Novel Mechanisms of ErbB-2-Mediated Breast Cancer Metastasis

Institution: Scripps Research Institute
Investigator(s): Richard Klemke, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6KB-0046 Award: $257,006
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2000)
ErbB-2 (also known as HER-2/neu) is a cell surface growth factor that sends signals into breast cancer cells to stimulate their differentiation and proliferation. Clinical studies indicate that the erbB-2 gene is one of the most frequently altered in human cancers. This is most notable in breast cancer where erbB-2 gene amplification and overabundance occurs in approximately 30% of patients. This is associated with a more malignant and metastatic phenotype resulting in poor clinical outcome. While there is extensive evidence linking erbB-2 to the development of cancer, there remain significant unanswered questions as to how erbB-2 actually causes cell changes associated with breast cancer. We are interested in the structure of the portion of the erbB-2 protein that extends inside of cells. We want to know how specific protein segments serve to ‘activate’ so-called signaling pathways inside of breast cancer cells. And, we want to be able to measure and correlate the ‘signaling’ changes with the biological properties of cell migration in the metastasis to other parts of the body.

Recent evidence indicates that too much erbB-2 leads to a self-activation process that involves addition of phosphate (autophosphorylation) on five tyrosine residues present in the carboxyl terminus of the protein. This could provide binding sites for other molecules to link erbB-2 to distinct signaling pathways and specific biological responses. However, the role of autophosphorylation in the development and spread of breast cancer has not been investigated. In this CBCRP-funded project we will study the relationship of erbB-2 autophosphorylation site(s) responsible for the activation of ERK and CAS/Crk signaling pathways in breast cancer cells. Our biological tests include the ability of the breast cancer cells to migrate and invade an artificial matrix compound. As control experiments we are able to alter the amount of erbB-2 in cells and introduce mutations into the protein sequence that eliminate the individual sites that can be phosphorylated.

Results from these studies will define the role of autophosphorylation of erbB-2 in mediating metastasis of breast cancer. As more information is gained, we can better target these regions of erbB-2 and the critical signaling proteins to combat this aspect of the disease. And, this information could be applicable to erbB-2 negative breast cancer, which although lacking this growth factor may use similar biological and molecular pathways via alternate means.

Final Report (2002)
ErbB-2 (also known as HER-2/neu) is a gene that causes breast cancer in humans. Clinical studies indicate that the erbB-2 gene is one of the most frequently (approx. 30%) altered in human breast cancer. This gene is also associated with cancers that have a more malignant and metastatic phenotype, which results in poor patient survival. Although traditionally considered a growth factor, we are interested in the ability of the erbB-2 gene to facilitate, in some way, the spread of cancer cells from the primary site (i.e., breast) to distant sites within the body, such as the lungs or bone. Recent evidence indicates that adhesive proteins in breast tissue may contribute to the development of cancer and its movement in the body.

In year one, we investigated how erbB-2 activates migration machinery of cells. Our work showed that CAS/Crk coupling and ERK activation are critical for the process of cell movement downstream of erbB-2 . ERK1 and 2 signaling promotes phosphorylation of myosin light chain kinase (MLCK) leading to activation of the actin/myosin "motor" to facilitate cell migration. We found that CAS/Crk is a second distinct signaling pathway that exists to regulate cell migration. In this case, tyrosine phosphorylation of the substrate domain of p130 CAS (CAS) and its association with c-Crk II (Crk) via its src-homology 2 (SH2) binding motif leads to the activation of the small GTPase Rac and membrane ruffling and cell migration. Importantly, we discovered that erbB-2 serves as a critical component that directly couples erbB family receptors to the cytoskeleton of migratory cells by facilitating the activation of these two pathways. In fact, without coupling to erbB-2, all erbB family receptors are severely impaired in their ability to regulate CAS/Crk coupling and ERK activation and, therefore, are deficient in their ability to mediate cell migration and invasion of the extracellular matrix.

In year two (final year), our work demonstrated that erbB-2 autophosphorylation within its kinase domain is critical for coupling erbB-2 and its effector molecules to the migration machinery of cells and invasion of the surrounding tissues. We have been able to demonstrate that amplification of erbB-2 at the cell surface leads to receptor dimerization and autophosphorylation of five tyrosine residues present in the carboxy terminal domain of erbB-2. This facilitates CAS/Crk coupling and ERK activation, two distinct signaling pathways that regulate the actin-myosin cytoskeleton of migratory cells. We also discovered that Abl tyrosine kinase is an important regulatory of CAS/Crk coupling in metastatic cells. In fact, we found that Abl serves as negative regulator of CAS/Crk coupling and thus serves to suppress migration. We found that Abl activity is reduced in metastatic cells compared to non-metastatic cells.

Future work is planned to investigate the role of these signaling pathways controlling invasion of the basement membrane in animal models. Our work will aid in developing targeted therapeutics designed to prevent the spread of breast cancer in patients.