The Role of a Newly Discovered Neuregulin in Breast Cancer

Institution: Scripps Research Institute
Investigator(s): Cary Hsing Chao Lai, Ph.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4IB-0055 Award: $87,472
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1998)
A number of protein factors and hormones affect the growth, survival and progression of breast cells from a normal to a malignant state. Our work focuses on finding molecules related to the molecule, neuregulin, which is known to activate the ErbB2 receptor that is expressed in roughly one-fourth of all human breast tumors. We believe that it is important to identify these molecules as their activities could affect the development of breast cancer and influence the growth rate of malignant cells.

We have identified a candidate novel neuregulin, AA238077, through a search of the databases that contain the DNA sequences of many genes of unknown function. When the sequence of AA238077 is compared with collections of known genes, it is most closely related to two of the previously identified distinct neuregulin genes, neuregulin-1 and neuregulin-3. While this similarity suggests that it too is a neuregulin, we must test its ability to activate ErbB2 and the related receptors ErbB3 and ErbB4 before its identity as a neuregulin-like molecule can be confirmed. We will accomplish this by first preparing the AA238077 protein in the laboratory and testing the ability of this protein to stimulate the activity of these receptors. Many breast cancer cells express one or more of the four ErbB receptors and we will use a set of tumor cell lines to assess the activity of AA238077.

The ability of neuregulin-1 to bind to breast cancer cells is currently being exploited as a way to target the specific destruction of ErbB2-expressing tumors. The general strategy is to couple a toxic substance to the neuregulin-1 molecule so that the breast tumor cells will be preferentially destroyed because they express more ErbB2 and other ErbB receptors than normal cells. Our previous identification and characterization of neuregulin-2 suggests that it binds to and activates certain breast cancer cells better than neuregulin-l. This suggests that each of the neuregulins, and perhaps AA238077, could be separately used to target different tumor populations. In addition, as it has been observed that neuregulin-1 can inhibit the ability of some breast cancer cells to divide, the distinct signaling properties of each of the neuregulins holds the promise that they may be useful to control the growth of different sets of breast tumors. Lastly, on a very practical level, it is important to know if any of the neuregulins is expressed (detectable) in the normal or cancerous breast as therapeutic strategies designed involving the use of these molecules could be affected by the presence of an as yet unidentified neuregulin-like molecule.

In order to best design therapies directed against breast cancer, it is important to identify as many of the molecular players involved. Although a complete understanding will certainly take many more years, we are in a position to make significant progress in finding the molecules that activate ErbB2 and the related receptors ErbB3 and ErbB4.

Final Report (1999)
The "neuregulins" ("NRGs") are a growing family of molecules that serve as ligands for the subfamily of receptor protein-tyrosine kinases known as the "ErbBs". The study of ErbB receptors is important to the study of breast cancer as one of these receptors, ErbB2, is present in high amounts in roughly 25% of tumors isolated from patients with breast carcinoma. The identification of novel neuregulin-like molecules is an important goal as these factors may be able to regulate the growth of tumor cells.

We have identified a novel molecule that represents the fourth member of the family, neuregulin-4 (NRG-4). This molecule is more closely related to the previously identified neuregulins, NRG-1, NRG-2 and NRG-3 than to any other molecules in the nucleic acid and protein databases. We used the AA238077 cDNA clone to screen a mouse liver cDNA library as this gene is expressed at higher levels in liver than in other tissues examined. Nucleotide sequence analysis of the positive clones confirmed the predicted protein sequence encoded by AA238077. The majority of cDNA clones appeared to be putative splice variants of NRG4 that encode short, novel proteins lacking EGF-like domains. By RT-PCR, subcloning and sequence analysis, the presence of these splice variants was confirmed in rat brain, liver, muscle and retina. The function of these predicted proteins is not understood. We have produced a recombinant GST-NRG-4 fusion protein containing the EGF-like domain and tested the ability of this protein to activate ErbB phosphorylation in breast tumor and ErbB transfected cell lines. We have determined that recombinant NRG4 can lead to the modest stimulation of ErbB4 tyrosine phosphorylation in the MDA-MB-361 breast tumor cell line. NRG-4 does not appear to stimulate ErbB receptor activation in breast cancer cell lines (MDA-MB-231, MDA-MB-453, MDA-MB-468) that do not express at least moderate levels of ErbB4.

The failure of NRG-4 to activate ErbB2 phosphorylation in these lines shows that it does not serve as a direct ligand for ErbB2. NRG-4 protein factor potently activates ErbB4 in NIH 3T3 cells that have been transfected with an ErbB4 expression vector. Its preference for activating ErbB4 is similar to that for NRG-3, which has been previously shown to be expressed in a subset of breast cancer cell lines. These studies suggest that NRG-4 can be considered a neuregulin because it exhibits amino acid sequence similarity to the other NRGs and because it activates ErbB4.