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Updated: Jun 4, 2026
Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers
Published on: December 5, 2017
Profiling the HER3/PI3K pathway in breast tumors using proximity-directed assays identifies correlations between
Ali Mukherjee1, Youssouf Badal, Xuan-Thao Nguyen
1Department of Oncology, Monogram Biosciences, South San Francisco, California, United States of America. amukherjee@monogrambio.com
Background:
The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples.
Methodology And Principal Findings:
Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation.
Significance:
This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.
Insights
New proximity assays measure HER3 signaling in FFPE tumor samples, overcoming limitations of current methods. These assays accurately assess pathway activation and protein interactions, aiding targeted cancer therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Accurate measurement of therapeutic targets and signaling complexes is crucial for targeted antineoplastic therapies.
- HER3 signaling is a key growth promoter and resistance mechanism in various tumors, particularly those expressing EGFR and HER2.
- Existing methods for studying these interactions are insufficient for formalin-fixed, paraffin-embedded (FFPE) tumor samples.
Purpose of the Study:
- To develop and validate proximity-directed assays for measuring protein interactions and phosphorylation in the HER3/PI3K/Akt pathway within FFPE samples.
- To assess the capability of these assays to provide insights into the functional state of the HER3 signaling pathway.
- To evaluate the utility of these assays in both preclinical models and human tumor samples.
Main Methods:
- Development of a panel of proximity-directed assays.
- Utilized FFPE breast cancer cell line and tumor models for validation.
- Analyzed ligand-dependent and independent pathway activation, protein-protein interactions, and phosphorylation levels.
- Investigated effects of HER2 inhibitors on the HER receptor pathway.
Main Results:
- Demonstrated ligand-dependent and independent activation of the HER3/PI3K/Akt pathway in breast cancer cell lines with strong correlations between key analytes.
- Observed novel molecular effects of HER2 inhibition beyond expected outcomes.
- Showcased preservation of HER3/PI3K complex formation in delayed fixation xenograft models, unlike unstable HER3 phosphorylation.
- Found correlations between HER3 phosphorylation and receptor interactions in breast tumor samples, suggesting phosphorylation is not always a necessary surrogate for activation.
Conclusions:
- The developed assay system quantitatively measures therapeutically relevant responses in HER3 signaling.
- Enables comprehensive molecular profiling of receptor networks in FFPE preclinical and tumor models.
- Provides a valuable tool for advancing targeted therapies and understanding resistance mechanisms.
