ITF2 prevents activation of the β-catenin-TCF4 complex in colon cancer cells and levels decrease with tumor progression.

Immunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of β-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice. Levels of ITF2, β-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of β-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes. Levels of β-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the β-catenin-TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation of the β-catenin-TCF4 complex by competing with TCF4 for β-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the β-catenin-TCF4 complex. In human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the β-catenin-TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.

Shin HW ，Choi H ，So D ，Kim YI ，Cho K ，Chung HJ ，Lee KH ，Chun YS ，Cho CH ，Kang GH ，Kim WH ，Park JW ... -  《-》

OVOL2, an Inhibitor of WNT Signaling, Reduces Invasive Activities of Human and Mouse Cancer Cells and Is Down-regulated in Human Colorectal Tumors.

Activation of WNT signaling promotes the invasive activities of several types of cancer cells, but it is not clear if it regulates the same processes in colorectal cancer (CRC) cells, or what mechanisms are involved. We studied the expression and function of OVOL2, a member of the Ovo family of conserved zinc-finger transcription factors regulated by the WNT signaling pathway, in intestinal tumors of mice and human beings. We analyzed the expression of OVOL2 protein and messenger RNA in CRC cell lines and tissue arrays, as well as CRC samples from patients who underwent surgery at Xiamen University in China from 2009 to 2012; clinical information also was collected. CRC cell lines (SW620) were infected with lentivirus expressing OVOL2, analyzed in migration and invasion assays, and injected into nude mice to assess tumor growth and metastasis. Tandem affinity purification was used to purify the OVOL2-containing complex from CRC cells; the complex was analyzed by liquid chromatography, tandem mass spectrometry, and immunoprecipitation experiments. Gene promoter activities were measured in luciferase reporter assays. We analyzed mice with an intestine-specific disruption of Ovol2 (Ovol2(flox/+) transgenic mice), as well as Apc(min/+) mice; these mice were crossed and analyzed. Analysis of data from patients indicated that the levels of OVOL2 messenger RNA were significantly lower in colon carcinomas than adenomas, and decreased significantly as carcinomas progressed from grades 2 to 4. Immunohistochemical analysis of a tissue array of 275 CRC samples showed a negative association between tumor stage and OVOL2 level. Overexpression of OVOL2 in SW620 cells decreased their migration and invasion, reduced markers of the epithelial-to-mesenchymal transition, and suppressed their metastasis as xenograft tumors in nude mice; knockdown of OVOL2 caused LS174T cells to transition from epithelial to mesenchymal phenotypes. OVOL2 bound T-cell factor (TCF)4 and β-catenin, facilitating recruitment of histone deacetylase 1 to the TCF4-β-catenin complex; this inhibited expression of epithelial-to-mesenchymal transition-related genes regulated by WNT, such as SLUG, in CRC cell lines. OVOL2 was a downstream target of WNT signaling in LS174T and SW480 cells. The OVOL2 promoter was hypermethylated in late-stage CRC specimens from patients and in SW620 cells; hypermethylation resulted in OVOL2 down-regulation and an inability to inhibit WNT signaling. Disruption of Ovol2 in Apc(min/+) mice increased WNT activity in intestinal tissues and the formation of invasive intestinal tumors. OVOL2 is a colorectal tumor suppressor that blocks WNT signaling by facilitating the recruitment of histone deacetylase 1 to the TCF4-β-catenin complex. Strategies to increase levels of OVOL2 might be developed to reduce colorectal tumor progression and metastasis.

Ye GD ，Sun GB ，Jiao P ，Chen C ，Liu QF ，Huang XL ，Zhang R ，Cai WY ，Li SN ，Wu JF ，Liu YJ ，Wu RS ，Xie YY ，Chan EC ，Liou YC ，Li BA ... -  《-》

TRIB3 Interacts With β-Catenin and TCF4 to Increase Stem Cell Features of Colorectal Cancer Stem Cells and Tumorigenesis.

Activation of Wnt signaling to β-catenin contributes to the development of colorectal cancer (CRC). Expression of tribbles pseudo-kinase 3 (TRIB3) is increased in some colorectal tumors and associated with poor outcome. We investigated whether increased TRIB3 expression promotes stem cell features of CRC cells and tumor progression by interacting with the Wnt signaling pathway. We performed studies with C57BL/6J-ApcMin/J mice injected with an adeno-associated virus vector that expresses a small hairpin RNA against Trib3 mRNA (ApcMin/J-Trib3KD) or a control vector (ApcMin/J-Ctrl). We created BALB/c mice that overexpress TRIB3 from an adeno-associated virus vector and mice with small hairpin RNA-mediated knockdown of β-catenin. The mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by histology, gene expression profiling, immunohistochemistry, and immunofluorescence. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive (LGR5Pos) and LGR5-negative (LGR5Neg) HCT-8 CRC cells, with or without knockdown or transgenic expression of TRIB3, were sorted and analyzed in sphere-formation assays. We derived organoids from human and mouse colorectal tumors to analyze the function of TRIB3 and test the effect of a peptide inhibitor. Wnt signaling to β-catenin was analyzed in dual luciferase reporter, chromatin precipitation, immunofluorescence, and immunoblot assays. Proteins that interact with TRIB3 were identified by immunoprecipitation. CRC cell lines were grown in nude mice as xenograft tumors. At 10 weeks of age, more than half the ApcMin/J-Ctrl mice developed intestinal high-grade epithelial neoplasia, whereas ApcMin/J-Trib3KD mice had no intestinal polyps and normal histology. Colon tissues from ApcMin/J-Trib3KD mice expressed lower levels of genes regulated by β-catenin and genes associated with cancer stem cells. Mice with overexpression of Trib3 developed more tumors after administration of azoxymethane and dextran sodium sulfate than BALB/c mice. Mice with knockdown of β-catenin had a lower tumor burden after administration of azoxymethane and dextran sodium sulfate, regardless of Trib3 overexpression. Intestinal tissues from mice with overexpression of Trib3 and knockdown of β-catenin did not have activation of Wnt signaling or expression of genes regulated by β-catenin. LGR5Pos cells sorted from HCT-8 cells expressed higher levels of TRIB3 than LGR5Neg cells. CRC cells that overexpressed TRIB3 had higher levels of transcription by β-catenin and formed larger spheroids than control CRC cells; knockdown of β-catenin prevented the larger organoid size caused by TRIB3 overexpression. TRIB3 interacted physically with β-catenin and transcription factor 4 (TCF4). TRIB3 overexpression increased, and TRIB3 knockdown decreased, recruitment of TCF4 and β-catenin to the promoter region of genes regulated by Wnt. Activated β-catenin increased expression of TRIB3, indicating a positive-feedback loop. A peptide (P2-T3A6) that bound β-catenin disrupted its interaction with TRIB3 and TCF4. In primary CRC cells and HCT-8 cells, P2-T3A6 decreased expression of genes regulated by β-catenin and genes associated with cancer stem cells and decreased cell viability and migration. Injection of C57BL/6J-ApcMin/J mice with P2-T3A6 decreased the number and size of tumor nodules and colon expression of genes regulated by β-catenin. P2-T3A6 increased 5-fluorouracil-induced death of CRC cells and survival times of mice with xenograft tumors. TRIB3 interacts with β-catenin and TCF4 in intestine cells to increase expression of genes associated with cancer stem cells. Knockdown of TRIB3 decreases colon neoplasia in mice, migration of CRC cells, and their growth as xenograft tumors in mice. Strategies to block TRIB3 activity might be developed for treatment of CRC.

Hua F ，Shang S ，Yang YW ，Zhang HZ ，Xu TL ，Yu JJ ，Zhou DD ，Cui B ，Li K ，Lv XX ，Zhang XW ，Liu SS ，Yu JM ，Wang F ，Zhang C ，Huang B ，Hu ZW ... -  《-》

BCL9-2 promotes early stages of intestinal tumor progression.

The roles of the 2 BCL9 and 2 Pygopus genes in Wnt to β-catenin signaling are not clear in vertebrates. We examined their expression and function in normal and tumor intestinal epithelia in mice and humans. Specific antibodies were generated to characterize the BCL9 and Pygopus proteins in normal intestine and in colon tumors. Targets of BCL9 and Pygopus in colon cancer cells were analyzed using small interfering RNA analysis. Transgenic mice were created that overexpressed BCL9-2 in intestine; these were crossed with APCMin/+ mice to create BCL9-2;APCMin/+ mice. BCL9 and Pygopus2 were expressed in all normal intestinal and colon cancer cells. BCL9-2 was detectable only in the villi, not in the crypts of normal intestine. BCL9-2 was up-regulated in adenomas and in almost all colon tumors, with a concomitant increase of Pygopus2, whereas levels of BCL9 were similar between normal and cancer cells. Transgenic overexpression of BCL9-2 in the intestine of BCL9-2; APCMin/+ mice increased formation of adenomas that progressed to invasive tumors, resulting in reduced survival time. Using small interfering RNA analysis, we found that BCL9s and Pygopus are not targets of Wnt in colon cancer cells, but Wnt signaling correlated with levels of BCL9-2. BCL9-2 regulated expression of β-catenin-dependent and -independent target genes that have been associated with early stages of intestinal tumorigenesis. BCL9-2 promotes early phases of intestinal tumor progression in humans and in transgenic mice. BCL9-2 increases the expression of a subset of canonical Wnt target genes but also regulates genes that are required for early stages of tumor progression.

Brembeck FH ，Wiese M ，Zatula N ，Grigoryan T ，Dai Y ，Fritzmann J ，Birchmeier W ... -  《-》

Clinicopathological significance of expression of p-c-Jun, TCF4 and beta-Catenin in colorectal tumors.

Takeda K ，Kinoshita I ，Shimizu Y ，Ohba Y ，Itoh T ，Matsuno Y ，Shichinohe T ，Dosaka-Akita H ... -  《BMC CANCER》