Geminin facilitates FoxO3 deacetylation to promote breast cancer cell metastasis
Lei Zhang, … , Y. Eugene Chin, Han You
Lei Zhang, … , Y. Eugene Chin, Han You
Published April 24, 2017
Citation Information: J Clin Invest. 2017;127(6):2159-2175. https://doi.org/10.1172/JCI90077.
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Research Article Cell biology Oncology

Geminin facilitates FoxO3 deacetylation to promote breast cancer cell metastasis

Abstract

Geminin expression is essential for embryonic development and the maintenance of chromosomal integrity. In spite of this protective role, geminin is also frequently overexpressed in human cancers and the molecular mechanisms underlying its role in tumor progression remain unclear. The histone deacetylase HDAC3 modulates transcription factors to activate or suppress transcription. Little is known about how HDAC3 specifies substrates for modulation among highly homologous transcription factor family members. Here, we have demonstrated that geminin selectively couples the transcription factor forkhead box O3 (FoxO3) to HDAC3, thereby specifically facilitating FoxO3 deacetylation. We determined that geminin–associated HDAC3 deacetylates FoxO3 to block its transcriptional activity, leading to downregulation of the downstream FoxO3 target Dicer, an RNase that suppresses metastasis. Breast cancer cells depleted of geminin or HDAC3 exhibited poor metastatic potential that was attributed to reduced suppression of the FoxO3-Dicer axis. Moreover, elevated levels of geminin, HDAC3, or both together with decreased FoxO3 acetylation and reduced Dicer expression were detected in aggressive human breast cancer specimens. These results underscore a prominent role for geminin in promoting breast cancer metastasis via the enzyme-substrate–coupling mechanism in HDAC3-FoxO3 complex formation.

Authors

Lei Zhang, Meizhen Cai, Zhicheng Gong, Bingchang Zhang, Yuanpei Li, Li Guan, Xiaonan Hou, Qing Li, Gang Liu, Zengfu Xue, Muh-hua Yang, Jing Ye, Y. Eugene Chin, Han You

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Figure 4

Geminin inhibits FoxO3 via recruitment of HDAC3 to deacetylate FoxO3.

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Geminin inhibits FoxO3 via recruitment of HDAC3 to deacetylate ...
(A) LM2 cells infected with the indicated lentiviral constructs were lysed and subjected to immunoprecipitation with FoxO3 or FoxO1 antibodies. The immunoprecipitates were then blotted with anti–Ac-Lys, anti-FoxO3, anti–Ac-FoxO, and anti-FoxO1 (left panel). Total lysates were analyzed by Western blotting (right panel). (B) Two-step coimmunoprecipitation identifies a FoxO3-geminin-HDAC3 complex. LM2 cell lysates were first precipitated with anti-geminin and eluted with the polypeptide against which the geminin antibody was raised. Elutes were then immunoprecipitated with anti‑HDAC3 or control IgG. Protein samples from each step were analyzed by immunoblotting with the indicated antibodies. (C) Geminin-depleted LM2 cells were reconstituted with the indicated geminin constructs. Cell lysates were subjected to immunoprecipitation with FoxO3 antibody. The immunoprecipitates were then blotted with the indicated antibodies. (D) FoxO3-depleted LM2 cells infected with lentivirus encoding the indicated shRNAs were lysed, and lysates were subjected to immunoprecipitation with anti-FLAG. The immunoprecipitates were then blotted with the indicated antibodies. (E) ChIP to analyze the occupancy of FLAG-FoxO3 (WT, 5KQ, 5KR) on Dicer promoter in LM2 cells. n = 3 independent experiments. ‡P < 0.001; **P < 0.01, 2-way ANOVA with Bonferroni’s post hoc test. Vec, vector. (F and G) LM2 cells were infected with the indicated lentiviral constructs. Cell lysates were extracted and subjected to Western blotting. (H–J) ChIP analysis for the presence of FoxO3 (H), GEM (I), or HDAC3 (J) at the Dicer promoter in LM2 cells with or without FoxO3 depletion.