Fructose promotes liver cancer via microbial acetate-induced O-GlcNAcylation

Abstract

High dietary fructose consumption is linked to multiple disease states including cancer. Zhou and colleagues recently reported a novel mechanism where high dietary fructose levels increase acetate production by the gut microbiome increasing post-translational modification O-GlcNAcylation in liver cells, which contribute to disease progression in mouse models of hepatocellular carcinoma.

Consumption of high levels of fructose contributes to the public health epidemic of obesity and obesity-related diseases including hepatocellular carcinoma (HCC) [1]. HCC accounts for over 90% of all liver cancer cases and is typically caused by a chronic pre-existing liver disease [2]. Increased fat deposits in the liver can lead to benign hepatic steatosis known as metabolic dysfunction-associated steatotic liver disease (MASLD) [2]. This condition can progress to a more severe condition of liver inflammation and fibrosis called metabolic dysfunction-associated steatohepatitis (MASH) and can further develop into HCC [2]. This disease progression has been linked to high fructose consumption [2, 3]. However, the mechanisms linking high fructose consumption to liver cancer are not well understood. Growing evidence has linked liver disease to communication with the intestinal microbiome. Recently in Cell Metabolism, Zhou et al. identified how a high-fructose diet in mice can increase microbiota-derived acetate that is utilized by cancer cells to enhance biosynthesis of the amino sugar uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) leading to hyper-O-GlcNAcylation and contributing to liver cancer cell growth and progression [4].

High-fructose diet enhances tumor progression in HCC mouse model and relies on increased O-GlcNAcylation.

Huang et al. demonstrated how supplementation of fructose in the diet for 10 weeks was sufficient to significantly increase tumor growth in both spontaneous and chemically (DEN/CCl₄)-induced HCC mouse models. The mice also suffered from significant weight gain and increased liver dysfunction. A high fructose consumption diet increased cell proliferation marker Ki67 in hepatocytes compared to control normal chow diet, reinforcing the idea that increased fructose consumption enhances tumor cell growth in HCC mice. The authors hypothesized that increased fructose consumption alters the metabolism in liver cancer cells; indeed, metabolomic profiling identified significant increases in various metabolites in the chemically induced HCC livers fed a high fructose diet, most notably, UDP-GlcNAc. UDP-GlcNAc is utilized by the enzyme O-linked N-acetylglucosamine transferase (OGT) which is responsible for adding O-GlcNAc groups to nuclear and cytoplasmic proteins. Both OGT and O-GlcNAc are found to be elevated in nearly all cancers and have been shown to contribute to cancer progression in HCC [5]. Fructose supplementation enhances O-GlcNAcylation within liver tumors cells and knock-down of OGT in these cells reversed the increase in Ki67 and decreased liver size and tumor volume in fructose-promoting HCC tumors compared to the controls. These results suggest that high fructose intake contributes to HCC tumor growth by enhancing O-GlcNAcylation.

Fructose-generated acetate increases glutamine metabolism and O-GlcNAcylation.

Previous studies have shown fructose metabolism in HCC cells is reduced [6], suggesting there may be an alternative mechanism by which fructose consumption leads to increased O-GlcNAcylation. The gut microbiome can convert fructose to acetate; in turn, acetate can be converted to acetyl-CoA in the liver [7]. Acetyl-CoA is essential for de novo glutamine synthesis, one of the major metabolites required to generate UDP-GlcNAc [5]. The authors confirmed this by supplementing the mice with acetate via the portal vein and observed increased O-GlcNAcylation in HCC cells. By tracing labelled carbons in fructose, the authors confirmed the carbons in fructose were supporting increased UDP-GlcNAc production via glutamine synthesis. Glutamine is synthesized under the regulation of the enzyme glutamine synthase (GLUL) [8]. Interestingly, GLUL is elevated in HCC tumor cells and genetic targeting of GLUL in the liver reduced the levels of fructose-derived glutamine, UDP-GlcNAc levels, total O-GlcNAcylation and reduced overall HCC tumor progression. The metabolic pathway of acetate conversion to acetyl-CoA, glutamine, UTP leading to increase in UDP-GlcNAc levels was found to be specific to HCC cells, but not in hepatocytes. It would be interesting to examine in future studies whether additional pathways that can also increase UDP-GlcNAc synthesis from acetyl-CoA, including acetylation of enzymes in the hexosamine biosynthetic pathways [9] which may also contribute to increased O-GlcNAcylation in HCC under high fructose-stress.

Fructose-mediated O-GlcNAcylation of elongation factor eEF1A1 and protease CAPNS1 promotes tumor growth in HCC

To understand mechanistically how hyper-O-GlcNAcylation contributes to HCC, the authors performed O-GlcNAcylomics to identify all O-GlcNAcylated proteins in their fructose-mediated HCC tumor models. This revealed two O-GlcNAcylated proteins, calpain small subunit 1 (CAPNS1) and eukaryotic elongation factor 1A1 eEF1A1, both of which have been shown to act as tumor promoters in HCC [10, 11]. Expression of CAPNS1 and eEF1A1 O-GlcNAcylation-site mutants blocked tumor growth and proliferation in vivo compared to expression of wildtype proteins. The O-GlcNAcylomics analysis identified many interesting pathways that are elevated in HCC upon high fructose diets. Most notably, the analysis showed that several key proteins involved in the cell death pathway of ferroptosis to be highly O-GlcNAcylated. This is quite interesting as ferroptosis is emerging as a critical cell death pathway in cancer, yet little is known on mechanisms connecting O-GlcNAcylation to regulation of ferroptosis. These results indicate fructose supplementation can promote HCC, in part, by increasing O-GlcNAcylation of eEF1A1 and CAPNS1 to drives tumor growth. However, other O-GlcNAcylated proteins may also contribute to this phenotype.

Acetate from microbiota enhances O-GlcNAcylation and promotes liver cancer in fructose supplemented mice

Lastly, the authors sought out to directly test whether the gut microbiota is required to generate fructose-derived acetate to promote HCC tumor progression. By adding a broad-spectrum antibiotic to the fructose-containing drinking water, 99% of the gut microbiota was depleted. The addition of the antibiotic to the drinking water decreased the levels of glutamine, UDP-GlcNAc, and total O-GlcNAcylation in cancer cells and reduced tumor growth. The supplementation of acetate to the antibiotic in the drinking water was able to restore the levels of metabolites and tumor burden. This important finding highlighted the relevance of the gut microbiota to generate acetate ultimately to enhance O-GlcNAcylation and promote HCC progression.

Concluding remarks and future perspectives

Overall, Zhou at al. report a novel mechanism by which the gut microbiota can generate fructose-derived acetate that is converted to acetyl-CoA for glutamine synthesis and the generation of UDP-GlcNAc, leading to hyper-O-GlcNAcylation of tumor-promoting proteins eEFA1 and CAPNS1 contributing to liver cancer progression (Figure 1). This study implicated a possible role of acyl-CoA synthetase short chain family member 1 (ACSS1), a key mitochondrial enzyme that converts acetate to acetyl-CoA and contributes to glutamine metabolism. ACSS1 levels have been found to be elevated in patients with HCC with poor outcomes; since ACSS1 knockout mice are viable [12], drugs targeting this enzyme may have minimal toxicities and may serve as a possible therapeutic target in regulating fructose-mediated HCC progression. On the other hand, targeting OGT or GLUL with small molecules may be problematic, as they both have critical functions in normal cells. These data raise additional interesting questions. Is this pathway specific to DEN/CCl4-induced liver cancer model or a general pathway in other HCC models? Can high levels of acetate from microbiome also contribute to progression of MASH or MASLD into HCC? Does fructose-derived acetate from the gut microbiota regulate other microbiome-dependent cancers including colon, gastric, and pancreatic cancers via a similar pathway? Further studies are needed to answer these questions and help advance our knowledge in understanding how diets in high fructose impacts the microbiome and contribute to disease states such as cancer.

Figure 1. Fructose supplementation induces HCC progression via O-GlcNAcylation of eEf1A1 and CAPNS1.

A chemically induced (DEN/CCl₄) HCC mouse model fed a high fructose diet increased progression of liver cancer via microbiota-generated acetate that is taken up by hepatocytes possibly via MCT1. Acetate is converted into Acetyl-CoA the mitochondria by the enzyme ACSS1 and acetyl-CoA enters the TCA cycle to produce glutamate and converted to glutamine by the GLUL. Glutamine is used to generate UDP-GlcNAc which serves as a substrate for OGT to O-GlcNAcylate eEF1A1 and CAPNS1 that drive growth and progression of HCC. Abbreviations: MCT1, monocarboxylate transporter 1: ACSS1, Acyl-CoA Synthetase Short Chain Family Member 1; TCA, tricarboxylic acid cycle; GLUL, glutamine synthase; UDP-GlcNAc, uridine diphospho-N-acetylglucosamine; OGT, O-GlcNAc transferase; eEF1A1, eukaryotic elongation factor 1A1; CAPNS1, calpain small subunit 1; HCC, hepatocellular carcinoma. Figure created with BioRender (https://biorender.com/).