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

Abstract

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(/TIS21) expression. Based on our observation that forced expression of BTG2(/TIS21) downregulated Twist1 protein expression without altering mRNA level, we investigated molecular mechanisms of the BTG2(/TIS21)-inhibited Twist1 translation in the triple negative breast cancer (TNBC) cells and in vivo BTG2(/TIS21)-knockout (KO) mice and human breast cancer tissues. (1) C-terminal domain of Twist1 and Box B of BTG2(/TIS21) interacted with each other, which abrogated Twist1 activity. (2) BTG2(/TIS21) inhibited translational initiation by depleting eIF4E availability via inhibiting 4EBP1 phosphorylation. (3) Expression of BTG2(/TIS21) maintained p-eIF2alpha that downregulates initiation of protein translation, confirmed by eIF2alpha-AA mutant expression and BTG2(/TIS21) knockdown in MEF cells. (4) cDNA microarray analysis revealed significantly higher expression of initiation factors-eIF2A, eIF3A, and eIF4G2-in the BTG2(/TIS21)-KO mouse than that in the wild type. (5) BTG2(/TIS21)-inhibited translation initiation lead to the collapse of polysome formation and the huge peak of 80s monomer in the BTG2(/TIS21) expresser, but not in the control. (6) mRNAs and protein expressions of elongation factors were also downregulated by BTG2(/TIS21) expression in TNBC cells, but much higher in both TIS21-KO mice and lymph node-positive human breast cancers. (7) BTG2(/TIS21)-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(/TIS21) gene in the regulation of Twist1 protein level. Altogether, the present study support our hypothesis that BTG2(/TIS21) is a promising target to combat with metastatic cancers with high level of Twist1 without BTG2(/TIS21) expression.