Role of the β-Catenin/REG Iα Axis in the Proliferation of Sessile Serrated Adenoma/Polyps Associated with Fusobacterium nucleatum

Abstract

Although sessile serrated adenoma/polyps (SSA/Ps) may arise through a pathway different from the traditional adenoma-carcinoma sequence, details of SSA/P tumorigenesis still remain unclear. Fusobacterium nucleatum (Fn) is frequently detected in colorectal cancer (CRC) tissues and may play a pivotal role in colorectal carcinogenesis. Here, we investigated the relationship between Fn and the β-catenin/REG Iα axis in SSA/Ps and their involvement in the proliferation of these lesions. Fn was detected in SSA/Ps by fluorescence in situ hybridization using a Fn-targeted probe, and expression of β-catenin, REG Iα and Ki67 was examined using immunohistochemistry. Sixteen of 30 SSA/P lesions (53.3%) were positive for Fn. Eighteen SSA/P lesions (60%) showed β-catenin immunoreactivity in the tumor cell nuclei. A significant majority of Fn-positive lesions showed nuclear expression of β-catenin (87.5%) and higher REG Iα scores and Ki67 labeling indices relative to Fn-negative lesions. The SSA/P lesions expressing β-catenin in nuclei had significantly higher REG Iα scores and Ki67 labeling indices than those expressing β-catenin on cytomembranes. The REG Iα score was positively correlated with the Ki67 labeling index in SSA/P lesions. The treatment with Wnt agonist SKL2001 promoted nuclear β-catenin translocation and enhanced REG Ia expression in Caco2 cells. Fn may play a role in the proliferation of SSA/P lesions through promotion of β-catenin nuclear translocation and REG Iα expression.

Keywords: Fusobacterium nucleatum; Ki67; REG; polyp; proliferation; sessile serrated adenoma; β-catenin.

Figure 1

Detection of Fusobacterium nucleatum (Fn) by fluorescence in situ hybridization. (A) Non-neoplastic colonic mucosa adjacent to a sessile serrated adenoma/polyp (SSA/P) lesion. (B,C) SSA/P lesions. The signals (red dots) for Fn are evident at the surface (B) or lamina propria (C) of the SSA/P lesions but are hardly evident in the non-neoplastic mucosa. Signals are indicated by white arrows.

Figure 2

Expression of REG Iα, β-catenin and Ki67 in SSA/P and its adjacent non-neoplastic mucosa in the colon. (A–D) Grading of REG Iα expression in SSA/P lesions and non-neoplastic colonic mucosa. (E–G) β-catenin expression pattern in SSA/P lesions and non-neoplastic colonic mucosa. Arrows indicate nuclear expression of β-catenin. (H,I) Expression of Ki67 in SSA/P lesions and non-neoplastic colonic mucosa.

Figure 3

Relationships among REG Iα, β-catenin, and Ki67 expression in SSA/P lesions. (A) Correlation between the REG Iα expression score and the Ki67 labeling index in SSA/P lesions. (B) Comparison of Ki67 labeling index between SSA/P lesions with cytomembrane-type β-catenin expression and those with nuclear-type expression. (C) Comparison of REG Iα expression score between SSA/P lesions with cytomembrane-type β-catenin expression and those with nuclear-type expression. Results are expressed as the mean ± SE. * p < 0.005, ** p < 0.001 vs. cytomembrane-type group.

Figure 4

Effect of Wnt agonist SKL2001 on nuclear β-catenin translocation and REG Iα expression in Caco2 cells. (A) Representative photograph showing nuclear β-catenin (red) translocation in Caco2 cells treated with Wnt agonist SKL2001. Caco2 cells (5 × 10⁴) were cultured in 12-well plates for 24 h and then treated with Wnt agonist SKL2001 (40 mM) for 24 h. After stimulation, the cells were fixed with methanol for 10 min at room temperature, incubated with primary anti-β-catenin antibody for 1 h, followed by TRITC-labeled secondary anti-rabbit antibodies for 1 h, nuclear counterstained using Antifade Mountant with DAPI (blue) and observed under Olympus BX53 microscope. Bar = 20 mm. (B) Change in REG Iα expression in in Caco2 cells treated with Wnt agonist SKL2001. Results are expressed as the mean ±SE. * p < 0.05 vs. control group.