Enterococcus faecalis promotes the progression of colorectal cancer via its metabolite: biliverdin

Abstract

Background: Enterococcus faecalis (Efa) has been shown to be a "driver bacteria" in the occurrence and development of colorectal cancer (CRC). This study aims to explore the effect of specific metabolites of Efa on CRC.

Methods: The pro-tumor effects of Efa were assessed in colonic epithelial cells. The tumor-stimulating molecule produced by Efa was identified using liquid chromatography mass spectrometry (LC-MS). The proliferative effect of metabolites on CRC cells in vitro was assayed as well. The concentration of vascular endothelial growth factor A (VEGFA) and interleukin-8 (IL-8) was determined using enzyme-linked immunosorbent assay (ELISA). Tubular formation assay of human umbilical vein endothelial cells (HUVEC) and cell migration assay were applied to study angiogenesis. Additionally, western blot analysis was used to investigate key regulatory proteins involved in the angiogenesis pathway. Tumor growth was assessed using mouse models with two CRC cells and human colon cancer organoid.

Results: Co-incubation with the conditioned medium of Efa increased the proliferation of cultured CRC cells. Biliverdin (BV) was determined as the key metabolite produced by Efa using LC-MS screening. BV promoted colony formation and cell proliferation and inhibited cell cycle arrest of cultured CRC cells. BV significantly increased the expression level of IL-8 and VEGFA by regulating the PI3K/AKT/mTOR signaling pathway, leading to the acceleration of angiogenesis in CRC. The up-regulation of proliferation and angiogenesis by BV were also confirmed in mice.

Conclusion: In conclusion, BV, as the tumor-stimulating metabolite of Efa, generates proliferative and angiogenic effects on CRC, which is mainly mediated by the activation of PI3K/AKT/mTOR.

Keywords: Angiogenesis; Colorectal cancer; Enterococcus faecalis; Interleukin-8; Vascular endothelial growth factor A.

Fig. 1

The conditioned medium and the metabolite of Efa promote the viability of CRC cells in vitro. A CCK-8 assay in CRC cells treated with the conditioned medium of EFa or E. coli ATCC 25922. B–D Metabolomics analysis of the conditioned medium (CM) with or without Efa. B: Principal Component Analysis (PCA) score plot of metabolomics. The orange dots represent the conditioned medium (CM) with Efa, and the blue dots represent the CM without Efa. C: Heatmap of differentially metabolites. The p-values were represented by a color scale from blue (relatively lower expression) to red (relatively higher expression). Each column represented individual sample, and each row represented a single metabolite. D: KEGG analysis for the various differentially expressed signal pathway. The dots represented various pathways. Pathways impact was represented by the area of each dots. The p-value was represented by a color scale from blue (relatively lower significance) to orange (relatively higher significance). E, F Proliferation ability of BV was determined by CCK8 and colony formation assay in CRC cells. G Proliferating cell nuclear antigen (PCNA) levels in CRC cells treated with or without BV was detected by western blot. H Flow cytometry showing the percentages of BV treated cells and control cells at different cell cycle phase. The histogram on the left is the control group, and the right is the BV treatment group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001

Fig. 2

BV stimulates CRC angiogenesis by secreting VEGFA. A, B The expression of VEGFA was detected by RT-qPCR and ELISA in HCT116, SW480 and SW620 cells cultured with BV. C Cell migration in HUVECs were examined by transwell assays after HUVECs were plated and cocultured with the CM from HCT116 and SW480 treated with BV. One representative image from three reproducible experiments is shown. Scale bar, 50 μm. Migrated HUVEC numbers are shown in the bar graph. D Tubule formation of HUVECs was shown in representative images after co-incubating with the CM from BV treated HCT116 and SW480. Scale bar, 100 μm. The increasing folds of tube formation is shown in the bar graph. **P < 0.01, ***P < 0.001, ****, P < 0.0001

Fig. 3

BV enhances VEGFA secretion and angiogenesis via activating PI3K/AKT/mTOR pathway. A The expression of PI3K/AKT/mTOR pathway members were detected by western blot in HCT116 and SW480 treated with or without BV. B Western blot analysis of PI3K/AKT/mTOR pathway members with or without the treatment of BV, LY294002 and Rapamycin in HCT116. C The concentration of VEGFA in the culture medium of control and BV treated HCT116 and SW480 cells with or without the absence of LY294002 and Rapamycin. D HUVEC migration and tube formation in representative images in the CM of HCT116 cocultured with BV with or without the absence of LY294002 and Rapamycin. Scale bar, 50 μm and 100 μm respectively. The migrated HUVEC numbers and increasing folds of tube formation are shown in the bar graph. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001

Fig. 4

BV promotes proliferation and angiogenesis in vivo. A Representative images of subcutaneous tumors in nude mice injected HCT116 cells treated with or without BV. Both the volume and weight of subcutaneous tumor were shown in the right panel. B PI3K/AKT/mTOR pathway was activated in mice tumor by western blot analysis. C The concentration of IL-8 and VEGFA in mice tumors were detected by ELISA. D The growth of human colon cancer organoids was assessed after 10 days of BV treatment or not. E IHC analysis demonstrated the expression of CD31 in subcutaneous tumors of nude mice. Bars of the right panel represent the microvascular density. Scale bar represents 100 μm. F, G The expression of Ki67 and CD31 in subcutaneous tumors of nude mice and organoid models were assessed by immunofluorescence staining. Representative images were shown. Scale bar represents 50 μm and 20 μm, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 5

Schematic model of Efa/BV/PI3K/AKT/mTOR/VEGFA axis in CRC. Efa promotes colon tumorigenesis by the secretion of BV. BV promotes growth and angiogenesis in CRC by regulating PI3K/AKT/mTOR/VEGFA pathways