Abstract
BackgroundThe 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 FindingsHerein, 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.SignificanceThis assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.
Highlights
A goal of modern molecular cancer diagnostics is to identify the underlying molecular signature of cancers on a patient-bypatient basis to guide the selection of an appropriate therapeutic regimen [1]
We described the use of a dual antibody-based, proximity-driven assay for the detection and quantitation of receptor dimers and protein complexes in the HER3/PI3K/Akt pathway
In matched FFPE sample and cell lysate studies, there is good qualitative agreement between the VeraTag assay and data obtained from traditional Western blotting or co-immunoprecipitation experiments
Summary
A goal of modern molecular cancer diagnostics is to identify the underlying molecular signature of cancers on a patient-bypatient basis to guide the selection of an appropriate therapeutic regimen [1]. The capability to measure individual proteins, protein trafficking and localization, protein-protein interactions and protein phosphorylation are key requisites to deduce pathway activation and correlate specific signaling events with biological outcomes such as cell growth and survival or resistance/sensitivity to therapeutic treatments [2]. Obtaining such measurements from formalin-fixed, paraffin-embedded (FFPE) samples is necessary since patient biopsies are routinely preserved in this format for histological assessment: biochemical techniques suitable for this sample type are severely limited. Our findings demonstrate a number of viable assays measuring HER3-complex formation that are useful for assessing HER3 activity in patient tumor samples
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