Tumor Suppression by Dystroglycan in Breast Epithelial Cells

Institution: Lawrence Berkeley National Laboratory
Investigator(s): John Muschler, Ph.D. - John Muschler, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7KB-0017 Award: $9,624
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

This is a collaboration with: 7KB-0017A -

Initial Award Abstract (2001)
Normal breast epithelial cells are organized as a single layer of cells with one side of the cell attached to a basement membrane. Cellular attachment to proteins present in the basement membrane is critical to breast cell physiology, providing both structural and behavioral cues to the cell. This cell-basement membrane interaction prevents uncontrolled cell growth. In breast cancer the cells grow as disorganized masses, attachment process is often absent, and growth control is not regulated. Specific molecular information on breast cell attachment is one potential key to controlling disease progression, and even for finding ways to prevent breast cancer. I have recently discovered that a molecule called "dystroglycan" (DG) is important for attachment and signaling growth arrest in breast cells. When we have studied breast tumor cells, it appears that dystroglycan is non-functional in the majority. Most importantly, we find that introducing dystroglycan function into breast cancer cells can restore many normal properties.

In the early stages of breast cancer, there is considerable evidence that restoring critical attachment function will serve to reverse the disease. Evidence indicates that loss of DG function is one part of cancer progression. Nothing is currently known about how dystroglycan functions in normal breast cells and how it’s function is undermined in cancer cells. We plan to use this CBCRP funding to study: (i) how dystroglycan acts in normal breast cells; and (ii) which functions of DG are critical to enact its tumor suppressor function. The long-term aim of this study is to understand how dystroglycan function is undermined in tumor cells, and identify methods to restore DG function to these cells. The experimental approaches are, first, to analyze the signaling pathways emanating from dystroglycan in normal cells. This will be accomplished by comparing signaling events in normal breast cells with cells where the dystroglycan gene has been genetically deleted. In addition, we will compare signaling events in a tumor cell line, where DG function is undermined, with the same cells where dystroglycan’s tumor suppressor function has been restored. These combined experiments will help us determine which specific signaling events from DG are critical to restore normal cell behavior to tumor cells.

The investigation of DG as a tumor suppressor is an entirely novel approach to understanding cancer. This research will shed light on a novel and important growth-regulatory mechanism within breast epithelial cells, and reveal some root causes of tumor cell behavior. But most importantly, the studies presented here can ultimately reveal strategies to restore critical aspects of dystroglycan function in tumor cells, giving us new methods to fight cancer cell proliferation and spreading.

Final Report (2003)
Cells sense their environment and through “receptors” located at their cell surface. These receptors bind to molecules outside the cell and then transmit instructions into the cell that regulate cell behavior. I am studying the function of a cell-surface molecule called “dystroglycan” (DG) that binds to the extracellular protein “laminin” and is believed to send information into the cell, telling it to adopt a particular structure (epithelial polarization) and to stop growing. I have previously shown that this molecule is functionally compromised in many tumor cell lines, and enhancement of its function can restore aspects of normal behavior to a tumor cell. Under funding from the CBCRP, I have begun to dissect the precise functions of this molecule in normal cells, and to determine why it does not function correctly in many tumor cells.

To examine the precise functions of DG in breast cells, we have created breast epithelial cells in which the DG gene can be selectively deleted via a technology called “cre-lox recombination”. We have achieved this using breast epithelial cells taken form a particular transgenic mouse line wherein the DG gene was target for destruction by a specific enzyme called “Cre”. We have obtained primary cultures from these mice and used these to create immortalized cell lines in culture. Using standard gene transfer technologies, we can now easily and efficiently remove the DG gene from functionally normal mammary epithelial cells, and compare the behavior of cells with and without DG. Using these methods we have determined that DG is essential for mediating multiple functions in response to basement membrane proteins: we have determined that signaled growth inhibition by specific basement membrane molecules such as laminin-1 and HSPGs; we have determined that DG is essential for signaling polarization in response to laminin-1; we have determined that DG is essential for the binding and organization of laminin-1 at the surface of breast epithelial cells. Through these genetic manipulations, we have also learned that other, yet unidentified, basement membrane receptors exist that can substitute for DG. Using this unique cell system, we are now in a position to dissect in greater detail the mechanisms of critical signaling functions through DG.

In parallel, we have investigated the cause for loss of DG function in breast cancer cells. We have revealed that a portion of the DG molecule is released from the cell surface, contributing to a loss of DG function. We have shown a role for enzymes known as proprotein convertases and metalloproteases in the loss of the laminin-binding portion of DG. Importantly we have also revealed that the sugar molecules that adorn DG (which are required for DG function) are altered in invasive carcinoma cells, and these alterations are the primary cause for loss of DG function in invasive breast cancer cells. These results have provided important insight into the defects that arise in cancer progression, contributing to loss of growth control and loss of tissue architecture. In addition, these insights have provided new direction to efforts aimed at restoring DG function in function in breast carcinoma cells.