Fig 1.
BPAF promotes ER+ breast cancer cell proliferation and migration.
A) MCF-7 and T47D ER+ breast cancer cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0, 0.1, 0.5, 1, or 5 μM) in phenol red-free medium with 5% C.S. FBS for 5 days. The percentage of viable cells in each cell line was determined with an MTT assay. B) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0, 0.5, or 1 μM) in phenol red-free medium with 5% C.S. FBS for 24 hours, followed by FACS analysis of the percentage of cells in G0/G1, S, and G2/M phases of the cell cycle. The average percentages of cells in each phase are graphed. C) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0, 1, or 5 μM) in phenol red-free medium with 5% C.S. FBS for 21 days. Then, the cells were fixed and stained with crystal violet. The graphs in the lower panels present the average number of colonies formed with representative images in the panels above. D) The migration of cells treated with BPAF (0 or 1 μM) for 24 hours was determined by a wound healing assay. The panel to the left shows MCF-7 cells at Day 0 and Day 4 after the initial wound was formed. Representative images were captured at 10× magnification and dashed lines indicate the wound boundaries. The panel to the right depicts the percent of the wound width that the cells migrated after 4 days. All values are presented as the means ± standard error of the mean (S.E.) (*P<0.05, **P<0.01 as compared to the corresponding controls).
Fig 2.
A) MCF-7 and T47D cells transiently transfected with the ERE luciferase reporter plasmid (MCF-7/ERE and T47D/ERE cells) were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0, 0.5, or 1 μM) in phenol red-free medium with 5% C.S. FBS for 24 hours. Cell lysates were used for the reporter assays to quantify the relative luciferase activities after each treatment. Values are presented as the means ± S.E. (**P<0.01). B) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0, 0.1, 0.5, 1 or 5 μM) in phenol red-free medium with 5% C.S. FBS for 30 minutes, followed by Western blotting analysis of the indicated markers involved in the ER signaling pathways.
Fig 3.
BPAF stimulates ErbB3/RTK signaling.
MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0, 0.1, 0.5, 1 or 5 μM) in phenol red-free medium with 5% C.S. FBS for 30 minutes, followed by Western blotting analysis of the indicated markers involved in the ErbB3/RTK signaling pathways.
Fig 4.
Disruption of the ER pathway blocks BPAF-induced cell growth and ER-mediated transcriptional activity.
A) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (1 μM) ± ICI-182,780 (2 μM) in phenol red-free medium with 5% C.S. FBS for 5 days. The average percentage of viable cells in each treatment group was determined with an MTT assay. B) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (1 μM) ± ICI-182,780 (2 μM) in phenol red-free medium with 5% C.S. FBS for 21 days, followed by fixation and staining with crystal violet. The graphs in the lower panels present the average number of colonies formed with representative images in the panels above. C) MCF-7/ERE and T47D/ERE cells transiently transfected with ERE luciferase reporters were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (1 μM) ± ICI-182,780 (2 μM) in phenol red-free medium with 5% C.S. FBS for 24 hours. The relative luciferase activities for each treatment group are graphed. All values are presented as the means ± S.E. (**P<0.01). D) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were pretreated with ICI-182,780 (2 μM) in phenol red-free medium with 5% C.S. FBS for 16 hours, followed by treatment with BPAF (1 μM) in phenol red-free medium with 5% C.S. FBS for 30 minutes. Western blotting analysis was performed on the indicated markers involved in the ER and ErbB3/RTK signaling pathways.
Fig 5.
Inhibition of EGFR and PI3K blocks BPAF-induced cell growth.
A) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (1 μM) ± Iressa (2 μM) in phenol red-free medium with 5% C.S. FBS for 5 days. The average percentage of viable cells in each treatment group was determined with an MTT assay. B) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (1 μM) ± Iressa (2 μM) in phenol red-free medium with 5% C.S. FBS for 21 days, followed by fixation and staining with crystal violet. The graphs in the lower panels present the average number of colonies formed with representative images in the panels above. C) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (1 μM) ± LY294002 (5 μM) in phenol red-free medium with 5% C.S. FBS for 5 days. The average percentage of viable cells in each treatment group was determined with an MTT assay. D) MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (1 μM) ± LY294002 (5 μM) in phenol red-free medium with 5% C.S. FBS for 21 days, followed by fixation and staining with crystal violet. The graphs in the lower panels present the average number of colonies formed with representative images in the panels above. All values are presented as the means ± S.E. (**P<0.01).
Fig 6.
BPAF upregulates the expression of ER and growth factor target genes.
MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0 or 1 μM) in phenol red-free medium with 5% C.S. FBS for 16 hours, followed by qPCR analysis of the indicated ER and growth factor gene targets. The fold changes for the BPAF-treated samples are graphed relative to the normalized values of the corresponding controls. Values are presented as the means ± S.E. (*P<0.05, **P<0.01 as compared to the corresponding controls).
Fig 7.
AREG knockdown impedes BPAF-mediated cell proliferation and ER transcriptional activity.
MCF-7 and T47D cells were transiently transfected with lentivirus-mediated AREG shRNA. PCR validation of AREG knockdown is shown in A. B) Control and AREG shRNA MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0, 0.05, 0.5, or 5 μM) in phenol red-free medium with 5% C.S. FBS for 5 days. The average percentage of viable cells in each treatment group was determined with an MTT assay. C) Control and AREG shRNA MCF-7 and T47D cells were transiently transfected with the ERE luciferase reporter plasmids, followed by serum starvation in phenol red-free medium for 48 hours. Then, the cells were exposed to BPAF (1 μM) in phenol red-free medium with 5% C.S. FBS for 24 hours. The relative luciferase activities for each treatment group are graphed. All values are presented as the means ± S.E. (**P<0.01).
Fig 8.
AREG knockdown blocks BPAF-mediated ER-RTK crosstalk.
Control and AREG shRNA MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0 or 1 μM) in phenol red-free medium with 5% C.S. FBS for 16 hours (A) and MCF-7 and T47D cells were treated with BPAF (1 μM) ± ICI-182,780 (2 μM) in phenol red-free medium with 5% C.S. FBS for 16 hours (B), followed by qPCR analysis of AREG, TFF1, and MYC, the ER/RTK target genes most significantly upregulated by BPAF. The fold changes for the treated samples are graphed relative to the normalized values of the corresponding controls. Values are presented as the means ± S.E. (*P<0.05, **P<0.01 as compared to the corresponding treatment groups). C) Control and AREG shRNA MCF-7 and T47D cells were serum-starved for 48 hours in phenol red-free medium. Then, cells were treated with BPAF (0 or 1 μM) in phenol red-free medium with 5% C.S. FBS for 30 minutes. Then, Western blotting analysis was performed on the indicated markers involved in the ER and ErbB3/RTK signaling pathways.