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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

BMC Cancer 1/2017

Development of a test that measures real-time HER2 signaling function in live breast cancer cell lines and primary cells

Zeitschrift:
BMC Cancer > Ausgabe 1/2017
Autoren:
Yao Huang, David J. Burns, Benjamin E. Rich, Ian A. MacNeil, Abhijit Dandapat, Sajjad M. Soltani, Samantha Myhre, Brian F. Sullivan, Carol A. Lange, Leo T. Furcht, Lance G. Laing
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12885-017-3181-0) contains supplementary material, which is available to authorized users.

Abstract

Background

Approximately 18–20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies.

Methods

A new biosensor-based test (CELxTM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2– breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor.

Results

The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2– cell line.

Conclusions

Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab.
Zusatzmaterial
Additional file 1: Table S1. Antibodies used in this study. All epitopes were extracellular with the exceptions of ER and PR. All antibodies were purchased from companies as listed who provided empirical demonstration of each of the antibodies for our applications. (DOCX 30 kb)
12885_2017_3181_MOESM1_ESM.docx
Additional file 2: Figure S1. The PI3K/AKT pathway significantly contributes to the ligand-driven HER2 signaling activities detected by CELx HSF tests. (A and B) SKBr3 cells were seeded in sensor plates and then treated with a serial titration of the PI3K/AKT pathway inhibitor GDC-0941 (0 nM to 2700 nM) two hours prior to maximal stimulation with NRG1b (800 pM) (A) or EGF (600 pM) (B). CELx curves are displayed using Baseline Delta CI values. The relative CELx signals were baseline subtracted to the time point (arrow) when the stimulus (EGF or NRG1b) was added and the signals induced by stimulus alone without the drug (GDC-0941) were set as baselines. Dose–response curves of GDC-0941 inhibition on NRG1b and EGF-driven HER2 signals are shown in the insets. (PDF 92 kb)
12885_2017_3181_MOESM2_ESM.pdf
Additional file 3: Figure S2. The MEK/ERK pathway does not significantly contribute to the ligand-driven HER2 signaling activities detected by CELx HSF tests. SKBr3 cells were seeded in sensor plates and then treated with a serial titration of the MEK/ERK pathway inhibitor trametinib (0 nM to 2700 nM) two hours prior to maximal stimulation with NRG1b (800 pM) (A) or EGF (600 pM) (B). CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. No trametinib dose-dependent inhibition on NRG1b or EGF-driven HER2 signals was detected (insets). (PDF 107 kb)
12885_2017_3181_MOESM3_ESM.pdf
Additional file 4: Figure S3. The p38 MAPK pathway does not significantly contribute to the ligand-driven HER2 signaling activities detected by CELx HSF tests. SKBr3 cells were seeded in sensor plates and then treated with a serial titration of the p38 MAPK pathway inhibitor doramapimod (0 nM to 810 nM) two hours prior to maximal stimulation with NRG1b (800 pM) (A) or EGF (600 pM) (B). CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. No doramapimod dose-dependent inhibition on NRG1b or EGF-driven HER2 signals was detected. (PDF 99 kb)
12885_2017_3181_MOESM4_ESM.pdf
Additional file 5: Figure S4. The JNK pathway does not significantly contribute to the ligand-driven HER2 signaling activities detected by CELx HSF tests. SKBr3 cells were seeded in sensor plates and then treated with a serial titration of the JNK pathway inhibitor SP600125 (0 nM to 2700 nM) two hours prior to maximal stimulation with NRG1b (800 pM) (A) or EGF (600 pM) (B). CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. No SP600125 dose-dependent inhibition on NRG1b or EGF-driven HER2 signals was detected. (PDF 98 kb)
12885_2017_3181_MOESM5_ESM.pdf
Additional file 7: Figure S5. Fluorescence flow cytometry showing two epithelial markers that delineate basal epithelial, stromal (fibroblast), progenitor epithelial, and luminal cells in primary cell R49 from short term culture. The image shows very few fibroblasts and significant luminal, basal and some progenitor populations. In contrast, a second image (right panel) is shown for the combined experimental runs of SKBr3 (luminal breast cancer reference) and MDA-MB-231 (basal epithelial breast cancer reference) cell lines demonstrating their more monoclonal character. (PDF 179 kb)
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Additional file 8: Figure S6. The MAPK pathway does not significantly contribute to the ligand-driven HER2 signaling activities detected by CELx HSF tests in breast cancer primary cells. Patient R54 breast tumor-derived primary cells (15,000 cells per well) pre-seeded in sensor plates were treated with a serial titration of the MEK/ERK pathway inhibitor trametinib (0 nM to 810 nM) two hours prior to stimulation with NRG1b (800 pM) (A) or EGF (600 pM) (B). No trametinib dose-dependent inhibition on NRG1b or EGF-driven HER2 signals was detected when data were subjected to dose–response inhibitory curve fitting. (PDF 44 kb)
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