The pleiotropic regulation of cyclin D1 by newly identified sesaminol-binding protein ANT2

M Watanabe,T Sakai,Y Iizumi,M Iizuka-Ohashi,Y Sowa,M Sukeno

doi:10.1038/oncsis.2017.10

Abstract

The expression of cyclin D1 is upregulated in various cancer cells by diverse mechanisms, such as increases in mRNA levels, the promotion of the translation by mammalian target of rapamycin complex 1 (mTORC1) signaling and the protein stabilization. We here show that sesaminol, a sesame lignan, reduces the expression of cyclin D1 with decreasing mRNA expression levels, inhibiting mTORC1 signaling and promoting proteasomal degradation. We subsequently generated sesaminol-immobilized FG beads to newly identify sesaminol-binding proteins. As a consequence, we found that adenine nucleotide translocase 2 (ANT2), the inner mitochondrial membrane protein, directly bound to sesaminol. Consistent with the effects of sesaminol, the depletion of ANT2 caused a reduction in cyclin D1 with decreases in its mRNA levels, mTORC1 inhibition and the proteasomal degradation of its protein, suggesting that sesaminol negatively regulates the function of ANT2. Furthermore, we screened other ANT2-binding compounds and found that the proliferator-activated receptor-γ agonist troglitazone also reduced cyclin D1 expression in a multifaceted manner, analogous to that of the sesaminol treatment and ANT2 depletion. Therefore, the chemical biology approach using magnetic FG beads employed in the present study revealed that sesaminol bound to ANT2, which may pleiotropically upregulate cyclin D1 expression at the mRNA level and protein level with mTORC1 activation and protein stabilization. These results suggest the potential of ANT2 as a target against cyclin D1-overexpressing cancers.

Highlights

Cyclin D1 is one of the most important cell cycle regulators in the G1 to S phases and is overexpressed in various cancers, such as breast cancer,[1,2] non-small-cell lung cancer,[1,3] melanoma,[4] pancreatic cancer[5] and colorectal cancer.[6]
In an attempt to elucidate the mechanisms by which sesaminol reduces the expression of cyclin D1 in a multifaceted manner, we newly identified the inner mitochondrial membrane protein, adenine nucleotide translocase 2 (ANT2), as a sesaminol direct binding protein using sesaminolimmobilized FG beads
DMSO-treated control. (c) Cyclin D1 and phosphorylated RB at serine 780 and serines 807/811 were analyzed by western blotting in MCF7 cells treated with sesaminol at the indicated concentrations for 24 h. β-Actin was used as a loading control. (d) Cyclin D1 and phosphorylated RB at serine 780 and serines 807/811 were analyzed by western blotting in MCF7 cells treated with 50 μM sesaminol for the indicated times. β-Actin was used as a loading control. (e) The IC50 values of normal human cell lines (MCF-10 A cells and WI-38 cells) and a variety of human cancer cell lines (MDA-MB-231 cells, SK-MEL-28 cells, A549 cells, RKO cells and MCF7 cells) treated with sesaminol were calculated

Summary

INTRODUCTION

Cyclin D1 is one of the most important cell cycle regulators in the G1 to S phases and is overexpressed in various cancers, such as breast cancer,[1,2] non-small-cell lung cancer,[1,3] melanoma,[4] pancreatic cancer[5] and colorectal cancer.[6]. Regarding the translational control of cyclin D1, mammalian target of rapamycin complex 1 (mTORC1) signaling is known to promote the translation of cyclin D1 mRNA.[17,18,19] The protein stability of cyclin D1 is mainly regulated via the ubiquitin proteasome system.[20,21,22] the expression of cyclin D1 is pleiotropically regulated in the progression of cancer. Our chemical biology approach revealed the noteworthy functions of the newly identified sesaminol-binding protein ANT2, which may pleiotropically upregulate cyclin D1 levels at the mRNA and protein levels with the activation of mTORC1 signaling and protein stabilization. ANT2 has potential as a target against cyclin D1-overexpressing cancers

RESULTS

DISCUSSION

MATERIALS AND METHODS