could lead to new insights into how breast cancer develops. Findings from this research were published in Cell Cycle 7(2008)2544 and Nature Reviews Cancer 8(2008)671.

Indole (I3C) Control of Breast Cancer by ER Downregulation
Studies have found that indole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables, such as broccoli, can slow the growth of human breast cancer cells because it has an effect on cell cycle regulators like estrogen receptor-alpha (ERα). Crystal Marconett, B.A., and colleagues at the University of California, Berkeley conducted studies that would illuminate how I3C affects ERα. Their work established the molecular mechanism I3C elicits to ablate ERα expression in hormone sensitive breast cancer cells, and demonstrated that ERα-dependent loss of other downstream gene targets—IGF1R and IRS1, critical regulators of growth factor signaling in breast cancer—accounted for the loss of proliferation. These research findings suggest that I3C could be pursued as a potential new breast cancer treatment. Findings from this research appeared in Molecular and Cellular Biology 21(2010)1166.

Tumor Suppressor 14-3-3sigma in Breast Cancer Progression
There are at least five breast cancer subtypes, with distinct genetics, response to chemotherapy, clinical outcome, and biology. Developing effective targeted cancer therapies requires learning more about the molecular basis of tumor progression of each breast cancer subtype. Aaron Boudreau, B.Sc., and colleagues at the Lawrence Berkeley National Laboratory recently identified a protein called 14-3-3sigma that becomes highly expressed during malignancy in a culture model of breast cancer progression. Mr. Boudreau’s research characterized a novel mechanism by which 14-3-3sigma regulates cell migration and invasion by regulating the homeostasis of the cell’s cytoskeleton. Their research also found that there are high levels of 14-3-3sigma in a specific subset of breast tumors that are associated with a poor clinical outcome. These findings suggest that targeting 14-3-3sigma may be an effective therapeutic strategy in a subset of breast tumors. Findings from this research were published in Cancer Metastasis Reviews 28(2009)167.

Dietary Metabolite Inhibition of Breast Cancer Cell Survival
Indole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables, such as broccoli, can slow the growth of human breast cancer cells. 3,3’-Diindolylmethane (DIM), the major acid condensation product of I3C, has been shown to have anticancer effects in breast cancer. DIM also inhibits Akt, a kinase whose signaling promotes proliferation, survival, and motility in breast cancer cells in vitro. Holly Nicastro, B.S., and colleagues at the University of California, Berkeley investigated whether DIM’s inhibition of Akt is partly responsible for DIM’s anti-proliferative/pro-apoptotic effects. They found that DIM inhibits proliferation, cell cycle progression and motility, and induces apoptosis in MDA-MB-231 breast cancer cells, which is consistent with Akt inhibition. They also showed that DIM inhibits Akt downstream of hepatocyte growth factor (HGF). And they found that DIM decreases activation of c-Met at several tyrosine residues, indicating decreased activation of the receptor. These findings suggest that DIM is a promising potential therapeutic option for breast cancers with aberrant HGF/c-Met/Akt signaling.

Dissecting the Role of Twist in Breast Cancer Metastasis
Several changes that occur in metastatic cancer cells resemble an evolutionarily conserved process in embryonic development called epithelial-mesenchymal transition (EMT). Recently, a gene-regulatory transcription factor called Twist was shown to play a prominent role in promoting EMT in mammalian breast cancer cells. Janine Low-Marchelli, B.S., and colleagues at the University of California, San Diego are investigating whether the Twist protein promotes breast cancer metastasis. Using a new technology called ChIP-Sequencing, they are trying to identify the genes that are under the direct control of Twist during angiogenesis (the growth of a tumor’s blood vessels). They found that a gene called semaphoring appears to be required for angiogenesis, but that it cannot promote angiogenesis on its own. They now intend to conduct additional research into semaphorin’s role in angiogenesis. This work could help lead to the development of prognostic tools and drugs that can more accurately predict and treat breast cancer metastasis.

Chemokine Receptor Signaling in Breast Cancer
Chemokines and their receptors play an important role in the immune system by guiding the migration of cells involved in routine immune surveillance and inflammatory responses. However, cancer cells also can use these proteins to facilitate metastasis and enhance tumor growth. Morgan O’Hayre, B.S., and colleagues at the University of California, San Diego are studying the role the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, play in breast cancer progression. (CXCR4 and CXCR7 receptors are not normally found in breast tissue, but they are often found in breast cancer.) Their studies demonstrated that while both CXCR4 and CXCR7 could accelerate primary tumor growth, CXCR4 appeared to have a stronger effect. They also demonstrated that presence of CXCR4 but not CXCR7 enhanced rates of metastasis to the lungs and lymph nodes. In addition, the research team identified a tumor suppressor protein, programmed cell death factor 4, as a novel target of CXCL12 signaling that may contribute to breast cancer cell growth. Ms. O’Hayre and her colleagues are continuing to conduct experiments on CXCR4 and CXCR7 and their role in breast cancer growth and metastasis. These findings could lead to the identification of new targets for new breast cancer treatments. Findings from this research appeared in Cell and Molecular Life Sciences 66(2009)1370, Methods and Enzymology 460(2009)331, and PLOS One 5(2010)e11716.

Maternal Embryonic Leucine Zipper Kinase in Mammary Tumors
The maternal leucine zipper kinase (Melk) gene is a potential marker of proliferating mammary epithelial progenitor cells that are highly expressed in multiple human cancers, including breast cancer. Robert Oshima, Ph.D., and colleagues at Sanford-Burnham Medical Research Institute in La Jolla used a genetically engineered mouse model to determine whether Melk played a role in breast cancer. They found that in their model Melk kinase activity was not required for mammary tumors. However, additional experiments using shRNA (short hairpin RNA) knockdown of Melk decreased the ability of cultured mammary tumors to form both tumors in vivo and tumorspheroid colonies in cell culture. Specifically, Melk shRNA decreased tumor frequency by six fold. This research suggests that Melk protein, but not kinase activity, may be important for mammary tumor formation. These findings could lead to a new target for new breast cancer therapies.

The Regulation of SATB1 in Metastatic Breast Cancer
Metastasis occurs when cancer cells travel through the body and create new tumors in other organs. Laurie Friesenhahn, Ph.D., and colleagues at the Lawrence Berkeley National Laboratory are studying a protein called SATB1 (Special AT Sequence Binding Protein 1), which regulates the tumor-initiating and metastatic potential of breast cancer cells. Not every cancer cell in a primary breast tumor has the SATB1 protein, and Dr. Friesenhahn and her team explored their hypothesis that cells with SATB1 are an aggressive, metastatic, sub-population of tumor-initiating cells. Their studies showed that cells with SATB1 are resistant to the widely used breast cancer chemotherapy drug fluorouracil. This finding supports the hypothesis that breast cancer cells that express SATB1 pose a greater risk of relapse to the patient. Dr. Friesenhahn and her team intend to continue to investigate the link between SATB1 expressing cells and chemo-resistance, using different chemotherapy drugs and different breast cancer cell lines. They also intend to investigate how cells initiate and sustain SATB1 expression. Their findings could lead to the development of new breast cancer therapies that target SATB1.

Role of Circadian Rhythm Gene Homolog PER3 in Breast Cancer
Studies suggest that disruption of day-night cycles—which occurs, for example, during night-shift work—can increase breast cancer risk. These day-night cycles, called circadian rhythms, are controlled by defined molecular pathways. Circadian rhythm genes show daily cycles in their gene expression and protein activity. Kuang-Yu Jen, M.D., Ph.D., and colleagues at the University of California, San Francisco previously discovered that mice deficient in one of the circadian rhythm genes, known as Period3 (Per3), are more susceptible to developing breast tumors following exposure to carcinogens. They have now demonstrated that the cancer susceptibility in Per3-deficient mice is likely not attributed to their acute ability to repair DNA damage. They also have shown that breast cancer tumors that express low amounts of PER3 are more likely to stop responding to anti-hormone treatment. Dr. Jen’s team intends to pursue additional research on PER3 levels in breast cancer. Findings from this research were published in the Journal of Clinical Oncology 28(2010)3770.

Understanding the Role of GATA3 in Breast Cancer
Despite recent advances in our understanding of breast cancer, patients who do not respond to treatment or who develop metastatic disease have a poor prognosis. Currently, the molecular basis for the metastases process remains largely unknown. Jonathan Chou, B.S., at the University of California, San Francisco, studied GATA3, a master regulatory transcription factor that specifies mammary cell differentiation. Because GATA3 is lost in breast cancer progression, Mr. Chou and his team were interested in investigating how it functions at the molecular and cellular level to prevent metastasis. Their studies found that GATA3 induces the expression of miR29b, a miRNA that has recently been shown to be a tumor suppressor, and they showed that miR29 family members regulate key factors involved in blood vessel recruitment and permeability, including vascular endothelial growth factor. They also showed that miR29b is lost during tumor progression in a mouse model of breast cancer, concomitant with the loss of GATA3. The laboratory is now investigating whether miR29b targets are important regulators of tumor metastasis, and whether miR29b expression promotes mammary cell differentiation. This research could lead to the development of new breast cancer treatments. Findings from this research appeared in the Journal of Cellular Physiology 222(2010)42.

Research Initiated in 2010

Complement-mediated Stem Cell Recruitment to Breast Cancer 
Ingrid Schraufstatter
Torrey Pines Institute for Molecular Studies
          
Inhibiting Mutation to Prevent and Treat Breast Cancer     
Floyd Romesberg
Scripps Research Institute

Local Adipocyte Function in Breast Cancer                  
Barbara Mueller                 
Torrey Pines Institute for Molecular Studies

Myeloperoxidase Mediated Protection in Breast Cancer       
Wanda Reynolds                
Sanford-Burnham Medical Research Institute

p97 as a Therapeutic Target in Breast Cancer Metastasis    
Martin Latterich
Proteomics Research Institute for Systems Medicine
      
Pharmacological Modulation of PP2A Activity in Breast Cancer
Daniel Bachovchin
Scripps Research Institute

Reelin Signaling Involvement in Breast Cancer Cell Migration
Ellen Carpenter
University of California, Los Angeles

The Role of Clim Proteins in Breast Cancer                 
Suman Verma
University of California, Irvine     

The Role of microRNAs in Triple-Negative Breast Cancer     
Leonard Kusdra
University of California, San Francisco              

The Role of Twist1 in Epithelial-mesenchymal Transition    
Jeff Tsai                    
University of California, San Diego

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

Control of BRCA2-mediated Homologous Recombination
Damon Meyer
University of California, Davis
Discovery of Fusion Genes in Breast Cancer
Jonathan Pollack
Stanford University

Finding BRCA1 Ubiquitinated Substrates in Breast Cancer
Charles Spruck
Sanford-Burnham Medical Research Institute

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

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

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

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

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

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

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

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

Proline Metabolism in Metastatic Breast Cancer
Adam Richardson
Sanford-Burnham Medical Research Institute

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

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

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

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

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

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

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

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

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