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/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.

TIS21/BTG2 inhibits breast cancer growth and progression by differential regulation of mTORc1 and mTORc2-AKT1-NFAT1-PHLPP2 signaling axis.

PURPOSE: It has been reported that PI3K/AKT pathway is altered in various cancers and AKT isoforms specifically regulate cell growth and metastasis of cancer cells; AKT1, but not AKT2, reduces invasion of cancer cells but maintains cancer growth. We propose here a novel mechanism of the tumor suppresser, TIS21/BTG2, that inhibits both growth and invasion of triple negative breast cancer cells via AKT1 activation by differential regulation of mTORc1 and mTORc2 activity. METHODS: Transduction of adenovirus carrying TIS21/BTG2 gene and transfection of short interfering RNAs were employed to regulate TIS21/BTG2 gene expression in various cell lines. Treatment of mTOR inhibitors and mTOR kinase assays can evaluate the role of mTORc in the regulation of AKT phosphorylation at S473 residue by TIS21/BTG2 in breast cancer cells. Open data and immunohistochemical analysis were performed to confirm the role of TIS21/BTG2 expression in various human breast cancer tissues. RESULTS: We observed that TIS21/BTG2 inhibited mTORc1 activity by reducing Raptor-mTOR interaction along with upregulation of tsc1 expression, which lead to significant reduction of p70S6K activation as opposed to AKT1S473, but not AKT2, phosphorylation via downregulating PHLPP2 (AKT1-specific phosphatase) in breast cancers. TIS21/BTG2-induced pAKTS473 required Rictor-bound mTOR kinase, indicating activation of mTORc2 by TIS21/BTG2 gene. Additionally, the TIS21/BTG2-induced pAKTS473 could reduce expression of NFAT1 (nuclear factor of activated T cells) and its target genes, which regulate cancer microenvironment. CONCLUSIONS: TIS21/BTG2 significantly lost in the infiltrating ductal carcinoma, but it can inhibit cancer growth via the TIS21/BTG2-tsc1/2-mTORc1-p70S6K axis and downregulate cancer progression via the TIS21/BTG2-mTORc2-AKT1-NFAT1-PHLPP2 pathway.

Inhibition of TNFα-interacting protein α (Tipα)-associated gastric carcinogenesis by BTG2/TIS21 via downregulating cytoplasmic nucleolin expression.

To understand the regulation of Helicobacter pylori (H. pylori)-associated gastric carcinogenesis, we examined the effect of B-cell translocation gene 2 (BTG2) expression on the biological activity of Tipα, an oncoprotein secreted from H. pylori. BTG2, the human ortholog of mouse TIS21 (BTG2/TIS21), has been reported to be a primary response gene that is transiently expressed in response to various stimulations. Here, we report that BTG2 is constitutively expressed in the mucous epithelium and parietal cells of the gastric gland in the stomach. Expression was increased in the mucous epithelium following H. pylori infection in contrast to its loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2/TIS21 significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulating tumor necrosis factor-α (TNFα) expression and Erk1/2 phosphorylation by reducing expression of nucleolin, a Tipα receptor. Chromatin immunoprecipitation proved that BTG2/TIS21 inhibited Sp1 expression and its binding to the promoter of the nucleolin gene. In addition, BTG2/TIS21 expression significantly reduced membrane-localized nucleolin expression in cancer cells, and the loss of BTG2/TIS21 expression induced cytoplasmic nucleolin availability in gastric cancer tissues, as evidenced by immunoblotting and immunohistochemistry. Higher expression of BTG2 and lower expression of nucleolin were accompanied with better overall survival of poorly differentiated gastric cancer patients. This is the first report showing that BTG2/TIS21 inhibits nucleolin expression via Sp1 binding, which might be associated with the inhibition of H. pylori-induced carcinogenesis. We suggest that BTG2/TIS21 is a potential inhibitor of nucleolin in the cytoplasm, leading to inhibition of carcinogenesis after H. pylori infection.

Inhibition of bladder cancer invasion by Sp1-mediated BTG2 expression via inhibition of DNA methyltransferase 1.

Significantly lower endogenous expression of B-cell translocation gene 2 (BTG2) was observed in human muscle-invasive bladder cancers (MIBC) than matched normal tissues and non-muscle invasive bladder cancers (NMIBC). BTG2 expression was inversely correlated with increased expression of the DNA methyltransferases DNMT1 and DNMT3a in MIBC, but not NMIBC, suggesting a potential role for BTG2 expression in muscle invasion of bladder cancer. Over 90% of tumor tissues revealed strong methylation at CpG islands of the BTG2 gene, compared with no methylation in the normal tissues, implying epigenetic regulation of BTG2 expression in bladder carcinogenesis. By using EJ bladder cancer cells and the demethylating agent decitabine, transcription of BTG2 was shown to be up-regulated by inhibiting DNMT1 expression via modification at CpG islands. DNMT1 binding to the BTG2 gene further regulated BTG2 expression by chromatin remodeling, such as H3K9 dimethylation and H3K4 trimethylation, and Sp1 activation. Induced BTG2 expression significantly reduced EJ cell tumorigenesis and invasiveness together with induction of G2 /M arrest. These results demonstrate an important role for the BTG2(/TIS21/PC3) gene in the progression of bladder cancers, and suggest that BTG2(/TIS21/PC3) is a promising epigenetic target for prevention of muscle invasion in human bladder cancers.