GLUT3 induced by AMPK/CREB1 axis is key for withstanding energy stress and augments the efficacy of current colorectal cancer therapies

Abstract

Cancer cells are usually characterized by hyperactive glucose metabolism, which can often lead to glucose scarcity; thus, alternative pathways to rewire cancer metabolism are required. Here, we demonstrated that GLUT3 was highly expressed in colorectal cancer (CRC) and negatively linked to CRC patient outcomes, whereas GLUT1 was not associated with CRC prognosis. Under glucose-limiting conditions, GLUT3 expedited CRC cell growth by accelerating glucose input and fuelling nucleotide synthesis. Notably, GLUT3 had a greater impact on cell growth than GLUT1 under glucose-limiting stress. Mechanistically, low-glucose stress dramatically upregulated GLUT3 via the AMPK/CREB1 pathway. Furthermore, high GLUT3 expression remarkably increased the sensitivity of CRC cells to treatment with vitamin C and vitamin C-containing regimens. Together, the results of this study highlight the importance of the AMPK/CREB1/GLUT3 pathway for CRC cells to withstand glucose-limiting stress and underscore the therapeutic potential of vitamin C in CRC with high GLUT3 expression.

Fig. 1

GLUT3 is highly expressed in CRC patient tissues and correlated with poor clinical outcomes. a The GEO dataset GSE4017 indicates SLC2A3 expression levels in specimens from CRC patients and healthy donors. b The GEO dataset GSE32323 indicates SLC2A3 expression levels in CRC tissues and paired adjacent normal tissues. c TCGA datasets indicate SLC2A3 expression levels in paired adjacent normal tissues and tumour tissues from CRC patients. d SLC2A1-4 expression between paired normal tissues and tumour tissues from patients derived from the FUSCC database. e Immunohistochemical staining of CRC tumour tissues and paired normal tissue microarrays from patients at the FUSCC using anti-GLUT3 antibody. f Difference in SLC2A1-4 expression between patients who died within three years and patients with long-term survival in TCGA database. g Kaplan–Meier analysis of SLC2A3 expression (median as the cut-off point) and overall survival in data from TCGA database. h Difference in SLC2A3 expression between patients who died within three years and patients with long-term survival in the FUSCC database. i Kaplan–Meier analysis of SLC2A3 expression (median as the cut-off point) and overall survival in data from TCGA database. j Representative immunohistochemical staining for GLUT3 protein expression in CRC patients with different intensity. k–n Kaplan–Meier analysis of GLUT3 expression and overall survival in the overall FUSCC cohort (k), patients with stage I-III CRC (l), patients with stage II CRC (m), and patients with stage III CRC (n)

Fig. 2

Glucose utilization mediated by GLUT3 is required to promote CRC growth in vitro and in vivo. a Western blot showing short hairpin RNA-mediated deletion of SLC2A3 in HCT116 and SW620 cells. NC, nontarget control; KD, knockdown. b Glucose uptake by HCT116 and SW620 cells with or without SLC2A3 silencing. c Glucose-induced proliferation of HCT116 and SW620 cells with or without SLC2A3 depletion. d Analysis of colony formation abilities of HCT116 and SW620 cells with or without SLC2A3 depletion. e ¹³C-glucose uptake by HCT116 xenografts with or without SLC2A3 deletion. f, g Subcutaneous tumour growth, xenograft tumour images and tumour weights of xenografts from HCT116 cells with or without SLC2A3 silencing in nude mice

Fig. 3

Enhanced fructose utilization mediated by GLUT3 promotes CRC growth in vitro and in vivo. a, b RT-PCR (a) and western blot analysis (b) showing SLC2A3 expression in RKO and DLD1 cells transfected with the control PCDH retrovirus or PCDH-GLUT3 retrovirus. c Glucose uptake by RKO and DLD1 cells with or without ectopic GLUT3 expression. d Glucose-induced proliferation of RKO and DLD1 cells with or without ectopic GLUT3 expression. e Analysis of the colony formation abilities of RKO and DLD1 cells with or without ectopic GLUT3 expression. f ¹³C-glucose uptake by RKO xenografts with or without ectopic GLUT3 expression. g, h Subcutaneous tumour growth, xenograft tumour images and tumour weights of xenografts from RKO cells with or without ectopic GLUT3 expression in nude mice

Fig. 4

GLUT3 has higher impact than GLUT1 on CRC cell growth under low-glucose conditions. a Western blot showing CRISP-Cas9-mediated deletion of SLC2A1 and SLC2A3 in RKO and SW620 cells. NC, nontarget control; KO, knockout. b Glucose-induced proliferation of RKO and SW620 cells with/without SLC2A1 or SLC2A3 ablation. c Analysis of the colony formation abilities of RKO and SW620 cells with/without SLC2A1 or SLC2A3 ablation. d EdU staining for RKO and SW620 cells with/without SLC2A1 or SLC2A3 deletion. e Cell cycle distribution of RKO cells with/without SLC2A1 or SLC2A3 deletion. f Cell cycle distribution of SW620 cells with/without SLC2A1 or SLC2A3 deletion

Fig. 5

In vivo glucose utilization mediated by GLUT3 uptake preferentially fuels nucleotide synthesis. a The influence of impaired glucose utilization induced by SLC2A3 silencing on glucose-derived metabolite production and the synthesis of nucleotides in HCT116 xenografts. b The impact of enhanced glucose utilization induced by GLUT3 overexpression on glucose-derived metabolite generation and the synthesis of nucleotides of RKO xenografts. c–f Production of ¹³C-labelled metabolites derived from 13C-glucose in HCT116 xenografts with or without SLC2A3 silencing. g–j Generation of ¹³C-labelled metabolites derived from 13C-fructose in RKO xenografts with or without ectopic GLUT3 expression. k–l Analysis of the colony formation abilities of HCT116 (k) and SW620 (l) cells with or without SLC2A3 silencing during nucleoside rescue

Fig. 6

Glucose deficiency activates AMPK and induces GLUT3 expression in CRC cells via CREB1. a mRNA expression of SLC2A3 in RKO and DLD1 cells cultured in 1-mM glucose-containing medium for 0–24 h. b Activation of AMPK in RKO and DLD1 cells cultured in 1-mM glucose-containing medium for 0–24 h. c mRNA expression of SLC2A3 in RKO and DLD1 cells treated with or without the AMPK activator AICAR. d Western blot of CREB1, phosphorylated CREB1 and GLUT3 in RKO and DLD1 cells treated with or without the AMPK activator AICAR. e mRNA expression of SLC2A3 in RKO and HCT116 cells transfected with or without AMPK siRNA. f Western blot of CREB1, phosphorylated CREB1 and GLUT3 in RKO and DLD1 cells treated with or without AMPK siRNA. g Expression of GLUT3 in RKO and HCT116 cells with CREB1 silencing and in RKO and DLD1 cells with enhanced CREB1 expression. h Ectopic CREB1 expression in RKO and DLD1 cells increased the transcriptional activity of the entire SLC2A3 promoter. i ChIP results showing that CREB1 can occupy the genomic region of the SLC2A3 promoter. j The influence of ectopic CREB1 expression on glucose uptake by RKO and DLD1 cells. k The influence of ectopic CREB1 expression on the proliferation of RKO and DLD1 cells cultured in complete medium containing 1-mM glucose for 48 h

Fig. 7

High GLUT3 expression in CRC provides a therapeutic opportunity. a Analysis of the apoptosis of RKO cells with or with ectopic GLUT3 expression treated with L-OHP alone, vitamin C alone or the two agents combined. b, c Analysis of the colony formation ability of RKO cells with or with ectopic GLUT3 expression treated with L-OHP alone, vitamin C alone or the two agents combined. d Analysis of cell proliferation inhibition in RKO cells with or with ectopic GLUT3 expression treated with L-OHP alone, vitamin C alone or the two agents combined. e Analysis of the apoptosis of DLD1 cells with or with ectopic GLUT3 expression treated with L-OHP alone, vitamin C alone or the two agents combined. f, g Analysis of the colony formation ability of DLD1 cells with or with ectopic GLUT3 expression treated with L-OHP alone, vitamin C alone or the two agents combined. h Analysis of cell proliferation inhibition in DLD1 cells with or with ectopic GLUT3 expression treated with L-OHP alone, vitamin C alone or the two agents combined. i, j In vivo analysis of the treatment effects of L-OHP alone, vitamin C alone and the two agents combined on xenografts from RKO and DLD1 cells with or without enhanced GLUT3 expression. k Immunohistochemical staining for Ki67 in xenografts from RKO cells with or without enhanced GLUT3 expression in nude mice treated with normal saline, L-OHP alone, vitamin C alone or the two agents combined