IGFBP-1 Expression Promotes Tamoxifen Resistance in Breast Cancer Cells via Erk Pathway Activation

Abstract

Insulin-like growth factor (IGF) system plays a significant role in many cellular processes, including proliferation, and survival. In estrogen receptor positive breast cancer, the level of circulating IGF-1 is positively associated with the incidence and at least 50% of cases have elevated IGF-1R signaling. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. Unfortunately, tamoxifen resistance is a frequent occurrence in patients receiving treatment and the molecular mechanisms that underlie tamoxifen resistance not adequately defined. It has recently been reported that the inhibition of IGF-1R activation and the proliferation of breast cancer cells upon tamoxifen treatment is mediated by the accumulation of extracellular insulin-like growth factor binding protein 1 (IGFBP-1). Elevated IGFBP-1 expression was observed in tamoxifen-resistant (Tam^R) MCF-7 and T-47D cells lines suggesting that the tamoxifen-resistant state is associated with IGFBP-1 accumulation. MCF-7 and T-47D breast cancer cells stably transfected with and IGFBP-1 expression vector were generated (MCF7-BP1 and T47D-BP1) to determine the impact of breast cancer cell culture in the presence of increased IGFBP-1 expression. In these cells, the expression of IGF-1R was significantly reduced compared to controls and was similar to our observations in tamoxifen-resistant MCF-7 and T-47D cells. Also similar to Tam^R breast cancer cells, MCF7-BP1 and T47D-BP1 were resistant to tamoxifen treatment, had elevated epidermal growth factor receptor (EGFR) expression, increased phospho-EGFR (pEGFR), and phospho-Erk (pErk). Furthermore, tamoxifen sensitivity was restored in the MCF7-BP1 and T47D-BP1 upon inhibition of Erk phosphorylation. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 inhibited pErk accumulation and increased tamoxifen sensitivity. Taken together, these data support the conclusion that IGFBP-1 is a key component of the development of tamoxifen resistance in breast cancer cells.

Keywords: EGFR; IGFBP-1; breast cancer; drug resistance; tamoxifen.

Figure 1

MCF7-TamR and T47D-TamR expressed more IGFBP-1 and the establishment of MCF7-BP1 and T47D-BP1 stable cell lines. Immunoblot analysis of IGFBP-1 expression in MCF-7 and T-47D stable cells. (A) Measurement of IGFBP-1 expression in MCF7-P and MCF7-TamR (left), and in T47D-P and T47D-TamR (right); (B) measurement of IGFBP-1 expression in MCF7-EV and MCF7-BP1 (left), and in T47D-EV and T47D-BP1 (right). The Coomassie blue staining indicated the even loading of the proteins from the concentrated media. Results are the representatives of 3 independent experiments. Extra-IGFBP-1: extracellular IGFBP-1; intra-IGFBP-1: intracellular IGFBP-1.

Figure 2

Expression of IGF-1R decreased in MCF7-BP1 and T47-BP1 cells. (A) Immunoblot analysis of IGF-1R protein expressions in MCF-7 and T-47D cells. (B) qRT-PCR analysis of IGF-1R mRNA levels in MCF-7 and T-47D cells. Results are the average of 3 independent experiments, and error bars are the standard error of the mean. *p < 0.05.

Figure 3

Sustained IGFBP-1 exposure increases EGFR signaling. (A) Immunoblot analysis of phospho-EGFR (Y1068), EGFR, phospho-Erk, and Erk in MCF-7 cells (top); measurement of relative cell number (%) in MCF-7 cells after 5 days of EGF treatment (bottom). (B) Immunoblot analysis of phospho-EGFR (Y1068), EGFR, phospho-Erk, and Erk in T-47D cells (top); measurement of relative cell number (%) in T-47D cells after 5 days of EGF treatment (bottom). Results are the average of 3 independent experiments, and error bars are the standard error of the mean. *p < 0.05.

Figure 4

Sustained IGFBP-1 exposure results in the development of tamoxifen resistance in breast cancer cells. (A) Measurement of relative cell number (%) in four different MCF-7 cell lines under 4-hydroxytamoxifen (4OHT) treatment for 5 days. (B) Measurement of relative cell number (%) in four different T-47D cell lines under 4-hydroxytamoxifen (4OHT) treatment for 5 days. Results are the average of 3 independent experiments, and error bars are the standard error of the mean. *p < 0.05.

Figure 5

MAPK inhibition reverses tamoxifen resistance in breast cancer cells. (A) Left: immunoblot analysis of phospho-Erk (pErk) of treated MCF7-EV; right: measurement of relative cell number (%) of MCF7-EV under different treatments for 5 days. (B) Left: immunoblot analysis of phospho-Erk (pErk) of treated MCF7-BP1; right: measurement of relative cell number (%) of MCF7-BP1 under different treatments for 5 days. (C) Left: immunoblot analysis of phospho-Erk (pErk) of treated T47D-EV; right: measurement of relative cell number (%) of T47D-EV under different treatments for 5 days. (D) Left: immunoblot analysis of phospho-Erk (pErk) of treated T47D-BP1; right: measurement of relative cell number (%) of T47D-BP1 under different treatments for 5 days. Results are the average of 3 independent experiments, and error bars are the standard error of the mean. *p < 0.05.

Figure 6

Knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 reduced the level of phospho-Erk and sensitized the cells to 4-OHT. (A) Immunoblot analysis of IGFBP-1, phospho-Erk, and Erk after transfected with non-targeting (NT) shRNA or IGFBP-1 shRNA in MCF7-BP1 (left); measurement of relative cell number (%) in MCF7-BP1 after transfected with NT shRNA or IGFBP-1 shRNA and treated with either vehicle or 1 μM 4-OHT (right). (B) Immunoblot analysis of IGFBP-1, phospho-Erk, and Erk after transfected with non-targeting (NT) shRNA or shRNA for IGFBP-1 in T47D-BP1 (left); measurement of relative cell number (%) in T47D-BP1 after transfected with NT shRNA or IGFBP-1 shRNA and treated with either vehicle or 1 μM 4-OHT (right). Results are the average of 3 independent experiments, and error bars are the standard error of the mean. *p < 0.05.

Figure 7

Knockdown of IGFBP-1 restores 4-OHT sensitivity in breast cancer cells. (A) Immunoblot analysis of IGFBP-1, phospho-Erk, and Erk after transfected with non-targeting (NT) shRNA or IGFBP-1 shRNA in MCF7-TamR (left); measurement of relative cell number (%) in MCF7-TamR after transfected with NT shRNA or IGFBP-1 shRNA and treated with either vehicle or 1 μM 4-OHT (right). (B) Immunoblot analysis of IGFBP-1, phospho-Erk, and Erk after transfected with non-targeting (NT) shRNA or shRNA for IGFBP-1 in T47D-TamR (left); measurement of relative cell number (%) in T47D-TamR after transfected with NT shRNA or IGFBP-1 shRNA and treated with either vehicle or 1 μM 4-OHT (right). Results are the average of 3 independent experiments, and error bars are the standard error of the mean. *p < 0.05.