Abstract
BackgroundThe mechanisms of endocrine resistance are complex, and deregulation of several oncogenic signalling pathways has been proposed. We aimed to investigate the role of the EGFR and Src-mediated STAT3 signalling pathway in tamoxifen-resistant breast cancer cells.MethodsThe ER-positive luminal breast cancer cell lines, MCF-7 and T47D, were used. We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. Cell viability was determined using an MTT assay, and protein expression levels were determined using western blot. Cell cycle and annexin V staining were analysed using flow cytometry.ResultsTamR cells showed decreased expression of estrogen receptor and increased expression of EGFR. TamR cells showed an acceleration of the G1 to S phase transition. The protein expression levels of phosphorylated Src, EGFR (Y845), and STAT3 was increased in TamR cells, while phosphorylated Akt was decreased. The expression of p-STAT3 was enhanced according to exposure time of tamoxifen in T47D cells, suggesting that activation of STAT3 can cause tamoxifen resistance in ER-positive breast cancer cells. Both dasatinib (Src inhibitor) and stattic (STAT3 inhibitor) inhibited cell proliferation and induced apoptosis in TamR cells. However, stattic showed a much stronger effect than dasatinib. Knockdown of STAT3 expression by siRNA had no effect on sensitivity to tamoxifen in MCF-7 cells, while that enhanced sensitivity to tamoxifen in TamR cells. There was not a significant synergistic effect of dasatinib and stattic on cell survival. TamR cells have low nuclear p21(Cip1) expression compared to MCF-7 cells and inhibition of STAT3 increased the expression of nuclear p21(Cip1) in TamR cells.ConclusionsThe EGFR and Src-mediated STAT3 signalling pathway is activated in TamR cells, and inhibition of STAT3 may be a potential target in tamoxifen-resistant breast cancer. An increase in nuclear p21(Cip1) may be a key step in STAT3 inhibitor-induced cell death in TamR cells.
Highlights
The mechanisms of endocrine resistance are complex, and deregulation of several oncogenic signalling pathways has been proposed
Overexpression To confirm the resistance to tamoxifen in tamoxifen-resistant breast cancer (TamR) cells, we performed a cell viability assay after treatment with various concentrations of 4-OH-TAM in MCF-7 and TamR cells
TamR cells had a cell cycle shift from G0/G1 to S phase compared with MCF-7 cells, indicating that G1/S cell cycle progression was increased in TamR cells (Fig. 1b)
Summary
The mechanisms of endocrine resistance are complex, and deregulation of several oncogenic signalling pathways has been proposed. A life-threatening disease that is among the most common cancers in women, is classified into subtypes according to the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) Among these subtypes, hormone receptor (HR)-positive breast cancer, referring to ER- and/or PR-positive tumours, accounts for 70% of all breast cancers, and endocrine therapy such as tamoxifen or aromatase inhibitors is an important therapeutic option in HR-positive breast cancer [1]. We investigated the roles of the EGFR and Src-mediated STAT3 signalling pathway in tamoxifen-resistant breast cancer (TamR) cells. We found that inhibition of STAT3 increased the expression of nuclear p21(Cip1) in TamR cells These findings suggest a potential role of the EGFR and Src-mediated STAT3 signalling pathway in maintaining cell survival in tamoxifen-resistant breast cancer and inhibition of STAT3 may be a more effective treatment strategy for tamoxifen-resistant breast cancer
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