miR-200c Targets a NF-κB Up-Regulated TrkB/NTF3 Autocrine Signaling Loop to Enhance Anoikis Sensitivity in Triple Negative Breast Cancer

<div><p>Anoikis is apoptosis initiated upon cell detachment from the native extracellular matrix. Since survival upon detachment from basement membrane is required for metastasis, the ability to resist anoikis contributes to the metastatic potential of breast tumors. miR-200c, a potent repressor of epithelial to mesenchymal transition, is expressed in luminal breast cancers, but is lost in more aggressive basal-like, or triple negative breast cancers (TNBC). We previously demonstrated that miR-200c restores anoikis sensitivity to TNBC cells by directly targeting the neurotrophic receptor tyrosine kinase, TrkB. In this study, we identify a TrkB ligand, neurotrophin 3 (NTF3), as capable of activating TrkB to induce anoikis resistance, and show that NTF3 is also a direct target of miR-200c. We present the first evidence that anoikis resistant TNBC cells up-regulate both TrkB and NTF3 when suspended, and show that this up-regulation is necessary for survival in suspension. We further demonstrate that NF-κB activity increases 6 fold in suspended TNBC cells, and identify RelA and NF-κB1 as the transcription factors responsible for suspension-induced up-regulation of TrkB and NTF3. Consequently, inhibition of NF-κB activity represses anoikis resistance. Taken together, our findings define a critical mechanism for transcriptional and post-transcriptional control of suspension-induced up-regulation of TrkB and NTF3 in anoikis resistant breast cancer cells.</p> </div

TrkB and NTF3 are up-regulated in suspended cells and miR-200c blocks this up-regulation.

<p>Cells were plated in suspension and harvested at the time points indicated. <b>A.</b> Immunoblot for TrkB expression, α-tubulin used as loading control. <b>B.</b> NTF3 ELISA performed on medium. <i>Points,</i> mean of three biological replicates, <i>bars</i>, SEM. Cells treated with transfection reagent only (mock), scrambled negative control (neg) or miR-200c mimic (200c) and 24 hrs later plated in suspension. <b>C.</b> Cells were harvested 24 hrs later and immunoblot performed for TrkB, α-tubulin used as loading control. <b>D.</b> NTF3 ELISA performed on medium at time points indicated. <i>Points,</i> mean of three biological replicates, <i>bars</i>, SEM.</p

Triple negative breast cancer cells are more anoikis resistant than luminal cells and miR-200c sensitizes aggressive cells to anoikis.

<p><b>A.</b> 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. <i>Columns</i>, mean of three biological replicates, <i>bars</i>, SEM. <b>B.</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. <i>Columns,</i> mean of five biological replicates, <i>bars,</i> SEM. <b>C.</b> 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. <i>Columns</i>, mean of three biological replicates, <i>bars</i>, SEM.</p

TrkB and NTF3 are required for anoikis resistance.

<p>BT549 cells stably selected for expression of shneg, shTrkB or shNTF3 constructs. <b>A.</b> Efficacy of TrkB knockdown. <i>Left</i>, immunoblot showing knockdown of TrkB, α-tubulin used as loading control, <i>right</i>, quantitation of immunoblot. <b>B.</b> Efficacy of NTF3 knockdown. NTF3 ELISA performed on medium. <i>Columns,</i> mean of three biological replicates, <i>bars</i>, SEM. <b>C.</b> Cell death ELISA performed on cells suspended for 24 hrs. <i>Columns,</i> mean of three biological replicates, <i>bars</i>, SEM. <b>D–G.</b> 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. <b>D.</b> Immunoblot for TrkB, α-tubulin used as loading control. <b>E.</b> NTF3 ELISA performed on medium. <i>Columns,</i> mean of three biological replicates, <i>bars</i>, SEM. shTrkB, <b>F</b>, and shNTF3, <b>G</b>, cells analyzed by cell death ELISA. <i>Columns,</i> mean of three biological replicates, <i>bars</i>, SEM.</p

Model of select signaling pathways active in anoikis sensitive or resistant breast cancer cells.

<p>This model summarizes our findings regarding signaling pathways activated in breast cancer cells following loss of ECM attachment.</p

NF-κB transcriptional activity increases in suspended TNBC cells. A.

<p>Cells were transfected with 3x NF-κB transcriptional response element reporter and a <i>Renilla</i> 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. <i>Columns,</i> mean of three biological replicates, <i>bars</i>, SEM. MDA-231, <b>B</b>, and BT549, <b>C</b>, cells were transfected with 3x NF-κB or mutant reporter and assayed as in A. Data presented relative to NF-κB attached condition. <i>Columns,</i> mean of three biological replicates, <i>bars</i>, SEM. <b>D.</b> 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). <i>Columns</i>, mean of three biological replicates, <i>bars</i>, SEM.</p

TrkB requires ligand to induce anoikis resistance. A.

<p>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>B</b>, and T47D, <b>C</b>, 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. <i>Columns</i>, mean of three biological replicates, <i>bars</i>, SEM.</p