Roles of ABCC1 and ABCC4 in Proliferation and Migration of Breast Cancer Cell Lines

Abstract

ABCC1 and ABCC4 utilize energy from ATP hydrolysis to transport many different molecules, including drugs, out of the cell and, as such, have been implicated in causing drug resistance. However recently, because of their ability to transport signaling molecules and inflammatory mediators, it has been proposed that ABCC1 and ABCC4 may play a role in the hallmarks of cancer development and progression, independent of their drug efflux capabilities. Breast cancer is the most common cancer affecting women. In this study, the aim was to investigate whether ABCC1 or ABCC4 play a role in the proliferation or migration of breast cancer cell lines MCF-7 (luminal-type, receptor-positive) and MDA-MB-231 (basal-type, triple-negative). The effects of small molecule inhibitors or siRNA-mediated knockdown of ABCC1 or ABCCC4 were measured. Colony formation assays were used to assess the clonogenic capacity, MTT assays to measure the proliferation, and scratch assays and Transwell assays to monitor the cellular migration. The results showed a role for ABCC1 in cellular proliferation, whilst ABCC4 appeared to be more important for cellular migration. ELISA studies implicated cAMP and/or sphingosine-1-phosphate efflux in the mechanism by which these transporters mediate their effects. However, this needs to be investigated further, as it is key to understand the mechanisms before they can be considered as targets for treatment.

Keywords: MRP1; MRP4; breast cancer; cAMP; invasion; migration; proliferation.

Figure 1

ABCC1 and ABCC4 are both expressed in MCF-7 and MDA-MB-231 breast cancer cells. Membrane extracts (80 μg protein/well) from MCF-7 and MDA-MB-231 cells were assayed by Western blot for the expression of (a) ABCC1 or (b) ABCC4, alongside Hela cells as a positive control. ABCC1 was detected using the anti-MRP1 EPR4658 antibody (1:1000). ABCC4 was detected using the anti-ABCC4 M₄I-10 antibody (1:100). The blue arrow indicates the band corresponding to ABCC1 (a) or ABCC4 (b). Comparable sample loading was monitored afterwards using the anti-tubulin primary antibody. Uncropped images can be found in Figure S3.

Figure 2

MK571 and Reversan affect the clonogenic capacity of breast cancer cells. 100 cells/well were seeded in 6-well plates and cultured at 37 °C for 24 h, after which cells were treated with the indicated concentrations of inhibitors. Control wells were untreated cells. After 7 days of culture, the colonies formed were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, the colonies counted and measured. Example results for MDA-MB-231 cells treated with (a) MK571 or (b) Reversan. Average results for the number (c,d) and size (e,f) of colonies for MCF-7 cells (c,e) and MDA-MB-231 (d,f). Data are mean ± sem, n ≥ 6. Data were analyzed using a one-way ANOVA with a Dunnett’s post hoc test; * p < 0.05, *** p < 0.001, and **** p < 0.0001 significantly lower than the untreated sample.

Figure 3

MK571 and Reversan affect the proliferation of breast cancer cells. Fifteen thousand MCF-7 cells (a,b) or 6000 MDA-MB-231 cells (c,d) were seeded in 24-well plates. After 4 h of culture, cells were treated with inhibitors, as detailed. Cell viability was assessed at 6, 12, 24, 48, and 72 h after treatment using an MTT assay and absorbance measured at 570 nm. Data are mean ± SD, n ≥ 6. Data were analyzed using a two-way ANOVA with a Dunnett’s post hoc test. *** p < 0.001 and **** p < 0.0001 significantly lower than the untreated sample.

Figure 4

MK571 decreases the rate of migration by MDA-MB-231 cells. Cells were seeded in 24-well plates to reach 100% confluency the day of the assay. A scratch across the monolayer of the cells was carefully made, and the medium was replaced with fresh prewarmed culture medium. Cells were treated with the inhibitors as described above. Three image positions were selected from each well, and images were taken at 1-h intervals using the Cell-IQ. Representative images of MDA-MB-231 scratch assay (a). Pink lines represent the scratch edges as defined by the Cell IQ software, and the blue lines are the distance measurement between the edges. Average results for MCF-7 (b) and MDA-MB-231 (c) cell migration in the presence of inhibitors. Data are mean ± sem, n ≥ 6. Data were analyzed using a two-way ANOVA with a Dunnett’s post hoc test. * p < 0.05 significantly lower than the untreated sample.

Figure 5

ABCC1 and ABCC4 can be successfully knocked down in breast cancer cells using siRNA. Gene knockdown in breast cancer cells was performed using the INTERFERin-siRNA transfection protocol, with two different ABCC1 siRNAs (#30 or #31), two different ABCC4 siRNAs (#34 or #35), or in combination (#30/#34 or #31/#35). A negative control siRNA was also used. The effectiveness of the knockdown was measured by both RT-qPCR and Western blotting. (a) Knockdown of ABCC1 in MCF-7 cells, (b) knockdown of ABCC4 in MCF-7 cells, (c) knockdown of ABCC1 in MDA-MB-231 cells, (d) knockdown of ABCC4 in MDA-MB231 cells and (e) double knockdown of both transporters in each cell line. Uncropped images can be found in Figure S4. RT-qPCR data are mean ± sem, n = 3. Data were analyzed using a one-way ANOVA with a Dunnett’s post hoc test. * p < 0.05 and ** p < 0.01 significantly lower than the negative siRNA sample.

Figure 6

Combined knockdown of ABCC1 and ABCC4 affects the clonogenic capacity of breast cancer cells. (a,b) Clonogenic capacity of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was analyzed using a colony formation assay. (c,d) Proliferation of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was analyzed using an MTT assay. Data are mean ± sem, n ≥ 6. Data were analyzed by an ANOVA with a Dunnett’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001significantly lower than with the negative siRNA treatment.

Figure 7

siRNA knockdown of ABCC4 affects the migration of breast cancer cells. (a,b) Migration of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was analyzed using a scratch assay. Data are mean ± sem, n ≥ 9. (c,d) Migration of breast cancer cells following the siRNA-mediated knockdown of ABCC1 or ABCC4 was also investigated using a cellular invasion assay. Data are mean ± sem, n ≥ 3. Data were analyzed by an ANOVA with a Dunnett’s post hoc test. * p < 0.05, ** p < 0.01, and *** p < 0.001 significantly lower than with the negative siRNA treatment.

Figure 8

Efflux of cAMP or S1P are possible mechanisms by which ABCC transporters affect breast cancer cells. (a) Western blot analysis of the expression of GPR55 in breast cancer cell lines. The blue arrow indicates the band corresponding to GPR55 (b) Efflux of cAMP from MDA-MB-231 breast cancer cells was assayed using an ELISA. Data are mean ± sem, n ≥ 2, each in duplicate. (c) Efflux of Prostaglandin E₂ (PGE₂) from MCF-7 breast cancer cells was assayed using an ELISA. Data are mean ± sem, n ≥ 2, each in duplicate. (d) Efflux of S1P from MDA-MB-231 breast cancer cells was assayed using an ELISA. Data are mean ± sem, n ≥ 3, each in duplicate. Data were analyzed by a one-way ANOVA with a Dunnett’s post hoc test. * p < 0.05 and ** p < 0.01, significantly lower than untreated or negative siRNA treatment.