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 G 2 /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.

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.

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.

TIS21 significantly lost in the infiltrating ductal carcinoma, but it can inhibit cancer growth via the TIS21-tsc1/2-mTORc1-p70S6K axis and downregulate cancer progression via the TIS21-mTORc2-AKT1-NFAT1-PHLPP2 pathway.

It has been known that cytoplasmic p53 regulates cell cycle arrest, apoptosis, autophage, and metabolism of human and animal cells in a transactivation-dependent manner. Wild type p53 induces senescence of EJ human bladder cancer cells, which carries oncogenic H-ras and mutant p53. Tumor supporessor, TIS21/BTG2/PC3, is one of the target genes regulated by wild type p53. Employing adenoviral vectors carrying p53(Ad-p53) and TIS21(Ad-TIS21) genes, we investigated effect of TIS21 on cancer cell senescence induced by p53 supplementation; Infection of EJ cells with p53 rapidly induced senescence phenotypes, such as cellular enlargement, inhibition of its growth, expressions of SA-[[Unsupported Character – Symbol Font ]]-galactosidase and SA-pErk1/2, and generation of reactive oxygen species, in addition to down-regulation of PARP and pRb expressions. A mechanism of the senescence was likely to be upregulations of H-ras and paxillin expressions by p53, evidenced by employing si-p53 RNA construct. On the contrary, the combined effect of p53 plus TIS21 significantly induced apoptosis of EJ cells rather than senescence, when evaluated by Annexin V expression, Calcein-AM and EhtD-1 double staining, increased expressions of proapoptotic genes and caspase 3 activity, whereas TIS21 alone failed to induce any changes, as compared with those of the control. p53 plus TIS21 significantly increased Bax and Apaf-1 expressions, whereas paxillin expression was clearly reduced as compared with those of p53 alone. When its mechanism was investigated, acetylation of p53 on K120 and K373 residues were found to be increased in addition to the significant translocation of p53 after coexpression of TIS21. We strongly suggest here that TIS21 is an endogenous cell death promoting gene at the downstream of p53 even in the expressions of oncogenic H-Ras and mutant p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 211. doi:10.1158/1538-7445.AM2011-211