Roles of the hexosamine biosynthetic pathway and pentose phosphate pathway in bile acid-induced cancer development

Abstract

Esophageal squamous cell carcinomas (ESCCs) as well as adenocarcinomas (EACs) were developed in rat duodenal contents reflux models (reflux model). The present study aimed to shed light on the mechanism by which bile acid stimulation causes cancer onset and progression. Metabolomics analyses were performed on samples of neoplastic and nonneoplastic tissues from reflux models, and K14D, cultivated from a nonmetastatic, primary ESCC, and ESCC-DR, established from a metastatic thoracic lesion. ESCC-DRtca2M was prepared by treating ESCC-DR cells with taurocholic acid (TCA) to accelerate cancer progression. The lines were subjected to comprehensive genomic analyses. In addition, protein expression levels of glucose-6-phosphate dehydrogenase (G6PD), nuclear factor kappa B (NF-κB) (p65) and O-linked N-Acetylglucosamine (O-GlcNAc) were compared among lines. Cancers developed in the reflux models exhibited greater hexosamine biosynthesis pathway (HBP) activation compared with the nonneoplastic tissues. Expression of O-GlcNAc transferase (OGT) increased considerably in both ESCC and EAC compared with nonneoplastic squamous epithelium. Conversely, cell line-based experiments revealed the greater activation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. G6PD overexpression in response to TCA exposure was observed. Both NF-κB (p65) and O-GlcNAc were expressed more highly in ESCC-DRtca2M than in the other cell lines. Moreover, ESCC-DRtca2M cells had additional chromosomal abnormalities in excess of ESCC-DR cells. Overall, glucose metabolism was upregulated in both esophageal cancer tissue and cell lines. While bile acids are not mutagenic, chronic exposure seems to trigger NF-κB(p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression. Glucose metabolism was upregulated in both esophageal cancer tissue and cell lines, and the HBP was activated in the former. The cell line-based experiments demonstrated upregulation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. While bile acids are not mutagenic, chronic exposure seems to trigger G6PD overexpression and NF-κB (p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression.

Keywords: NF-κB; bile acids; hexosamine biosynthesis pathway; metabolomics analyses; pentose phosphate pathway.

Figure 1

A duodenal contents reflux model. B, bile duct; D, duodenum; E, esophagus; J, jejunum. Following total gastrectomy, the upper jejunum and lower esophagus were connected by end‐to‐side anastomosis. The procedure allows duodenal fluid to flow back into the esophagus

Figure 2

Histological image of esophageal tumor induced in the duodenal fluid reflux model. A, Esophageal squamous cell carcinomas: Note the infiltration nests with keratinization. B, Adenosquamous carcinoma: Note the tumor contains a coexisting neoplastic squamous component with hyperkeratinization on the left side and adenocarcinomatous component with well formed glands on the right side. C, Mucinous adenocarcinoma: Note the atypical glands lined by mucus‐secreting epithelia surrounding groups of extracellular mucin. Scale line = 500 μm

Figure 3

Metabolomics analysis of rat esophageal tissues. A, Clustering analysis. The hierarchical clustering analysis identified 3 clusters corresponding to mucinous adenocarcinomas (Muc‐1 to Muc‐3), esophageal squamous cell carcinomas (ESCC) (ESCC‐1 to ESCC‐3), and nonneoplastic samples [N‐1(Muc) to N‐3(Muc) and N‐1(ESCC) to N‐3(ESCC)]. B, PCA. Blue circle: Mucinous adenocarcinomas (n = 3), red circle: ESCCs (n = 3), green circle: nonneoplastic tissues from mucinous adenocarcinomas (n = 3), orange circle: nonneoplastic tissues from ESCCs (n = 3). Metabolites were organized into 3 groups in both PCA and clustering analysis: adenocarcinoma, squamous cell carcinoma, and nonneoplastic tissue. C, HBP activation in esophageal cancer tissue, (ND: Not detected). PPP upregulation is not apparent, while HBP activation is evident. UDP‐GlcNAc production notably increased in ESCCs. D, Representative metabolites in rat esophageal tissues. Error bars indicate SD (n = 3). Representative metabolites in cancer tissues in the HBP, such as NAcGlcNP, GlcNAc, and GlcNAc‐P, increased considerably compared with in nonneoplastic tissues. UDP‐GlcNAc production significantly increased in ESCCs (Welch's t test, P = .009)

Figure 4

O‐GlcNAc transferase (OGT) expression in rat tissues. Immunohistochemical staining of OGT revealed weak nuclear positivity in nonneoplastic squamous epithelium of the upper esophagus of the reflux model (A). Conversely, the staining of OGT revealed strong nuclear and weakly cytoplasmic positivity in both ESCC and mucinous adenocarcinoma cells (B,C). Scale line = 200 μm

Figure 5

Cytological findings for all lineages. K14D (A) cells were larger than ESCC‐DR (B) or ESCC‐DRtca2M cells (C). K14D and ESCC‐DR cells were characterized by sheet‐like proliferation; ESCC‐DRtca2M cells exhibited cytoplasmic projections and few intercellular junctions, which increased their migratory potential. Scale line = 200 μm

Figure 6

Metabolomics analysis of ESCC lines. A, Clustering analysis.The hierarchical clustering analysis identified 3 clusters, which were divided by a bold dotted line, corresponding to the K14D, ESCC‐DRtca2M, and ESCC‐DR and ESCC‐DRtca24h lines. The clusters of ESCCDRtca24h and ESCC‐DR were similar. B, PCA. Blue circle: ESCC‐DR (n = 3), red circle: ESCC‐DRtca24h (n = 3), green circle: ESCC‐DRtca2M (n = 3), orange circle: K14D (n = 3). Metabolites were organized into 3 groups in the PCA: K14D, ESCC‐DRtca2M, and ESCC‐DR and ESCC‐DRtca24h lines. A shift of ESCC‐DRtca24h cluster from ESCC‐DR cluster in the direction of ESCC‐DRtca2M cluster occurred. C, Bile acid exposure upregulates the pentose phosphate pathway (ND: Not detected). Compared with other lines, ESCC‐DRtrca2M cells had considerably lower levels of glycolysis products (G6P, F6P, F1,6P), and much higher levels of PPP‐related metabolites (6‐PG, Ru5P, R5P). Conversely, most metabolites in the HBP were not detected. D, Representative metabolites in ESCC lines. Error bars indicate SD (n = 3). Although the volume of G6P associated with glycolysis was the lowest in ESCC‐DRtca2M cells, Ru5P, and R5P in ESCC‐DRtca2M cells were much higher than in ESCC‐DR, ESCC‐DRtca24h, and K14D cells. 6‐PG in ESCC‐DRtca2M cells was considerably higher than in the other cell lines

Figure 7

Western blot analysis of esophageal cell lines. G6PD expression was significantly higher in ESCC‐DRtca2M and ESCC‐DRtca24h cells than in K14D and ESCC‐DR cells. Both NF‐κB(p65) and O‐GlcNAc protein expression increased with increasing malignancy. However, no marked correlation was observed between NF‐κB2 (p100/p52) and cancer progression

Figure 8

Comparative genomic hybridization microarray analysis of esophageal cell lines. A, K14D, B, ESCC‐DR, C, ESCC‐DRtca2M, D, ESCC‐DR vs ESCC‐DRtca2M. Upward and downward changes indicate copy number gain and deletion, respectively. Numerous chromosome abnormalities were detected in all the 3 cell lines (K14D, ESCC‐DR, ESCC‐DRtca2M) (A‐C). Subsequently, as a test, ESCC‐DRtca cells were analyzed concerning ESCC‐DR (control). D, Indicates that the tumor progression caused by continuous TCA stimulation is associated with numerous additional chromosome abnormalities