TRIB3 Interacts with Beta-catenin and TCF4 to Increase Stem Cell Features of Colorectal Cancer Stem Cells and Tumorigenesis

Fang Hua, Shuang Shang, Yu-Wei Yang, Hai-Zeng Zhang, Tian-Lei Xu, Jiao-Jiao Yu, Dan-Dan Zhou, Bing Cui, Ke Li, Xiao-Xi Lv, Xiao-Wei Zhang, Shan-Shan Liu, Jin-Mei Yu, Feng Wang, Cheng Zhang, Bo Huang, Zhuo-Wei Hu
Gastroenterology 2018 October 23

BACKGROUND & AIMS: Activation of Wnt signaling to beta-catenin contributes to development of colorectal cancer (CRC). Expression of tribbles pseudokinase 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.

METHODS: 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 beta-catenin. The mice were given azoxymethane followed by dextran sodium sulfate (DSS) to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by histology, gene expression profiling, immunohistochemistry and immunofluorescence. 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 beta-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.

RESULTS: At 10 weeks of age, more than half of 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 beta-catenin and genes associated with cancer stem cells. Mice with overexpression of TRIB3 developed more and more tumors following administration of azoxymethane and DSS than BALB/c mice. Mice with knockdown of beta-catenin had a lower tumor burden following administration of azoxymethane and DSS, regardless of TRIB3 overexpression. Intestinal tissues from mice with overexpression of TRIB3 and knockdown of beta-catenin did not have activation of Wnt signaling or expression of genes regulated by beta-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 beta-catenin and formed larger spheroids than control CRC cells; knockdown of beta-catenin prevented the larger organoid size caused by TRIB3 overexpression. TRIB3 interacted physically with beta-catenin and TCF4. TRIB3 overexpression increased, and TRIB3 knockdown reduced, recruitment of TCF4 and beta-catenin to promoter region of genes regulated by Wnt. Activated beta-catenin increased expression of TRIB3, indicating a positive-feedback loop. A peptide (P2-T3A6) that bound beta-catenin disrupted its interaction with TRIB3 and TCF4. In primary CRC cells and HCT-8 cells, P2-T3A6 reduced expression of genes regulated by beta-catenin and genes associated with cancer stem cells, and decreased cell viability and migration. Injection of C57BL/6J-ApcMin /J mice with P2-T3A6 reduced the number and size of tumor nodules and colon expression of genes regulated by beta-catenin. P2-T3A6 increased 5-fluorouracil-induced death of CRC cells and survival times of mice with xenograft tumors.

CONCLUSION: TRIB3 interacts with beta-catenin and TCF4 in intestine cells to increase expression of genes associated with cancer stem cells. Knockdown of TRIB3 reduces 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.

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