The Dysregulation of Polyamine Metabolism in Colorectal Cancer Is Associated with Overexpression of c-Myc and C/EBPβ rather than Enterotoxigenic Bacteroides fragilis Infection

Abstract

Colorectal cancer is one of the most common cancers in the world. It is well known that the chronic inflammation can promote the progression of colorectal cancer (CRC). Recently, a number of studies revealed a potential association between colorectal inflammation, cancer progression, and infection caused by enterotoxigenic Bacteroides fragilis (ETBF). Bacterial enterotoxin activates spermine oxidase (SMO), which produces spermidine and H2O2 as byproducts of polyamine catabolism, which, in turn, enhances inflammation and tissue injury. Using qPCR analysis, we estimated the expression of SMOX gene and ETBF colonization in CRC patients. We found no statistically significant associations between them. Then we selected genes involved in polyamine metabolism, metabolic reprogramming, and inflammation regulation and estimated their expression in CRC. We observed overexpression of SMOX, ODC1, SRM, SMS, MTAP, c-Myc, C/EBPβ (CREBP), and other genes. We found that two mediators of metabolic reprogramming, inflammation, and cell proliferation c-Myc and C/EBPβ may serve as regulators of polyamine metabolism genes (SMOX, AZIN1, MTAP, SRM, ODC1, AMD1, and AGMAT) as they are overexpressed in tumors, have binding site according to ENCODE ChIP-Seq data, and demonstrate strong coexpression with their targets. Thus, increased polyamine metabolism in CRC could be driven by c-Myc and C/EBPβ rather than ETBF infection.

Figure 1

(a) Enterotoxigenic B. fragilis (ETBF) DNA copy number per 50 ng of total extracted DNA in paired samples of colorectal cancer (logarithmic scale). (b) SMOX expression level relatively to two reference genes: RPN1 and GUSB. The samples with high rates of ETBF colonization tend to have higher expression of SMOX, especially in normal tissue (compared to the other norms). However, no statistically significant correlation between SMOX expression and ETBF colonization was observed.

Figure 2

Results of the qPCR expression analysis of genes involved in polyamine metabolism and inflammation regulation in paired colorectal cancer samples. Cell color indicates expression level change in tumor compared to normal: increase (orange) and decrease (blue). Genes are rearranged according to the similarity of expression profiles. Samples with a high concentration of enterotoxigenic B. fragilis DNA (>1000 copies per 1 ng of total DNA) are marked with an asterisk.

Figure 3

Results of coexpression analysis for genes participating in polyamine metabolism. Pearson correlation coefficients between the expression levels changes of genes participating in polyamine metabolism and inflammation across 50 colorectal cancer samples are presented. Cell color reflects these values (green: positive, brown: negative). Normalized ChIP-Seq score (according to ENCODE data) is indicated with blue bars.

Figure 4

Classic path of polyamine metabolism consists of the following: (1) arginine is converted to ornithine through the action of ARG (arginase) in the urea cycle; (2) putrescine is formed from the reaction of ornithine decarboxylation catalyzed by ODC1 (ornithine decarboxylase-1). OAZ can bind to ODC1 to form OAZ-ODC1 complex and subsequently reduce polyamine synthesis. AZIN1 (antizyme inhibitor-1) brakes the ODC1-OAZ complex and liberates ODC1; (3) AMD1 (S-adenosylmethionine decarboxylase) decarboxylates S-adenosylmethionine (SAM) to decarboxylated SAM (dcSAM); (4) dcSAM provides aminopropyl groups to putrescine to produce spermidine by spermidine synthase (SRM) and spermine by spermine synthase (SMS). MTA (methylthioadenosine) is generated as a byproduct. Spermine can be recycled back to spermidine directly by spermine oxidase (SMOX). Spermine and spermidine can be recycled to spermidine and putrescine by spermidine/spermine-N1-acetyltransferase (SAT1) followed by oxidation by polyamine oxidase (PAOX) [101]. MTA can be processed to the methionine: MTA phosphorylase (MTAP) catalyzes the cleavage of MTA yielding 5-methylthioribose-1-phosphate (MTRu-P), which is further metabolized to DHKMP (1,2-dihydro-3-keto-5-methylthiopentene). ADI (acireductone dioxygenase) catalyzes DHKMP to 2-oxo-4-methylthiobutyrate (KMTB) and transamination of KMTB results in formation of methionine [–104].