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Insulin receptor substrate-1 involvement in epidermal growth factor receptor and insulin-like growth factor receptor
Janice M Knowlden1, Helen E Jones, Denise Barrow
1Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3XF, UK.
Abstract:
Classically the insulin receptor substrate-1 (IRS-1) is an essential component of insulin-like growth factor type 1 receptor (IGF-IR) signalling, providing an interface between the receptor and key downstream signalling cascades. Here, however, we show that in tamoxifen-resistant MCF-7 (Tam-R) breast cancer cells, that are highly dependent on epidermal growth factor receptor (EGFR) for growth, IRS-1 can interact with EGFR and be preferentially phosphorylated on tyrosine (Y) 896, a Grb2 binding site. Indeed, phosphorylation of this site is greatly enhanced by exposure of these cells, and other EGFR-positive cell lines, to EGF. Importantly, while IGF-II promotes phosphorylation of IRS-1 on Y612, a PI3-K recruitment site, it has limited effect on Y896 phosphorylation in Tam-R cells. Furthermore, EGF and IGF-II co-treatment, reduces the ability of IGF-II to phosphorylate Y612, whilst maintaining Y896 phosphorylation, suggesting that the EGFR is the dominant recruiter of IRS-1 in this cell line. Significantly, challenge of Tam-R cells with the EGFR-selective tyrosine kinase inhibitor gefitinib, for 7 days, reduces IRS-1/EGFR association and IRS-1 Y896 phosphorylation, while promoting IRS-1/IGF-IR association and IRS-1 Y612 phosphorylation. Furthermore, gefitinib significantly enhances IGF-II-mediated phosphorylation of IRS-1 Y612 and AKT in Tam-R cells. Importantly, induction of this pathway by gefitinib can be abrogated by inhibition/downregulation of the IGF-IR. Our data would therefore suggest a novel association exists between the EGFR and IRS-1 in several EGFR-positive cancer cell lines. This association acts to promote phosphorylation of IRS-1 at Y896 and drive MAPK signalling whilst preventing recruitment of IRS-1 by the IGF-IR and inhibiting signalling via this receptor. Treatment with gefitinib alters the dynamics of this system, promoting IGF-IR signalling, the dominant gefitinib-resistant growth regulatory pathway in Tam-R cells, thus, potentially limiting its efficacy.
Insights
Insulin receptor substrate-1 (IRS-1) interacts with epidermal growth factor receptor (EGFR) in tamoxifen-resistant breast cancer cells, promoting MAPK signaling. Gefitinib treatment shifts IRS-1 signaling to the insulin-like growth factor type 1 receptor (IGF-IR) pathway.
Area of Science:
- Oncology
- Molecular Cell Biology
- Signal Transduction
Background:
- Insulin receptor substrate-1 (IRS-1) is a key mediator in insulin-like growth factor type 1 receptor (IGF-IR) signaling.
- Tamoxifen-resistant (Tam-R) breast cancer cells rely heavily on epidermal growth factor receptor (EGFR) for growth.
Purpose of the Study:
- To investigate the novel interactions between IRS-1 and EGFR in Tam-R breast cancer cells.
- To elucidate the downstream signaling consequences of IRS-1 and EGFR association.
- To determine the effect of EGFR inhibition on IRS-1 signaling dynamics.
Main Methods:
- Cell culture of tamoxifen-resistant MCF-7 (Tam-R) and other EGFR-positive breast cancer cell lines.
- Stimulation with EGF, IGF-II, and co-treatments.
- Treatment with gefitinib, an EGFR-selective tyrosine kinase inhibitor.
- Analysis of IRS-1 phosphorylation at specific tyrosine residues (Y896, Y612) via Western blotting or similar techniques.
- Assessment of protein-protein interactions (IRS-1/EGFR, IRS-1/IGF-IR) using co-immunoprecipitation.
- Investigation of downstream signaling pathways (MAPK, AKT) and the role of IGF-IR.
Main Results:
- IRS-1 interacts with EGFR in Tam-R cells, leading to preferential phosphorylation at Y896 (a Grb2 binding site), enhanced by EGF.
- IGF-II primarily phosphorylates IRS-1 at Y612 (a PI3-K recruitment site), with limited Y896 phosphorylation.
- Co-treatment with EGF and IGF-II favors Y896 phosphorylation, indicating EGFR dominance in IRS-1 recruitment.
- Gefitinib treatment reduces IRS-1/EGFR association and Y896 phosphorylation, while increasing IRS-1/IGF-IR association and Y612 phosphorylation.
- Gefitinib enhances IGF-II-mediated phosphorylation of IRS-1 Y612 and AKT, a pathway dependent on IGF-IR.
Conclusions:
- A novel association between EGFR and IRS-1 exists in EGFR-positive cancer cells, promoting MAPK signaling via Y896 phosphorylation and inhibiting IGF-IR signaling.
- Gefitinib treatment disrupts this EGFR-IRS-1 interaction, promoting IGF-IR signaling, which represents a dominant gefitinib-resistant growth pathway in Tam-R cells.
- Understanding this signaling interplay is crucial for predicting and potentially overcoming resistance to EGFR-targeted therapies in breast cancer.
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