A novel ent-kaurane diterpenoid executes antitumor function in colorectal cancer cells by inhibiting Wnt/β-catenin signaling.

Aberrant activation of Wnt signaling pathway is crucial for the onset and progression of human colorectal cancer (CRC). Owing to the persistent dependence on Wnt signaling for growth and survival, inhibition of this pathway is an attractive approach for new therapies. 11α, 12α-epoxyleukamenin E (EPLE) is a novel ent-kaurane diterpenoid that we previously isolated from Salvia cavaleriei, exhibiting antitumor activities in a variety of cancer cells. Herein, we found that whereas sparing normal human colon mucosal epithelial cells, EPLE selectively inhibited the proliferation of CRC cell lines as well as primary tumor cells. Mechanistically, we demonstrated EPLE exerted its function through suppressing Wnt signaling pathway, as evidenced by EPLE-mediated downregulation of Wnt target genes such as c-Myc, Axin2 and Survivin. Consistently, luciferase reporter assays showed that the EPLE directly blocked Wnt/β-catenin-mediated transcriptional activation. In combination of co-immunoprecipitation and protein structure-based analyses, we determined that the EPLE entered the interface of β-catenin/TCF4 complexes and blocked their interaction that is required for β-catenin-mediated transcriptional activation. Moreover, overexpression of β-catenin alleviated the cytotoxicity of EPLE in CRC cells, supporting Wnt signaling is a major and specific target of EPLE. Combined treatments of EPLE and 5-fluorouracil, the first-line chemotherapy for CRC patients, achieved a synergistic effect. More importantly, EPLE hampered tumor development in a CRC xenograft model. Our data thus establish EPLE as a novel inhibitor of Wnt signaling that holds great promise as a potential candidate for further preclinical evaluation for CRC treatments.

Ye Q ，Yao G ，Zhang M ，Guo G ，Hu Y ，Jiang J ，Cheng L ，Shi J ，Li H ，Zhang Y ，Liu H ... -  《-》

Henryin, an ent-kaurane diterpenoid, inhibits Wnt signaling through interference with β-catenin/TCF4 interaction in colorectal cancer cells.

Li X ，Pu J ，Jiang S ，Su J ，Kong L ，Mao B ，Sun H ，Li Y ... -  《PLoS One》

Preclinical evaluation of destruxin B as a novel Wnt signaling target suppressing proliferation and metastasis of colorectal cancer using non-invasive bioluminescence imaging.

In continuation to our studies toward the identification of direct anti-cancer targets, here we showed that destruxin B (DB) from Metarhizium anisopliae suppressed the proliferation and induced cell cycle arrest in human colorectal cancer (CRC) HT29, SW480 and HCT116 cells. Additionally, DB induced apoptosis in HT29 cells by decreased expression level of anti-apoptotic proteins Bcl-2 and Bcl-xL while increased pro-apoptotic Bax. On the other hand, DB attenuated Wnt-signaling by downregulation of β-catenin, Tcf4 and β-catenin/Tcf4 transcriptional activity, concomitantly with decreased expression of β-catenin target genes cyclin D1, c-myc and survivin. Furthermore, DB affected the migratory and invasive ability of HT29 cells through suppressed MMPs-2 and -9 enzymatic activities. We also found that DB targeted the MAPK and/or PI3K/Akt pathway by reduced expression of Akt, IKK-α, JNK, NF-κB, c-Jun and c-Fos while increased that of IκBα. Finally, we demonstrated that DB inhibited tumorigenesis in HT29 xenograft mice using non-invasive bioluminescence technique. Consistently, tumor samples from DB-treated mice demonstrated suppressed expression of β-catenin, cyclin D1, survivin, and endothelial marker CD31 while increased caspase-3 expression. Collectively, our data supports DB as an inhibitor of Wnt/β-catenin/Tcf signaling pathway that may be beneficial in the CRC management.

Yeh CT ，Rao YK ，Ye M ，Wu WS ，Chang TC ，Wang LS ，Wu CH ，Wu AT ，Tzeng YM ... -  《-》

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 ... -  《-》

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 ... -  《-》