Discovering Novel Cell-ECM Interactions in Breast Cells

Institution: California Pacific Medical Center Research Institute
Investigator(s): John Muschler, Ph.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10IB-0067 Award: $159,939
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2004)
Normal breast epithelial cells contact a complex mixture of secreted "extracellular matrix" (ECM) proteins called the basement membrane (BM). Cell contact with the BM is a critical regulator of breast cell physiology, providing cues that direct the organization of cells into tissues and prevent uncontrolled cell growth. These cues are communicated to the cells by sensor proteins, known as "receptors", which bind to particular BM proteins and send "signals" into the cell. Alterations in cell-BM communications are known to occur in cancer cells, permitting unregulated growth and metastasis in the presence of the BM. Yet, detailed knowledge of cell-BM interactions in normal cells and cancer cells is lacking. Known BM receptors operating in breast cells include "integrins" and "dystroglycan", but evidence from our laboratory indicates that other important receptors remain to be discovered.

We hypothesize that, in addition to dystroglycan and integrin signaling, there exist undiscovered cell-BM interactions that are important regulators of epithelial architecture, the functions of which are lost in cancer cells, thereby favoring cancer progression. We will generate mammary epithelial cells lacking both dystroglycan and β1 integrin receptor expression, and employ this unique cell system to characterize the novel BM receptors functioning in mammary epithelial cells. We propose that elucidation of these novel signaling mechanisms in normal breast cells will advance our understanding of how cell-BM interactions regulate normal cell behavior, and will ultimately lead to a better understanding, diagnosis and treatment of breast cancers.

To reveal the functions of novel BM receptors we will create breast epithelial cells wherein most of the known BM receptors (specifically dystroglycan and β1 integrins) have been removed from the otherwise normal cells. The removal of known BM receptors will be accomplished by a state-of-the-art method of genetic manipulation using specific transgenic mice already provided to us by collaborators. Once the known receptors have been removed, we will test our hypothesis that these cell can still respond to BM proteins through the use of other, unidentified receptors. We will then employ our novel experimental cell system in biochemical studies to characterize and identify the unknown receptors.

Although cell-BM interactions have been studied extensively in breast epithelial cells, we still have an incomplete understanding of the cell-BM interactions that regulate normal breast epithelial architecture and growth. In addition, we have a poor understanding of how these important signaling mechanisms are compromised in cancer cells. My research is revealing important roles for mediators of cell-BM interactions that have not been previously investigated in breast epithelial cells. My laboratory is developing an array of unique tools for the dissection of these signals. With these unique tools, and unique insights, we are opening entirely new avenues of investigation in the field of cell-BM interactions in normal and tumorigenic breast epithelial cells.


Final Report (2005)
Normal breast epithelial cells contact a complex mixture of secreted “extracellular matrix” proteins called the basement membrane (BM). These cells use proteins, known as “receptors”, to sense the BM, and this communication serves as a critical regulator of breast cell physiology, providing cues that direct the organization of cells into tissues and prevent uncontrolled cell growth. Alterations in cell-BM communications are known to occur in cancer cells, permitting unregulated growth and metastasis in the presence of the BM. Yet, detailed knowledge of cell-BM interactions in normal cells and cancer cells is lacking. Known BM receptors operating in breast cells include “integrins” and “dystroglycan”. However, evidence from our laboratory indicates that, in addition to dystroglycan and integrin signaling, there exist undiscovered cell-BM interactions that are important regulators of epithelial architecture, the functions of which are lost in cancer cells, thereby favoring cancer progression.

Through this grant, we proposed to create mammary epithelial cells lacking both dystroglycan and ß1 integrin receptor expression, and to employ these unique cell systems to reveal novel BM receptors functioning in mammary epithelial cells. The removal of known BM receptors from normal breast epithelial cells has been achieved, as proposed, although success was not guaranteed. To achieve this, we generated a new strain of transgenic mice that permitted the simultaneous deletion of both dystroglycan and ß1 integrin receptor expression. Breast epithelial tissues from this new strain were isolated and manipulated to achieve the genetic elimination of known receptor expression. In addition, we succeeded, with considerable patience, in establishing from these mice at least 6 new breast epithelial cell lines that can grown in indefinitely in the laboratory and be manipulated easily without further need for using animals. Having created these tools, we have begun testing our hypothesis that these cells can still respond to BM proteins through the use of other, unidentified receptors. Preliminary results are validating our hypothesis, as described in the accompanying report. While generating these tools, and as an alternative model system, we also examined human breast cancer cell lines to investigate mechanisms of cell responses to BM that are independent of integrin and dystroglycan function, and here too we have found evidence that important responses to the BM can be mediated mechanisms that are independent of known pathways. Therefore, this work has validated our model for discovering novel defects in cancer cells, and allowed us to generate unique and valuable research tools that are necessary to advance this investigation.

The transgenic animals and unique breast epithelial cell lines generated here represent an array of unique tools for the dissection of the signaling mechanisms mediating cell-BM interactions. With these tools now in hand, we are continuing these investigations in our laboratory at full speed. We are currently testing the ability of our customized cells, fully lacking integrin and dystroglycan expression, to polarize, growth arrest, and alter gene expression in response to a mixture of BM proteins and to purified BM components. We will then work to identify the undiscovered signaling pathways remaining in these cells, and move our investigations towards studying these mechanisms in cancer cells. Using preliminary data generated from this project, we are also applying for larger grant support to continue and expand this work. The ultimate elucidation of these novel signaling mechanisms in normal breast cells will advance our understanding of how cell-BM interactions regulate normal cell behavior. Most importantly, this knowledge should lead to the identification of molecular defects that contribute to breast cancer progression, and will ultimately lead to a better understanding, diagnosis and treatment of breast cancers.