KLF8 is associated with poor prognosis and regulates glycolysis by targeting GLUT4 in gastric cancer

Abstract

Krüppel-like transcription factor (KLF) family is involved in tumorigenesis in different types of cancer. However, the importance of KLF family in gastric cancer is unclear. Here, we examined KLF gene expression in five paired liver metastases and primary gastric cancer tissues by RT-PCR, and immunohistochemistry was used to study KLF8 expression in 206 gastric cancer samples. The impact of KLF8 expression on glycolysis, an altered energy metabolism that characterizes cancer cells, was evaluated. KLF8 showed the highest up-regulation in liver metastases compared with primary tumours among all KLF members. Higher KLF8 expression associated with larger tumour size (P < 0.001), advanced T stage (P = 0.003) and N stage (P < 0.001). High KLF8 expression implied shorter survival outcome in both TCGA and validation cohort (P < 0.05). Silencing KLF8 expression impaired the glycolysis rate of gastric cancer cells in vitro. Moreover, high KLF8 expression positively associated with SUVmax in patient samples. KLF8 activated the GLUT4 promoter activity in a dose-dependent manner (P < 0.05). Importantly, KLF8 and GLUT4 showed consistent expression patterns in gastric cancer tissues. These findings suggest that KLF8 modulates glycolysis by targeting GLUT4 and could serve as a novel biomarker for survival and potential therapeutic target in gastric cancer.

Keywords: KLF8; Warburg effect; gastric cancer; prognosis.

Figure 1

KLF8 is a potential biomarker for gastric cancer. A, Heatmap of KLF family genes in five paired primary gastric cancer tissues and liver metastasis using RT‐PCR. Results were normalized by Z‐score. Each column represents a specimen denoted on the above and each role represents a gene which is denoted on the right. Red colour indicates up‐regulated genes and green colour indicates down‐regulated genes. P: primary gastric cancer tissue; M: corresponding liver metastasis. KLF8 was significantly up‐regulated in liver metastases than corresponding primary tumours. B, X‐tile analyses of 5 years overall survival were performed using patient data from TCGA to determine the optimal cut‐off values for KLF8 mRNA. The gastric cancer patients were divided into training and validation sets. X‐tile plots of training sets are shown in the left panels, with plots of matched validation sets shown in the smaller inset. The optimal cut‐off values highlighted by the black circles in left panels are shown in histograms of the entire cohort (middle panels), and Kaplan‐Meier plots are displayed in right panels. P values were determined using the cut‐off values defined in training sets and applying them to validation sets. High KLF8 expression was associated with shorter overall survival (P = 0.006)

Figure 2

Overexpression of KLF8 is correlated with poor survival for gastric cancer patients. KLF8 expression was detected by immunohistochemical staining in gastric cancer tissues. KLF8 was mainly localized in the nuclei of tumour cells. A, Examples of high (i) or low (ii) KLF8 expression in gastric cancer, and high (iii) or low (iv) KLF8 expression in normal gastric tissues. Magnification, 400×. B, The percentage of high KLF8 expression was significantly higher in gastric cancer than normal controls (P = 0.001). (C, D) Patients were divided into KLF8 high and low expression groups and Kaplan‐Meier analyses were performed to evaluate the correlation of KLF8 expression and survival. (C) Overexpression of KLF8 was correlated with worse OS (χ ² = 19.15, P < 0.001). (D) Overexpression of KLF8 was correlated with worse DFS (χ ² = 17.58, P < 0.001)

Figure 3

KLF8 promotes glycolysis in gastric cancer. KLF8 expression was silenced by shRNA in AGS and MGC803 cells, and knockdown efficacy was confirmed by Western blotting (A) and RT‐PCR (B). Knockdown of KLF8 expression significantly decreased glucose consumption (C), lactate production (D) and ATP production (E). Patients with high KLF8 expression exhibited higher SUVmax value than those with low KLF8 expression (P = 0.008). *P < 0.05

Figure 4

KLF8 promotes glycolysis by targeting glucose transporter GLUT4. (A) Silencing KLF8 up‐regulated or down‐regulated several rate‐limiting genes in glycolysis process, and the most significantly decreased was GLUT4 in AGS cells. sh = shKLF8, c: control. 1, 2, 3 means repeated three times. (B) RT‐PCR and (C) Western blot analysis confirmed that knockdown of KLF8 expression decreased GLUT4 expression in both transcriptional and protein levels in AGS and MGC803 cells. (D) Silencing KLF8 expression decreased GLUT4 expression on cell membranes. (E) KLF8 and GLUT4 show consistent expression patterns in gastric cancer tissues. High KLF8 expression is often accompanied by high GLUT4 expression, and GLUT4 is always expressed at low levels when KLF8 is expressed at low levels. (F) KLF8 was positively correlated with GLUT4 expression in the TCGA database (P < 0.001). (G) Dual luciferase assay indicated that KLF8 activated the GLUT4 promoter activity in a dose‐dependent manner. *P < 0.05

Figure 5

GLUT4 expression in gastric cancer. (A) X‐tile analysed indicated that high GLUT4 expression predicted poor survival in gastric cancer (P = 0.045). GLUT4 expression was further analysed in the validation cohort using IHC staining. The results demonstrated that patients with higher GLUT4 expression levels had worse OS (B) and DFS (C) rates than did patients with lower GLUT4 expression levels. (D) A positive relationship between GLUT4 and KLF8 expression was also observed in the validation cohort (P < 0.001). GLUT4 was knocked down in AGS and MGC803 cells, and the knockdown effect was determined by Western blot (E) and RT‐PCR (F) analysis. Silencing GLUT4 markedly inhibited glucose consumption (G), lactate production (H) and ATP production (I) in both AGS and MGC803 cells (P < 0.05). *P < 0.05