Abstract B27: Transfection of mutant P53 promotes membrane translocation of GLUT1 and increases glucose uptake in MCF10A human breast epithelial cells
Authors: Ahn Sung Gwe . , Shin Hyekyoung , June Bae Soong , Changik Yoon , Woo Lee Hak , Joon Jeong 
Published: August 2018

Background: High rate of glycolysis exhibited by cancer cells plays a crucial role in carcinogenesis and tumor progression. Previous study showed that gain-of-function (GOF) by mutant P53 drives the Warburg effect via GLUT1 membrane translocation in malignancies. Also, in the patients with a preoperative 18F-fluorodeoxy glucose positron emission tomography (FDG-PET), which enables to estimate glucose uptake by tumor as standardized uptake value (SUV), we identified high SUV as a poor prognostic marker. In this study, we wondered whether P53 promotes GLUT1 membrane translocation and increases glucose uptake in human normal breast epithelial cells using MCF10A cells.

Methods: We generated mutant-P53 MCF10A cell line, which include sequence variants (R175H, R273H, and R248W). First, to generate stable P53 knockdown cell lines, lentiviral transduction particles from TRC-pLKO-U6 shP53 were infected into MCF10A. To generate mutant P53 producing MCF10A, pcDNA mutant P53 was transfected using lipofectamin (Invitrogen) into stable P53 knockdown cell lines. Glucose uptake assay and confocal imaging studies with 2NDBG were performed. Confocal imaging system was used to detect GLUT1 membrane expression. In case-matched patients according to P53 mutation (n=114), we compared SUV. Mutational analysis of exons 5-9 of the P53 gene was carried out using Sanger sequencing.

Results: Glucose uptake assay showed that the level of glucose uptake was increased in P53 mutant MCF10A cell lines (R175H or R273H, and R248W) with a 1.5-1.8 fold change compared with wild-type MCF10A. The increase of glucose uptake by mutant-P53 was reproducible with the experiments with 2NBGD. The mRNA expressional level of LDHA was also upregulated by mutant P53. In contrast, the mRNA expression of GLUT1, hexokinase 2, PKM1, PKM2, and G6PD was not elevated by P53 mutation. The confocal imaging studies captured translocation to membrane of GLUT1 in mutant P53-MCF10A. However, in wild-type MCF10A, GLUT1 was diffusely expressed in cytoplasm. In patients, the mean SUV of p53-mutational group was significantly higher than that of wild-type p53-group (7.49 vs. 5.44, P=0.013). Compared with the group with intact p53, recurrence-free survival (RFS) was significantly reduced in the group with p53 mutation (P =0.017). In multivariate analysis, p53 mutational status carried prognostic significance (hazard ratio 3.73, 95% CI 1.15-12.07) independent of tumor size, nodal status, and estrogen receptor status.

Conclusion: We showed that P53 mutation promotes GLUT1 translocation to membrane, which consequently induces glucose influx in MCF10A cells, elucidating that P53 mutation contributes to the increase of SUV in patients with breast cancer.

Citation Format: Sung Gwe . Ahn, Hyekyoung Shin, Bae Soong June, Yoon Changik, Lee Hak Woo, Jeong Joon. Transfection of mutant P53 promotes membrane translocation of GLUT1 and increases glucose uptake in MCF10A human breast epithelial cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B27.