Pathway-Specific Gene Expression in Breast Cancer Cells

Institution: University of California, Davis
Investigator(s): Colleen Sweeney, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7KB-0085S Award: $339,287
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2001)
The identification of the genes involved in breast tumor progression will be necessary to understand the disease and for the establishment of novel drug targets and therapies. Much effort has gone into finding genetic defects or mutations associated with cancer. However, in breast cancer it appears that defects in hereditary genes, such as BRCA1 and BRCA2 account for only about 10% of total incidence. Thus, sporadic breast cancer is associated with newly-produced genetic defects that occurs only in the tumor cells. While information on tumor suppressors (e.g. p53) and oncogenes (e.g. Her-2) are very helpful in categorizing breast cancer, there remains a huge 'black box' of genetic defects with no defined association with the key clinical properties of the disease. Because of the many sub-types of clinical breast cancer, it appears that complex associations of genetic defects as well as subtle changes in the amounts of normal genes are both involved. Until recently, the role of specific genes and their products has been carried out largely on a one-gene-at-a-time basis. New methods, however, permit simultaneous profiling of the expression of thousands of genes in tumor samples. This provides information on the amounts of gene products present (i.e. RNA or gene duplication) and the absence of gene products (i.e., mutation or gene loss). Results from such studies indicate that there are hundreds of gene expression differences between normal breast tissue and tumors.

In this project we plan to elucidate genes critical to breast cancer progression. We will focus on those genes associated with known cellular growth, survival and motility pathways. In other words, the pathways that underlie the clinical issues of cancer proliferation, resistance to cell death and drug treatment, and spread in the body (metastasis). The two protein signaling pathways of particular interest to us are the ras/Erk pathway, which has been implicated in tumor cell proliferation; and the PI3-kinase/Akt pathway, which has been implicated in tumor cell survival and motility. Our plan is to genetically modulate these two pathways by introducing blocking genes into breast cancer cells. These artificially mutated cells gain many of the characteristics of tumor cells. We can confirm these properties using biological (e.g. motility assays) and biochemical/genetic tests. Then, we can make extracts from the cells and use special 2,000 gene cDNA arrays to assay for the presence and changes in amounts of all known cancer-associated genes. The list of genes to be analyzed includes those involved in cell growth, cell division, and invasion processes. We can also use commercially available arrays (Affymetrix) to expand and confirm the data we obtain using our custom-made cancer cDNA arrays. In other experiments we can use specific drugs to modulate ras/Erk and PI3-kinase/Akt and determine the effect on the breast cancer cells' genetic profile.

These studies will result in a very large data set of pathway-specific gene targets that will be made publicly available to the breast cancer and broader scientific communities through the worldwide web. It is anticipated that the data set will include many genes known to be involved in breast tumor progression, and many more that have not been previously implicated. The identification of novel genes involved in breast cancer progression is anticipated to spawn an array of studies directed toward assessing the utility of such genes as targets for anti-cancer therapies.

Final Report (2004)
An important process used by tumors in their progression to metastatic disease is the aberrant activation of cellular signaling pathways, which in turn leads to aberrant expression of cellular genes. Some of these genes are critical to the tumor’s progression but many of them have not yet been linked to breast cancer. The goal of this project was to use Affymetrix chip technology to identify genes that are targets of the Ras/Erk pathway and the PI-3 kinase/Akt pathway, two signaling pathways known to regulate breast tumor cell growth, motility, invasion and survival. In identifying targets of these two pathways, we aimed to enrich for genes that are involved in breast tumor progression. Two human breast cancer cell lines, MCF-7 and BT474, representative of an early and late phenotype of breast cancer, respectively, were used in this project.

We first determined the best conditions for signaling pathway inhibition using drugs that specifically inhibited the desired pathway without affecting other signaling pathways or cellular viability. Using these conditions, we have identified multiple gene targets of both signaling pathways in both breast cancer cell lines. These targets include growth factors, cell adhesion molecules, angiogenesis factors, matrix metalloproteases, signaling proteins and survival factors, amongst others. While some have been previously implicated in breast cancer, others have not and may represent new targets for breast cancer therapy. To confirm the validity of our Affymetrix analysis, several genes from each cell line were chosen and expression was examined using an independent technique, quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR verified the results we obtained from our Affymetrix experiments, indicating that the genes we identified are likely genuine targets of the Ras/Erk and PI-3 kinase/Akt pathways.

Future studies utilizing “knock-down” technology will determine which of these genes are critically involved in breast cancer cell proliferation and survival.

Note: The CBCRP also funded a graduate student in Dr. Sweeney’s laboratory, Ms. Melanie Funes-Duran, through a “diversity supplement” to this grant. Ms. Funes-Duran conducted research from 2002-2004 on a transmembrane sialomucin protein complex, called MUC4. This complex has an uncertain role in tumor progression and metastasis. It was found that MUC4 enhances the formation of a ErbB2-ErbB3 heterodimer complex, which increases the binding of an EGF-like protein, called neuregulin1?. The net effect is to “upregulate” activity of certain elements of the PI-3K pathway, thus increasing the growth of tumor cells.

Symposium Abstract (2003)
The ErbB family of receptor tyrosine kinases (RTKs) plays important roles in cellular processes such as proliferation, differentiation, cell growth, migration and cell survival. It is comprised of four members ErbB1 (epidermal growth factor receptor EGFR/ HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4). EGF-like growth factor binding induces the dimerization of the RTKs, followed by subsequent cross phosphorylation on cytoplasmic tyrosine residues. The phosphorylated tyrosine residues then act as docking sites for intracellular signaling proteins, initiating a variety of signaling cascades that disseminate signals to the nucleus and cytoskeleton. It has been noted that ErbB2 and its heterodimeric partner ErbB3 are up regulated in many human breast cancers. Recently the membrane mucin Muc4 was also found to be overexpressed in human breast cancers. These findings have been correlated to poor patient prognosis. ASGP-2, the transmem-brane domain of the membrane mucin, Muc4, interacts specifically with ErbB2. The studies presented here demonstrate that Muc4 selectively enhances signaling, through the ErbB2/ErbB3 heterodimer, by activation of the phosphatidylinositol-3-Kinase (PI3K) cell survival pathway, but inhibits PI3K signaling through ErbB1 (EGFR).

Stabilization of the E3 ubiquitin ligase Nrdp1 by the deubiquitinating enzyme USP8.
Periodical:Molecular and Cellular Biology
Index Medicus: Mol Cell Biol
Authors: Wu X, Yen L, Irwin L, Sweeney C, Carraway KL 3rd.
Yr: 2004 Vol: 24 Nbr: 17 Abs: Pg:7748-7757

The Leucine-rich Repeat Protein LRIG1 Is a Negative Regulator of ErbB Family Receptor Tyrosine Kinases.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Laederich MB, Funes-Duran M, Yen L, Ingalla E, Wu X, Carraway KL 3rd, Sweeney C.
Yr: 2004 Vol: 279 Nbr: 45 Abs: Pg:47050-6

The mucin Muc4 potentiates neuregulin signaling by increasing the cell surface populations of ErbB2 and ErbB3
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Funes M, Miller JK, Lai C, Carraway KL 3rd, Sweeney C.
Yr: 2006 Vol: Nbr: May 11 Abs: Pg: