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p185HER2 signal transduction in breast cancer cells
G K Scott1, J M Dodson, P A Montgomery
1Cancer Research Institute, University of California, San Francisco 94143.
Abstract:
A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2-overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neu-overexpressing breast cancer cells (BT-474 and SK-Br-3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56-kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5- stimulated phosphorylation of p185HER2 and ptyr56 was a 5-10-fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity and a 2-fold induction of inositol 1,4,5-trisphosphate 3'-kinase activity. The increased phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and also co-eluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonistic-like signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.
Insights
The monoclonal antibody 4D5 activates signaling in HER2-overexpressing breast cancer cells, leading to phosphorylation of p185HER2 and a novel protein, ptyr56. This antibody shows potential for targeted breast cancer therapy by modulating receptor activity.
Area of Science:
- Oncology
- Molecular Biology
- Signal Transduction
Background:
- The monoclonal antibody 4D5 targets the extracellular domain of p185HER2.
- p185HER2 overexpression is linked to aggressive breast cancer.
- Understanding HER2-mediated signaling is crucial for targeted therapies.
Purpose of the Study:
- To investigate signal transduction pathways activated by the antibody 4D5.
- To identify phosphotyrosyl protein substrates associated with p185HER2 upon 4D5 binding.
- To explore the therapeutic potential of 4D5 in HER2-overexpressing breast cancer.
Main Methods:
- Utilized a partially agonistic monoclonal antibody (4D5) against p185HER2.
- Exposed HER2/neu-overexpressing breast cancer cells (BT-474, SK-Br-3) to 4D5.
- Analyzed protein phosphorylation, mRNA induction, and enzyme activities using immunoprecipitation and immunoaffinity chromatography.
Main Results:
- 4D5 induced rapid phosphorylation of p185HER2 and a 56-kDa phosphotyrosyl protein (ptyr56) in overexpressing cells.
- Observed 5-10 fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity.
- Demonstrated that phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and co-eluted with ptyr56.
Conclusions:
- Short-term 4D5 activation of p185HER2 mimics agonistic signaling of growth factor receptors.
- ptyr56 is identified as a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.
- These findings support 4D5 as a potential therapeutic agent for HER2-overexpressing breast cancers.