ABCB1 and ABCC11 confer resistance to eribulin in breast cancer cell lines

Abstract

This study aimed to elucidate the mechanisms underlying the resistance of breast cancer to eribulin. Seven eribulin-resistant breast cancer cell lines (MCF7/E, BT474/E, ZR75-1/E, SKBR3/E, MDA-MB-231/E, Hs578T/E, and MDA-MB-157/E) were established. mRNA and protein expression of ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 11 (ABCC11) increased in all eribulin-resistant cell lines compared to the parental cell lines. When ABCB1 or ABCC11 expression was inhibited by small interfering RNA in MCF7/E, BT474/E, and MDA-MB-231/E cells, eribulin sensitivity was partially restored. Moreover, eribulin resistance was attenuated additively by inhibiting ABCB1 and ABCC11 in MCF7/E cells. Additionally, overexpression of exogenous ABCB1 or ABCC11 in HEK293T cells conferred resistance to eribulin. MCF7/E and MDA-MB-231/E cells showed cross-resistance to paclitaxel, doxorubicin, and fluorouracil. Inhibition of ABCB1 partially restored paclitaxel and doxorubicin sensitivity. Partial restoration of fluorouracil sensitivity was induced by inhibiting ABCC11 in MCF7/E and MDA-MB-231/E cells. Both ABCB1 and ABCC11 are involved in the development of eribulin resistance in breast cancer cells in vitro regardless of the breast cancer subtype. Thus, ABCB1 and ABCC11 expression may be used as a biomarker for predicting the response to eribulin in patients with breast cancer. Concomitant inhibition of ABCB1 and ABCC11 might help enhance the antitumor effects of eribulin.

Keywords: ABCB1; ABCC11; breast cancer; drug resistance; eribulin.

Figure 1. Sensitivity to eribulin in eribulin-resistant breast cancer cells and their parental cells

Eribulin-resistant breast cancer cell lines were obtained by culturing MCF7, BT474, ZR75-1, SKBR3, MDA-MB-231, Hs578T, and MDA-MB-157 cells with stepwise-increasing doses of eribulin for more than 6 months. Sensitivity to eribulin was assayed by using the WST assay. Each cell line with “/E” indicates an established eribulin-resistant cell line. Closed circles (●) indicate parental cells, whereas open circles (○) indicate eribulin-resistant cells. The error bars represent the standard error of the value obtained in the experiments performed in triplicate.

Figure 2. mRNA and protein expression of ABCB1 and ABCC11 in eribulin-resistant breast cancer cells and their parental cells

A. ABCB1 mRNA expression quantitated by real-time RT-PCR. β-actin was used as an internal control. “P” indicates parental cell line and “E” indicates eribulin-resistant cell line. The error bars represent the standard error of the value obtained in the experiments performed in triplicate. B. ABCB1 protein expression analyzed with western blotting. β-actin was used as a loading control. C. ABCC11 mRNA expression quantitated by real-time RT-PCR. D. ABCC11 protein expression analyzed by western blotting. *P < 0.05 for parental cell line vs. eriburin-resistant cell line.

Figure 3. Effects of ABCB1 or ABCC11 knockdown in eribulin-resistant breast cancer cells

The expression of ABCB1 and ABCC11 in MCF7/E, BT474/E, and MDA-MB231/E cells was inhibited by siRNA, and the sensitivity to eribulin was tested using WST assays. A. ABCB1 mRNA expression quantitated by real-time RT-PCR (upper panel) and representative results of western blot analyses (lower panel) in MCF7/E, BT474/E, and MDA-MB-231/E cells transfected with siRNA targeting ABCB1 (si-ABCB1) or control siRNA (si-control). *P < 0.05 for si-control vs. si-ABCB1. β-actin was used as a loading control. The error bars represent the standard error of the value obtained in the experiments performed in triplicate. B. Sensitivity to eribulin was measured in eribulin-resistant cells transfected with siRNA (si-control or si-ABCB1) and the parental cells. Closed circles (●) indicate parental cells, whereas open circles (○) indicate eribulin-resistant cells transfected with si-control; closed triangles (▲) indicate eribulin-resistant cells transfected with si-ABCB1, respectively. C. ABCC11 mRNA expression quantitated by real-time RT-PCR (upper panel) and representative results of the western blot analyses (lower panel) in MCF7/E, BT474/E, and MDA-MB-231/E cells transfected with siRNA targeting ABCC11 (si-ABCC11) or control siRNA (si-control). *P < 0.05 for si-control vs. si-ABCC11. β-actin was used as a loading control. D. Sensitivity to eribulin was measured in eribulin-resistant cells transfected with siRNA (si-control or si-ABCC11) and the parental cells. Closed circles (●) indicate parental cells, open circles (○) indicate eribulin-resistant cells transfected with si-control, and open triangles (D) indicate eribulin-resistant cells transfected with si-ABCC11. E. ABCB1 and ABCC11 mRNA expression quantitated by real-time RT-PCR (upper panel) and representative results of the western blot analyses (lower panel) in MCF7/E cells transfected with siRNA targeting ABCB1 (si-ABCB1), ABCC11 (si-ABCC11), both ABCB1 and ABCC11 (si-ABCB1 + si-ABCC11), or control siRNA (si-control). *P< 0.05. F. Sensitivity to eribulin was measured in the parental MCF7 cells and eribulin-resistant MCF7/E cells transfected with each siRNA. Closed circles (●) indicate parental MCF7 cells, open circles (○) indicate MCF7/E cells transfected with si-control, closed triangles (∆) indicate MCF7/E cells transfected with si-ABCB1, open triangles (∆) indicate MCF7/E cells transfected with si-ABCC11, and open squares (□) indicate MCF7/E cells transfected with both si-ABCB1 and si-ABCC11. The error bars represent the standard error of the value obtained in the experiments performed in triplicate.

Figure 4. Effects of ABCB1 or ABCC11 overexpression on the sensitivity of HEK293T cells to eribulin, paclitaxel, doxorubicin, and fluorouracil

A plasmid expressing Flag-ABCB1 (pcDNA3.1-FLAG-ABCB1) or Flag-ABCC11 (pcDNA3.1-FLAG-ABCC11) or an empty vector (pcDNA3.1) was transfected into HEK293T cells, and we tested whether overexpression of ABCB1 or ABCC11 altered the sensitivity to eribulin, paclitaxel, doxorubicin, and fluorouracil in the transiently transfected HEK293T cells. A. ABCB1 and ABCC11 protein expression in HEK-293T cells transfected with the plasmid DNA 24 h after transfection. Exogenously expressed ABCB1 and ABCC11 were detected by the specific antibodies for ABCB1 and ABCC11, and also the anti-FLAG antibody. The sensitivity to eribulin B. paclitaxel C. doxorubicin D. and fluorouracil E. in HEK293T cells transfected with the empty vector (HEK293T-vector) or the expression vectors for ABCB1 (HEK293T-ABCB1) or ABCC11 (HEK293T-ABCC11) was determined 72 h after transfection. Closed circles (●) indicate HEK293T-vector, open circles (○) indicate HEK293T-ABCB1, and closed triangles (▲) indicate HEK293T-ABCC11. The error bars represent the standard error of the value obtained in the experiments performed in triplicate.

Figure 5. Cross-resistance to paclitaxel, doxorubicin, and fluorouracil in eribulin-resistant breast cancer cells

Sensitivity to paclitaxel A. doxorubicin B. and fluorouracil C. in eribulin-resistant and parental MCF7 and MDA-MB231 cells was determined by WST assay. Closed circles (●) indicate parental cells, whereas open circles (○) indicate eribulin-resistant cells. The error bars represent the standard error of the value obtained in experiments performed in triplicate.

Figure 6. Effects of ABCB1 and ABCC11 knockdown on cross-resistance to paclitaxel, doxorubicin, and fluorouracil in eribulin-resistant breast cancer cells

The expression of ABCB1 and ABCC11 in MCF7/E and MDA-MB231/E cells was inhibited by siRNA, and the sensitivity to paclitaxel, doxorubicin, and fluorouracil was tested by WST assay. Sensitivity to paclitaxel A. doxorubicin B. and fluorouracil C. in eribulin-resistant cells transfected with si-ABCB1 or si-control and the parental cells. Sensitivity to paclitaxel. D. doxorubicin E. and fluorouracil F. in eribulin-resistant cells transfected with si-ABCC11 or si-control and the parental cells. Closed circles (●) indicate parental cells, open circles (○) indicate eribulin-resistant cells transfected with si-control, and closed triangles (▲) indicate eribulin-resistant cells transfected with si-ABCB1 (A, B, and C) or with si-ABCC11 (D, E, and F), respectively.