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

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.

失巢凋亡（anoikis）是指细胞脱离天然细胞外基质（extracellular matrix）后触发的细胞凋亡。由于脱离基底膜后的细胞存活是肿瘤转移的必要前提，因此抵抗失巢凋亡的能力可增强乳腺肿瘤的转移潜能。miR-200c是一种强效的上皮间质转化（epithelial to mesenchymal transition）抑制因子，在腔型乳腺癌中有所表达，但在侵袭性更强的基底样型或三阴性乳腺癌（TNBC）中发生缺失。我们此前的研究已证实，miR-200c可通过直接靶向神经营养受体酪氨酸激酶TrkB，恢复三阴性乳腺癌细胞对失巢凋亡的敏感性。本研究中，我们鉴定出一种TrkB配体——神经营养因子3（NTF3），其可激活TrkB以诱导失巢凋亡抵抗，并证实NTF3同时也是miR-200c的直接靶标。我们首次提供实验证据表明，耐失巢凋亡的三阴性乳腺癌细胞在悬浮培养状态下会上调TrkB与NTF3的表达，且该上调过程对细胞的悬浮存活至关重要。我们进一步证实，悬浮状态下的三阴性乳腺癌细胞中核因子κB（NF-κB）活性提升6倍，并鉴定出RelA与NF-κB1是负责介导悬浮诱导的TrkB与NTF3上调的转录因子。因此，抑制核因子κB活性可有效抑制失巢凋亡抵抗。综上，我们的研究结果阐明了在耐失巢凋亡的乳腺癌细胞中，悬浮状态诱导的TrkB与NTF3上调的转录及转录后调控关键机制。