The NF-κB signalling pathway regulates GLUT6 expression in endometrial cancer

Highlights

• Mutations or amplifications in GLUT6 occur more frequently in endometrial cancer than all other malignancies

• GLUT6 gene expression positively correlates with genes of the NF-κB signalling pathway in endometrial cancer

• A transcription factor PCR array identifies RELA as a positive regulator of GLUT6 gene expression

• Constitutively-active RELA increases, while dominant-negative RELA decreases GLUT6 expression in endometrial cells

• TNFα stimulation of endometrial cells exclusively increases GLUT6 expression without altering other glucose transporters

Abstract

Background

Gene and protein expression of the glucose transporter GLUT6 are elevated in multiple cancers, including endometrial cancer. However, the extrinsic and intrinsic mechanisms that regulate GLUT6 expression in this malignancy are unknown. Herein we investigate the potential mechanisms regulating GLUT6 expression in endometrial cancer.

Methods

Data mining of the GLUT6 gene (SLC2A6) in The Cancer Genome Atlas (TCGA) PanCan datasets was performed in cBioPortal. A transcriptome PCR array was used to identify regulators of GLUT6 expression. The role of RELA in regulating GLUT6 gene and protein expression was investigated by overexpressing constitutively active and dominant-negative RELA in endometrial cells. Endometrial cells were treated with the pro-inflammatory cytokine TNFα and the expression of RELA, IκBα, TNFα, and GLUT6 were examined by Western blotting and RT-qPCR.

Results

GLUT6 is altered in 1% of all cancer samples (157 of 10, 967 samples) within TCGA datasets including 4.7% of uterine (endometrial) cancers. GLUT6 expression was positively co-expressed with multiple members of the NF-κB signalling pathway including NFKB2, RELB, NFKBIE, and TNF in endometrial cancer samples. A transcriptome PCR array identified RELA as the top potential transcriptional regulator of GLUT6 expression. Overexpression of constitutively active RELA increased GLUT6 gene expression in normal endometrial epithelial cells (hUE-Ts), while overexpression of dominant-negative RELA decreased GLUT6 expression in cancerous RL95–2 endometrial cells. TNFα treatment activated canonical NF-κB signalling and increased the expression of GLUT6, but not that of other glucose transporters (GLUTs 1, 3, 4, 8, 10, or 12) in endometrial cells.

Conclusions

TNFα is a cytokine that is commonly increased in obesity-related endometrial cancer and the findings herein support a potential mechanism whereby TNFα may contribute to endometrial cancer initiation or progression by increasing GLUT6 expression. Furthermore, we identified RELA, an important downstream mediator of the TNFα signalling cascade, as a regulator of GLUT6 expression in endometrial cells. Future studies are warranted to determine how GLUT6 expression affects endometrial tumourigenesis or cancer progression.

Introduction

Endometrial cancer is the most common gynecological cancer in the developed world and the fifth most common cancer in women worldwide [1,2]. The incidence of endometrial cancer is increasing due to declining fertility rates, increased life expectancy, sedentary lifestyles and increased incidence of obesity and type 2 diabetes [3,4]. Obesity is the greatest identified risk factor for endometrial cancer with over 55% of cases attributed to excess body weight [[5], [6], [7]] and obese women have a 6-fold higher relative risk of endometrial cancer-related death compared to lean women [8,9]. Although the precise mechanisms are unclear, a number of obesity-related factors are suspected to promote endometrial tumour growth and/or progression. These include increased/unopposed estrogen [10], altered serum adipokines [11], insulin resistance with associated hyperglycemia and hyperinsulinemia [12] and chronic inflammation [13].

Elevated levels of proinflammatory cytokines such as tumour necrosis factor alpha (TNFα) have been identified in the sera of endometrial cancer patients, and have been linked to an increased risk of endometrial cancer [[14], [15], [16], [17], [18], [19], [20]]. Furthermore, high secretion of TNFα by cancerous endometrial epithelial cells cultured in vitro is associated with advanced-stage disease and worse overall survival for the patients from which they were derived [21]. TNFα activates the canonical nuclear factor kappa light chain enhancer of activated B cells (NF-κB) signalling pathway by activating IκB kinase (IKK) which phosphorylates IκB, targeting it for ubiquitination and degradation. Loss of IκB protein frees the NF-κB complex which then translocates to the nucleus where it directly binds to the promoter or enhancer regions of target genes to regulate their expression. The NF-κB complex is a homo- or heterodimer formed by the REL-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52, of which the heterodimeric RELA-NFKB1 complex is the most abundant. Once inside the nucleus, post-translational modifications, such as the phosphorylation of RELA/p65 at Ser536, increase NF-κB transcriptional activity and modulate DNA binding and/or oligomerization [22,23].

In a previous study from our laboratory, we compared the transcriptome of endometrial tissue from equally obese women with and without cancer [24]. We found that the glucose transporter GLUT6 (SLC2A6) was upregulated in malignant tissue by ~37-fold, more so than any other glucose transporter. We validated that GLUT6 protein was upregulated in malignant tissue compared to matched benign tissue of an independent cohort of endometrial cancer patients [24]. GLUT6 was also highly expressed in 7 human endometrial cancer cell lines, but only lowly expressed in normal endometrial epithelial or stromal cell lines. Importantly, knockdown of GLUT6 in endometrial cancer cells decreased glucose metabolism and induced cell death [24]. Results from this study suggested that GLUT6 may be beneficial for endometrial tumour growth and/or survival. Furthermore, since GLUT6 is not widely expressed in the human body [25] and loss of GLUT6 does not result in any deleterious effects to mouse physiology, whole-body glucose metabolism [26] or macrophage inflammatory function [27,28], GLUT6 represents an attractive therapeutic target for endometrial cancer. However, it is unclear what factor/s drive GLUT6 expression in endometrial cancer. The aim of this study was to therefore identify factors that regulate GLUT6 expression to gain a better understanding of the mechanisms underpinning GLUT6 upregulation in endometrial cancer.