P1212 Effects of Enterococcus faecalis-derived Gelatinase E on HCT116 colorectal cancer cells

Abstract

Abstract Background The leading cause of death in colorectal cancer (CRC) patients is metastasis to crucial organs, such as the lung, brain and liver. The tumour microenvironment (TME) influences metastasis heavily. One important component in the TME of CRC is the microbiota. Microbiota can affect several pathways related to the origin of metastasis and reorganization of the extracellular matrix. One of the possible contributors to early CRC metastasis is Enterococcus faecalis. E. faecalis produces Gelatinase E (GelE), a quorum-sensing protein with collagenolytic properties. The primary goal of our study was to determine the effect of GelE on CRC behaviour, and especially on migration and invasion in HCT-116 human CRC cells. Moreover, we aimed to uncover the underlying pathways by which GelE influences CRC cells. Methods Extracellular GelE was purified from E. faecalis strain V583 from the culture medium. HCT-116 cells were cultured and exposed to different concentrations of GelE. We performed a viability assay using CCK-8 to assess potential toxicity of GelE. To test effects of GelE on collagen structure, we measured stiffness of collagen-I. A transwell assay was used to assess the influence of GelE on invasive capacity of cells through a collagen-I matrix. GelE exposed cells were assessed for shape and motility markers using immunofluorescent staining for β-catenin (cell shape) and loss of E-cadherin (cell motility marker). For mechanistic insight of GelE exposure on HCT116, we performed proteomics analysis on the cells. Results GelE treatment did not affect relative cell viability of HCT116 cells up to a concentration of 100 μg/ml. Exposure to GelE caused a concentration-dependent increase in HCT-116 cell invasion through collagen-I. The immunostainings showed a downregulation of E-cadherin, indicating an induction of cell motility. Immunostaining for β-catenin showed a statistically significant effect on cell shape, becoming more elongated, an additional indication of cell motility. Proteomics showed possible mechanistic pathways that explain the increased motility including activation of the SMAD pathway, RhoGTPAse activation and activation the urokinase-plasminogen system. Reactome database analysis on the proteomics data also revealed an increase in cell-cycle related pathways. Conclusion Our results indicate that E. faecalis-derived GelE drives HCT-116 invasion with an elongated motility. Therefore, drugs inhibiting the activity of GelE could decrease the risk of metastasis in patients with a high E. faecalis V583 abundance in the TME.