Reelin Signaling Involvement in Breast Cancer Cell Migration

Institution: University of California, Los Angeles
Investigator(s): Ellen Carpenter, Ph.D. -
Award Cycle: 2010 (Cycle 16) Grant #: 16IB-0110 Award: $149,493
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2010)

The reelin signaling pathway is a critical regulator of cell positioning in the developing central nervous system. Reelin, a large secreted extracellular glycoprotein, binds to several classes of cell surface receptors including the low-density lipoprotein receptors, ApoER2 and VLDLR, and a31 integrin. Reelin signaling induces phosphorylation of the intracellular adaptor protein Dab1, triggering a cascade that modulates cytoskeletal activity and ultimately regulates cell migration. Several recent studies have suggested that members of the reelin signaling pathway may also function outside the nervous system, particularly in the developing mammary gland.

In this project, we propose to determine whether reelin signaling affects the migration and behavior of normal and metastatic mammary epithelial cells (MECs). First, using mouse primary cell cultures and tissue transplantation approaches, we will determine whether MEC migration and invasion into the stroma is regulated by reelin signaling. We will test both wild-type cells and cells in which reelin signaling is disrupted. These studies will provide a direct demonstration of the requirement for reelin signaling in regulating MEC migration. In our second aim, we will examine whether components of the reelin signaling pathway are differentially expressed in breast cancer cell lines compared to pre-malignant cells or MECs. We will also determine if the presence of reelin affects the migration of breast cancer cells using a variety of pre-malignant and malignant human cell lines. Finally, we will determine whether mammary tumor cells implanted into mutant mice that lack reelin signaling show increased metastatic behavior.

The regulation of cell positioning and migration is important for understanding how an embryo develops, but it is also critical for understanding the biology of cancer cells. Thus we hope that our studies will open up potential avenues for cancer treatment by modulating reelin signaling activity.




Final Report (2012)

The studies conducted for this proposal addressed the basic cellular mechanisms that breast cancer cells invoke during their transition to metastasis. The reelin signaling pathway has long been recognized as a critical regulator of cell positioning in the developing central nervous system. In this pathway, the extracellular matrix protein reelin binds to the cell surface receptors ApoER2 and VLDLR, activating an intracellular cascade initiated by phosphorylation of the Dab1 adaptor protein and ending with the inhibition of the tau protein, which stabilizes the cytoskeleton, causing a cell to stop migrating. Prior studies from my laboratory showed that reelin and Dab1 are also expressed in the developing mammary gland, suggesting that reelin signaling may also regulate the migration of cells in the mammary gland.

During the first year of this study, we demonstrated that disrupting the reelin signaling pathway disrupts the normal development of the ductal network of the mammary gland. We demonstrated that this disruption is intrinsic to the mammary gland using transplantation studies. In these studies, we collected mammary gland tissue from Dab1 mutant mice and transplanted it into wildt-ype hosts. The transplanted tissue failed to regenerate, suggesting that the ability to respond to reelin signaling is a necessary component of the normal mammary gland. We also determined that the presence of reelin protein inhibits the migratory ability of wild-type mammary gland cells, but that mammary gland cells collected from Dab1 mutant mice failed to respond to the presence of reelin.

Next, we obtained 8 human breast cancer cell lines representative of different types of breast cancer. We designed PCR primers for human components of the reelin signaling pathway and have established that many of these pathway components are reduced or absent in the breast cancer cell lines. These studies also revealed that Dab1 is alternatively spliced in the mammary gland, a novel result. In addition, we obtained a mouse breast cancer cell line, the 4T1 line. We found that these cells failed to respond to reelin in Boyden chamber migration assays. We have transplanted cells from this line into reeler and Dab1 mutant mice. Surprisingly, we found that the metastatic migration of this line is almost totally inhibited in both Dab1 and reeler mutant mice. This contradicted our expectation that loss of reelin signaling would accelerate metastatic migration and suggests that loss of reelin directly affects interactions made by the breast cancer cells with the extracellular matrix.

Thus, we have completed the majority of the proposed aims in the original application and have made observations that will lead to future experiments addressing the role of reelin signaling in regulating interactions between breast cancer cells and the extracellular matrix.

Publications:
Reelin Deficiency Delays Mammary Tumor Growth and Metastatic Progression



Disruption of reelin signaling alters mammary gland morphogenesis.
Periodical:Development
Index Medicus:
Authors: Khialeeva E, Lane TF, Carpenter EM
Yr: 2011 Vol: 138 Nbr: 4 Abs: Pg:767-76