Cytoskeletal Regulation of Invading Breast Cells

Institution: University of California, San Francisco
Investigator(s): Catherine Jacobson, Ph.D. -
Award Cycle: 2007 (Cycle 13) Grant #: 13FB-0115 Award: $20,123
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2007)
Many types of early breast cancer lesions are characterized by a loss of acinar organization (i.e., the berry-shaped terminal regions of mammary ducts), including loss of cell-cell adhesions, loss of cell polarity, increased proliferation, and invasion into the surrounding tissue, all of which are precursors to metastatic breast cancer. These same cellular behaviors were originally described as normal processes during embryonic development, and were collectively referred to as ‘epithelial-to-mesenchymal transition’ (EMT). Although EMT is widely regarded as a mechanism for tumor invasion, epithelial tumors rarely undergo a complete EMT. In fact, many tumors appear to partially and sometimes reversibly acquire characteristics of the mesenchymal phenotype. Invasion relies on the polarization of the cell’s internal supporting framework, called the cytoskeleton. One aspect of cytoskeletal polarization required for other cell types, such as fibroblasts and neurons, to move on a two-dimensional surface, is the reorientation of the microtubule-organizing center (MTOC) and the nucleus along the direction of migration. The mechanism underlying this MTOC and nucleus reorientation during the invasion of tumor-like breast epithelial cells is not known, and new information in this topic might prove to be very useful in designing targeted therapies to prevent the earliest processes in breast cancer.

In breast cancer, cells leave the organized structures of acini and invade the surrounding tissue. Because the molecular mechanisms underlying invasion are difficult to study in whole animals, we will use a cell-based model of this process. Mouse epithelial breast cells grown in a three-dimensional (3-D) matrix spontaneously form acini, thus mimicking several aspects of the glandular architecture of the normal breast in vivo. Treatment with growth factors induces these cells to leave the organized acinar structure and invade the surrounding extracellular matrix. Using this 3-D migration of mammary epithelial cells away from organized acini in a 3D matrix as a model of tumor invasion, I will examine the role that MTOC reorientation plays during invasion. First, I will examine the spatial reorientation of both the MTOC and the nucleus in cells that are induced to leave an intact acinus by visualizing fluorescently tagged proteins associated with these two structures in “real time.” In my second aim, I will test the function of proteins proposed to regulate MTOC reorientation in other cell types, including the well-known polarity protein complex consisting of the Rho GTPase Cdc42 and its downstream effectors Par6/aPKC [i.e., mammalian equivalent of the C. elegans, a nematode, partitioning-defective (Par) proteins], as well as the molecular motor, termed cytoplasmic dynein, and its proposed regulator, called Lis1.

Research aimed at understanding the basic biology of breast cancer cells will collectively have a great impact on the development of new, effective therapies for this devastating disease. The goal of this proposal is to contribute to this effort by elucidating the cell biology of one of the earliest steps in the invasion of tumor cells into the surrounding tissue.

Final Report (2008)
*Note: The PI resigned the grant after four months.

Many types of early breast cancer lesions are characterized by a loss of acinar organization (i.e., secretary, exocrine glands with a luminal and basement membrane polarity), including loss of cell-cell adhesions, increased proliferation, and invasion into the surrounding tissue. These changes are all precursors to metastatic breast cancer. The detailed steps of how breast cells begin to migrate away from the acini in three dimensions and invade the surrounding tissue are not known. We used the mouse mammary epithelial cell line EpH4, cultured in three dimensions, as a model for the conversion of normal breast cells into invasive tumor-like cells. This breast cell line is unique in that it is the only breast cell line shown to form normal acini in culture that can then be induced with a growth factor to undergo migration.

In the four months that I worked on this project, I was able to establish the culture of EpH4 cells as acini composed of a single layer of cells, which could then be induced to undergo a migratory phenotype upon growth factor treatment. It was crucial to optimize the migration of cells for live cell imaging. This means that I had to be able to induce enough cells to migrate within a certain time period in order to capture them during the microscopic imaging process. However, too many migrating cells would interfere with the imaging. Furthermore, I had to be able to restrict migration to a single plane of imaging. I accomplished this by using a combination of Matrigel (an artificial adhesion surface and migration media) and extracellular matrix components commonly used in the Mostov (mentor’s) lab.