Metabolic modeling of host–microbe interactions for therapeutics in colorectal cancer

The onset of colorectal cancer (CRC) is often attributed to gut bacterial dysbiosis, and thus gut microbiota are highly relevant in devising treatment strategies. Certain gut microbes, like Enterococcus spp., exhibit remarkable anti-neoplastic and probiotic properties, which can aid in silver nanoparticle (AgNPs) induced reactive oxygen species (ROS)-based CRC treatment. However, the effects of AgNPs on gut microbial metabolism have not been reported thus far. In this study, a detailed systems-level understanding of ROS metabolism in Enterococcus durans (E. durans), a representative gut microbe, was gained using constraint-based modeling, wherein, the critical association between ROS and folate metabolism was established. Experimental studies involving low AgNP concentration treatment of E. durans cultures confirmed these modeling predictions (an increased extracellular folate concentration by 52%, at the 9th h of microbial growth, was observed). Besides, the computational studies established various metabolic pathways involving amino acids, energy metabolites, nucleotides, and SCFAs as the key players in elevating folate levels on ROS exposure. The anti-cancer potential of E. durans was also studied through MTT analysis of HCT 116 cells treated with microbial culture (AgNP treated) supernatant. A decrease in cell viability by 19% implicated the role of microbial metabolites (primarily folate) in causing cell death. The genome-scale modeling approach was then extended to extensively model CRC metabolism, as well as CRC–E. durans interactions in the context of CRC treatment, using tissue-specific metabolic models of CRC and healthy colon. These findings on further validation can facilitate the development of robust and effective cancer therapy.

HPLC analysis of intracellular folate levels in AgNP-treated bacterial culture showed a maximum folate concentration at the 6 th hour of microbial growth. The specific intracellular folic acid concentration was 45.53 ± 0.012 nmol/g-cell in AgNP-treated culture, which was 49% higher compared to control. The optimal-cytotoxic concentration was around 0.5 μM folic acid, which resulted in decrease of viability by 13.89% when compared with the appropriate control. The reduction in cell viability at lower concentrations of folic acid was statistically significant and exhibited Hormesis effect 1 .
Hence, this biphasic dose response phenomenon or the Hormesis effect, exhibited by folic acid needs to be studied further to establish its modality as an anti-cancer drug.  The association between SCFAs and folic acid metabolism was captured for the first time through flux analysis of the ROS expanded microbe model.

Supplementary Discussion
The colon-microbe and CRC-microbe integrated models consisted of 13589 and 14698 reactions, respectively. As per the modeling analysis, there were a total of 104 bacteria secreted metabolites that could be taken up by the host (CRC) cell, of which, 56 metabolites were predominantly required for increasing flux through CRC biomass reaction. It was found that any increment in the biomass flux was directly proportional to the uptake fluxes (mmol/g-DW/h) of these metabolites.
These significant metabolites included 20 amino acids, vitamins, and other micro-nutrients, which were the participating species in the biomass reaction, and therefore, can explain the increased biomass flux values. However, the other 48 metabolites showed no quantitative effects on the net biomass flux. The specific interactions among various microbe secreted metabolites, as well as their cumulative effects on the flux through biomass reaction were not considered in our modeling studies, which could possibly justify the reason why no significant quantitative change was observed in the biomass flux.
To understand the metabolic differences between models, flux span ratios were calculated, where FSr is defined as the difference between maximum and minimum flux.
Based on the FSr values (0.8>FSr>2), the following pathways were found to be affected: Both ASPCT and DHORTS are part of the CAD protein, and the gene coding the latter is highly expressed in many tumors. DHORDH is required for the formation of the pyrimidine ring 2 . It is the only enzyme that can catalyze the conversion of dihydroorotate to orotate, therefore, it is of utmost importance for synthesizing uridine monophosphate (UMP) and the inhibition of this enzyme causes suppression of the de novo pathway.
Moreover, the DHORDH catalyzed reaction is coupled ubiquinone reduction to generate ubiquinol 3

Fatty acid metabolism:
Cancer pathogenesis involves increased expression of genes encoding monoacylglycerol lipase, an enzyme of lipid metabolic pathway that releases free FA from lipid stores 5 . The rate controlling step in FAO is the import of fatty acid into the mitochondria catalyzed by tissue-specific isoforms of Carnitine O-Palmitoyltransferase (CPT). Enhanced activity of CPT increases FAO and ATP production and protects cells from glucose deprivation or hypoxia induced cytotoxicity 6 . Acyl-CoA dehydrogenases (ACADs) catalyzes the first and rate-determining step of peroxisomal betaoxidation of fatty acids 7 , whereas ECH catalyzes the second step of the mitochondrial FAO. Enoyl Coenzyme A Hydratase (ECH) metabolizes fatty acids to generate acetyl CoA and ATP by hydrolyzing the double bond between the second and third carbons on 2-trans/cis-enoyl-CoA 8 .
The role of ECHS1 has been implicated in breast, prostate, colon, and liver cancer, as per the literature 9 . In addition, the role of ECH has been identified in signal transduction, where it acts as a novel interacting protein of signal transducer and activator of transcription 3 (STAT3) 10 . Acetyl CoA acyltransferase catalyzes the final step of FAO, wherein, acetyl CoA is released and the CoA ester of a fatty acid two carbons shorter is formed 11 . The acetyl CoA is then consumed as substrate in the energy pathways. Upon analysis of the CRC-microbe and CRC models, these enzyme catalyzed reactions associated with fatty acid oxidation showed increased fluxes, thus capturing the significance of fatty acid oxidation in cancer in these metabolic models.

Amino acid metabolism:
Valine (Val), leucine (Leu) and isoleucine (Ile) are the branched-chain aliphatic amino acids (BCAAs), and their degradation products include acetyl Co A, which is an important substrate for fatty acid synthesis 12 . Besides their role as respiratory substrates, these amino acids also play a structural and signaling role 13 . The CRC-microbe integrated metabolic model showed an increase in fluxes for various enzyme catalyzed reactions associated with BCAAs degradation, thereby supporting increased BCAAs catabolism in cancer cell.

Energy metabolism:
L-Alanine:2-Oxoglutarate Aminotransferase (also known as Alanine aminotransferase, ALT) is an important enzyme, which catalyzes the formation of pyruvate and glutamate by transferring an amino group from alanine to alpha-ketoglutarate in alanine cycle 14 . Pyruvate is a critical metabolite, which participates in variety of metabolic pathways like glucose, amino acids, and lipid metabolism 15 . Glutamate serves as a precursor metabolite in glutamine formation. Glutamine again being a key metabolite, is a 1-carbon (1-C) donor in nucleic acid and amino acid synthesis.
The increased activity of ALT, in terms of metabolic flux was captured in CRC-microbe model.

Steroid metabolism:
Recent studies have emphasized the importance of sex hormones (specifically estrogen) in breast cancer pathogenesis 16 . Increased serum levels of estradiol (E2), the active form of estrogen, has been reported in colon cancer 17 . In the CRC-microbe model, steroid sulfatase (STS) and Hydroxysteroid (