Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis
Anju Singh, … , Geoffrey Girnun, Shyam Biswal
Anju Singh, … , Geoffrey Girnun, Shyam Biswal
Published June 10, 2013
Citation Information: J Clin Invest. 2013;123(7):2921-2934. https://doi.org/10.1172/JCI66353.
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Research Article Oncology

Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis

Abstract

The mechanisms by which deregulated nuclear factor erythroid-2–related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and 13C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.

Authors

Anju Singh, Christine Happel, Soumen K. Manna, George Acquaah-Mensah, Julian Carrerero, Sarvesh Kumar, Poonam Nasipuri, Kristopher W. Krausz, Nobunao Wakabayashi, Ruby Dewi, Laszlo G. Boros, Frank J. Gonzalez, Edward Gabrielson, Kwok K. Wong, Geoffrey Girnun, Shyam Biswal

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Figure 1

NRF2 reprograms glucose metabolism to promote tumor growth.

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NRF2 reprograms glucose metabolism to promote tumor growth. (A) NRF2 act...
(A) NRF2 activity is essential for the growth of KEAP1-deficient cancer cells. A549 (KEAP1G333C) cells stably transduced with doxycycline-inducible Nrf2 shRNA were implanted into nude mice. Two weeks after tumor implantation, mice were randomly allocated to 2 groups (n = 13–15 per group) and administered vehicle or doxycycline in the drinking water. Tumor volume was recorded. Data are presented as the mean tumor volume ± SEM. *P < 0.05 relative to the vehicle-treated group. (B) Possible 13C (red circles) labeling in the intermediates of the pentose cycle, lactate, and glutamate using [1,2-13C2] glucose as the single tracer. (C) Comparison of 13CO2 production by A549-Luc shRNA and A549-Nrf2 shRNA cultures. *P < 0.0000. (D) Decrease in 13C-labeled extracellular lactate in the culture medium from A549-Nrf2 shRNA cells. *P < 0.004. (E) Pentose cycle activity relative to glycolysis measured by m1/m2 ratios in lactate in the media. (F) Determination of C2-labeled through C5-labeled glutamate levels indicating substrate (OAA and acetyl CoA) entry into the TCA cycle. *P = 0.006 relative to the cells expressing Luc shRNA. (G) Reduced PDH flux in A549-Nrf2 shRNA cells indicating a significant decrease in the TCA cycle flux. *P < 0.0001 relative to cells expressing Luc shRNA. (H) Comparison of 14C-labeled RNA ribose fraction in Luc shRNA and Nrf2 shRNA cells. *P < 0.05. (I) Relative 13CO2 production by MEF cells. Keap1–/– cells demonstrated a significantly higher glucose oxidation rate. *P < 0.00 relative to WT. (J) Relative RNA ribose synthesis (mRNA and rRNA) through direct glucose oxidation and the nonoxidative steps of the pentose cycle in MEF cells. *P < 0.01 relative to WT.