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Purpose: To investigate the role and the underlying mechanism of scaffold attachment factor B (SAFB) in the progression of colorectal cancer (CRC). Experimental Design: SAFB expression was analyzed in the Cancer Outlier Profile Analysis of Oncomine and in 175 paraffin-embedded archived CRC tissues. Gene Ontology analyses were performed to explore the mechanism of SAFB in CRC progression. Western blot, RT-PCR, luciferase assay, and chromatin immunoprecipitation (ChIP) were used to detect the regulation of transforming growth factor-β–activated kinase 1 (TAK1) and NF-κB signaling by SAFB. The role of SAFB in invasion, metastasis, and angiogenesis was investigated using in vitro and in vivo assays. The relationship between SAFB and TAK1 was analyzed in CRC tissues. Results: SAFB was downregulated in CRC tissues, and low expression of SAFB was significantly associated with an aggressive phenotype and poorer survival of CRC patients. The downregulation of SAFB activated NF-κB signaling by targeting the TAK1 promoter. Ectopic expression of SAFB inhibited the development of aggressive features and metastasis of CRC cells both in vitro and in vivo. The overexpression of TAK1 could rescue the aggressive features in SAFB-overexpressed cells. Furthermore, the expression of SAFB in CRC tissues was negatively correlated with the expression of TAK1- and NF-κB–related genes. Conclusions: Our results show that SAFB regulated the activity of NF-κB signaling in CRC by targeting TAK1. This novel mechanism provides a comprehensive understanding of both SAFB and the NF-κB signaling pathway in the progression of CRC and indicates that the SAFB–TAK1–NF-κB axis is a potential target for early therapeutic intervention in CRC progression. Clin Cancer Res; 23(22); 7108–18. ©2017 AACR.

Background Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/β-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce. Methods Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR. Results TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1. Conclusion This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.