Fig 2: A proposed model of the mechanism of FBW7-mediated regulation of migration via the HIF1a/CEACAM5 axis in colorectal cancer.

Fig 3: FBW7 negatively regulates CEACAM5 in colorectal cancer. Overexpression of FBW7 downregulated CEACAM5 in both HCT116 and LoVo cells, which was determined by both qRT-PCR (a) and Western blotting (b). (c) Representative figures of the correlation of FBW7 and CEACAM5 were evaluated by IHC staining of TMA (scale bar, 50µm). (d) A negative correlation of FBW7 and CEACAM5 was validated (?2=9.817, P=0.002). *P<0.05.

Fig 4: Overexpression of CEACAM5 reverses the inhibitory effects of FBW7 on migration in colon cancer cells. CEACAM expression was restored in FBW7-transfected HCT-116 and LoVo cells and the restore efficacy was determined by Western blotting (a) and qRT-PCR (b). Transwell migration assay indicated that overexpression of CEACAM5 fully abolished suppression effect on cell migration of FBW7 in both HCT-116 and LoVo cells (c). *P<0.05.

Fig 5: FBW7 regulates CEACAM5 expression in a HIF1a-dependent manner. The well-known substrates of FBW7, HIF1a and c-Myc, were then transfected into HCT116 and LoVo cells. We found that HIF1a could promote CEACAM5 expression (a), while c-Myc could not (b). (c)Promoter region of CEACAM5 was then analyzed and we found that it harbored one E-box elements AGCGTG from -1 to -5bp. (d) Dual luciferase assay indicated HIF1a and FBW7 altered CEACAM5 promoter activity in a dosage dependent way. When co-transfected with FBW7 and siRNA against HIF1a, FBW7 lost its inhibition effect on CEA promoter. (e) Chromatin immunoprecipitation (ChIP) assay showed that HIF1a occupied the E-boxes in the CEACAM5 promoter region. (f) IHC staining of TMA of colorectal cancer indicated a positive correlation between HIF1a and CEACAM5 (scale bar, 50µm). *P<0.05, **P>0.05.