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A specific anti-cyclin D1 intrabody represses breast cancer cell proliferation by interrupting the cyclin D1–CDK4 interaction

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Abstract

Background

Cyclin D1 overexpression may contribute to development of various cancers, including breast cancer, and thus may serve as a key cancer diagnostic marker and therapeutic target. In our previous study, we generated a cyclin D1-specific single-chain variable fragment antibody (ADκ) from a human semi-synthetic single-chain variable fragment library. ADκ specifically interacted with recombinant and endogenous cyclin D1 proteins through an unknown molecular basis to inhibit HepG2 cell growth and proliferation.

Results

Here, using phage display and in silico protein structure modeling methods combined with cyclin D1 mutational analysis, key residues that bind to ADκ were identified. Notably, residue K112 within the cyclin box was required for cyclin D1–ADκ binding. In order to elucidate the molecular mechanism underlying ADκ anti-tumor effects, a cyclin D1-specific nuclear localization signal-containing intrabody (NLS-ADκ) was constructed. When expressed within cells, NLS-ADκ interacted specifically with cyclin D1 to significantly inhibit cell proliferation, induce G1-phase arrest, and trigger apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. Moreover, the NLS–ADκ–cyclin D1 interaction blocked binding of cyclin D1 to CDK4 and inhibited RB protein phosphorylation, resulting in altered expression of downstream cell proliferation-related target genes.

Conclusion

We identified amino acid residues in cyclin D1 that may play key roles in the ADκ–cyclin D1 interaction. A nuclear localization antibody against cyclin D1 (NLS-ADκ) was constructed and successfully expressed in breast cancer cells. NLS-ADκ exerted tumor suppressor effects via blocking the binding of CDK4 to cyclin D1 and inhibiting phosphorylation of RB. The results presented here demonstrate anti-tumor potential of intrabody-based cyclin D1-targeted breast cancer therapy.

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Abbreviations

scFv:

Single-chain variable fragment

NLS:

Nuclear localization signal

E. coli :

Escherichia coli

RB:

Retinoblastoma tumor suppressor protein

CDK4:

Cyclin-dependent kinase 4

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Acknowledgements

We thank Dr. Ye Sun of the School of Life Sciences, Jilin University, Changchun, China, for his assistance and suggestion with the molecular model.

Funding

This work was supported by the Natural Science Foundation of Jilin Province, China (Grant No. 20210101003JC) and the National Natural Science Foundation of China (Grant Nos. 31570934, 31170882, 81903102).

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Author notes

  1. Jialiang Zhao and Yan Wu have equally contributed to this work.

Authors and Affiliations

  1. Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, 130012, China

    Jialiang Zhao, Yan Wu, Tong Xiao, Cheng Cheng, Tong Zhang, Ziyang Gao, Siyuan Hu, Ze Ren, Xinze Yu, Fang Yang & Guiying Li

  2. Medical Research Center, Binzhou Medical University Hospital, Binzhou, 256600, China

    Yan Wu

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  1. Jialiang Zhao
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Contributions

JZ and YW designed and performed all experiments and various analyses. TX, CC, and TZ performed cell experiments, data collection, and analysis. ZG and SH constructed and extracted plasmids and performed data collection and analysis. ZR and XY performed protein preparation. JZ, YW, FY, and GL designed the experiments, interpreted the data, and wrote the manuscript. GL contributed to the study conception and design, supervised the study, and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Fang Yang or Guiying Li.

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Zhao, J., Wu, Y., Xiao, T. et al. A specific anti-cyclin D1 intrabody represses breast cancer cell proliferation by interrupting the cyclin D1–CDK4 interaction. Breast Cancer Res Treat 198, 555–568 (2023). https://doi.org/10.1007/s10549-023-06866-7

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