# E. coli Biomolecules Increase Glycolysis and Invasive Potential in Lung Adenocarcinoma

## TL;DR

E. coli biomolecules boost glycolysis and invasiveness in lung cancer cells, suggesting a microbial influence on cancer progression.

## Contribution

This study identifies E. coli biomolecules as direct drivers of metabolic and invasive changes in lung adenocarcinoma cells.

## Key findings

- E. coli biomolecules upregulate glycolytic enzymes and increase glucose uptake and lactate production in LUAD cells.
- Exposure to E. coli enhances the invasive potential of LUAD cells, as shown by Boyden chamber assays.
- TLR4 inhibition does not block the effects of E. coli biomolecules on glycolysis or invasion.

## Abstract

Crosstalk between our cells and the microbes within our bodies has long been known to play a role in normal physiology and disruption of this crosstalk can lead to pathological conditions. We recently provided evidence that the microbes within lung cancers are enriched for methionine-producing constituents. Cancer cells can utilize this methionine under conditions of limiting nutrients. Herein, we expand these studies to demonstrate that biomolecules produced by bacteria can influence multiple signaling and metabolic pathways within lung cancer cells. By furthering our understanding of how microbes can influence tumorigenic and metastatic processes, we may be able to design novel therapeutics for treating lung cancer.

Introduction: Recent studies have discovered that lung cancer subtypes possess distinct microbiome profiles within their tumor microenvironment. Additionally, the tumor-associated microbiome exhibits altered bacterial pathways, suggesting that certain bacterial families are more capable of facilitating tumor progression than others. We hypothesize that there exists a crosstalk between lung adenocarcinoma (LUAD) cells and bacterial cells. Methods and Materials: RNA sequencing (RNA-seq) was performed on LUAD cell lines to explore the paracrine signaling effects of bacterial biomolecules. Based on our RNA-seq data, we investigated glycolysis by measuring glucose uptake and lactate production, invasive potential through invasion assays, and epithelial-to-mesenchymal transition (EMT) markers. Since lipopolysaccharides (LPS), abundant on the cell walls of Gram-negative bacteria, can activate toll-like receptor 4 (TLR4), we inhibited TLR4 with C34 to assess its relationship with the observed phenotypic changes. To identify the bacterial biomolecules responsible for these changes, we treated the media with RNAse enzyme, charcoal or dialyzed away molecules larger than 3 kDa. Results and Discussion: RNA-seq revealed 948 genes upregulated in the presence of E. coli biomolecules. Among these, we observed increased expression of Hexokinase II (HKII), JUN proto-oncogene, and Snail Family Transcriptional Repressor 1. We verified the elevation of glycolytic enzymes through Western blot and saw elevation of 2-deoxyglucose uptake and lactate production in LUAD cell lines incubated in E. coli biomolecules. In addition to E. coli elevating glycolysis in LUAD cell lines, E. coli exposure enhanced invasive potential as demonstrated by Boyden chamber assays. Notably, inhibition of TLR4 did not reduce the impact of E. coli biomolecules on glycolysis or the invasive potential of LUAD. Modulating the E. coli-supplemented media with RNAse enzyme or dextran-coated charcoal or using a spin column to remove biomolecules smaller than 3 kDa resulted in changes in HKII and Claudin protein expression. These findings suggest a direct relationship between E. coli and LUAD, wherein several cancer hallmarks are upregulated. Future studies should further investigate these bacterial biomolecules and their role in the tumor microenvironment to fully understand the impact of microbial shifts on cancer progression.