Invasive Fusobacterium nucleatum activates beta-catenin signaling in colorectal cancer via a TLR4/P-PAK1 cascade

Abstract

The underlying mechanism of Fusobacterium nucleatum (Fn) in the carcinogenesis of colorectal cancer (CRC) is poorly understood. Here, we examined Fn abundance in CRC tissues, as well as β-catenin, TLR4 and PAK1 protein abundance in Fn positive and Fn negative CRCs. Furthermore, we isolated a strain of Fn (F01) from a CRC tissue and examined whether Fn (F01) infection of colon cancer cells activated β-catenin signaling via the TLR4/P-PAK1/P-β-catenin S675 cascade. Invasive Fn was abundant in 62.2% of CRC tissues. TLR4, PAK1 and nuclear β-catenin proteins were more abundant within Fn-positive over Fn-negative CRCs (P < 0.05). Fn and its lipopolysaccharide induced a significant increase in TLR4/P-PAK1/P-β-catenin S675/C-myc/CyclinD1 protein abundance, as well as in the nuclear translocation of β-catenin. Furthermore, inhibition of TLR4 or PAK1 prior to challenge with Fn significantly decreased protein abundance of P-β-catenin S675, C-myc and Cyclin D1, as well as nuclear β-catenin accumulation. Inhibition of TLR4 significantly decreased P-PAK1 protein abundance, and for the first time, we observed an interaction between TLR4 and P-PAK1 using immunoprecipitation. Our data suggest that invasive Fn activates β-catenin signaling via a TLR4/P-PAK1/P-β-catenin S675 cascade in CRC. Furthermore, TLR4 and PAK1 could be potential pharmaceutical targets for the treatment of Fn-related CRCs.

Keywords: Fusobacterium nucleatum; colorectal cancer; p21-activated kinase 1; toll-like receptor 4; β-catenin signaling.

Figure 1. Biome analysis of human colorectal cancer samples

(A) Relative abundance of phyla present in colorectal cancer samples (n = 14). Data shown represent the most abundant phyla, whereas low abundant and unclassified OTUs are grouped as “Others.” (B) Hierarchically clustered heat map analysis of the highly represented bacterial taxa (genus level) in 14 colorectal cancer samples. The relative percentages of the bacterial families are indicated by varying color intensities.

Figure 2. Invasive Fn in CRC tissues is associated with an activated β-catenin signaling pathway and TLR4/PAK1 protein abundance

The upper panel shows that abundant invasive Fn in CRC tissue is associated with a high abundance of TLR4 and PAK1 and with β-catenin nuclear accumulation. The lower panel demonstrates that both TLR4 and PAK1 are absent in a Fn-negative CRC, and that cytoplasmic but not nuclear accumulation of β-catenin was seen in the tissue. Invasive Fn was detected by fluorescence in situ hybridization. 200× magnification.

Figure 3. Fn activates the β-catenin signaling pathway in SW480 cells through the TLR4/P-PAK1/P-β-catenin S675 cascade

(A-E) Western blots showing that the levels of TLR4, P-PAK1, P-β-catenin S675 and C-myc gradually increase when SW480 cells are challenged with Fn (F01) over increasing time periods. (F-J) The levels of TLR4, P-PAK1, P-β-catenin S675 and C-myc also gradually increase when SW480 cells are challenged with Fn (ATCC10953) over increasing time periods. (K-O) The levels of P-PAK1, P-β-catenin S675 and C-myc do not significantly increase when SW480 cells are challenged with E. coli for increasing time periods, although TLR4 protein significantly increases. T-PAK1, total PAK1; P-PAK1, phosphorylated PAK1. Bar diagrams represent the results obtained after densitometric scanning from three different experiments. Bars represent the mean ± SD. *, P< 0.05, compared with control group (0 h).

Figure 4. Fn activates nuclear accumulation of β-catenin and P-β-catenin S675 in SW480 cells

(A, B) Fn (F01) significantly induces nuclear accumulation of total β-catenin over increasing time periods in SW480 cells. (C, D) Fn (F01) significantly induces nuclear accumulation of P-β-catenin S675 over increasing time periods in SW480 cells. (D, E) TAK-242 and IPA-3 inhibited the nuclear translocation of P-β-catenin S675 induced by Fn (F01) in SW480 cells. Three random 200× magnification fields per sample were evaluated, and the average percentage of β-catenin nuclear accumulation per field was calculated. Bars represent the mean ± SD. *, P< 0.05.

Figure 5. LPS of Fn could be the main reason for activation of the β-catenin signaling pathway through the TLR4/P-PAK1/P-β-catenin S675 cascade

(A-G) Western blots showing that TLR4, P-PAK1, P-β-catenin S675, C-myc and Cyclin D1 levels gradually increase when SW480 cells are challenged with LPS extracted from Fn (F01) over increasing time periods. Bar diagrams represent the results obtained after densitometric scanning from three different experiments. Bars represent the mean ± SD. *, P< 0.05, as compared with control group (0 h).

Figure 6. Activation of the β-catenin signaling pathway by Fn (F01) can be inhibited by both the TLR4 inhibitor (TAK-242) and PAK1 inhibitor (IPA-3)

(A-E) Western blots showing that P-PAK1, P-β-catenin S675 and C-myc levels significantly decrease when SW480 cells are treated with TAK-242 prior to Fn (F01) challenge. (F-J) Western blots showing that P-PAK1, P-β-catenin S675 and C-myc levels significantly decrease when SW480 cells are treated with IPA-3 prior to Fn (F01) challenge. (K-O) Western blots showing that P-PAK1, P-β-catenin S675 and C-myc levels significantly decrease when SW480 cells are treated with both TAK-242 and IPA-3 prior to Fn (F01) challenge. (P) Cell lysates from Fn (F01) challenged SW480 cells were subjected to immunoprecipitation with anti-TLR4 agarose beads. DMSO was used as controls. Total lysates (Input), IgG and anti-TLR4 immunoprecipitations were subjected to western blots with P-PAK1 antibodies. Bar diagrams represent the results obtained after densitometric scanning from three different experiments. Bars represent the mean ± SD. *, P< 0.05.