Mechanisms of Gefitinib-mediated reversal of tamoxifen resistance in MCF-7 breast cancer cells by inducing ERα re-expression

OPEN SUBJECT AREAS: BREAST CANCER HORMONE RECEPTORS Received 3 June 2014 Accepted 15 December 2014 Published 3 February 2015 Correspondence and Mechanisms of Gefitinib-mediated reversal of tamoxifen resistance in MCF-7 breast cancer cells by inducing ERa re-expression Xia Zhang1*, Bin Zhang2*, Jie Liu1, Jiwei Liu2, Changzheng Li1, Wei Dong3, Shu Fang1, Minmin Li1, Bao Song1, Bo Tang4, Zhehai Wang1 & Yang Zhang5 1 Department of Oncology, Shandong Cancer Hospital and Institute, Jinan, Shandong 250117, P. R. China, 2Department of Oncology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, P. R. China, 3Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan, Shandong 250117, P. R. China, 4Department of Hepatobiliary Surgery, Guilin Medical University, Affiliated Hospital, Guilin, Guangxi 541001, P. R. China, 5Department of Oncology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116027, P. R. China. requests for materials should be addressed to Y.Z. (zydl@medmail. com.cn); Z.W. (wzhai8778@sina. com) or B.T. () * These authors contributed equally to this work. Estrogen receptor (ER)-positive breast cancer patients may turn ER-negative and develop acquired drug resistance, which compromises the efficacy of endocrine therapy. By investigating the phenomenon that gefitinib can re-sensitise tamoxifen (TAM)-resistant MCF-7 breast cancer cells (MCF-7/TAM) to TAM, the present study verified that gefitinib could reverse the acquired drug resistance in endocrine therapy and further explored the underlying mechanism.ERa-negative MCF-7/TAM cells were established. Upon treating the cells with gefitinib, the mRNA and protein levels of ERa and ERb, as well as the expression of molecules involved in the MAPK pathway, were examined using the RT-PCR and immunocytochemistry. The RT-PCR results showed that the mRNA levels of ERa and ERb in MCF-7/TAM cells were up-regulated following gefitinib treatment; specifically, ERa was re-expressed, and ERb expression was up-regulated. The expression of molecules involved in the MAPK pathway, including RAS, MEK1/2, and p-ERK1/2, in MCF-7/ TAM cells was significantly up-regulated, compared with MCF-7 cells. After the gefitinib treatment, the expression levels of MEK1/2 and p-ERK1/2 were significantly down-regulated. ERa loss is the primary cause for TAM resistance. Gefitinib reverses TAM resistance primarily by up-regulating the ERa mRNA level and inducing the re-expression of ERa. The MAPK pathway plays a key role in ERa re-expression. A s the primary therapeutic regimen for treating hormone receptor-positive breast cancers, endocrine therapy demonstrates an efficacy of approximately 50–60%. In clinical practice, TAM is the most commonly used endocrine therapeutic drug and has been shown to reduce the relapse and mortality rates of ER-positive breast cancers by 40–50% and 30–35%, respectively1. However, primary or acquired drug resistance is the primary cause of the compromised efficacy of endocrine therapy. Approximately 30–50% of ER-positive metastatic breast cancer patients effectively respond to initial TAM treatment. However, almost all patients ultimately develop acquired drug resistance, leading to disease progression or death, which significantly compromises the efficacy of endocrine therapy2,3. Studies of the mechanism underlying endocrine resistance and the corresponding intervention approaches have attracted substantial attention recently. In clinical practice, replacing endocrine therapeutic drugs or switching to chemotherapy is the most common strategy adopted once endocrine resistance has been developed. However, the ER status becomes negative when endocrine resistance occurs; thus, the treatment efficacy cannot be completely restored to the level of the initial treatment by switching to a different endocrine therapeutic drug4,5. Studies have demonstrated that re-expression of the ER, which is the gold standard for selecting patients for endocrine therapy, can re-sensitise breast cancer cells to endocrine therapy6,7. Therefore, searching for approaches to re-express the ER in endocrine resistant cells, investigating the mechanisms underlying ER re-expression, and screening for effective drugs to reverse endocrine resistance are key strategies for enhancing the efficacy of endocrine therapy for breast cancer. As a small-molecule tyrosine kinase inhibitor, Gefitinib is commonly used in molecularly targeted therapy for lung cancer8–10. Gefitinib has also recently been used in studies of combination endocrine therapy for breast SCIENTIFIC REPORTS | 5 : 7835 | DOI: 10.1038/srep07835 1 www.nature.com/scientificreports Figure 1 | Dose curve and time course of the effect of Gefitinib on the proliferation of MCF-7 and MCF-7/TAM cells. (A): MCF-7 and MCF-7/TAM cells were treated with different doses of Gefitinib. The absorbance values determined at 492 nm in an ELISA analyser demonstrated that Gefitinib at different concentration gradients exhibited differing degrees of inhibition on cell proliferation. (B): The absorbance values of MCF-7/TAM cells treated with different concentrations of Gefitinib for 24 h or 48 h. Effects on MCF-7 and MCF-7/TAM cell proliferation in different time and different doses of Gefitinib for detection of CCK-8. (C): the effect of different doses of Gefitinib to inhibit the proliferation of M0 and M/T group, there showed inhibitory concentrations of Gefitinib in two kinds of cells for the proliferation. (D): different concentrations of Gefitinib changes in the range of 24 h, 48 h absorbance in group M/T cells. The experiments consisted of the following groups: the MCF-7 group (M0), the MCF-7/TAM group (M/T), and the MCF7/TAM-Gefitinib (10 mg/ml) group (G10). All the experiments were repeated three times. cancer11,12. Endocrine resistance is co-regulated by the signalling networks of both the ER and epidermal growth factor receptor (EGFR), and up-regulation of the MAPK signal converts ER-positive cells into ER-negative cells13. Oh et al. found that the transient transfection of MCF-7 cells with active human epidermal growth factor receptor 2 (Her-2), MEK, Raf, or ligand-activated EGFR could down-regulate the mRNA and protein expression of the ER. Moreover, the application of MEK inhibitors was shown to stimulate ER re-expression in breast cancer cells, with these cells subsequently regaining their sensitivity to selective ER antagonists14. However, Bayliss et al. demonstrated that ER re-expression does not always result in effective responses to endocrine therapy15, as certain cancer cells fail to reexpress ER upon inhibition of the MAPK pathway. Therefore, ER reexpression in ER-negative breast cancer cells for re-sensitisation to endocrine therapy and the mechanism underlying how ER reexpression relates to the MAPK pathway remain crucial questions in endocrine therapy for breast cancers. In addition, the physiological (...truncated)