S-phase kinase-associated protein 2 positively controls mitotic arrest deficient 2 in lung cancer cells
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Abstract
Background. Mitotic arrest deficient 2 (Mad2) is a key component of spindle assembly checkpoint and overexpressed in human lung cancers, but the mechanism of the deregulation of Mad2 in lung cancer is largely unknown. We aim to investigate the regulation of Mad2 by S-phase kinase-associated protein 2 (Skp2) in human lung cancer cells.
Methods. Human lung cancer A549 and NCI-H1975 cells were transfected with MAD2 and SKP2 siRNAs or plasmids to silence or overexpress MAD2 and SKP2. Flavopiridol and HLM006474 were used to inhibit cyclin dependent kinases (CDKs) and E2F1, respectively. mRNA and protein levels were determined by real-time PCR and Western blot, respectively. Cell cycle progression was assayed by flow cytometery.
Results. Knockdown of Skp2 by siRNA decreased Mad2 mRNA and protein levels in A549 and NCI-H1299 cells, accompanied with upregulation of p27 but decrease of the phosphorylation of retinoblastoma (Rb). In contrast, ectopic overexpression of Skp2 increased Mad2 mRNA and protein levels and phosphorylation of Rb, while decreased p27. Pharmacological inhibition of CDK1/2 by flavopiridol or E2F1 with HLM006474 led to downregulation of Mad2 expression, and prevented the increase of Mad2 expression by Skp2. Accordingly, silencing of either Mad2 or Skp2 impaired the mitosis arrest in response to nocadazole.
Conclusion. SKP2 positively regulates the gene expression of MAD2 through p27-CDKs-E2F1 signaling pathway, suggesting that deregulation of Skp2 may lead to upregulation of Mad2 via enhancing the activity of CDKs in human lung cancers. Our findings may provide an explanation of the simultaneous upregulation of MAD2 and SKP2 in lung cancer and potential targets for the development of molecular targeted therapy for lung cancers.
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2016. S-phase kinase-associated protein 2 positively controls mitotic arrest deficient 2 in lung cancer cells. PeerJ Preprints 4:e2298v1 https://doi.org/10.7287/peerj.preprints.2298v1Author comment
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Supplemental Information
The raw data of the MAD2 mRNA detected by qRT-PCR
Additional Information
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Tonghai Huang conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Lin Yang conceived and designed the experiments, performed the experiments, analyzed the data, reviewed drafts of the paper.
Guangsuo Wang performed the experiments, reviewed drafts of the paper.
Guanggui Ding analyzed the data, reviewed drafts of the paper.
Bin Peng analyzed the data, reviewed drafts of the paper.
Yuxin Wen analyzed the data, reviewed drafts of the paper.
Zheng Wang conceived and designed the experiments, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Data Deposition
The following information was supplied regarding data availability:
The raw data has been supplied as a supplementary file.
Funding
The authors received no funding for this work.