Translational downregulation of Twist1 expression by antiproliferative gene, B-cell translocation gene 2, in the triple negative breast cancer cells.

Twist1, a key transcription factor regulating epithelial-mesenchymal transition and cancer metastasis, is highly expressed in invasive cancers in contrast to the loss of BTG2 expression. Based on our observation that forced expression of BTG2 downregulated Twist1 protein expression without altering mRNA level, we investigated molecular mechanisms of the BTG2-inhibited Twist1 translation in the triple negative breast cancer (TNBC) cells and in vivo BTG2-knockout (KO) mice and human breast cancer tissues. (1) C-terminal domain of Twist1 and Box B of BTG2 interacted with each other, which abrogated Twist1 activity. (2) BTG2 inhibited translational initiation by depleting eIF4E availability via inhibiting 4EBP1 phosphorylation. (3) Expression of BTG2 maintained p-eIF2α that downregulates initiation of protein translation, confirmed by eIF2α-AA mutant expression and BTG2 knockdown in MEF cells. (4) cDNA microarray analysis revealed significantly higher expression of initiation factors-eIF2A, eIF3A, and eIF4G2-in the BTG2-KO mouse than that in the wild type. (5) BTG2-inhibited translation initiation lead to the collapse of polysome formation and the huge peak of 80s monomer in the BTG2 expresser, but not in the control. (6) mRNAs and protein expressions of elongation factors were also downregulated by BTG2 expression in TNBC cells, but much higher in both TIS21-KO mice and lymph node-positive human breast cancers. (7) BTG2-mediated Twist1 loss was not due to the protein degradation by ubiquitination and autophagy activation. (8) Twist1 protein level was significantly higher in various organs of TIS21-KO mice compared with that in the control, indicating the in vivo role of BTG2 gene in the regulation of Twist1 protein level. Altogether, the present study support our hypothesis that BTG2 is a promising target to combat with metastatic cancers with high level of Twist1 without BTG2 expression.