Fusobacterium nucleatum has recently emerged as a significant participant in colorectal (CRC) and other cancers and has been termed an 'oncomicrobe'. Commonly found in the oral microbiome as a commensal, it morphs into an opportunitistic pathogen in CRC.  However, it is not clear as to why or how this happens and if the effects of intracellular infection lead to long- lasting epigenetic modifications within host cells.

Most in vitro studies have previously investigated host-microbial interactions of this bacterium in normoxia conditions. Since F. nucleatum is an anaerobe, we hypothesized that tumor hypoxia could be a conducive niche for bacterial localization. There are several challenges in dissecting this response in vivo including the lack of effective methods to simultaneously measure oxygen levels and microbial abundance in live tissue. Thus, we set out to investigate if we can determine host response to F. nucleatum infection in a controlled in vitro system, where we can modulate tumor microenvironmental features such as hypoxia. We employed a multiomic approach to analyze H3K27ac ChIPseq data and bulk RNAseq data and revealed changes to the global epigenome and transcriptome of a host CRC cell line (HCT116) in response to infection in different oxygen environments. 

It is well known that hypoxia conditioning drastically impacts cancer cell phenotype. Several signaling pathways are impacted in hypoxia resulting in altered metabolism,  increased epithelial-mesenchymal transition, and drug resistance. We wanted to find out if this altered phenotype facilitates intracellular F. nucleatum invasion and promotes bacterial persistence within the cell. We have previously published in Science Signaling that HCT116 cells secrete cytokines IL-8 and CXCL1 specifically upon infection with F. nucleatum and that these cytokines can promote host cell migration. In this study, we discovered that this secretion is increased two-fold in hypoxic HCT116 cells, possibly accelerating the migratory response of the cells. We further observed increased bacterial invasion under hypoxia through flow cytometry experiments. 

This prompted us to investigate if the signaling pathways in high and low oxygen are altered upon F. nucleatum infection. Controlling for cell type, bacterial strain, and experimental conditions, we subjected HCT116 cells to differing oxygen levels. We provided the cells 24 hours of additional incubation time in their oxygen treatment environment following infection to capture stable levels of mRNA and histone acetylation before extracting these cells for analysis. 

We analyzed active enhancers (through identifying regions labeled by H3K27ac which do not intersect with promoters) and found increased enhancer activity in hypoxic cells. In addition, we observed a varied response to F. nucleatum infection in normoxia and hypoxia.  Several cancer-related pathways were triggered upon hypoxia treatment and F. nucleatum infection. The RNAseq data paralleled this response and using Gene Set Variation Analysis, we discovered that F. nucleatum infection in normoxia activates the same and related hallmark pathways that are induced by just hypoxia conditioning of the cells. F. nucleatum infection in hypoxia showed that the pathways are only mildly activated over hypoxia conditioning alone, likely since they are already active.  Furthermore, infection in hypoxia downregulated pathways related to the peroxisome which may indicate active subversion of host recognition and degradation. Thus, we show that F. nucleatum impacts a multitude of pathways that can impact cancer progression and the activation of pathways similar to hypoxia conditioning could also imply that F. nucleatum prefers a host phenotype similar to a hypoxic conditioned cell. In addition, the analysis of enriched transcription factor motifs revealed differential regulation of several transcription factors that are involved in cancer progression. 

In conclusion, our study revealed that TME factors such as hypoxia can modulate the host cell response to F. nucleatum infection. These findings will influence the development of improved models to study the tumor microbiome and host-microbe interactions in physiologically relevant conditions. There is much that remains to be uncovered to understand microbial localization within tumors and how this colonization can affect cancer progression to result in poor patient outcomes. F. nucleatum infection influences sustained epigenetic reprogramming of cancer cells and promotes the development of more aggressive cell phenotypes similar to hypoxia effects on the cells. The recent discovery of strain and clade specificity of cancer-promoting F. nucleatum and enrichment of other microbial species within CRC and other cancers substantiate a need for improved in vitro models to dissect accurate host-microbial interactions, which can be harnessed to develop therapeutic strategies to target the tumor microbiome.  

Check out the paper here: https://www.nature.com/articles/s42003-024-06201-w

(Blog post image: F. nucleatum infection of HCT116 cells. Credits: SEM: LaDeidra Monet Roberts | Pseudocolor: Barath Udayasuryan)