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Membrane protein CNNM4–dependent Mg2+ efflux suppresses tumor progression
Yosuke Funato, … , Kazuya Kikuchi, Hiroaki Miki
Yosuke Funato, … , Kazuya Kikuchi, Hiroaki Miki
Published December 1, 2014; First published October 27, 2014
Citation Information: J Clin Invest. 2014;124(12):5398-5410. https://doi.org/10.1172/JCI76614.
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Categories: Research Article Oncology

Membrane protein CNNM4–dependent Mg2+ efflux suppresses tumor progression

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Abstract

Intracellular Mg2+ levels are strictly regulated; however, the biological importance of intracellular Mg2+ levels and the pathways that regulate them remain poorly understood. Here, we determined that intracellular Mg2+ is important in regulating both energy metabolism and tumor progression. We determined that CNNM4, a membrane protein that stimulates Mg2+ efflux, binds phosphatase of regenerating liver (PRL), which is frequently overexpressed in malignant human cancers. Biochemical analyses of cultured cells revealed that PRL prevents CNNM4-dependent Mg2+ efflux and that regulation of intracellular Mg2+ levels by PRL and CNNM4 is linked to energy metabolism and AMPK/mTOR signaling. Indeed, treatment with the clinically available mTOR inhibitor rapamycin suppressed the growth of cancer cells in which PRL was overexpressed. In ApcΔ14/+ mice, which spontaneously form benign polyps in the intestine, deletion of Cnnm4 promoted malignant progression of intestinal polyps to adenocarcinomas. IHC analyses of tissues from patients with colon cancer demonstrated an inverse relationship between CNNM4 expression and colon cancer malignancy. Together, these results indicate that CNNM4-dependent Mg2+ efflux suppresses tumor progression by regulating energy metabolism.

Authors

Yosuke Funato, Daisuke Yamazaki, Shin Mizukami, Lisa Du, Kazuya Kikuchi, Hiroaki Miki

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Figure 1

Identification of CNNM4 as a PRL-interacting protein.

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Identification of CNNM4 as a PRL-interacting protein.
(A) Lysates of SW4...
(A) Lysates of SW480 cells stably expressing FLAG-PRL1 or GFP (control) were subjected to IP with anti-FLAG antibody. The precipitated proteins were separated by SDS-PAGE and visualized by silver staining. The band indicated by an arrow was excised, and the trypsin-digested peptides were subjected to mass spectrometry. (B) Lysates of COS7 cells transfected with CNNM4-Myc and either FLAG-GFP (G) or FLAG-PRL1/2/3 were subjected to IP and immunoblot analyses with the indicated antibodies. (C) COS7 cells transfected with the indicated constructs were treated with H2O2 (10 minutes), and their lysates were subjected to IP and immunoblot analyses under both reducing and nonreducing conditions. (D) HEK293 cells were treated with H2O2 (200 μM, 10 minutes), and the lysates were subjected to IP and immunoblot analyses. (E) HEK293 cells were transfected with control or PRL1 siRNA (#1), and the lysates were subjected to IP and immunoblot analyses. (F) Recombinant GST or GST-PRL3 proteins immobilized on beads were mixed with purified recombinant CNNM4 proteins under reduced (with 5 mM DTT) or oxidized (with 100 or 1,000 μM H2O2) conditions. The proteins were separated by SDS-PAGE and visualized by Coomassie staining. Representative immunoblots/gels from 3 independent experiments are shown in each panel. Mr, relative molecular mass.
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