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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-eIF2α that downregulates initiation of protein translation, confirmed by eIF2α-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.
Highlights
Epithelial–mesenchymal transition (EMT) is a process trans-differentiating epithelial cells to mesenchymal cells, Metastasis of epithelial tumors takes place when the cancer cells lose cell–cell contact inhibition in contrast to obtaining migration capacity along with EMT process[2].Twist[1], a basic helix-loop-helix transcription factor, plays an important role in the embryonal development as a master regulator of morphogenesis[3] and in EMT process during tumor invasion and metastasis[4]
Downregulation of Twist[1] protein expression by BTG2/TIS21 gene To explore regulation of Twist[1] expression by BTG2/TIS21 gene, transfection analysis was performed in 293TN cells for 48 h with v5-Twist[1] and BTG2-HA gene, and the cells were analyzed by immunoblotting
BTG2/TIS21-regulated TWIST1 protein level was dependent on the concentration of BTG2/TIS21 gene (Supplementary Fig. S2A)
Summary
Epithelial–mesenchymal transition (EMT) is a process trans-differentiating epithelial cells to mesenchymal cells, Metastasis of epithelial tumors takes place when the cancer cells lose cell–cell contact inhibition in contrast to obtaining migration capacity along with EMT process[2].Twist[1], a basic helix-loop-helix transcription factor, plays an important role in the embryonal development as a master regulator of morphogenesis[3] and in EMT process during tumor invasion and metastasis[4]. Among the several tumor suppressors, BTG2/TIS21 gene was primarily focused due to its targeting activity on actin nucleator, Dia[19]. Overexpression of BTG2/TIS21 has been significantly associated with G2/M arrest by interacting with cyclin B1-Cdc[2] complex[25], and with cell death by inducing MnSOD expression via nuclear factor-κB activation[26,27]. BTG2/TIS21 high expression in breast cancer increases survival rate[28], whereas loss of BTG2 expression induces breast tumor progression that responds to ErbB/ HER inhibitor lapatinib[29]. Overexpression of BTG2/TIS21 inhibits invadopodia formation in the TNBC, MDA-MB-231, cells[30] and reduces cancer invasion to muscle layer and lymph nodes of human bladder and breast cancers[9,20]. Constitutive expression of BTG2/TIS21 in the ductal carcinoma in situ (DCIS), but not in the infiltrating ductal carcinoma, strongly suggests that BTG2 might be an important barrier to block cancer progression from DCIS to infiltrating cancers[31], and the notion is well supported by the report that p53 deficiency-enhanced metastatic potential of breast cancer is linked to BTG2/TIS21 loss in the primary and metastatic sites of TNBC-PDX model[32]
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