Figure 1.
Triple negative breast cancer cells are more anoikis resistant than luminal cells and miR-200c sensitizes aggressive cells to anoikis.
A. Cells were plated attached or suspended for 24 hrs prior to staining with DAPI and propidium iodide (PI). Representative images of suspended cells are shown, scale bar 50 µm. Quantitation of data in A, presented as a ratio of PI to DAPI, with each cell line normalized to the attached condition. Shown relative to MDA-231 cell line. Columns, mean of three biological replicates, bars, SEM. B. Cells treated with transfection reagent only (mock), scrambled negative control (neg) or miR-200c mimic (200c) and 48 hrs later harvested for qRT-PCR analysis of miR-200c levels. Data normalized to U6 levels and presented relative to MDA-231 mock transfection condition. Columns, mean of five biological replicates, bars, SEM. C. Cells as in B and 24 hrs later plated in suspension. After 24 hrs in suspension, a cell death ELISA was performed. Data normalized to attached condition and shown relative to MDA-231 mock transfection. Columns, mean of three biological replicates, bars, SEM.
Figure 2.
TrkB requires ligand to induce anoikis resistance. A.
MCF7 and T47D cells were stably selected for expression of empty vector (EV) or TrkB. Immunoblot showing TrkB expression, α-tubulin used as loading control. MCF7, B, and T47D, C, cells were plated suspended in increasing concentrations of BDNF or NTF3. Cells were harvested 24 hrs later and apoptosis assayed by cell death ELISA, data normalized to attached condition and shown relative to EV conditions. Columns, mean of three biological replicates, bars, SEM.
Figure 3.
NTF3 is a direct target of miR-200c. A
. Regions of the 3′ UTR where miR-200c is predicted to bind. B. Hec50 cells transfected with NTF3 luciferase constructs and 24 hrs later treated with transfection reagent only (mock), scrambled negative control (neg), miR-200c mimic (200c), miR-200c antagomiR alone (α200c) or in conjunction with miR-200c (α200c+200c) and luciferase assay performed. Columns, mean of five biological replicates, bars, SEM. C. Cells transfected with miRNA constructs and 48 hrs later medium collected for analysis by NTF3 ELISA. Columns, mean of three biological replicates, bars, SEM.
Figure 4.
TrkB and NTF3 are required for anoikis resistance.
BT549 cells stably selected for expression of shneg, shTrkB or shNTF3 constructs. A. Efficacy of TrkB knockdown. Left, immunoblot showing knockdown of TrkB, α-tubulin used as loading control, right, quantitation of immunoblot. B. Efficacy of NTF3 knockdown. NTF3 ELISA performed on medium. Columns, mean of three biological replicates, bars, SEM. C. Cell death ELISA performed on cells suspended for 24 hrs. Columns, mean of three biological replicates, bars, SEM. D–G. Cells treated with transfection reagent only (mock), scrambled negative control (neg) or miR-200c mimic (200c) and 24 hrs later plated in suspension. Cells were harvested 24 hrs later for analysis. D. Immunoblot for TrkB, α-tubulin used as loading control. E. NTF3 ELISA performed on medium. Columns, mean of three biological replicates, bars, SEM. shTrkB, F, and shNTF3, G, cells analyzed by cell death ELISA. Columns, mean of three biological replicates, bars, SEM.
Figure 5.
TrkB and NTF3 are up-regulated in suspended cells and miR-200c blocks this up-regulation.
Cells were plated in suspension and harvested at the time points indicated. A. Immunoblot for TrkB expression, α-tubulin used as loading control. B. NTF3 ELISA performed on medium. Points, mean of three biological replicates, bars, SEM. Cells treated with transfection reagent only (mock), scrambled negative control (neg) or miR-200c mimic (200c) and 24 hrs later plated in suspension. C. Cells were harvested 24 hrs later and immunoblot performed for TrkB, α-tubulin used as loading control. D. NTF3 ELISA performed on medium at time points indicated. Points, mean of three biological replicates, bars, SEM.
Figure 6.
NF-κB transcriptional activity increases in suspended TNBC cells. A.
Cells were transfected with 3x NF-κB transcriptional response element reporter and a Renilla control and 24 hrs later plated in suspension. Cells were harvested at time points indicated and dual luciferase assay performed. Data normalized to attached time point and presented relative to MCF7 attached condition. Columns, mean of three biological replicates, bars, SEM. MDA-231, B, and BT549, C, cells were transfected with 3x NF-κB or mutant reporter and assayed as in A. Data presented relative to NF-κB attached condition. Columns, mean of three biological replicates, bars, SEM. D. BT549 cells were grown on coverslips (attached), or in suspension and spun onto slides. Immunocytochemistry was performed for RelA or NF-κB1 (left), and the percentage of nuclear staining at each time point was quantitated (right). Columns, mean of three biological replicates, bars, SEM.
Figure 7.
NF-κB transcriptionally up-regulates TrkB and NTF3 in suspended cells. A.
BT549 cells were plated in suspension for 2 hrs and harvested for ChIP analysis. Following precipitation with antibodies against NF-κB1 and RelA, SYBR green qRT-PCR was performed for sites in the TrkB (left) and NTF3 (right) promoters. PLK1 used as a positive control for increased RelA binding in suspended cells. Data normalized to input controls and presented as a ratio of suspended over attached conditions. Columns, mean of three biological replicates, bars, SEM. B–G BT549 cells stably selected for empty vector (EV) or genetic NF-κB inhibition through mutant IκBα (mIκBα). B. Characterization of mIκBα cells, immunoblot of IκBα, α-tubulin used as loading control. Numbers represent amount of IκBα normalized to α-tubulin. C. Cells were transfected with 3×NF-κB transcriptional response element reporter and a Renilla control and 24 hrs later plated in suspension. Cells were harvested at time points indicated and dual luciferase assay performed. Data normalized to attached time point and presented relative to EV condition. Points, mean of three biological replicates, bars, standard error of the mean. D. Cells were plated in suspension and RNA was harvested at time points indicated. SYBR green qRT-PCR was performed for TrkB and NTF3. Data normalized to actin and presented relative to attached. Points, mean of three biological replicates, bars, SEM. E. Cells were plated in suspension for 24 hrs and harvested for immunoblot analysis of TrkB, α-tubulin used as loading control. F. NTF3 ELISA performed on medium at time points indicated. Points, mean of three biological replicates, bars, SEM. G. BT549 (left) and MDA-231 (right) cells were plated in suspension and harvested at the time points indicated for analysis by Cell Death ELISA. Points, mean of three biological replicates, bars, SEM.
Figure 8.
Model of select signaling pathways active in anoikis sensitive or resistant breast cancer cells.
This model summarizes our findings regarding signaling pathways activated in breast cancer cells following loss of ECM attachment.