Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways
Yasuhito Onodera, Jin-Min Nam, Mina J. Bissell
Yasuhito Onodera, Jin-Min Nam, Mina J. Bissell
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Research Article Oncology

Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways

Abstract

There is a considerable resurgence of interest in the role of aerobic glycolysis in cancer; however, increased glycolysis is frequently viewed as a consequence of oncogenic events that drive malignant cell growth and survival. Here we provide evidence that increased glycolytic activation itself can be an oncogenic event in a physiologically relevant 3D culture model. Overexpression of glucose transporter type 3 (GLUT3) in nonmalignant human breast cells activated known oncogenic signaling pathways, including EGFR, β1 integrin, MEK, and AKT, leading to loss of tissue polarity and increased growth. Conversely, reduction of glucose uptake in malignant cells promoted the formation of organized and growth-arrested structures with basal polarity, and suppressed oncogenic pathways. Unexpectedly and importantly, we found that unlike reported literature, in 3D the differences between “normal” and malignant phenotypes could not be explained by HIF-1α/2α, AMPK, or mTOR pathways. Loss of epithelial integrity involved activation of RAP1 via exchange protein directly activated by cAMP (EPAC), involving also O-linked N-acetylglucosamine modification downstream of the hexosamine biosynthetic pathway. The former, in turn, was mediated by pyruvate kinase M2 (PKM2) interaction with soluble adenylyl cyclase. Our findings show that increased glucose uptake activates known oncogenic pathways to induce malignant phenotype, and provide possible targets for diagnosis and therapeutics.

Authors

Yasuhito Onodera, Jin-Min Nam, Mina J. Bissell

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Figure 8

HBP supports oncogenic signaling and malignant phenotype through O-GlcNAcylation.

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HBP supports oncogenic signaling and malignant phenotype through O-GlcNA...
(A) Western blot of GFPT and OGT in S1 cells, T4-2 cells, and T4-2 cells reverted with 2DG or different signaling inhibitors (see Figure 1A). The same lamin A/C blots also served as a control for Figure 1, C and E. (B–H) T4-2 cells were cultured in 3D lrECM with or without inhibitors of GFPT (20 μM AZS or DON), O-GlcNAc transferase (5 mM BADGP), or N-glycosylation (10 ng/ml TM). (B) Confocal IF images. Green, α6 integrin; red, nuclei. Scale bars: 20 μm. (C) Cell number at the colony midsection (black bars) and EdU incorporation per cell (white bars). (D) Percent colonies with basal polarity. (E) Glucose uptake (black bars) and lactate release (white bars). (F) Relative oxygen consumption rate. (G and H) Western blot of signaling intermediates (G) and GLUT3 and metabolic enzymes (H). Lamin A/C served as a control for both G and H. (I) mRNA expression of EGFR, ITGB1, and SLC2A3 in S1 cells, T4-2 cells, and T4-2 cells reverted with metabolic inhibitors. Expression level of each gene was normalized to 18S ribosomal RNA. (J) Total O-GlcNAc level in S1 cells, T4-2 cells, and T4-2 cells reverted with DON, BADGP, or low glucose (low gluc; 0.175 mM). Asterisks denote O-GlcNAc bands specifically increased in T4-2 cells. In C–F and I, data are mean ± SD of triplicate experiments.