Glucose Enhances The Stability Of β-catenin Via The Hexosamine Biosynthetic Pathway In Endometrial Cells

EC is the most common cancer of the female genital tract in the United States with approximately 52,630 new cases and 8,590 deaths occurring in 2014. With economic development and westernization of lifestyles, the occurrence of EC has also risen rapidly in Asian countries. Although it is widely known that EC is more prevalent among diabetic and/or obese women, little is known about the underlying mechanism. Excessive exposure to carcinogens, including insulin, estrogens, insulin-like growth factors, leptin, and adiponectin, may contribute to the development of EC in diabetic and/or obese women. However, elevated serum glucose levels, an early symptom of diabetes and obesity, may directly regulate tumor-related signaling pathways, especially meeting the high glucose need of cancer cells.Objective:Endometrial cancer(EC) is one of the most common gynecological malignancies in the world. Associations between fasting glucose levels(greater than 5.6 mmol/L) and the risk of cancer fatality have been reported. However, the underlying link between glucose metabolic disease and EC remains unclear. In the present study, we explored the influence of elevated glucose levels on the Wnt/β-catenin pathway in EC.Methods and Results:1. β-catenin expression is associated with glucose concentration in EC cells.AN3CA(A) and HEC-1-B(B) cells were maintained with 0, 5.5, or 25 mM glucose, and the levels of β-catenin expression and O-GlcNAcylation were determined by westernblot.Higher O-GlcNAcylation and β-catenin expression levels were observed for 25 mM glucose than for 0 and 5.5mM glucose(“high glucose effects”).2.Glucose modulates β-catenin via the hexosamine pathway.We explored the HBP using DON and Azaserine, inhibitors of the HBP rate-limiting enzyme GFAT(glutamine: fructose-6-phosphate amidotransferase). We found that DON and Azaserine reversed the “high glucose effects” on the expression of β-catenin in the cell lines. Additionally, GlcNH2(glucosamine), which can directly enter the HBP downstream of GFAT, was able to replicate the “high glucose effects” in low glucose medium;3.HBP enhances the expression of β-catenin by elevating its O-GlcNAcylation levels. PUGNAc is an inhibitor of O-GlcNAcase(OGA) that mediates the removal of O-GlcNAc from proteins. We cultured AN3 CA and HEC-1-B cells in 0 and 5.5 mM glucose with or without PUGNAc(50 μM). Results showed that PUGNAc elevated the expression of β-catenin as well as increased the overall level of O-GlcNAcylation4.Glucose-induced β-catenin elevation enhances the activation of Wnt signaling.To investigate whether the glucose-induced β-catenin elevation could activate the Wnt signaling pathway, we used the TOP/FOP FLASH assay. High glucose treatment enhances Wnt reporter gene activation in cell lines, and PUGNAc augments this effect. Meanwhile, these phenomena could be reversed by adding Azaserine and DON.5.Glucose-induced elevation of β-catenin O-GlcNAcylation increases nuclear expression of β-catenin and activates downstream target genes.AN3CA and HEC-1-B cells were cultured in 0, 5.5 or 25 mM glucose with or without Azaserine(50 μM). It showed that higher glucose concentrations led to an increase in β-catenin in the nucleus and Azaserine reversed this phenomenon. Consistent with this result, the expression of Wnt target genes(Axin2 and CyclinD1) were increased following the increase of glucose concentration, which could be reversed by adding Azaserine and DON. Additionally, PUGNAc elevated the expression of Axin2 and Cyclin D16.Femal SD rats were randomly divided into 5 groups: normal control group, fasting group, high suger high fat group, glucosamine group and glucose group. Rats in high glucose and high fat group were given high glucose and high fat diet. Other groups were given normal diet. And after 8 weeks’ feeding endometrium of normal control group and high suger high fat group were collected. Fasting group, glucosamine group and glucose group were fasted for 24 h and force-fed with glucose or glucosamine for 3 hours. And then endometrium were collected, homogenized, and analyzed by Western blotting and RT-PCR.The expression of β-catenin of high suger high fat group was significantly higher than that in the normal control group and O-GlcNAc levels also increased. GLU group and GLN group has an elevated expression of β-catenin and increased level of O-GlcNAcylation compared with FASTING group. But there is no significant difference between GLU group and GLN group. The hexosamine biosynthetic pathway(HBP) and O-GlcNAcylation drive the expression of β-catenin and the abnormal aggregation of β-catenin can further activate downstream target genes.ConclusionElevated glucose levels, via HBP, increase the O-GlcNAcylation level, thereby inducing the over expression of β-catenin and subsequent transcription of the target genes in endometrial cancer cells. In addition, high blood glucose level induces an increased expression of β-catenin and O-GlcNAc levels in endometrial cells of femal SD rats and the elevated β-catenin can active transcription of target genes in the endometrium.